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Sample records for two-photon fluorescence microscope

  1. Multiphoton microscopic imaging of adipose tissue based on second-harmonic generation and two-photon excited fluorescence.

    Science.gov (United States)

    Huang, Zufang; Zhuo, Shuangmu; Chen, Jianxin; Chen, Rong; Jiang, Xingshan

    2008-01-01

    The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.

  2. Deep two-photon microscopic imaging through brain tissue using the second singlet state from fluorescent agent chlorophyll α in spinach leaf.

    Science.gov (United States)

    Shi, Lingyan; Rodríguez-Contreras, Adrián; Budansky, Yury; Pu, Yang; Nguyen, Thien An; Alfano, Robert R

    2014-06-01

    Two-photon (2P) excitation of the second singlet (S₂) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the "tissue optical window" (650 to 950 nm). S₂ state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach leaf under a thick layer of freshly sliced rat brain tissue in combination with 2P microscopic imaging. Strong emission at the peak wavelength of 685 nm under the 2P S₂ state of Chl α enabled the imaging depth up to 450 μm through rat brain tissue.

  3. MULTIPHOTON MICROSCOPIC IMAGING OF MOUSE INTESTINAL MUCOSA BASED ON TWO-PHOTON EXCITED FLUORESCENCE AND SECOND HARMONIC GENERATION

    Directory of Open Access Journals (Sweden)

    REN'AN XU

    2013-01-01

    Full Text Available Multiphoton microscopy (MPM, based on two-photon excited fluorescence and second harmonic generation, enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents. In this paper, we used MPM to image the microstructures of the mucosa in fresh, unfixed, and unstained intestinal tissue of mouse. The morphology and distribution of the main components in mucosa layer such as columnar cells, goblet cells, intestinal glands, and a little collagen fibers were clearly observed in MPM images, and then compared with standard H&E images from paired specimens. Our results indicate that MPM combined with endoscopy and miniaturization probes has the potential application in the clinical diagnosis and in vivo monitoring of early intestinal cancer.

  4. Microscopic imaging of glyceraldehyde-induced tissue glycation with intrinsic second harmonic generation and two-photon fluorescence contrasts

    Science.gov (United States)

    Hwang, Yu Jer; Granelli, Joseph; Tirumalasetty, Manasa; Lyubovitsky, Julia

    2013-02-01

    The bioinspired approaches to tissue strengthening and preservation rely on non-toxic cross-linking agents one of which is glyceraldehyde. In this study we used multiphoton microscopy that employs second harmonic generation (SHG) contrast to evaluate collagen microstructures and two-photon fluorescence (TPF) contrast to monitor progression of cross-linking upon treatment of tissues with glyceraldehyde. We examined collagen hydrogels assembled at 37 °C and 27 °C, bovine scleral and corneal tissues, skin as well as rat tail tendons. The results show a different effect of glyceraldehyde on collagen microstructures within the above tissues. This effect depends on the original microstructural assembly of collagen within a specific tissue. Our data suggests that epidermis (in skin and cornea) will protect collagen from cross-linking with glyceraldehyde. The work highlights benefits of monitoring progression of collagen cross-linking and effects of cross-linking on fiber microstructures as imaged with SHG and TPF signals.

  5. Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

    2016-03-01

    Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

  6. A new approach to dual-color two-photon microscopy with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Rebane Aleks

    2010-02-01

    Full Text Available Abstract Background Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA efficiency. Results Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. Conclusion Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

  7. [Intensity loss of two-photon excitation fluorescence microscopy images of mouse oocyte chromosomes].

    Science.gov (United States)

    Zhao, Feng-Ying; Wu, Hong-Xin; Chen, Die-Yan; Ma, Wan-Yun

    2014-07-01

    As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed. Mouse oocytes with a large diameter, which play an important role in clinical and biological fields, have obvious absorption and scattering effects. In the present paper, we performed compensation for two-photon fluorescence images of mouse oocyte chromosomes. Using volume as a parameter, the attenuation degree of these chromosomes was also studied. The result of our data suggested that there exists a severe axial intensity loss in two-photon microscopic images of mouse oocytes due to the absorption and scattering effects. It is necessary to make compensation for these images of mouse oocyte chromosomes obtained from two-photon microscopic system. It will be specially needed in studying the quantitative three-dimensional information of mouse oocytes.

  8. A fluorescent benzothiazole probe with efficient two-photon absorption

    Science.gov (United States)

    Echevarria, Lorenzo; Moreno, Iván; Camacho, José; Salazar, Mary Carmen; Hernández, Antonio

    2012-11-01

    In this work, we report the two-photon absorption of 2-[4-(dimethylamino)phenyl]-1,3-benzothiazole-6-carbonitrile (DBC) in DMSO solution pumping at 779 nm with a 10 ns pulse laser-Nd:YAG system. The obtained two-photon absorption cross-section in DBC (407 ± 18 GM) is considerably high. Because DBC is a novel compound and have high values of fluorescence quantum yield, this result is expected to have an impact in biomolecules detection, diagnosis and treatment of cancer. Similar structures have previously been reported to show remarkable antitumour effects.

  9. Two-photon excited fluorescence microendoscopic imaging using a GRIN lens

    Science.gov (United States)

    Yan, Wei; Peng, Xiao; Lin, Danying; Wang, Qi; Gao, Jian; Zhou, Jie; Ye, Tong; Qu, Junle; Niu, Hanben

    2015-03-01

    With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in traditional fluorescence microscopes have limited the imaging applications mostly to superficial tissues. To overcome this disadvantage, researchers have developed the graded-index (GRIN) probes with small diameters for imaging internal organs of small animals in a minimally invasive fashion. Here, we present the development of a fluorescence endoscopic imaging system based on a GRIN lens using two-photon excitation. Experimental results showed that this system could perform dynamic fluorescence microendoscopic imaging and monitor the blood flow in anesthetized living mice using two-photon excitation.

  10. [Two-photon excitation fluorescence of 5-ALA induced PpIX in DHL cells].

    Science.gov (United States)

    Huang, Zu-Fang; Chen, Rong; Li, Yong-Zeng; Chen, Guan-Nan; Chen, Xian-Ling; Feng, Shang-Yuan; Jia, Pei-Min

    2008-11-01

    Two-photon fluorescence microscopy is a novel imaging technique, which is primarily sensitive to a specimen's response coming from an in-focus plane, thus has low photo-bleaching and photo-damage to biological samples. 5-ALA induced production of PpIX in DHL cells was excited by 820 nm femtosecond laser; two-photon excitation fluorescence of single cell was obtained in Lambda mode of laser scanning confocal microscope. The specific fluorescence intensity of PpIX which accumulated in DHL cells was measured at 2, 4 and 10 mmol x L(-1) concentration of 5-ALA with different incubation time, which reflected the kinetics of 5-ALA accumulated in DHL cells. Accumulation of PpIX in DHL cells was a dynamic change process. Biphasic alterations of PpIX accumulation were noted: PpIX content enhanced with the increasing time and reached the maximal value around 3 h, however PpIX content decreased in the subsequent incubation time. Results indicate that two-photon fluorescence based on laser scanning microscope can be a useful technology for studying the kinetics of 5-ALA induced PpIX production in DHL cells and other leukemia cells.

  11. Rational Design of Fluorescent Phthalazinone Derivatives for One- and Two-Photon Imaging.

    Science.gov (United States)

    Yang, Lingfei; Zhu, Yuanjun; Shui, Mengyang; Zhou, Tongliang; Cai, Yuanbo; Wang, Wei; Xu, Fengrong; Niu, Yan; Wang, Chao; Zhang, Jun-Long; Xu, Ping; Yuan, Lan; Liang, Lei

    2016-08-22

    Phthalazinone derivatives were designed as optical probes for one- and two-photon fluorescence microscopy imaging. The design strategy involves stepwise extension and modification of pyridazinone by 1) expansion of pyridazinone to phthalazinone, a larger conjugated system, as the electron acceptor, 2) coupling of electron-donating aromatic groups such as N,N-diethylaminophenyl, thienyl, naphthyl, and quinolyl to the phthalazinone, and 3) anchoring of an alkyl chain to the phthalazinone with various terminal substituents such as triphenylphosphonio, morpholino, triethylammonio, N-methylimidazolio, pyrrolidino, and piperidino. Theoretical calculations were utilized to verify the initial design. The desired fluorescent probes were synthesized by two different routes in considerable yields. Twenty-two phthalazinone derivatives were synthesized and their photophysical properties were measured. Selected compounds were applied in cell imaging, and valuable information was obtained. Furthermore, the designed compounds showed excellent performance in two-photon microscopic imaging of mouse brain slices.

  12. Development of a two photon microscope for tracking Drosophila larvae

    Science.gov (United States)

    Karagyozov, Doycho; Mihovilovic Skanata, Mirna; Gershow, Marc

    Current in vivo methods for measuring neural activity in Drosophila larva require immobilization of the animal. Although we can record neural signals while stimulating the sensory organs, we cannot read the behavioral output because we have prevented the animal from moving. Many research questions cannot be answered without observation of neural activity in behaving (freely-moving) animals. Our project aims to develop a tracking microscope that maintains the neurons of interest in the field of view and in focus during the rapid three dimensional motion of a free larva.

  13. Plasmonic-enhanced two-photon fluorescence with single gold nanoshell

    Science.gov (United States)

    Zhang, TianYue; Lu, GuoWei; Shen, HongMing; Perriat, P.; Martini, M.; Tillement, O.; Gong, QiHuang

    2014-06-01

    Single gold nanoshell with mutilpolar plasmon resonances is proposed to enhance two-photon fluorescence efficiently. The single emitter single nanoshell configuration is studied systematically by employing the finite-difference time-domain method. The emitter located inside or outside the nanoshell at various positions leads to a significantly different enhancement effect. The fluorescent emitter placed outside the nanoshell can achieve large fluorescence intensity given that both the position and orientation of the emission dipole are optimally controlled. In contrast, for the case of the emitter placed inside the nanoshell, it can experience substantial two-photon fluorescence enhancement without strict requirements upon the position and dipole orientations. Metallic nanoshell encapsulating many fluorescent emitters should be a promising nanocomposite configuration for bright two-photon fluorescence label. The results provide a comprehensive understanding about the plasmonic-enhanced two-photon fluorescence behaviors, and the nanocomposite configuration has great potential for optical detecting, imaging and sensing in biological applications.

  14. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-01

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2.

  15. Development and design of advanced two-photon microscope used in neuroscience

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    Doronin, M. S.; Popov, A. V.

    2016-08-01

    This work represents the real steps to development and design advanced two-photon microscope by efforts of laboratory staff. Self-developed microscopy system provides possibility to service it and modify the structure of microscope depending on highly specialized experimental design and scientific goals. We are presenting here module-based microscopy system which provides an opportunity to looking for new applications of this setup depending on laboratories needs using with galvo and resonant scanners.

  16. Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

    Science.gov (United States)

    Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

    2017-07-01

    The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

  17. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins.

    OpenAIRE

    2000-01-01

    We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotr...

  18. Two-photon fluorescence and confocal reflected light imaging of thick tissue structures

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    Kim, Ki H.; So, Peter T. C.; Kochevar, Irene E.; Masters, Barry R.; Gratton, Enrico

    1998-04-01

    The technology of two-photon excitation has opened a window of opportunity for developing non-invasive medical diagnostic tools capable of monitoring thick tissue biochemical states. Using cellular endogenous chromophores, (beta) -nicotinamide- adenine dinucleotide phosphate [NAD(P)H], the cellular metabolic rates in living human skin were determined. Although important functional information can be obtained from the fluorescence spectroscopy of endogenous chromophores, these chromophores are rather poor contrast enhancing agent for mapping cellular morphology. First, most endogenous chromophores are confined to the cellular cytoplasm which prevents the visualization of other cellular organelles. Second, there is significant variability in the distribution and the quantum yield of endogenous chromophores which depends on tissue biochemistry but prevents consistent comparison of cellular morphology. On the other hand, the deep tissue cellular morphology has been imaged with excellent resolution using reflected light confocal microscopy. In reflected light microscopy, the image contrast originates from the index of refraction differences of the cellular structures. The organelle boundaries with significant index differences such as the plasma membrane and the nucleus envelope can be consistently visualized. A combination of morphological and functional information is required for a thorough tissue study. This presentation describes the development of a new microscope which is capable of simultaneously collecting both two-photon fluorescence and confocal reflected light signals. Promising biomedical applications include the non-invasive diagnosis of skin cancer and the study of wound healing.

  19. Two-photon fluorescence probes for imaging of mitochondria and lysosomes.

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    Yang, Wanggui; Chan, Pui Shan; Chan, Miu Shan; Li, King Fai; Lo, Pik Kwan; Mak, Nai Ki; Cheah, Kok Wai; Wong, Man Shing

    2013-04-28

    Novel biocompatible cyanines show not only a very large two-photon cross-section of up to 5130 GM at 910 nm in aqueous medium for high-contrast and -brightness two-photon fluorescence live cell imaging but also highly selective subcellular localization properties including localization of mitochondria and lysosomes.

  20. Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy.

    Science.gov (United States)

    Jiang, Xingshan; Zhong, Jiazhao; Liu, Yuchun; Yu, Haibo; Zhuo, Shuangmu; Chen, Jianxin

    2011-01-01

    Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging. Copyright © 2011 Wiley Periodicals, Inc.

  1. Two-photon fluorescence imaging and femtosecond laser microsurgery to study drosophila dorsal closure

    Science.gov (United States)

    Thayil K. N., Anisha; Pereira, Andrea; Mathew, Manoj; Artigas, David; Martín Blanco, Enrique; Loza-Alvarez, Pablo

    2008-02-01

    Dorsal closure is a key morphogenic process that occurs at the last stages of Drosophila melanogaster embryogenesis. It involves a well coordinated rearrangement and movement of tissues that resemble epithelial wound healing in mammals. The cell dynamics and intracellular signaling pathways that accompany hole closure are expected to be similar during would healing providing a model system to study epithelial healing. Here we demonstrate the use of two-photon fluorescence microscope together with femtosecond laser ablation to examine the epithelial wound healing during embryonic dorsal closure. By using tightly focused NIR femtosecond pulses of subnanojoule energy we are able to produce highly confined microsurgery on the epithelial cells of a developing embryo. We observed that drosophila epidermis heals from the laser wounds with increased activity of actin near the wound edges.

  2. Quantitative optical biomarkers of lung cancer based intrinsic two-photon excited fluorescence signal

    Science.gov (United States)

    Li, Jingwen; Zhan, Zhenlin; Lin, Hongxin; Zuo, Ning; Zhu, Xiaoqin; Xie, Shusen; Chen, Jianxin; Zhuo, Shuangmu

    2016-10-01

    Alterations in the elastic fibers have been implicated in lung cancer. However, the label-free, microscopic imaging of elastic fibers in situ remains a major challenge. Here, we present the use of intrinsic two-photon excited fluorescence (TPEF) signal as a novel means for quantification of the elastic fibers in intact fresh human lung tissues. We obtained the TPEF images of elastic fibers from ex vivo the human lung tissues. We found that three features, including the elastic fibers area, the elastic fibers orientation, the elastic fibers structure, provide the quantitative identification of lung cancer and the direct visual cues for cancer versus non-cancer areas. These results suggest that the TPEF signal can be used as the label-free optical biomarkers for rapid clinical lung diagnosis and instant image-guided surgery.

  3. Cyanines as new fluorescent probes for DNA detection and two-photon excited bioimaging.

    Science.gov (United States)

    Feng, Xin Jiang; Wu, Po Lam; Bolze, Frédéric; Leung, Heidi W C; Li, King Fai; Mak, Nai Ki; Kwong, Daniel W J; Nicoud, Jean-François; Cheah, Kok Wai; Wong, Man Shing

    2010-05-21

    A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized, among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.

  4. Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

    NARCIS (Netherlands)

    Broess, K.; Borst, J.W.; Amerongen, van H.

    2009-01-01

    This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of

  5. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.

    Science.gov (United States)

    Day, Richard N; Tao, Wen; Dunn, Kenneth W

    2016-11-01

    Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.

  6. Two-photon STED spectral determination for a new V-shaped organic fluorescent probe with efficient two-photon absorption.

    Science.gov (United States)

    Belfield, Kevin D; Bondar, Mykhailo V; Morales, Alma R; Padilha, Lazaro A; Przhonska, Olga V; Wang, Xuhua

    2011-10-24

    Two-photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two-photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two-photon absorption (2PA), and stimulated emission properties of a new fluorene-based compound, (E)-2-{3-[2-(7-(diphenylamino)-9,9-diethyl-9H-fluoren-2-yl)vinyl]-5-methyl-4-oxocyclohexa-2,5-dienylidene} malononitrile (1), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two-photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open-aperture Z-scan and relative two-photon-induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ~1700 GM was observed in the main, long wavelength, one-photon absorption band. One- and two-photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump-probe technique, resulting in relatively high two-photon stimulated emission depletion cross sections (~1200 GM). A potential application of 1 in bioimaging was demonstrated through one- and two-photon fluorescence microscopy images of HCT 116 cells incubated with micelle-encapsulated dye.

  7. Monitoring photosensitizer uptake using two photon fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Yeh, Shu-Chi Allison; Diamond, Kevin R; Patterson, Michael S; Nie, Zhaojun; Hayward, Joseph E; Fang, Qiyin

    2012-01-01

    Photodynamic Therapy (PDT) provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin(®) at various intracellular components in the Mat-LyLu (MLL) cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin(®) was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns) compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05).

  8. Monitoring Photosensitizer Uptake Using Two Photon Fluorescence Lifetime Imaging Microscopy

    Directory of Open Access Journals (Sweden)

    Shu-Chi Allison Yeh, Kevin R. Diamond, Michael S. Patterson, Zhaojun Nie, Joseph E. Hayward, Qiyin Fang

    2012-01-01

    Full Text Available Photodynamic Therapy (PDT provides an opportunity for treatment of various invasive tumors by the use of a cancer targeting photosensitizing agent and light of specific wavelengths. However, real-time monitoring of drug localization is desirable because the induction of the phototoxic effect relies on interplay between the dosage of localized drug and light. Fluorescence emission in PDT may be used to monitor the uptake process but fluorescence intensity is subject to variability due to scattering and absorption; the addition of fluorescence lifetime may be beneficial to probe site-specific drug-molecular interactions and cell damage. We investigated the fluorescence lifetime changes of Photofrin® at various intracellular components in the Mat-LyLu (MLL cell line. The fluorescence decays were analyzed using a bi-exponential model, followed by segmentation analysis of lifetime parameters. When Photofrin® was localized at the cell membrane, the slow lifetime component was found to be significantly shorter (4.3 ± 0.5 ns compared to those at other locations (cytoplasm: 7.3 ± 0.3 ns; mitochondria: 7.0 ± 0.2 ns, p < 0.05.

  9. Two photon fluorescence imaging of lipid membrane domains and potentials using advanced fluorescent probes

    Science.gov (United States)

    Kilin, Vasyl; Darwich, Zeinab; Richert, Ludovic; Didier, Pascal; Klymchenko, Andrey; Mély, Yves

    2013-02-01

    Biomembranes are ordered and dynamic nanoscale structures critical for cell functions. The biological functions of the membranes strongly depend on their physicochemical properties, such as electrostatics, phase state, viscosity, polarity and hydration. These properties are essential for the membrane structure and the proper folding and function of membrane proteins. To monitor these properties, fluorescence techniques and notably, two-photon microscopy appear highly suited due to their exquisite sensitivity and their capability to operate in complex biological systems, such as living cells and tissues. In this context, we have developed multiparametric environment-sensitive fluorescent probes tailored for precise location in the membrane bilayer. We notably developed probes of the 3-hydroxychromone family, characterized by an excited state intramolecular proton transfer reaction, which generates two tautomeric emissive species with well-separated emission bands. As a consequence, the response of these probes to changes in their environment could be monitored through changes in the ratios of the two bands, as well as through changes in the fluorescence lifetimes. Using two-photon ratiometric imaging and FLIM, these probes were used to monitor the surface membrane potential, and were applied to detect apoptotic cells and image membrane domains.

  10. Single- and two-photon fluorescence recovery after photobleaching.

    Science.gov (United States)

    Sullivan, Kelley D; Majewska, Ania K; Brown, Edward B

    2015-01-05

    Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.

  11. Two-photon-excited fluorescence spectroscopy of atomic fluorine at 170 nm

    Science.gov (United States)

    Herring, G. C.; Dyer, Mark J.; Jusinski, Leonard E.; Bischel, William K.

    1988-01-01

    Two-photon-excited fluorescence spectroscopy of atomic fluorine is reported. A doubled dye laser at 286-nm is Raman shifted in H2 to 170 nm (sixth anti-Stokes order) to excite ground-state 2P(0)J fluorine atoms to the 2D(0)J level. The fluorine atoms are detected by one of two methods: observing the fluorescence decay to the 2PJ level or observing F(+) production through the absorption of an additional photon by the excited atoms. Relative two-photon absorption cross sections to and the radiative lifetimes of the 2D(0)J states are measured.

  12. A High Performance, Cost-Effective, Open-Source Microscope for Scanning Two-Photon Microscopy that Is Modular and Readily Adaptable

    Science.gov (United States)

    Rosenegger, David G.; Tran, Cam Ha T.; LeDue, Jeffery; Zhou, Ning; Gordon, Grant R.

    2014-01-01

    Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems. PMID:25333934

  13. A high performance, cost-effective, open-source microscope for scanning two-photon microscopy that is modular and readily adaptable.

    Directory of Open Access Journals (Sweden)

    David G Rosenegger

    Full Text Available Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems.

  14. Enhanced-locality fiber-optic two-photon-fluorescence live-brain interrogation

    Energy Technology Data Exchange (ETDEWEB)

    Fedotov, I. V.; Doronina-Amitonova, L. V. [International Laser Center, Physics Department, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 1430125 (Russian Federation); Kurchatov Institute National Research Center, Moscow (Russian Federation); Sidorov-Biryukov, D. A.; Fedotov, A. B. [International Laser Center, Physics Department, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 1430125 (Russian Federation); Anokhin, K. V. [Kurchatov Institute National Research Center, Moscow (Russian Federation); P.K. Anokhin Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow (Russian Federation); Kilin, S. Ya. [B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk (Belarus); Sakoda, K. [National Institute for Materials Science, 1-1 Namiki, Tsukuba 305-0044 (Japan); Zheltikov, A. M. [International Laser Center, Physics Department, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 1430125 (Russian Federation); Department of Physics and Astronomy, Texas A and M University, College Station, Texas 77843 (United States); Center of Photochemistry, Russian Academy of Sciences, ul. Novatorov 7a, Moscow 117421 (Russian Federation)

    2014-02-24

    Two-photon excitation is shown to substantially enhance the locality of fiber-based optical interrogation of strongly scattering biotissues. In our experiments, a high-numerical-aperture, large-core-are fiber probe is used to deliver the 200-fs output of a 100-MHz mode-locked ytterbium fiber laser to samples of live mouse brain, induce two-photon fluorescence of nitrogen–vacancy centers in diamond markers in brain sample. Fiber probes with a high numerical aperture and a large core area are shown to enable locality enhancement in fiber-laser–fiber-probe two-photon brain excitation and interrogation without sacrificing the efficiency of fluorescence response collection.

  15. Design and performance of an ultra-flexible two-photon microscope for in vivo research

    Science.gov (United States)

    Mayrhofer, Johannes M.; Haiss, Florent; Haenni, Dominik; Weber, Stefan; Zuend, Marc; Barrett, Matthew J. P.; Ferrari, Kim David; Maechler, Philipp; Saab, Aiman S.; Stobart, Jillian L.; Wyss, Matthias T.; Johannssen, Helge; Osswald, Harald; Palmer, Lucy M.; Revol, Vincent; Schuh, Claus-Dieter; Urban, Claus; Hall, Andrew; Larkum, Matthew E.; Rutz-Innerhofer, Edith; Zeilhofer, Hanns Ulrich; Ziegler, Urs; Weber, Bruno

    2015-01-01

    We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance. PMID:26600989

  16. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N.; Wientjes, Emilie; Amerongen, van Herbert

    2016-01-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was ac

  17. Sensing for intracellular thiols by water-insoluble two-photon fluorescent probe incorporating nanogel

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xudong; Zhang, Xin; Wang, Shuangqing; Li, Shayu [Beijing National Laboratory for Molecular Sciences, Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hu, Rui, E-mail: hurui@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Li, Yi, E-mail: yili@mail.ipc.ac.cn [Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Yang, Guoqiang, E-mail: gqyang@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China)

    2015-04-15

    Highlights: • A novel “turn-on” two-photon fluorescent probe based on a π-conjugated triarylboron luminogen was designed and synthesized. • Fast, selective and sensitive detection of biothiols in 100% aqueous solution by simply loaded on a nanogel. • Single-photon and two-photon fluorescent bioimaging of biothiols in NIH/3T3 fibroblasts. - Abstract: A novel “turn-on” two-photon fluorescent probe containing a π-conjugated triarylboron luminogen and a maleimide moiety DMDP-M based on the photo-induced electron transfer (PET) mechanism for biothiol detection was designed and synthesized. By simply loading the hydrophobic DMDP-M on a cross-linked Pluronic{sup ®} F127 nanogel (CL-F127), a probing system DMDP-M/CL-F127 was established, which shows quick response, high selectivity and sensitivity to cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous phase. The DMDP-M/CL-F127 system presented the fastest response to Cys with a rate constant of 0.56 min{sup −1}, and the detection limit to Cys was calculated to be as low as 0.18 μM. The DMDP-M/CL-F127 system has been successfully applied to the fluorescence imaging of biothiols in NIH/3T3 fibroblasts either with single-photon or two-photon excitation because of its high biocompatibility and cell-membrane permeability. The present work provides a general, simple and efficient strategy for the application of hydrophobic molecules to sensing biothiols in aqueous phase, and a novel sensing system for intracellular biothiols fitted for both single-photon and two-photon fluorescence imaging.

  18. Fluorenyl porphyrins for combined two-photon excited fluorescence and photosensitization

    Science.gov (United States)

    Mongin, Olivier; Hugues, Vincent; Blanchard-Desce, Mireille; Merhi, Areej; Drouet, Samuel; Yao, Dandan; Paul-Roth, Christine

    2015-04-01

    The two-photon absorption (2PA), the luminescence and the photosensitization properties of porphyrin-cored fluorenyl dendrimers and meso-substituted fluorenylporphyrin monomer, dimer and trimer are described. In comparison with model tetraphenylporphyrin, these compounds combine enhanced (non-resonant) 2PA cross-sections in the near infrared and enhanced fluorescence quantum yields, together with maintained singlet oxygen generation quantum yields. 'Semi-disconnection' between fluorenyl groups and porphyrins (i.e. direct meso substitution) proved to be more efficient than non-conjugated systems (based on efficient FRET between fluorenyl antennae and porphyrins). These results are of interest for combined two-photon imaging and photodynamic therapy.

  19. Two-photon fluorescent sensor for K+ imaging in live cells (Conference Presentation)

    Science.gov (United States)

    Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D.

    2016-03-01

    It is difficult to overstate the physiological importance of potassium for life as its indispensable roles in a variety of biological processes are widely known. As a result, efficient methods for determining physiological levels of potassium are of paramount importance. Despite this, relatively few K+ fluorescence sensors have been reported, with only one being commercially available. A new two-photon excited fluorescent K+ sensor is reported. The sensor is comprised of three moieties, a highly selective K+ chelator as the K+ recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (physiological metal cations. Upon binding K+, the sensor switches from non-fluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K+ sensing in living cells.

  20. Functional screening of intracardiac cell transplants using two-photon fluorescence microscopy.

    Science.gov (United States)

    Tao, Wen; Soonpaa, Mark H; Field, Loren J; Chen, Peng-Sheng; Firulli, Anthony B; Shou, Weinian; Rubart, Michael

    2012-08-01

    Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, its intrinsic renewal rate is insufficient to compensate for myocyte loss during cardiac disease. Transplantation of donor cardiomyocytes or cardiomyogenic stem cells is considered a promising strategy for reconstitution of cardiac mass, provided the engrafted cells functionally integrate with host myocardium and actively contribute to its contractile force. The authors previously developed a two-photon fluorescence microscopy-based assay that allows in situ screening of donor cell function after intracardiac delivery of the cells. This report reviews the techniques of two-photon fluorescence microscopy and summarizes its application for quantifying the extent to which a variety of donor cell types stably and functionally couple with the recipient myocardium.

  1. Diagnostics of MCF plasmas using Lyman-{alpha} fluorescence excited by one or two photons

    Energy Technology Data Exchange (ETDEWEB)

    Voslamber, D

    1998-11-01

    Laser-induced Lyman-{alpha} fluorescence of the hydrogen isotopes is investigated with regard to diagnostic applications in magnetically confined fusion plasmas. A formal analysis is presented for two excitation schemes: one-photon and Doppler-free two-photon excitation. The analysis includes estimates of the expected experimental errors arising from the photon noise and from the sensitivity of the observed fluorescence signals to variations of the plasma and laser parameters. Both excitation schemes are suitable primarily for application in the plasma edge, but even in the plasma bulk of large machines they can still be applied in combination with a diagnostic neutral beam. The two-photon excitation scheme is particularly attractive because it involves absorption spectra that are resolved within the Doppler width. This implies a large diagnostic potential and in particular offers a way to measure the deuterium-tritium fuel mix in fusion reactors. (author) 37 refs.

  2. In vitro imaging of thyroid tissues using two-photon excited fluorescence and second harmonic generation.

    Science.gov (United States)

    Huang, Zufang; Li, Zuanfang; Chen, Rong; Lin, Juqiang; Li, Yongzeng; Li, Chao

    2010-08-01

    To evaluate the feasibility of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to discriminate the normal, nodular goiter and papillary cancerous thyroid tissue. In total, 45 fresh thyroid specimens (normal, 15; nodular goiter, 12; and papillary cancerous, 18) from 31 subjects were directly imaged by the TPEF and SHG combination method. The microstructure of follicle and collagen structure in thyroid tissue were clearly identified, morphologic changes between normal, nodular goiter, and papillary cancerous thyroid tissue were well characterized by using two-photon excitation fluorescence. SHG imaging of the collagen matrix also revealed the differences between normal and abnormal. Our preliminary study suggests that the TPEF and SHG combination method might be a useful tool in revealing pathologic changes in thyroid tissue.

  3. Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

    Science.gov (United States)

    Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

    2014-02-01

    Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

  4. Cell flow analysis with a two-photon fluorescence fiber probe

    Science.gov (United States)

    Chang, Yu-Chung; Ye, Jing Yong; Thomas, Thommey P.; Baker, James R., Jr.; Norris, Theodore B.

    2010-11-01

    We report the use of a sensitive double-clad fiber (DCF) probe for in situ cell flow velocity measurements and cell analysis by means of two-photon excited fluorescence correlation spectroscopy (FCS). We have demonstrated the feasibility to use this fiber probe for in vivo two-photon flow cytometry previously. However, because of the viscosity of blood and the non-uniform flow nature in vivo, it is problematic to use the detected cell numbers to estimate the sampled blood volume. To precisely calibrate the sampled blood volume, it is necessary to conduct real time flow velocity measurement. We propose to use FCS technique to measure the flow velocity. The ability to measure the flow velocities of labeled cells in whole blood has been demonstrated. Our two-photon fluorescence fiber probe has the ability to monitor multiple fluorescent biomarkers simultaneously. We demonstrate that we can distinguish differently labeled cells by their distinct features on the correlation curves. The ability to conduct in situ cell flow analysis using the fiber probe may be useful in disease diagnosis or further comprehension of the circulation system.

  5. A spirobifluorene-based two-photon fluorescence probe for mercury ions and its applications in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Haibo, E-mail: xiaohb@shnu.edu.cn; Zhang, Yanzhen; Zhang, Wu; Li, Shaozhi; Tan, Jingjing; Han, Zhongying

    2017-05-01

    A novel spirobifluorene derivative SPF-TMS, which containing dithioacetal groups and triphenylamine units, was synthesized. The probing behaviors toward various metal ions were investigated via UV/Vis absorption spectra as well as one-photon fluorescence changes. The results indicated that SPF-TMS exhibits high sensitivity and selectivity for mercury ions. The detection limit was at least 8.6 × 10{sup −8}M, which is excellent comparing with other optical sensors for Hg{sup 2+}. When measured by two-photon excited fluorescence technique in THF at 800 nm, the two-photon cross-section of SPF-TMS is 272 GM. Especially, upon reaction with mercury species, SPF-TMS yielded another two-photon dye SPF-DA. Both SPF-TMS and SPF-DA emit strong two-photon induced fluorescence and can be applied in cell imaging by two-photon microscopy. - Highlights: • We report a spirobifluorene-based molecule as two-photon fluorescent probe with large two-photon cross-section. • The molecule has exclusive selectivity and sensitivity for mercury species. • The molecule has large two-photon emission changes before and after addition of Hg{sup 2+}. • Both the probe and the mercury ion-promoted reaction product can be applied in cell imaging by two-photon microscopy.

  6. Novel xenon calibration scheme for two-photon absorption laser induced fluorescence of hydrogen

    Science.gov (United States)

    Elliott, Drew; Scime, Earl; Short, Zachary

    2016-11-01

    Two photon absorption laser induced fluorescence (TALIF) measurements of neutral hydrogen and its isotopes are typically calibrated by performing TALIF measurements on krypton with the same diagnostic system and using the known ratio of the absorption cross sections [K. Niemi et al., J. Phys. D 34, 2330 (2001)]. Here we present the measurements of a new calibration method based on a ground state xenon scheme for which the fluorescent emission wavelength is nearly identical to that of hydrogen, thereby eliminating chromatic effects in the collection optics and simplifying detector calibration. We determine that the ratio of the TALIF cross sections of xenon and hydrogen is 0.024 ± 0.001.

  7. Enhanced two-photon excited fluorescence from imaging agents using true thermal light

    Science.gov (United States)

    Jechow, Andreas; Seefeldt, Michael; Kurzke, Henning; Heuer, Axel; Menzel, Ralf

    2013-12-01

    Two-photon excited fluorescence (TPEF) is a standard technique in modern microscopy, but is still affected by photodamage to the probe. It has been proposed that TPEF can be enhanced using entangled photons, but this has proven challenging. Recently, it was shown that some features of entangled photons can be mimicked with thermal light, which finds application in ghost imaging, subwavelength lithography and metrology. Here, we use true thermal light from a superluminescent diode to demonstrate TPEF that is enhanced compared to coherent light, using two common fluorophores and luminescent quantum dots, which suit applications in imaging and microscopy. We find that the TPEF rate is directly proportional to the measured degree of second-order coherence, as predicted by theory. Our results show that photon bunching in thermal light can be exploited in two-photon microscopy, with the photon statistic providing a new degree of freedom.

  8. Two-photon light-sheet nanoscopy by fluorescence fluctuation correlation analysis

    Science.gov (United States)

    Chen, Xuanze; Zong, Weijian; Li, Rongqin; Zeng, Zhiping; Zhao, Jia; Xi, Peng; Chen, Liangyi; Sun, Yujie

    2016-05-01

    Advances in light-sheet microscopy have enabled the fast three-dimensional (3D) imaging of live cells and bulk specimens with low photodamage and phototoxicity. Combining light-sheet illumination with super-resolution imaging is expected to resolve subcellular structures. Actually, such kind of super-resolution light-sheet microscopy was recently demonstrated using a single-molecule localization algorithm. However, the imaging depth and temporal resolution of this method are limited owing to the requirements of precise single molecule localization and reconstruction. In this work, we present two-photon super-resolution light-sheet imaging via stochastic optical fluctuation imaging (2PLS-SOFI), which acquires high spatiotemporal resolution and excellent optical sectioning ability. 2PLS-SOFI is based on non-linear excitation of fluctuation/blinking probes using our recently developed fast two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM), which enables both deep penetration and thin sheet of light. Overall, 2PLS-SOFI demonstrates up to 3-fold spatial resolution enhancement compared with conventional two-photon light-sheet (2PLS) microscopy and about 40-fold temporal resolution enhancement compared with individual molecule localization-selective plane illumination microscopy (IML-SPIM). Therefore, 2PLS-SOFI is promising for 3D long-term, deep-tissue imaging with high spatiotemporal resolution.

  9. Conventional and photonic crystal fiber based two-photon fluorescence biosensing

    Science.gov (United States)

    Myaing, Mon Thiri

    Optical fiber probes are widely used in the biomedical field for applications such as optical microscopy, endoscopy, and optical biopsy. Due to their flexibility and small size, optical fibers offer a minimally invasive light interface for imaging and spectroscopic analysis of internal tissue. The development of fluorescent probes for studies of biological processes has increased the importance of developing optical methods for quantitative, in vivo diagnosis. In this dissertation, we discuss the development of a novel two-photon optical fiber fluorescence (TPOFF) probe for real time, in vivo, quantitative fluorescence measurements in biological samples. In order to understand and optimize two-photon excitation through an optical fiber, pulse propagation effects must be considered. We found a simple phenomenological scaling behavior for the energy dependence of the pulse width for negatively pre-chirped pulses propagating in a normally dispersive fiber. As a consequence of this scaling behavior, the dependence of two-photon fluorescence (TPF) on the pulse intensity becomes sub-quadratic. The TPOFF probe employs a scheme where the same single-mode fiber (SMF) is used for both the excitation and collection of TPF. Using this fiber probe, we show quantification of tumor fluorescence both ex vivo and in vivo. In ex vivo measurements of tumors developed from cells expressing the green fluorescence protein (GFP), the TPOFF probe detected fluorescence from tumors with as little as 0.3% GFP cells. These results were similar to flow cytometry analysis of isolated cells from the tumors. The TPOFF measurements of GFP tumors in live, anesthetized mice showed a linear relationship between the measured fluorescence and the percentage of GFP expressing cells. The TPOFF probe was also used in targeted binding experiments of Herceptin antibody and folic acid-dendrimer nanoparticle conjugates. To improve the sensitivity of the TPOFF probe, a double-clad photonic crystal fiber (DCF

  10. Fluorescence enhancement of asCP595 is due to consecutive absorbance of two photons

    Science.gov (United States)

    Savitsky, Alexander P.; Agranat, Michail B.; Lukyanov, Konstantin A.; Schuttrigkeit, Tanja; von Feilitzsch, Till; Kompa, Christian; Michel-Beyerle, Maria-Elisabeth

    2004-06-01

    Colored proteins are widely used as gene markers in biotechnology. Chromophores result from autocatalytic posttranslational reactions involving several amino acids. The protein asCP595 was isolated for the first time from the coral as a weakly fluorescent chromoprotein with a fluorescence maximum at 595 nm. Strong illumination in the blue wing of the low energy absorption band results in a superlinear increase of the fluorescence yield and shifts its fluorescence spectrum by about 10 nm to the red. Time resolved fluorescence measurements using excitation pulses with 10 ps duration revealed a multiexponential decay pattern with time constants in the range from 20 ps to 2.1 ns. The ratio of amplitudes related to the different time constants depends on the intensity of illumination favoring the ns component at high intensities. Transient absorption measurements using ultrashort excitation pulses (150 fs, 1 kHz repetition rate) did not reveal excited states with nanosecond lifetimes as observed in fluorescence upon excitation using 10 ps pulses. This observation leads to the notion that within 10 ps a second photon is absorbed by a state not yet populated within 150 fs. As a consequence we propose two different excited singlet states operative in asCP595, one with low fluorescence quantum yield peaking at 595 nm and one with high fluorescence quantum yield peaking at 605 nm which is populated via the consecutive absorption of two photons at high excitation intensities.

  11. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

    2011-07-01

    Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

  12. Single & Two-photon Excited Fluorescence of Two New Compounds with 2-Benzothiazolyl as Electron Acceptor

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Two new D-π-A type compounds, where electron-donor D is tertiary amino group, electron-acceptor A is 2-benzothiazolyl and π is two conjugated styryl units, have been synthesized.They are named as trans, trans-2-{4-[4-(N, N-diethylamino)styryl]styryl}-1, 3-benzothiazole and trans, trans-2-{4-[4-(N, N-diphenylamino)styryl]styryl}-1, 3-benzothiazole.Both compounds show strong two-photon excited fluorescence in yellow-orange region when excited by a femtosecond laser at 800 nm.

  13. Two-photon microscopes and in vivo multiphoton tomographs--powerful diagnostic tools for tissue engineering and drug delivery.

    Science.gov (United States)

    Schenke-Layland, Katja; Riemann, Iris; Damour, Odile; Stock, Ulrich A; König, Karsten

    2006-09-15

    Near-infrared multiphoton microscopes and in vivo femtosecond laser tomographs are novel powerful diagnostic tools for intra-tissue drug screening and high-resolution structural imaging applicable to many areas of biomedical research. Deep tissue cells and extracellular matrix (ECM) compartments can be visualized in situ with submicron resolution without the need for tissue processing. In particular, the reduced fluorescent coenzyme NAD(P)H, flavoproteins, keratin, melanin, and elastin are detected by two-photon excited autofluorescence, whereas myosin, tubulin and the ECM protein collagen can be imaged additionally by second harmonic generation (SHG). Therefore, these innovative multiphoton technologies have been used to probe architecture and state of a variety of native tissues, as well as of tissue-engineered constructs, giving insights on the interaction between scaffolds and seeded cells in vitro prior implantation. Moreover, non-invasive 4-D multiphoton tomographs are employed in clinical studies to examine the diffusion behavior, the intra-tissue accumulation of topically applied cosmetic and pharmaceutical components, and their interaction with skin cells.

  14. One- and Two-photon Excited Fluorescence of Zinc(Ⅱ), Cadmium(Ⅱ) Complexes Containing Phenothiazine Ligand

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A new ligand, 10-ethylphenothiazinyl - 3 - yl - methylene thiosemicarbazon (HL) and its complexes ML2 (M=Zn2+, Cd2+), which exhibit intensive two-photon excited (TPE) fluorescence at 800 nm laser pulses in femtosecond regime, were synthesized and characterized.The measured power dependence of the fluorescence signals provided direct evidence for TPE.All of them exhibited a large two-photon absorptive cross section and, more importantly from the application point of view, high photochemical/photothermal stability.

  15. A compact two photon light sheet microscope for applications in neuroscience

    DEFF Research Database (Denmark)

    Piksarv, Peeter; Marti, Dominik; Le, Tuan

    2016-01-01

    We present a compact setup for two photon light sheet microscopy. By using pulsed Airy beam illumination we demonstrate eight-fold increase of the FOV compared to Gaussian light sheet with the same axial resolution....

  16. A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues.

    Science.gov (United States)

    Jin, Qiang; Feng, Lei; Wang, Dan-Dan; Dai, Zi-Ru; Wang, Ping; Zou, Li-Wei; Liu, Zhi-Hong; Wang, Jia-Yue; Yu, Yang; Ge, Guang-Bo; Cui, Jing-Nan; Yang, Ling

    2015-12-30

    In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

  17. Two-photon cryomicroscope

    Science.gov (United States)

    Breunig, H. G.; Köhler, C.; König, K.

    2012-03-01

    We report on a new two-photon cryomicroscope which consist of a compact laser-scanning microscope combined with a motorized heating and freezing stage. Samples can be cooled down to -196 °C (77 K) and heated up to 600 °C (873 K) with adjustable heating/freezing rates between 0.01 K / min and 150 K / min. Two-photon imaging is realized by near infrared femtosecond-laser pulse excitation. The abilities of the two-photon cryomicroscope are illustrated in several measurements: imaging of fluorescent microspheres inside a piece of ice illustrates the feasibility of deep-microscopic imaging inside frozen sample. The temperature-dependent structural integrity of collagen is monitored by detection of second harmonic generation signals from porcine cornea. The measurements reveal also the dependence of the collagendenaturation temperature on hydration state of the cornea collagen. Furthermore, the potential of the two-photon cryomicroscope for optimization of freezing and thawing procedures as well as to evaluate the viability of frozen cells and tissue is discussed.

  18. Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator.

    Science.gov (United States)

    Matsumoto, Naoya; Inoue, Takashi; Matsumoto, Akiyuki; Okazaki, Shigetoshi

    2015-07-01

    We demonstrate fluorescence imaging with high fluorescence intensity and depth resolution in which depth-induced spherical aberration (SA) caused by refractive-index mismatch between the medium and biological sample is corrected. To reduce the impact of SA, we incorporate a spatial light modulator into a two-photon excitation fluorescence microscope. Consequently, when fluorescent beads in epoxy resin were observed with this method of SA correction, the fluorescence signal of the observed images was ∼27 times higher and extension in the direction of the optical axes was ∼6.5 times shorter at a depth of ∼890 μm. Thus, the proposed method increases the depth observable at high resolution. Further, our results show that the method improved the fluorescence intensity of images of the fluorescent beads and the structure of a biological sample.

  19. Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

    Science.gov (United States)

    Krasieva, Tatiana B.; Stringari, Chiara; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Gratton, Enrico; Tromberg, Bruce J.

    2013-03-01

    Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

  20. Cytosolic NADH-NAD+ Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

    Science.gov (United States)

    Mongeon, Rebecca; Venkatachalam, Veena

    2016-01-01

    Abstract Aim: Cytosolic NADH-NAD+ redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD+ redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. Results: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD+ ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Innovation: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Conclusion: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD+ ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553–563. PMID:26857245

  1. A fluorescence scanning electron microscope

    Directory of Open Access Journals (Sweden)

    Takaaki Kanemaru

    2010-01-01

    Full Text Available Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM and an electron microscope (EM. In the current study, a scanning electron microscope (SEM (JEOL JXA8600 M was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM. In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

  2. A two-photon fluorescent probe with a large turn-on signal for imaging hydrogen sulfide in living tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Kaibo [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082 (China); Lin, Weiying, E-mail: weiyinglin2013@163.com [Institute of Fluorescent Probes for Biological Imaging, University of Jinan, Jinan, Shandong 250022 (China); State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082 (China); Tan, Li; Cheng, Dan [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082 (China)

    2015-01-01

    Highlights: • A two-photon fluorescent probe for sensing H{sub 2}S was developed. • The probe shows a large turn on signal (120-fold enhancement). • The probe is suitable for fluorescence imaging of H{sub 2}S in living cells and tissues. • The probe was capable of detecting H{sub 2}S up to 170 μm depth in live tissues. - Abstract: A two-photon fluorescence turn-on H{sub 2}S probe GCTPOC–H{sub 2}S based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive H{sub 2}S recognition site, dinitrophenyl ether was constructed. The probe GCTPOC–H{sub 2}S exhibits desirable properties such as high sensitivity, high selectivity, functioning well at physiological pH and low cytotoxicity. In particular, the probe shows a 120-fold enhancement in the presence of Na{sub 2}S (500 μM), which is larger than the reported two-photon fluorescent H{sub 2}S probes. The large fluorescence enhancement of the two-photon probe GCTPOC–H{sub 2}S renders it attractive for imaging H{sub 2}S in living tissues with deep tissue penetration. Significantly, we have demonstrated that the probe GCTPOC–H{sub 2}S is suitable for fluorescence imaging of H{sub 2}S in living tissues with deep penetration by using two-photon microscopy. The further application of the two-photon probe for the investigation of biological functions and pathological roles of H{sub 2}S in living systems is under progress.

  3. A novel Kalman filter based video image processing scheme for two-photon fluorescence microscopy

    Science.gov (United States)

    Sun, Wenqing; Huang, Xia; Li, Chunqiang; Xiao, Chuan; Qian, Wei

    2016-03-01

    Two-photon fluorescence microscopy (TPFM) is a perfect optical imaging equipment to monitor the interaction between fast moving viruses and hosts. However, due to strong unavoidable background noises from the culture, videos obtained by this technique are too noisy to elaborate this fast infection process without video image processing. In this study, we developed a novel scheme to eliminate background noises, recover background bacteria images and improve video qualities. In our scheme, we modified and implemented the following methods for both host and virus videos: correlation method, round identification method, tree-structured nonlinear filters, Kalman filters, and cell tracking method. After these procedures, most of noises were eliminated and host images were recovered with their moving directions and speed highlighted in the videos. From the analysis of the processed videos, 93% bacteria and 98% viruses were correctly detected in each frame on average.

  4. Identification of calcifications in intracranial neoplasms using two photon excitation fluorescence microscopy

    Science.gov (United States)

    Lin, Peihua; Wang, Xingfu; Wu, Zanyi; Fang, Na; Li, Lianhuang; Kang, Dezhi; Chen, Jianxin

    2016-10-01

    Calcifications within brain tumors may be an indicator of a relatively long survival because a long time is required for the formation of calcium deposits, and may present a novel biomarker associated with response and improved outcome of therapy. In this paper, we describe the use of two-photon excitation fluorescent (TPEF) microscopy combined second harmonic generation (SHG) microscopy for high-resolution imaging that can be applied in identification of intratumoral calcifications. Our results demonstrate that the calcification has stronger TPEF signal than the area around it and the emission spectra shows the difference between the two areas clearly. The TPEF image of calcified region corresponds well with the corresponding H&E stained image. In this work, we present that the label-free imaging technique is able to distinguish the calcified mass lesions in intracranial neoplasms reliably.

  5. Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

    Science.gov (United States)

    Gasecka, Alicja; Han, Tsai-Jung; Favard, Cyril; Cho, Bong Rae; Brasselet, Sophie

    2009-01-01

    Abstract We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains. PMID:19917241

  6. Controlling and tracking of colloidal nanostructures through two-photon fluorescence

    Science.gov (United States)

    Mondal, Dipankar; Goswami, Debabrata

    2016-12-01

    Multiphoton absorbing dye-coated trapped spherical bead at the focal plane of femtosecond optical tweezers shows nonlinear optical (NLO) phenomena. One such NLO process of two-photon fluorescence (TPF) has been used for the background-free imaging of a femtosecond laser-trapping event. Due to the high peak powers of femtosecond laser pulses with low average powers, it is possible to not only trap single nanospheres, but encourage optically directed self-assembly. The TPF signatures of trapped particles show evidence of such a directed self-assembly process which, in turn, can provide information about the structural dynamics during the process of cluster formation. We are able to trap and characterize structure and dynamics in 3D until pentamer formation from the decay characteristics of trapping at the focal plane.

  7. Ag@Aggregation-induced emission dye core/shell nanostructures with enhanced one- and two-photon fluorescence

    Science.gov (United States)

    Wang, Cheng; Li, Yang; Xu, Qiujin; Luo, Liang

    2017-10-01

    Combining plasmonic nanostructures with two-photon fluorescence materials is a promising way to significantly enhance two-photon fluorescence. Ag@1,4-bis(2-cyano-2-phenylethenyl) benzene (BCPEB) core/shell nanostructures were fabricated by simply incubating the isolated Ag nanoparticles with BCPEB microrods in ethanol. BCPEB was chosen as the fluorescent organic molecule owing to the aggregation-induced-emission (AIE) nature which would reduce the emission loss as being practically applied in solid phase. By utilizing the match of the extinction spectrum of Ag nanoparticles and BCPEB's absorption band, the target Ag@BCPEB core/shell nanostructures showed an enhanced one-photon (12×) fluorescence, integrating with SERS signal as well. Moreover, the resultant second harmonic generation of Ag nanoparticles under two-photon excitation also well matched with the absorption band of BCPEB, and significant enhanced two-photon (17×) fluorescence was obtained. The confocal images of NIH-3T3 cells with these nanostructures under one- and two-photon excitation showed good contrast and brightness for bio-imaging.

  8. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N; Wientjes, Emilie; van Amerongen, Herbert

    2016-09-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes.

  9. Combined influences of chromatic aberration and scattering in depth-resolved two-photon fluorescence endospectroscopy.

    Science.gov (United States)

    Wu, Yicong; Li, Xingde

    2010-10-27

    The influence of chromatic aberration of an objective lens in two-photon fluorescence (TPF) endospectroscopy of scattering media has been systematically investigated through both experiments and numerical simulations. Experiments were carried out on a uniform 3D scattering gelatin phantom embedded with TiO(2) granules (to mimic tissue scattering) and fluorescein-tagged polystyrene beads. It was found that fluorescence spectral intensity and lineshape varied as a function of depth when measured with a gradient-index (GRIN) lens which has severe chromatic aberration. The spectral distortion caused by the chromatic aberration became diminishing as the imaging depth increased. Ray tracing analysis and Monte Carlo simulations were carried out to study the interplay of chromatic aberration and scattering in the depth-resolved TPF spectra. The simulation results suggest that the collected fluorescence signals from deeper layers included more out-of-focus photons that experienced a few or multiple scatterings, which diminish the influence of chromatic aberration on the measured TPF spectra. The simulated collection efficiencies of TPF at different wavelengths and depths can be used to properly recover the true depth-resolved TPF spectra of a relatively uniform scattering medium.

  10. Multimodal second harmonic generation and two photon fluorescence imaging of microdomain calcium contraction coupling in single cardiomyocytes

    Science.gov (United States)

    Chan, James; Awasthi, Samir; Izu, Leighton; Mao, Ziliang; Jian, Zhong; Landas, Trevor; Lerner, Aaron; Shimkunas, Rafael; Woldeyesus, Rahwa; Bossuyt, Julie; Wood, Brittani; Chen, Yi-Je; Matthews, Dennis; Lieu, Deborah; Chiamvimonvat, Nipavan; Lam, Kit; Chen-Izu, Ye

    2016-11-01

    The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.

  11. Pressure broadening of atomic oxygen two-photon absorption laser induced fluorescence

    Science.gov (United States)

    Marinov, Daniil; Drag, Cyril; Blondel, Christophe; Guaitella, Olivier; Golda, Judith; Klarenaar, Bart; Engeln, Richard; Schulz-von der Gathen, Volker; Booth, Jean-Paul

    2016-12-01

    Atomic oxygen, considered to be a determining reactant in plasma applications at ambient pressure, is routinely detected by two-photon absorption laser induced fluorescence (TALIF). Here, pressure broadening of the (2p 4 3 P 2  →  3p 3 P J=0,1,2) two-photon transition in oxygen atoms was investigated using a high-resolution TALIF technique in normal and Doppler-free configurations. The pressure broadening coefficients determined were {γ{{\\text{O}2}}}   =  0.40  ±  0.08  cm-1/bar for oxygen molecules and {γ\\text{He}}   =  0.46  ±  0.03 cm-1/bar for helium atoms. These correspond to pressure broadening rate constants k\\text{PB}{{\\text{O}2}}   =  9 · 10-9 cm3 s-1 and k\\text{PB}\\text{He}   =  4 · 10-9 cm3 s-1, respectively. The well-known quenching rate constants of O(3p 3 P J ) by O2 and He are at least one order of magnitude smaller, which signifies that non-quenching collisions constitute the main line-broadening mechanism. In addition to providing new insights into collisional processes of oxygen atoms in electronically excited 3p 3 P J state, reported pressure broadening parameters are important for quantification of oxygen TALIF line profiles when both collisional and Doppler broadening mechanisms are important. Thus, the Doppler component (and hence the temperature of oxygen atoms) can be accurately determined from high resolution TALIF measurements in a broad range of conditions.

  12. Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.

    Science.gov (United States)

    Alexander, Nathan S; Palczewska, Grazyna; Stremplewski, Patrycjusz; Wojtkowski, Maciej; Kern, Timothy S; Palczewski, Krzysztof

    2016-07-01

    Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)].

  13. Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

    Science.gov (United States)

    Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

    2016-08-01

    Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

  14. A two-photon laser induced fluorescence diagnostic with improved sensitivity, localization, and measurement rate

    Science.gov (United States)

    Elliott, Drew; Scime, Earl; Short, Zachary

    2016-10-01

    A two-photon absorption laser induced fluorescence diagnostic has been developed for measuring neutrals in fusion plasmas. Implementation of this diagnostic on the HIT-SI3 spheromak has demonstrated the sensitivity of the diagnostic and shown that measurements taken over several plasma pulses are possible. These measurements yielded an unexpected loss of signal when complex collection optics were utilized. Simulations show that this loss of signal can be explained by chromatic aberrations caused by the disparate Kr and D emission. This loss of signal has been addressed with the development of a new calibration scheme involving xenon gas. The Xe calibration scheme emission occurs at 656.00 nm while the deuterium emission is 656.09 nm. This nearly identical emission allows for advanced optical techniques such as confocal collection/injection and spatial filtering to be employed without loss of signal. Spatial filtering has been demonstrated to decrease noise while improving measurement localization, while confocal collection/injection allows for probing and measuring to occur through one viewport. The Xe scheme also allows for a Doppler-free hydrogen measurement. Doppler-free measurements eliminate the need to scan the laser spectrally thus greatly increasing the rate of measurement.

  15. Zn2+ responsive two-photon fluorescent probes based on branch structure: a computational investigation

    Science.gov (United States)

    Huang, Shuang; Yang, Bao-Zhu; Guo, Jing-Fu; Ren, Ai-Min

    2015-03-01

    A series of zinc ion fluorescent probes on the basis of multi-branched ligands were investigated in theory. The three-branched ligand TPPA (N,N,N‧,N‧-tetraphenyl-p-phenylenediamine) has better three-dimensional spatial localisation, which can detect zinc at the parts per million level. The complex coordinated with Zn2+ can show a significant improvement in two-photon absorption (TPA) cross-section in the near-infrared (NIR) excitation region. The calculated results reveal that the stability and sensitivity of Zn2+ complexes will be enhanced by increasing the number of branches. The selectivity of double phenyl-p-phenylenediamine (DPPA) ligand to Zn2+ will be better compared to Cd2+. With regard to the studied ligands single phenyl-p-phenylenediamine (SPPA), two connected single phenyl-p-phenylenediamine (2CSPPA), DPPA and TPPA, λEMmax shows a red-shift and ƒEM gets stronger upon the addition of Zn2+. Most of the molecules exhibit TPA peaks in the NIR region. The theoretical investigations demonstrate that DPPA-Zn2+ shows good TPA activity at a telecommunication wavelength.

  16. Increasing efficiency of two-photon excited fluorescence and second harmonic generation using ultrashort pulses

    Science.gov (United States)

    Tang, Shuo; Krasieva, Tatiana B.; Chen, Zhongping; Tempea, Gabriel; Tromberg, Bruce J.

    2006-02-01

    Multiphoton microscopy (MPM) has become an important tool for high-resolution and non-invasive imaging in biological tissues. However, the efficiencies of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) are relatively low because of their nonlinear nature. Therefore, it is critical to optimize laser parameters for most efficient excitation of MPM. Reducing the pulse duration can increase the peak intensity of excitation and thus potentially increase the excitation efficiency. In this paper, a multiphoton microscopy system using a 12 fs Ti:Sapphire laser is reported. With adjustable dispersion pre-compensation, the pulse duration at the sample location can be varied from 400 fs to sub-20 fs. The efficiencies of TPEF and SHG are studied for the various pulse durations, respectively. Both TPEF and SHG are found to increase proportionally to the inverse of the pulse duration for the entire tested range. To transmit most of the SHG and TPEF signals, the spectral transmission widow of the detection optics needs to be carefully considered. Limitation from phase-matching in SHG generation is not significant because the effective interaction length for SHG is less than 10 μm at the focal depth of the objectives. These results are important in improving MPM excitation efficiency using ultrashort pulses. MPM images from human artery wall are also demonstrated.

  17. Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

    Science.gov (United States)

    Bukara, Katarina; Jovanić, Svetlana; Drvenica, Ivana T.; Stančić, Ana; Ilić, Vesna; Rabasović, Mihailo D.; Pantelić, Dejan; Jelenković, Branislav; Bugarski, Branko; Krmpot, Aleksandar J.

    2017-02-01

    The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells' shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

  18. Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo

    Science.gov (United States)

    Krasieva, Tatiana B.; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Tromberg, Bruce J.

    2012-03-01

    The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant melanoma diagnostics. The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation. We acquired TPEF emission spectra (λex=1000 nm) of melanin from several specimens, including human hair, malignant melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted from the eumelanin with broad maximum at 640-680nm. We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm. The measured OMI for a melanoma cell line MNT-1 was 1.6+/-0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown) showed an anticipated change in melanins production ratio and had OMI of 0.55+/-0.05 and 0.17+/-0.02, respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05 and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI. These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization both in vitro and in vivo, including pigmented lesions.

  19. Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy.

    Science.gov (United States)

    Norlén, L; Plasencia, I; Bagatolli, L

    2008-12-01

    Skin moisturization is largely a function of stratum corneum barrier capacity, which in turn is a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix [J. Invest. Dermatol.18, 433 (1952); AIChE J. 21, 985 (1975); Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001)]. Three unsolved key questions with respect to this lipid matrix' structural organization [Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001); J. Invest. Dermatol.118, 897 (2002); J. Invest. Dermatol.118, 899 (2002)] are: i) whether the lipid matrix is constituted by a single-gel phase or by co-existing solid (crystalline or gel) domains, ii) whether a separate fluid (liquid crystalline) phase is present and iii) whether the local pH has a direct effect on the lipid matrix' phase behaviour. Using an array of complementary visual-related biophysical techniques (e.g. atomic force microscopy and confocal/two-photon excitation fluorescence microscopy), it was recently shown that reconstituted membranes composed of extracted decontaminated human stratum corneum lipids do not form a fluid phase, but exclusively a single-gel phase that segregates into co-existing microscopic domains below pH 6 [Biophys. J.93, 3142 (2007)]. It was further shown that the role of cholesterol is related to dispersion of ceramide-enriched domains. This effect is counteracted by the presence of free fatty acids, which mix with skin ceramides but not with cholesterol.

  20. Kinetics and subcellular localization of 5-ALA-induced PpIX in DHL cells via two-photon excitation fluorescence microscopy.

    Science.gov (United States)

    Chen, Rong; Huang, Zufang; Chen, Guannan; Li, Yongzeng; Chen, Xianlian; Chen, Jianxin; Zeng, Haishan

    2008-04-01

    Two-photon excitation fluorescence (TPEF) microscopy was used to measure the 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence in follicular lymphoma DHL cells. Kinetics of 5-ALA-induced PpIX accumulation in DHL cells under various 5-ALA concentrations was studied. We found that during the course of continuous incubation with 5-ALA, the relationship between the DHL cell fluorescence signal and the incubation time showed a biphasic variation. Initially the PpIX signal increased with the incubation time and reached the maximal value at about 3 h, and then it decreased with time during the subsequent incubation period. By labeling the 5-ALA incubated DHL cells with different organelle-specific fluorescence probes: Rhodamine 123 (for mitochondria), DioC6(3) (for endoplasmic reticulum) and LysoTracker Green (for lysosomes) respectively, we found that 5-ALA-induced PpIX was primarily localized in endoplasmic reticulum and mitochondria; its concentration in the lysosome was much lower. The results suggested that 5-ALA could potentially be an effective photosensitizer in photodynamic purging of DHL cells. Two-photon excitation fluorescence microscope is a useful tool for studying 5-ALA-induced PpIX subcellular localization.

  1. A two-photon fluorescent probe for exogenous and endogenous superoxide anion imaging in vitro and in vivo.

    Science.gov (United States)

    Li, Run-Qing; Mao, Zhi-Qiang; Rong, Lei; Wu, Nian; Lei, Qi; Zhu, Jing-Yi; Zhuang, Lin; Zhang, Xian-Zheng; Liu, Zhi-Hong

    2017-01-15

    Herein, we report a novel quinoline derivative-based two-photon fluorescent probe 6-(dimethylamino)quinoline-2-benzothiazoline (HQ), which is capable of tracking superoxide anion in organisms with specific "turn-on" fluorescence response based on extension of π-conjugations and moderate ICT process. The probe exhibited favorable photophysical properties, a broad linear range and high photostability. It can specifically detect superoxide anion with a significant fluorescence enhancement and great linearity from 0 to 500μM in PBS buffer. Furthermore, HQ shows low cytotoxicity and excellent photostability toward living cells and organisms, which was able to monitor endogenous superoxide anion fluxes in living cells and in vivo. For the first time, endogenous superoxide anion in lung inflammation was visualized successfully by using HQ through two-photon microscopy, and the probe HQ shows great potential for fast in-situ detecting of inflammatory response in live organisms.

  2. Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

    Directory of Open Access Journals (Sweden)

    Xinyue Zhu

    2015-01-01

    Full Text Available A reaction-based two-photon (TP ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM.

  3. Two Photon Absorption Laser Induced Fluorescence for Neutral Hydrogen Profile Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Scime, Earl E. [West Virginia Univ., Morgantown, WV (United States)

    2016-09-23

    The magnitude and spatial dependence of neutral density in magnetic confinement fusion experiments is a key physical parameter, particularly in the plasma edge. Modeling codes require precise measurements of the neutral density to calculate charge-exchange power losses and drag forces on rotating plasmas. However, direct measurements of the neutral density are problematic. In this work, we proposed to construct a laser-based diagnostic capable of providing spatially resolved measurements of the neutral density in the edge of plasma in the DIII-D tokamak. The diagnostic concept is based on two-photon absorption laser induced fluorescence (TALIF). By injecting two beams of 205 nm light (co or counter propagating), ground state hydrogen (or deuterium or tritium) can be excited from the n = 1 level to the n = 3 level at the location where the two beams intersect. Individually, the beams experience no absorption, and therefore have no difficulty penetrating even dense plasmas. After excitation, a fraction of the hydrogen atoms decay from the n = 3 level to the n = 2 level and emit photons at 656 nm (the Hα line). Calculations based on the results of previous TALIF experiments in magnetic fusion devices indicated that a laser pulse energy of approximately 3 mJ delivered in 5 ns would provide sufficient signal-to-noise for detection of the fluorescence. In collaboration with the DIII-D engineering staff and experts in plasma edge diagnostics for DIII-D from Oak Ridge National Laboratory (ORNL), WVU researchers designed a TALIF system capable of providing spatially resolved measurements of neutral deuterium densities in the DIII-D edge plasma. The laser systems were specified, purchased, and assembled at WVU. The TALIF system was tested on a low-power hydrogen discharge at WVU and the plan was to move the instrument to DIII-D for installation in collaboration with ORNL researchers. After budget cuts at DIII-D, the DIII-D facility declined to support

  4. Calcium silicate cement-induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two-photon fluorescence microscopy.

    Science.gov (United States)

    Atmeh, A R; Chong, E Z; Richard, G; Boyde, A; Festy, F; Watson, T F

    2015-02-01

    Two-photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate-based restorative material (Biodentine(TM) ) and a glass ionomer cement (GIC:​Fuji​IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate-free tetracycline, and tetracycline-free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8-week storage at 37 °C, dentine samples were imaged using two-photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine(TM) in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra- and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement-free or PBS-free media, or with GIC. It could therefore be concluded that Biodentine(TM) induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate-rich media, which was selectively detectable using the tetracycline labelling.

  5. Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging.

    Science.gov (United States)

    Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S

    2015-11-01

    In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated.

  6. Development and design of up-to-date laser scanning two-photon microscope using in neuroscience

    Science.gov (United States)

    Doronin, Maxim; Popov, Alexander

    2017-02-01

    Today one of the main areas of application of two-photon microscopy is biology. This is due to the fact that this technique allows to obtain 3D images of tissues due to laser focus change, that is possible due to substantially greater penetration depth on the main wavelength into biological tissues. Self-developed microscopy system provides possibility to service it and modify the structure of microscope depending on highly specialized experimental design and scientific goals. This article may be regarded as a quick reference to laboratory staff who are wishing to develop their own microscopy system for self-service and modernization of the system and in order to save the lab budget.

  7. A Selective Imidazoline-2-thione-Bearing Two-Photon Fluorescent Probe for Hypochlorous Acid in Mitochondria.

    Science.gov (United States)

    Xu, Qingling; Heo, Cheol Ho; Kim, Jin A; Lee, Hye Sue; Hu, Ying; Kim, Dayoung; Swamy, Kunemadihalli Mathada Kotraiah; Kim, Gyoungmi; Nam, Sang-Jip; Kim, Hwan Myung; Yoon, Juyoung

    2016-06-21

    Hypochlorite (OCl(-)) plays a key role in the immune system and is involved in various diseases. Accordingly, direct detection of endogenous OCl(-) at the subcellular level is important for understanding inflammation and cellular apoptosis. In the current study, a two-photon fluorescent off/on probe (PNIS) bearing imidazoline-2-thione as an OCl(-) recognition unit and triphenylphosphine (TPP) as a mitochondrial-targeting group was synthesized and examined for its ability to image mitochondrial OCl(-) in situ. This probe, based on the specific reaction between imidazoline-2-thione and OCl(-), displayed a selective fluorescent off/on response to OCl(-) with the various reactive oxygen species in a physiological medium. PNIS was successfully applied to image of endogenously produced mitochondrial OCl(-) in live RAW 264.7 cells via two-photon microscopy.

  8. Two-photon excited fluorescence of intrinsic fluorophores enables label-free assessment of adipose tissue function

    Science.gov (United States)

    Alonzo, Carlo Amadeo; Karaliota, Sevasti; Pouli, Dimitra; Liu, Zhiyi; Karalis, Katia P.; Georgakoudi, Irene

    2016-08-01

    Current methods for evaluating adipose tissue function are destructive or have low spatial resolution. These limit our ability to assess dynamic changes and heterogeneous responses that occur in healthy or diseased subjects, or during treatment. Here, we demonstrate that intrinsic two-photon excited fluorescence enables functional imaging of adipocyte metabolism with subcellular resolution. Steady-state and time-resolved fluorescence from intracellular metabolic co-factors and lipid droplets can distinguish the functional states of excised white, brown, and cold-induced beige fat. Similar optical changes are identified when white and brown fat are assessed in vivo. Therefore, these studies establish the potential of non-invasive, high resolution, endogenous contrast, two-photon imaging to identify distinct adipose tissue types, monitor their functional state, and characterize heterogeneity of induced responses.

  9. Two-color two-photon excited fluorescence of indole: Determination of wavelength-dependent molecular parameters

    Energy Technology Data Exchange (ETDEWEB)

    Herbrich, Sebastian; Al-Hadhuri, Tawfik; Gericke, Karl-Heinz, E-mail: k.Gericke@tu-bs.de [Institut für Physikalische und Theoretische Chemie, TU Braunschweig, Hans-Sommer-Straße 10, 38106 Braunschweig (Germany); Shternin, Peter S., E-mail: pshternin@gmail.com; Vasyutinskii, Oleg S., E-mail: osv@pms.ioffe.ru [Ioffe Institute, Politekhnicheskaya 26, 194021 St. Petersburg (Russian Federation); St. Petersburg Polytechnic University, Politekhnicheskaya 29, St. Petersburg 195251 (Russian Federation); Smolin, Andrey G. [Ioffe Institute, Politekhnicheskaya 26, 194021 St. Petersburg (Russian Federation)

    2015-01-14

    We present a detailed study of two-color two-photon excited fluorescence in indole dissolved in propylene glycol. Femtosecond excitation pulses at effective wavelengths from 268 to 293.33 nm were used to populate the two lowest indole excited states {sup 1}L{sub a} and {sup 1}L{sub b} and polarized fluorescence was then detected. All seven molecular parameters and the two-photon polarization ratio Ω containing information on two-photon absorption dynamics, molecular lifetime τ{sub f}, and rotation correlation time τ{sub rot} have been determined from experiment and analyzed as a function of the excitation wavelength. The analysis of the experimental data has shown that {sup 1}L{sub b}–{sup 1}L{sub a} inversion occurred under the conditions of our experiment. The two-photon absorption predominantly populated the {sup 1}L{sub a} state at all excitation wavelengths but in the 287–289 nm area which contained an absorption hump of the {sup 1}L{sub b} state 0-0 origin. The components of the two-photon excitation tensor S were analyzed giving important information on the principal tensor axes and absorption symmetry. The results obtained are in a good agreement with the results reported by other groups. The lifetime τ{sub f} and the rotation correlation time τ{sub rot} showed no explicit dependence on the effective excitation wavelength. Their calculated weighted average values were found to be τ{sub f} = 3.83 ± 0.14 ns and τ{sub rot} = 0.74 ± 0.06 ns.

  10. Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols.

    Science.gov (United States)

    Mueller-Harvey, Irene; Feucht, Walter; Polster, Juergen; Trnková, Lucie; Burgos, Pierre; Parker, Anthony W; Botchway, Stanley W

    2012-03-16

    Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ~1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ(2)=1.9-3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.

  11. Tracking of mercury ions in living cells with a fluorescent chemodosimeter under single- or two-photon excitation

    Energy Technology Data Exchange (ETDEWEB)

    Lu Zhoujun [State Key Lab for Advanced Photonic Materials and Devices, Department of Optical Science and Engineering, Fudan University, Shanghai 200433 (China); Wang Peinan [State Key Lab for Advanced Photonic Materials and Devices, Department of Optical Science and Engineering, Fudan University, Shanghai 200433 (China)], E-mail: pnwang@fudan.edu.cn; Zhang Yu [State Key Lab for Advanced Photonic Materials and Devices, Department of Optical Science and Engineering, Fudan University, Shanghai 200433 (China); Chen Jiyao; Zhen Shen [Department of Physics, Fudan University, Shanghai 200433 (China); Leng Bing; Tian He [Labs for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237 (China)

    2007-08-10

    Tracking of Hg{sup 2+} in solutions as well as in living cells was conducted with a fluorescent chemodosimeter by measuring the spectral shift of its fluorescence under single- or two-photon excitation. The spectral hypsochromic shifts of this chemodosimeter when reacting with Hg{sup 2+} were found to be about 50 nm in acetonitrile/water solutions and 32 nm in Euglena gracilis 277 living cells. This chemodosimeter shows high sensitivity and selectivity, and is not influenced by the pH values. It can signal Hg{sup 2+} in solutions down to the ppb range under either single-photon excitation (SPE) at 405 nm or two-photon excitation (TPE) at 800 nm. However, with low cellular chemodosimeter concentrations, the SPE spectra were disturbed by the auto-fluorescence from the native fluorophore in the cell, while the TPE spectra were still of high quality since the two-photon absorption cross section of this chemodosimeter is much larger than that of the native fluorophores in the cell.

  12. A two-photon activatable amino acid linker for the induction of fluorescence.

    Science.gov (United States)

    Friedrich, Felix; Klehs, Kathrin; Fichte, Manuela A H; Junek, Stephan; Heilemann, Mike; Heckel, Alexander

    2015-10-28

    A new one- and two-photon activatable fluorophore based on ATTO565 was developed using a photolabile linker that simultaneously acts as a quencher. It is especially interesting for protein and peptide applications because it can be incorporated by standard peptide chemistry. The application of the new fluorogenic construct in super-resolution microscopy of antibody conjugates is shown.

  13. One- and two-photon induced fluorescence spectroscopy enabling the detection of localized aflatoxin contamination in individual maize kernels

    Science.gov (United States)

    Smeesters, L.; Meulebroeck, W.; Raeymaekers, S.; Thienpont, H.

    2016-04-01

    The presence of carcinogenic aflatoxins in food and feed products is a major worldwide problem. To date, the aflatoxin contamination can only be detected by the use of destructive sample-based chemical analyses. Therefore, we developed an optical setup able to detect the localized aflatoxin contamination in individual maize kernels, on the basis of one- and two- photon induced fluorescence spectroscopy. Our developed optical configuration comprises a tunable titanium-sapphire laser (710nm-830nm) in combination with second harmonic wavelength generation (355nm-415nm), enabling the measurement of both one- and two-photon induced fluorescence spectra. Moreover, an accurate scanning of the kernel's surface was induced by the use of automated translation stages, allowing to study the localized maize contamination. First, the operation of the setup is validated by the characterization of pure aflatoxin B1 powder. Second, the fluorescence spectra of healthy (maize kernels (>70ppb aflatoxin B1) were measured, after excitation with 365nm, 730nm, 750nm and 780nm. For both the one- and two- photon induced fluorescence processes, the presence of the aflatoxin inside the contaminated maize kernels influenced the intrinsic fluorescence signals. Based on the fluorescence spectrum between 400nm and 550nm, we defined a detection criterion to identify the contaminated maize kernels. Furthermore, we demonstrate the sensing of the localized contamination level, indicating both contaminated maize kernels with a high contamination level in a limited surface area (as small as 1mm2) as with a lower contamination spread over a large surface area (up to 20mm2). As a result, our developed measurement methodology allows the identification of the localized aflatoxin contamination, paving the way to the non-destructive, real-time and high-sensitive industrial scanning-based detection of aflatoxins in food products.

  14. A new endoplasmic reticulum-targeted two-photon fluorescent probe for imaging of superoxide anion in diabetic mice.

    Science.gov (United States)

    Xiao, Haibin; Liu, Xiao; Wu, Chuanchen; Wu, Yaohuan; Li, Ping; Guo, Xiaomeng; Tang, Bo

    2017-05-15

    Excessive or unfolded proteins accumulation in endoplasmic reticulum (ER) will cause ER stress, which has evolved to involve in various metabolic diseases. In particular, ER stress plays an important role in the pathogenesis of diabetes. Both ER stress and course of diabetes accompany oxidative stress and production of reactive oxygen species (ROS), among which superoxide anion (O2(•-)) is the first produced ROS and has been recognized as cell signaling mediator involved in the physiological and pathological process of diabetes. Hence, the development of effective monitoring methods of O2(•-) in live cells and in vivo is of great importance for ascertaining the onset and progress of related diseases. Herein, a new endoplasmic reticulum-targeted two-photon fluorescent probe termed ER-BZT is designed and synthesized for imaging of O2(•-). The probe ER-BZT shows high sensitivity, selectivity, stability, and low cytotoxicity. Based on these superior properties, the rise of O2(•-) levels in endoplasmic reticulum induced with different stimuli is visualized by one- and two-photon fluorescence imaging. Most importantly, by utilizing ER-BZT, the two-photon fluorescence imaging results demonstrate that the endogenous O2(•-) concentration in abdominal or hepatic tissue of diabetic mice is higher than that in normal mice. Meanwhile, after treated with metformin, a broad-spectrum antidiabetic drug, the diabetic mice exhibit depressed O2(•-) level. The proposed two-photon probe, ER-BZT might serve as perfect tool to image the O2(•-) fluctuations and study the relevance between O2(•-) and various diseases in live cells and in vivo.

  15. Dynamic characterization of hydrophobic and hydrophilic solutes in oleic-acid enhanced transdermal delivery using two-photon fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Te-Yu; Yang, Chiu-Sheng; Chen, Yang-Fang [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Tsai, Tsung-Hua [Department of Dermatology, Far Eastern Memorial Hospital, New Taipei City, Taiwan (China); Dong, Chen-Yuan, E-mail: cydong@phys.ntu.edu.tw [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Center for Quantum Science and Engineering, National Taiwan University, Taipei, Taiwan (China); Center for Optoelectronic Biomedicine, National Taiwan University, Taipei, Taiwan (China)

    2014-10-20

    In this letter, we propose an efficient methodology of investigating dynamic properties of sulforhodamine B and rhodamine B hexyl ester molecules transporting across ex-vivo human stratum corneum with and without oleic acid enhancement. Three-dimensional, time-lapse fluorescence images of the stratum corneum can be obtained using two-photon fluorescence microscopy. Furthermore, temporal quantifications of transport enhancements in diffusion parameters can be achieved with the use of Fick's second law. Dynamic characterization of solutes transporting across the stratum corneum is an effective method for understanding transient phenomena in transdermal delivery of probe molecules, leading to improved delivery strategies of molecular species for therapeutic purposes.

  16. The use of one- and two- photon induced fluorescence spectroscopy for the optical characterization of carcinogenic aflatoxins

    Science.gov (United States)

    Smeesters, L.; Meulebroeck, W.; Raeymaekers, S.; Thienpont, H.

    2014-09-01

    Carcinogenic and toxic contaminants in food and feed products are nowadays mostly detected by destructive, time-consuming chemical analyses, like HPLC and LC-MS/MS methods. However, as a consequence of the severe and growing regulations on food products by the European Union, there arose an increased demand for the ultra-fast, high-sensitive and non-destructive detection of contaminants in food and feed products. Therefore, we have investigated fluorescence spectroscopy for the characterization of carcinogenic aflatoxins. With the use of a tunable titanium-sapphire laser in combination with second and third harmonic wavelength generation, both one- and two-photon induced fluorescence excitation wavelengths could be generated using the same setup. We characterized and compared the one- and two-photon induced fluorescence spectra of pure aflatoxin powder, after excitation with 365nm and 730nm respectively. Moreover, we investigated the absolute fluorescence intensity as function of the excitation power density. Afterwards, we applied our characterization setup to the detection of aflatoxins in maize grains. The fluorescence spectra of both healthy and contaminated maize samples were experimentally characterized. In addition to the fluorescence spectrum of the pure aflatoxin, we observed an unwanted influence of the intrinsic fluorescence of the maize. Depending on the excitation wavelength, a varying contrast between the fluorescence spectra of the healthy and contaminated samples was obtained. After a comparison of the measured fluorescence signals, a detection criterion for the optical identification of the contaminated maize samples could be defined. As a result, this illustrates the use of fluorescence spectroscopy as a valuable tool for the non-destructive, real-time and high-sensitive detection of aflatoxins in maize.

  17. Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

    Directory of Open Access Journals (Sweden)

    Mély Yves

    2008-09-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

  18. Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

    Science.gov (United States)

    Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

    2015-04-29

    Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

  19. Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

    Energy Technology Data Exchange (ETDEWEB)

    Mueller-Harvey, Irene, E-mail: i.mueller-harvey@reading.ac.uk [Chemistry and Biochemistry Laboratory, Food Production and Quality Research Division, School of Agriculture, Policy and Development, University of Reading, P O Box 236, Reading RG6 6AT (United Kingdom); Feucht, Walter, E-mail: walter.feucht@gmail.com [Department of Plant Sciences, Technical University of Munich (TUM), Wissenschaftszentrum Weihenstephan (WZW), D-85354 Freising (Germany); Polster, Juergen, E-mail: j.polster@wzw.tum.de [Department of Physical Biochemistry, Technical University of Munich (TUM), Wissenschaftszentrum Weihenstephan (WZW), D-85354 Freising (Germany); Trnkova, Lucie, E-mail: lucie.trnkova@uhk.cz [University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 50003 Hradec Kralove (Czech Republic); Burgos, Pierre, E-mail: pierre.burgos@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom); Parker, Anthony W., E-mail: tony.parker@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom); Botchway, Stanley W., E-mail: stan.botchway@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer This fluorescence lifetime imaging microscopy (FLIM) technique for flavanols overcomes autofluorescence interference in cells. Black-Right-Pointing-Pointer Plant flavanols differed in their lifetimes. Black-Right-Pointing-Pointer Dissolved and bound flavanols revealed contrasting lifetime changes. Black-Right-Pointing-Pointer This technique will allow studying of flavanol trafficking in live cells. - Abstract: Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were {approx}1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime ({tau}{sub 2} = 1.9-3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there

  20. Theoretical investigation on ratiometric two-photon fluorescent probe for Zn2+ detection based on ICT mechanism

    Science.gov (United States)

    Huang, Shuang; Yang, Bao-Zhu; Ren, Ai-Min

    2016-06-01

    OPA (one-photon absorption), TPA (two-photon absorption) and fluorescence properties of a free ligand L upon coordination with Zn2+, and the regeneration with CN- were investigated in theory. According to our research, OPA spectra of ligand L show red-shift binding with Zn2+ while blue-shift with CN-. The fluorescence spectra and TPA wavelength are shifted in the same situation as those of OPA spectra. The value of TPA cross-section decreased at first, and then increased to 1813 GM for [L-Zn(CN)4]2-. Intramolecular charge transfer (ICT) mechanism was investigated by natural bond orbital (NBO) analysis. It demonstrates that L is hopeful to be a good ratiometric fluorescent probe for zinc ion detection in solution, and it can regenerate after CN- was introduced.

  1. Deep-red polymer dots with bright two-photon fluorescence and high biocompatibility for in vivo mouse brain imaging

    Science.gov (United States)

    Alifu, Nuernisha; Sun, Zezhou; Zebibula, Abudureheman; Zhu, Zhenggang; Zhao, Xinyuan; Wu, Changfeng; Wang, Yalun; Qian, Jun

    2017-09-01

    With high contrast and deep penetration, two-photon fluorescence (2PF) imaging has become one of the most promising in vivo fluorescence imaging techniques. To obtain good imaging contrast, fluorescent nanoprobes with good 2PF properties are highly needed. In this work, bright 2PF polymer dots (P dots) were applied for in vivo mouse brain imaging. Deep-red emissive P dots with PFBT as the donor and PFDBT5 as the acceptor were synthesized and used as a contrast agent. P dots were further encapsulated by poly(styrene-co-maleic anhydride) (PSMA) and grafted with poly(ethylene glycol) (PEG). The P dots-PEG exhibit large two-photon absorption (2PA) cross-sections (δ≥8500 g), good water dispersibility, and high biocompatibility. P dots-PEG was further utilized first time for in vivo vascular imaging of mouse ear and brain, under 690-900 nm femtosecond (fs) laser excitation. Due to the large 2PA cross-section and deep-red emission, a large imaging depth ( 720 μm) was achieved.

  2. Mean cell size and collagen orientation from 2D Fourier analysis on confocal laser scanning microscopy and two-photon fluorescence microscopy on human skin in vivo

    Science.gov (United States)

    Lucassen, Gerald W.; Bakker, Bernard L.; Neerken, Sieglinde; Hendriks, Rob F. M.

    2003-07-01

    We present results from 2D Fourier analysis on 3D stacks of images obtained by confocal laser scanning reflectance microscopy (CLSM) and two-photon fluorescence microscopy (2PM) on human skin in vivo. CLSM images were obtained with a modified commercial system (Vivascope1000, Lucid Inc, excitation wavelength 830 nm) equipped with a piezo-focusing element (350 μm range) for depth positioning of the objective lens. 2PM was performed with a specially designed set-up with excitation wavelength 730 nm. Mean cell size in the epidermal layer and structural orientation in the dermal layer have been determined as a function of depth by 2D Fourier analysis. Fourier analysis on microscopic images enables automatic non-invasive quantitative structural analysis (mean cell size and orientation) of living human skin.

  3. Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas

    Science.gov (United States)

    Alexander, Nathan S.; Palczewska, Grazyna; Palczewski, Krzysztof

    2015-01-01

    Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE. PMID:26309765

  4. Noninvasive two-photon fluorescence microscopy imaging of mouse retina and RPE through the pupil of the eye

    Science.gov (United States)

    Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Hunter, Jennifer J.; Williams, David R.; Alexander, Nathan S.; Palczewski, Krzysztof

    2014-01-01

    Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions. PMID:24952647

  5. Enhanced two photon fluorescence microfluidic sensor based on dual cladding photonic-crystal fiber

    Science.gov (United States)

    Amitonova, Lyubov; Fedotov, Ilya; Fedotov, Andrey; Zheltikov, Aleksei

    2012-11-01

    The architecture of photonic-crystal fibers (PCFs) suggests a variety of strategies for optical sensing. A combination of TPA approaches with capabilities of fiber-optic probes offers numerous advantages, suggesting a convenient format for beam delivery, facilitating manipulation of excitation radiation, and allowing this excitation to be applied locally and selectively. In this work, we show that a PCF with a special design can realize different protocols of optical sensing, simultaneously serving, whenever necessary, for the collection and on-line monitoring of liquid-phase samples. Specially designed PCF is shown to substantially increase the guided-wave luminescent response from molecules excited through two-photon absorption (TPA) by femtosecond near-infrared laser pulses. Biophotonic implications of this waveguide TPL-response enhancement include fiber-format solutions for online monitoring of drug delivery and drug activation, interrogation of neural activity, biosensing, endoscopy, and locally controlled singlet oxygen generation in photodynamic therapy. This work was supported by the Russian Foundation for Basic Research, project 11-04-12185-ofi-m.

  6. Nonlinear spectral imaging of human hypertrophic scar based on two-photon excited fluorescence and second-harmonic generation.

    Science.gov (United States)

    Chen, G; Chen, J; Zhuo, S; Xiong, S; Zeng, H; Jiang, X; Chen, R; Xie, S

    2009-07-01

    A noninvasive method using microscopy and spectroscopy for analysing the morphology of collagen and elastin and their biochemical variations in skin tissue will enable better understanding of the pathophysiology of hypertrophic scars and facilitate improved clinical management and treatment of this disease. To obtain simultaneously microscopic images and spectra of collagen and elastin fibres in ex vivo skin tissues (normal skin and hypertrophic scar) using a nonlinear spectral imaging method, and to compare the morphological structure and spectral characteristics of collagen and elastin fibres in hypertrophic scar tissues with those of normal skin, to determine whether this approach has potential for in vivo assessment of the pathophysiology of human hypertrophic scars and for monitoring treatment responses as well as for tracking the process of development of hypertrophic scars in clinic. Ex vivo human skin specimens obtained from six patients aged from 10 to 50 years old who were undergoing skin plastic surgery were examined. Five patients had hypertrophic scar lesions and one patient had no scar lesion before we obtained his skin specimen. A total of 30 tissue section samples of 30 mum thickness were analysed by the use of a nonlinear spectral imaging system consisting of a femtosecond excitation light source, a high-throughput scanning inverted microscope, and a spectral imaging detection system. The high-contrast and high-resolution second harmonic generation (SHG) images of collagen and two-photon excited fluorescence (TPEF) images of elastin fibres in hypertrophic scar tissues and normal skin were acquired using the extracting channel tool of the system. The emission spectra were analysed using the image-guided spectral analysis method. The depth-dependent decay constant of the SHG signal and the image texture characteristics of hypertrophic scar tissue and normal skin were used to quantitatively assess the amount, distribution and orientation of their

  7. Two-photon absorption laser induced fluorescence measurement of atomic oxygen density in an air atmospheric pressure plasma jet

    Science.gov (United States)

    Conway, Jim; Gogna, Gurusharan; Daniels, Stephen

    2016-09-01

    Two-photon Absorption Laser Induced Fluorescence (TALIF) is used to measure atomic oxygen number density [O] in an air Atmospheric Pressure Plasma Jet (APPJ). A novel technique based on photolysis of O2 is used to calibrate the TALIF system ensuring the same species (O) is probed during calibration and measurement. As a result, laser intensity can be increased outside the TALIF quadratic laser power region without affecting calibration reliability as any high intensity saturation effects will be identical for calibration and experiment. Higher laser intensity gives stronger TALIF signals helping overcome weak TALIF signals often experienced at atmospheric pressure due to collisional quenching. O2 photo-dissociation and two-photon excitation of the resulting [O] are both achieved within the same laser pulse. The photolysis [O] is spatially non-uniform and time varying. To allow valid comparison with [O] in a plasma, spatial and temporal correction factors are required. Knowledge of the laser pulse intensity I0(t), and wavelength allows correction factors to be found using a rate equation model. The air flow into the jet was fixed and the RF power coupled into the system varied. The resulting [O] was found to increase with RF power.

  8. Label-free imaging of brain and brain tumor specimens with combined two-photon excited fluorescence and second harmonic generation microscopy

    Science.gov (United States)

    Jiang, Liwei; Wang, Xingfu; Wu, Zanyi; Du, Huiping; Wang, Shu; Li, Lianhuang; Fang, Na; Lin, Peihua; Chen, Jianxin; Kang, Dezhi; Zhuo, Shuangmu

    2017-10-01

    Label-free imaging techniques are gaining acceptance within the medical imaging field, including brain imaging, because they have the potential to be applied to intraoperative in situ identifications of pathological conditions. In this paper, we describe the use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy in combination for the label-free detection of brain and brain tumor specimens; gliomas. Two independently detecting channels were chosen to subsequently collect TPEF/SHG signals from the specimen to increase TPEF/SHG image contrasts. Our results indicate that the combined TPEF/SHG microscopic techniques can provide similar rat brain structural information and produce a similar resolution like conventional H&E staining in neuropathology; including meninges, cerebral cortex, white-matter structure corpus callosum, choroid plexus, hippocampus, striatum, and cerebellar cortex. It can simultaneously detect infiltrating human brain tumor cells, the extracellular matrix collagen fiber of connective stroma within brain vessels and collagen depostion in tumor microenvironments. The nuclear-to-cytoplasmic ratio and collagen content can be extracted as quantitative indicators for differentiating brain gliomas from healthy brain tissues. With the development of two-photon fiberscopes and microendoscope probes and their clinical applications, the combined TPEF and SHG microcopy may become an important multimodal, nonlinear optical imaging approach for real-time intraoperative histological diagnostics of residual brain tumors. These occur in various brain regions during ongoing surgeries through the method of simultaneously identifying tumor cells, and the change of tumor microenvironments, without the need for the removal biopsies and without the need for tissue labelling or fluorescent markers.

  9. Enhanced two-photon fluorescence imaging and therapy of cancer cells via Gold@bridged silsesquioxane nanoparticles.

    Science.gov (United States)

    Croissant, Jonas; Maynadier, Marie; Mongin, Olivier; Hugues, Vincent; Blanchard-Desce, Mireille; Chaix, Arnaud; Cattoën, Xavier; Wong Chi Man, Michel; Gallud, Audrey; Gary-Bobo, Magali; Garcia, Marcel; Raehm, Laurence; Durand, Jean-Olivier

    2015-01-21

    A two-photon photosensitizer with four triethoxysilyl groups is synthesized through the click reaction. This photosensitizer allows the design of bridged silsesquioxane (BS) nanoparticles through a sol-gel process; moreover, gold core BS shells or BS nanoparticles decorated with gold nanospheres are synthesized. An enhancement of the two-photon properties is noted with gold and the nanoparticles are efficient for two-photon imaging and two-photon photodynamic therapy of cancer cells.

  10. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.

    Science.gov (United States)

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine.

  11. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    Science.gov (United States)

    Jahn, Karolina; Hille, Carsten

    2014-01-01

    For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+)-free and Ca(2+)-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+)-dependent way, unraveling in vitro dissociation constants K(D) of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca(2+)-free and Ca(2+)-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D) of 180 nM was determined. Thus, quantitative [Ca(2+)]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+)]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+) indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  12. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    Directory of Open Access Journals (Sweden)

    Karolina Jahn

    Full Text Available For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR and Asante Calcium Green (ACG for two-photon (2P-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+-free and Ca(2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+-dependent way, unraveling in vitro dissociation constants K(D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns and long (2.44 ns decay time components attributable to the Ca(2+-free and Ca(2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D of 180 nM was determined. Thus, quantitative [Ca(2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  13. Fluorescent detection and imaging of Hg{sup 2+} using a novel phenanthroline derivative based single- and two-photon excitation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xian, E-mail: zhangx@qlu.edu.cn; Li, Long-long; Liu, Ying-kai

    2016-02-01

    A novel phenanthroline derivative, 4-[4-(N-methyl)styrene]-imidazo[4,5-f][1,10]phenanthroline-benzene iodated salt (MSIPBI), was synthesized, and the linear absorption and fluorescent spectra of MSIPBI in different solvents were investigated. The photophysical properties in unbound and in ligand–metal complexes were evaluated by UV absorption and one- and two-photon fluorescent spectra, and the quantum yields, two-photon active cross-sections and the binding constant of dye–metal were calculated. The results indicated that MSIPBI has a large Stokes shift (more than 167 nm), and the dye was selective and sensitive for the detection of Hg{sup 2+} with a two-photon active cross-section of 55.5 GM in tris–HCl buffer solution at 800 nm. Furthermore, the results of the fluorescence microscopy imaging indicated that MSIPBI is an efficient fluorescent probe for the detection of Hg{sup 2+} in living cells by one- and two-photon excitation. Moreover, the experiments of determination Hg{sup 2+} in river water and tap water were finished. - Highlights: • A novel phenanthroline derivative (MSIPBI) has been synthesized. • The dye of MSIPBI was selective and sensitive to detect Hg{sup 2+}. • MSIPBI has a large Stokes shift (≥ 167 nm). • Hg{sup 2+} in living cells was successfully imaged by one- and two-photon excitation.

  14. Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.

    Science.gov (United States)

    Peti-Peterdi, János; Morishima, Shigeru; Bell, P Darwin; Okada, Yasunobu

    2002-07-01

    Recently, multiphoton excitation fluorescence microscopy has been developed that offers important advantages over confocal imaging, particularly for in vivo visualization of thick tissue samples. We used this state-of-the-art technique to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney. This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal hemodynamics. We report, for the first time, on high-resolution three-dimensional morphology and Z-sectioning through isolated, perfused kidney glomeruli, tubules, and JGA. Time-series images show how alterations in tubular fluid composition cause striking changes in single-cell volume of the unique macula densa tubular epithelium in situ and how they also affect glomerular filtration through alterations in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. This imaging approach to the study of isolated, perfused live tissue with multiphoton microscopy may be applied to other biological systems in which multiple cell types form a functionally integrated syncytium.

  15. Photolytic-interference-free, femtosecond, two-photon laser-induced fluorescence imaging of atomic oxygen in flames

    Science.gov (United States)

    Kulatilaka, Waruna D.; Roy, Sukesh; Jiang, Naibo; Gord, James R.

    2016-02-01

    Ultrashort-pulse lasers are well suited for nonlinear diagnostic techniques such as two-photon laser-induced fluorescence (TPLIF) because the signals generated scale as the laser intensity squared. Furthermore, the broad spectral bandwidths associated with nearly Fourier-transform-limited ultrashort pulses effectively contribute to efficient nonlinear excitation by coupling through a large number of in-phase photon pairs, thereby producing strong fluorescence signals. Additionally, femtosecond (fs)-duration amplified laser systems typically operate at 1-10 kHz repetition rates, enabling high-repetition-rate imaging in dynamic environments. In previous experiments, we have demonstrated utilization of fs pulses for kilohertz (kHz)-rate, interference-free imaging of atomic hydrogen (H) in flames. In the present study, we investigate the utilization of fs-duration pulses to photolytic-interference-free TPLIF imaging of atomic oxygen (O). In TPLIF of O, photodissociation of vibrationally excited carbon dioxide (CO2) is known to be the prominent interference that produces additional O atoms in the medium. We have found that through the use of fs excitation, such interferences can be virtually eliminated in premixed laminar methane flames, which paves the way for two-dimensional imaging of O at kHz data rates. Such measurements can provide critical data for validating complex, multidimensional turbulent-combustion models as well as for investigating flame dynamics in practical combustion devices.

  16. Endogenous two-photon fluorescence imaging elucidates metabolic changes related to enhanced glycolysis and glutamine consumption in precancerous epithelial tissues.

    Science.gov (United States)

    Varone, Antonio; Xylas, Joanna; Quinn, Kyle P; Pouli, Dimitra; Sridharan, Gautham; McLaughlin-Drubin, Margaret E; Alonzo, Carlo; Lee, Kyongbum; Münger, Karl; Georgakoudi, Irene

    2014-06-01

    Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.

  17. Functional double-shelled silicon nanocrystals for two-photon fluorescence cell imaging: spectral evolution and tuning

    Science.gov (United States)

    Chandra, Sourov; Ghosh, Batu; Beaune, Grégory; Nagarajan, Usharani; Yasui, Takao; Nakamura, Jin; Tsuruoka, Tohru; Baba, Yoshinobu; Shirahata, Naoto; Winnik, Françoise M.

    2016-04-01

    Functional near-IR (NIR) emitting nanoparticles (NPs) adapted for two-photon excitation fluorescence cell imaging were obtained starting from octadecyl-terminated silicon nanocrystals (ncSi-OD) of narrow photoluminescence (PL) spectra having no long emission tails, continuously tunable over the 700-1000 nm window, PL quantum yields exceeding 30%, and PL lifetimes of 300 μs or longer. These NPs, consisting of a Pluronic F127 shell and a core made up of assembled ncSi-OD kept apart by an octadecyl (OD) layer, were readily internalized into the cytosol, but not the nucleus, of NIH3T3 cells and were non-toxic. Asymmetrical field-flow fractionation (AF4) analysis was carried out to determine the size of the NPs in water. HiLyte Fluor 750 amine was linked via an amide link to NPs prepared with Pluronic-F127-COOH, as a first demonstration of functional NIR-emitting water dispersible ncSi-based nanoparticles.Functional near-IR (NIR) emitting nanoparticles (NPs) adapted for two-photon excitation fluorescence cell imaging were obtained starting from octadecyl-terminated silicon nanocrystals (ncSi-OD) of narrow photoluminescence (PL) spectra having no long emission tails, continuously tunable over the 700-1000 nm window, PL quantum yields exceeding 30%, and PL lifetimes of 300 μs or longer. These NPs, consisting of a Pluronic F127 shell and a core made up of assembled ncSi-OD kept apart by an octadecyl (OD) layer, were readily internalized into the cytosol, but not the nucleus, of NIH3T3 cells and were non-toxic. Asymmetrical field-flow fractionation (AF4) analysis was carried out to determine the size of the NPs in water. HiLyte Fluor 750 amine was linked via an amide link to NPs prepared with Pluronic-F127-COOH, as a first demonstration of functional NIR-emitting water dispersible ncSi-based nanoparticles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01437b

  18. Fs-transient absorption and fluorescence upconversion after two- photon excitation of carotenoids in solution and in LHC II

    CERN Document Server

    Wall, P J; Fleming, G R

    2000-01-01

    With time resolved two-photon techniques we determined the lifetime and two-photon spectrum of the forbidden S/sub 1/ state of beta - carotene (9+or-0.2 ps), lutein (15+or-0.5 ps) and the energy transferring carotenoids in LHC II (250+or-50 fs). (7 refs).

  19. Volumetric label-free imaging and 3D reconstruction of mammalian cochlea based on two-photon excitation fluorescence microscopy

    Science.gov (United States)

    Zhang, Xianzeng; Geng, Yang; Ye, Qing; Zhan, Zhenlin; Xie, Shusen

    2013-11-01

    The visualization of the delicate structure and spatial relationship of intracochlear sensory cells has relied on the laborious procedures of tissue excision, fixation, sectioning and staining for light and electron microscopy. Confocal microscopy is advantageous for its high resolution and deep penetration depth, yet disadvantageous due to the necessity of exogenous labeling. In this study, we present the volumetric imaging of rat cochlea without exogenous dyes using a near-infrared femtosecond laser as the excitation mechanism and endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. We find that TPEF exhibits strong contrast, allowing cellular and even subcellular resolution imaging of the cochlea, differentiating cell types, visualizing delicate structures and the radial nerve fiber. Our results further demonstrate that 3D reconstruction rendered with z-stacks of optical sections enables better revealment of fine structures and spatial relationships, and easily performed morphometric analysis. The TPEF-based optical biopsy technique provides great potential for new and sensitive diagnostic tools for hearing loss or hearing disorders, especially when combined with fiber-based microendoscopy.

  20. Indole-based cyanine as a nuclear RNA-selective two-photon fluorescent probe for live cell imaging.

    Science.gov (United States)

    Guo, Lei; Chan, Miu Shan; Xu, Di; Tam, Dick Yan; Bolze, Frédéric; Lo, Pik Kwan; Wong, Man Shing

    2015-05-15

    We have demonstrated that the subcellular targeting properties of the indole-based cyanines can be tuned by the functional substituent attached onto the indole moiety in which the first example of a highly RNA-selective and two-photon active fluorescent light-up probe for high contrast and brightness TPEF images of rRNA in the nucleolus of live cells has been developed. It is important to find that this cyanine binds much stronger toward RNA than DNA in a buffer solution as well as selectively stains and targets to rRNA in the nucleolus. Remarkably, the TPEF brightness (Φσmax) is dramatically increased with 11-fold enhancement in the presence of rRNA, leading to the record high Φσmax of 228 GM for RNA. This probe not only shows good biocompatibility and superior photostability but also offers general applicability to various live cell lines including HeLa, HepG2, MCF-7, and KB cells and excellent counterstaining compatibility with commercially available DNA or protein trackers.

  1. Spatially and Temporally Resolved Atomic Oxygen Measurements in Short Pulse Discharges by Two Photon Laser Induced Fluorescence

    Science.gov (United States)

    Lempert, Walter; Uddi, Mruthunjaya; Mintusov, Eugene; Jiang, Naibo; Adamovich, Igor

    2007-10-01

    Two Photon Laser Induced Fluorescence (TALIF) is used to measure time-dependent absolute oxygen atom concentrations in O2/He, O2/N2, and CH4/air plasmas produced with a 20 nanosecond duration, 20 kV pulsed discharge at 10 Hz repetition rate. Xenon calibrated spectra show that a single discharge pulse creates initial oxygen dissociation fraction of ˜0.0005 for air like mixtures at 40-60 torr total pressure. Peak O atom concentration is a factor of approximately two lower in fuel lean (φ=0.5) methane/air mixtures. In helium buffer, the initially formed atomic oxygen decays monotonically, with decay time consistent with formation of ozone. In all nitrogen containing mixtures, atomic oxygen concentrations are found to initially increase, for time scales on the order of 10-100 microseconds, due presumably to additional O2 dissociation caused by collisions with electronically excited nitrogen. Further evidence of the role of metastable N2 is demonstrated from time-dependent N2 2^nd Positive and NO Gamma band emission spectroscopy. Comparisons with modeling predictions show qualitative, but not quantitative, agreement with the experimental data.

  2. Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

    Directory of Open Access Journals (Sweden)

    Ortrud Uckermann

    2015-01-01

    Full Text Available Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

  3. Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

    Science.gov (United States)

    Uckermann, Ortrud; Galli, Roberta; Beiermeister, Rudolf; Sitoci-Ficici, Kerim-Hakan; Later, Robert; Leipnitz, Elke; Neuwirth, Ales; Chavakis, Triantafyllos; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2015-01-01

    Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes. PMID:26355949

  4. Two-photon excited endogenous fluorescence for label-free in vivo imaging ingestion of disease-causing bacteria by human leukocytes

    Science.gov (United States)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-02-01

    Real time and in vivo monitoring leukocyte behavior provides unique information to understand the physiological and pathological process of infection. In this study, we demonstrate that two-photon excited reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides imaging contrast to distinguish granulocyte and agranulocyte. By using spectral and time-resolved NADH fluorescence, we study the immune response of human neutrophils against bacterial infection (Escherichia coli). The two-photon excited NADH fluorescence images clearly review the morphological changes from resting neutrophils (round shape) to activated neutrophils (ruffle shape) during phagocytosis. The free-tobound NADH ratio of neutrophils decreases after ingesting disease-causing pathogen: Escherichia coli. This finding may provide a new optical tool to investigate inflammatory processes by using NADH fluorescence in vivo.

  5. A bistriphenylamine-substituted spirobifluorene derivative exhibiting excellent nonlinearity/transparency/thermal stability trade-off and strong two-photon induced blue fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Hongyao [Department of Chemistry, Shanghai Normal University, Shanghai 200234 (China); Xiao, Haibo, E-mail: xiaohb@shnu.edu.cn [Department of Chemistry, Shanghai Normal University, Shanghai 200234 (China); Ding, Lei [Department of Chemistry, Shanghai Normal University, Shanghai 200234 (China); Zhang, Chun; Ren, Aiming [State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023 (China); Li, Bo [Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai 200241 (China)

    2015-02-01

    A spirobifluorene-bridged donor/donor chromophore, 2,7-bis-(4-(N,N-diphenylamino)phen-1-yl)-9,9′-spirobifluorene (SPF-TP), was found to combine excellent transparency in the near UV–visible region (λ{sub cut-off} ≤ 420 nm), large two-photon absorption cross-section (4.5 × 10{sup 3}GM) and high thermal stability (T{sub d} = 501 °C). In comparison to the reported two-photon absorption molecules, SPF-TP represents the best thermal stability so far described in the literature. The main electronic factors explaining the high two-photon absorption activities of SPF-TP were analyzed by theoretical calculations. Cyclic voltammograms were employed to explore the causes of the excellent transparency of SPF-TP. It was found that the spiroconjugation effect is responsible for the excellent nonlinearity/transparency/thermal stability trade-off in SPF-TP. In addition, SPF-TP is also a good two-photon induced blue fluorescent material with high fluorescence quantum yield (Φ = 0.90, in THF). - Highlights: • We report a molecule exhibiting excellent transparency. • The two-photon absorption cross-section is as large as 4.5 × 10{sup 3}GM. • The molecule exhibits excellent thermal stability. • The molecule is a good two-photon induced blue fluorescent material. • The spiroconjugation effect explains the excellent properties.

  6. Center for Fluorescence Spectroscopy: advanced studies of fluorescence dynamics, lifetime imaging, clinical sensing, two-photon excitation, and light quenching

    Science.gov (United States)

    Lakowicz, Joseph R.; Malak, Henryk M.; Gryczynski, Ignacy; Szmacinski, Henryk; Kusba, Jozef; Akkaya, Engin; Terpetschnig, Ewald A.; Johnson, Michael L.

    1994-08-01

    The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation. Both time-domain (TD) and frequency- domain (FD) measurements (10 MHz to 10 Ghz) are available, with a wide range of excitation and emission wavelengths (UV to NIR). The data can be used to recover distances and site-to-site diffusion in protein, interactions between macromolecules, accessibility of fluorophores to quenchers, and the dynamic properties of proteins, membranes and nucleic acids. Current software provides for analysis of multi-exponential intensity and anisotropy decays, lifetime distribution, distance distributions for independent observation of fluorescence donors and acceptors, transient effects in collisional quenching, phase-modulation spectra and time-resolved emission spectra. Most programs provide for global analysis of multiple data sets obtained under similar experimental conditions. Data can be analyzed on-site by connection with the CFS computers through the internet. During six years of operation we have established scientific collaborations with over 30 academic and industrial groups in the United States. These collaborations have resulted in 63 scientific papers.

  7. Emission turn-on and solubility turn-off in conjugated polymers: one- and two-photon-induced removal of fluorescence-quenching solubilizing groups.

    Science.gov (United States)

    Schelkle, Korwin M; Becht, Steffy; Faraji, Shirin; Petzoldt, Martin; Müllen, Klaus; Buckup, Tiago; Dreuw, Andreas; Motzkus, Marcus; Hamburger, Manuel

    2015-01-01

    The synthesis of highly efficient two-photon uncaging groups and their potential use in functional conjugated polymers for post-polymerization modification are reported. Careful structural design of the employed nitrophenethyl caging groups allows to efficiently induce bond scission by a two-photon process through a combination of exceptionally high two-photon absorption cross-sections and high reaction quantum yields. Furthermore, π-conjugated polyfluorenes are functionalized with these photocleavable side groups and it is possible to alter their emission properties and solubility behavior by simple light irradiation. Cleavage of side groups leads to a turn-on of the fluorescence while solubility of the π-conjugated materials is drastically reduced.

  8. Evaluation of the oxidative stress of psoriatic fibroblasts based on spectral two-photon fluorescence lifetime imaging

    Science.gov (United States)

    Kapsokalyvas, Dimitrios; Barygina, Victoria; Cicchi, Riccardo; Fiorillo, Claudia; Pavone, Francesco S.

    2013-02-01

    Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Metabolic activity is increased in the epidermis and the dermis. Oxidative stress is high mainly due to reactive oxygen species (ROS) originating from the skin environment and cellular metabolism. We employed a custom multiphoton microscope coupled with a FLIM setup to image primary culture fibroblast cells from perilesional and lesional psoriatic skin in-vitro. Twophoton excited fluorescence images revealed the morphological differences between healthy and psoriatic fibroblasts. Based on the spectral analysis of the NADH and FAD components the oxidative stress was assessed and found to be higher in psoriatic cells. Furthermore the fluorescence lifetime properties were investigated with a TCSPC FLIM module. Mean fluorescence lifetime was found to be longer in psoriatic lesional cells. Analysis of the fast (τ1) and slow (τ2) decay lifetimes revealed a decrease of the ratio of the contribution of the fast (α1) parameter to the contribution of the slow (α2) parameter. The fluorescence in the examined part of the spectrum is attributed mainly to NADH. The decrease of the ratio (α1)/ (α2) is believed to correlate strongly with the anti-oxidant properties of NADH which can lead to the variation of its population in high ROS environment. This methodology could serve as an index of the oxidative status in cells and furthermore could be used to probe the oxidative stress of tissues in-vivo.

  9. N-doped carbon dots derived from bovine serum albumin and formic acid with one- and two-photon fluorescence for live cell nuclear imaging.

    Science.gov (United States)

    Tan, Mingqian; Li, Xintong; Wu, Hao; Wang, Beibei; Wu, Jing

    2015-12-01

    Carbon dots with both one- and two-photon fluorescence have drawn great attention for biomedical imaging. Herein, nitrogen-doped carbon dots were facilely developed by one-pot hydrothermal method using bovine serum albumin and formic acid as carbon sources. They are highly water-soluble with strong fluorescence when excited with ultraviolet or near infrared light. The carbon dots have a diameter of ~8.32 nm and can emit strong two-photon induced fluorescence upon excitation at 750 nm with a femtosecond laser. X-ray photoelectron spectrometer analysis revealed that the carbon dots contained three components, C, N and O, corresponding to the peak at 285, 398 and 532 eV, respectively. The Fourier-transform infrared spectroscopy analysis revealed that there are carboxyl and carboxylic groups on the surface, which allowed further linking of functional molecules. pH stability study demonstrated that the carbon dots are able to be used in a wide range of pH values. The fluorescence mechanism is also discussed in this study. Importantly, these carbon dots are biocompatible and highly photostable, which can be directly applied for both one- and two-photon living cell imaging. After proper surface functionalization with TAT peptide, they can be used as fluorescent probes for live cell nuclear-targeted imaging.

  10. Two-photon microscopy for chemical neuroscience.

    Science.gov (United States)

    Ellis-Davies, Graham C R

    2011-04-20

    Microscopes using non-linear excitation of chromophores with pulsed near-IR light can generate highly localized foci of molecules in the electronic singlet state that are concentrated in volumes of less than one femtoliter. The three-dimensional confinement of excitation arises from the simultaneous absorption of two IR photons of approximately half the energy required for linear excitation. Two-photon microscopy is especially useful for two types of interrogation of neural processes. First, uncaging of signaling molecules such as glutamate, as stimulation is so refined it can be used to mimic normal unitary synaptic levels. In addition, uncaging allows complete control of the timing and position of stimulation, so the two-photon light beam provides the chemical neuroscientist with an "optical conductor's baton" which can command synaptic activity at will. A second powerful feature of two-photon microscopy is that when used for fluorescence imaging it enables the visualization of cellular structure and function in living animals at depths far beyond that possible with normal confocal microscopes. In this review I provide a survey of the many important applications of two-photon microscopy in these two fields of neuroscience, and suggest some areas for future technical development.

  11. Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

    Science.gov (United States)

    Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

    2016-03-01

    Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

  12. Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection

    Science.gov (United States)

    Eibl, Matthias; Karpf, Sebastian; Hakert, Hubertus; Weng, Daniel; Huber, Robert

    2017-02-01

    Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. We present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.

  13. Breaking the diffraction-limited resolution barrier in fiber-optical two-photon fluorescence endoscopy by an azimuthally-polarized beam.

    Science.gov (United States)

    Gu, Min; Kang, Hong; Li, Xiangping

    2014-01-10

    Although fiber-optical two-photon endoscopy has been recognized as a potential high-resolution diagnostic and therapeutic procedure in vivo, its resolution is limited by the optical diffraction nature to a few micrometers due to the low numerical aperture of an endoscopic objective. On the other hand, stimulated emission depletion (STED) achieved by a circularly-polarized vortex beam has been used to break the diffraction-limited resolution barrier in a bulky microscope. It has been a challenge to apply the STED principle to a fiber-optical two-photon endoscope as a circular polarization state cannot be maintained due to the birefringence of a fiber. Here, we demonstrate the first fiber-optical STED two-photon endoscope using an azimuthally-polarized beam directly generated from a double-clad fiber. As such, the diffraction-limited resolution barrier of fiber-optical two-photon endoscopy can be broken by a factor of three. Our new accomplishment has paved a robust way for high-resolution in vivo biomedical studies.

  14. Fluorescence Detection of H5N1 Virus Gene Sequences Based on Optical Tweezers with Two-Photon Excitation Using a Single Near Infrared Nanosecond Pulse Laser.

    Science.gov (United States)

    Li, Cheng-Yu; Cao, Di; Kang, Ya-Feng; Lin, Yi; Cui, Ran; Pang, Dai-Wen; Tang, Hong-Wu

    2016-04-19

    We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging, respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16-19 pM with good selectivity for one-base mismatch, which is approximately 1 order of magnitude lower than the most existing fluorescence-based analysis method. Besides, because of the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.

  15. Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

    Science.gov (United States)

    Adur, J.; Pelegati, V. B.; de Thomaz, A. A.; Bottcher-Luiz, F.; Andrade, L. A. L. A.; Almeida, D. B.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

  16. Fluorescence Talbot microscope using incoherent source

    Science.gov (United States)

    Sun, Yangyang; Pang, Shuo

    2016-08-01

    Fluorescence Talbot microscope is a scalable field-of-view (FOV) imaging platform, which takes advantage of the phase sensitivity of the self-image of a periodic structure. Such a system can maintain the microscopic resolution and extend the FOV for the whole slide (15 mm×15 mm) scanning. Previously reported Talbot fluorescence systems, tabletop and on-chip device alike, rely on the coherence of the illumination source, limiting their potential applications in low-resource setting environment. A more cost-effective setup using a light-emitting diode, which has an area of 4 mm2 and a full width at half maximum of 16 nm in wavelength, is demonstrated. Compared to the illumination that is spatially filtered by a single pinhole, our system has achieved an illumination intensity that is 357 times higher. The reconstructed image quality is comparable to that of a 10× microscope objective. Various samples, such as fluorescent beads, green fluorescence protein-labeled HeLa cells, and a mouse kidney slide, were reconstructed by the system.

  17. Effect of detergents on the physico-chemical properties of skin stratum corneum: A two-photon excitation fluorescence microscopy study

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Brewer, Jonathan R.; Pashkovski, Eugene

    2014-01-01

    performed using two-photon excitation fluorescence microscopy. Fluorescent images of fluorescence reporters sensitive to proton activity and hydration of SC were obtained in excised skin and examined in presence and absence of SCM and SDS detergents. RESULTS: Hydration of the intercellular lipid matrix......OBJECTIVE: Understanding the structural and dynamical features of skin is critical for advancing innovation in personal care and drug discovery. Synthetic detergent mixtures used in commercially available body wash products are thought to be less aggressive towards the skin barrier when compared...... to conventional detergents. The aim of this work is to comparatively characterize the effect of a mild synthetic cleanser mixture (SCM) and sodium dodecyl sulphate (SDS) on the hydration state of the intercellular lipid matrix and on proton activity of excised skin stratum corneum (SC). METHOD: Experiments were...

  18. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

    Directory of Open Access Journals (Sweden)

    Kurt F Ahrens

    2012-03-01

    Full Text Available A fluorescent voltage sensor protein Flare was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular visual crescent in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo.

  19. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

    Science.gov (United States)

    Ahrens, Kurt F.; Heider, Barbara; Lee, Hanson; Isacoff, Ehud Y.; Siegel, Ralph M.

    2012-01-01

    A fluorescent voltage sensor protein “Flare” was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular region of visual cortex in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo. PMID:22461770

  20. Miniaturized integration of a fluorescence microscope

    Science.gov (United States)

    Ghosh, Kunal K.; Burns, Laurie D.; Cocker, Eric D.; Nimmerjahn, Axel; Ziv, Yaniv; Gamal, Abbas El; Schnitzer, Mark J.

    2013-01-01

    The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals towards relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including semiconductor light source and sensor. This device enables high-speed cellular-level imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens. PMID:21909102

  1. Technique of Hadamard transform microscope fluorescence image analysis

    Institute of Scientific and Technical Information of China (English)

    梅二文; 顾文芳; 曾晓斌; 陈观铨; 曾云鹗

    1995-01-01

    Hadamard transform spatial multiplexed imaging technique is combined with fluorescence microscope and an instrument of Hadamard transform microscope fluorescence image analysis is developed. Images acquired by this instrument can provide a lot of useful information simultaneously, including three-dimensional Hadamard transform microscope cell fluorescence image, the fluorescence intensity and fluorescence distribution of a cell, the background signal intensity and the signal/noise ratio, etc.

  2. Two-photon excited fluorescence from higher electronic states of chlorophylls in photosynthetic antenna complexes a new approach to detect strong excitonic chlorophyll a/b coupling

    CERN Document Server

    Leupold, D; Ehlert, J; Irrgang, K D; Renger, G; Lokstein, H

    2002-01-01

    Stepwise two-photon excitation of chlorophyll a and b in the higher plant main light-harvesting complex (LHC II) and the minor complex CP29 (as well as in organic solution) with 100-fs pulses in the Q/sub y/ region results in a weak blue fluorescence. The dependence of the spectral shape of the blue fluorescence on excitation wavelength offers a new approach to elucidate the long-standing problem of the origin of spectral "chlorophyll forms" in pigment-protein complexes, in particular the characterization of chlorophyll a/b-heterodimers. As a first result we present evidence for the existence of strong chlorophyll a/b-interactions (excitonically coupled transitions at 650 and 680 nm) in LHC II at ambient temperature. In comparison with LHC II, the experiments with CP29 provide further evidence that the lowest energy chlorophyll a transition (at ~680 nm) is not excitonically coupled to chlorophyll b. (22 refs).

  3. In vivo quantitative visualization of hypochlorous acid in the liver using a novel selective two-photon fluorescent probe

    Science.gov (United States)

    Wang, Haolu; Jayachandran, Aparna; Gravot, Germain; Liang, Xiaowen; Thorling, Camilla A.; Zhang, Run; Liu, Xin; Roberts, Michael S.

    2016-11-01

    Hypochlorous acid (HOCl) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOCl, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOCl concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 μM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOCl molecules. The cellular concentration of HOCl was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOCl, and rapid "off-on" fluorescence. It is suitable for dynamic quantitatively monitoring HOCl generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.

  4. An organic dye with very large Stokes-shift and broad tunability of fluorescence: Potential two-photon probe for bioimaging and ultra-sensitive solid-state gas sensor

    Energy Technology Data Exchange (ETDEWEB)

    He, Tingchao; Tian, Xiaoqing; Lin, Xiaodong, E-mail: linxd@szu.edu.cn, E-mail: hdsun@ntu.edu.sg [College of Physics Science and Technology, Shenzhen University, Shenzhen 518060 (China); Wang, Yue; Zhao, Xin; Sun, Handong, E-mail: linxd@szu.edu.cn, E-mail: hdsun@ntu.edu.sg [Division of Physics and Applied Physics, and Centre for Disruptive Photonic Technologies (CDPT), School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 (Singapore); Gao, Yang; Grimsdale, Andrew C. [School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798 (Singapore)

    2016-01-04

    Light-emitting nonlinear optical molecules, especially those with large Stokes shifts and broad tunability of their emission wavelength, have attracted considerable attention for various applications including biomedical imaging and fluorescent sensors. However, most fluorescent chromophores have only limited potential for such applications due to small Stokes shifts, narrow tunability of fluorescence emissions, and small optical nonlinearity in highly polar solvents. In this work, we demonstrate that a two-photon absorbing stilbene chromophore exhibits a large two-photon absorption action cross-section (ηδ = 320 GM) in dimethylsulfoxide (DMSO) and shows broad fluorescence tunability (125 nm) by manipulating the polarity of the surrounding medium. Importantly, a very large Stokes shift of up to 227 nm is achieved in DMSO. Thanks to these features, this chromophore can be utilized as a two-photon probe for bioimaging applications and in an ultrasensitive solid-state gas detector.

  5. Nonlinear spectral imaging of human normal skin, basal cell carcinoma and squamous cell carcinoma based on two-photon excited fluorescence and second-harmonic generation

    Science.gov (United States)

    Xiong, S. Y.; Yang, J. G.; Zhuang, J.

    2011-10-01

    In this work, we use nonlinear spectral imaging based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) for analyzing the morphology of collagen and elastin and their biochemical variations in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and normal skin tissue. It was found in this work that there existed apparent differences among BCC, SCC and normal skin in terms of their thickness of the keratin and epithelial layers, their size of elastic fibers, as well as their distribution and spectral characteristics of collagen. These differences can potentially be used to distinguish BCC and SCC from normal skin, and to discriminate between BCC and SCC, as well as to evaluate treatment responses.

  6. The translated conceptual survey of physics / stablization of the focal plane in two photon excitation fluorescence microscopy

    Science.gov (United States)

    Wada, Asma

    As a reflection of my career to be an effective college physics teacher, my thesis is in two parts. The first is in education research, the focus of this part is to have a tool to evaluate pedagogies I have learned at the school and plan to apply in my classrooms back home. Consequently, this resulted in the development of the translated conceptual survey of physics ( TCSP). (TCSP) was designed by combining some questions from the Force Conceptual Inventory (FCI), and the Conceptual Survey of Electricity and Magnetism (CSEM) to assess student's understanding of basic concepts of Newtonian mechanics and electricity and magnetism in introductory physics. The idea of developing this questionnaire is to use it in classrooms back home as a part of a long term objective to implement what has been realized in the area of education research to improve the quality of teaching physics there. The survey was initially written in English, validated with interviews with native English speakers, translated into Arabic, and then validated via an interview with a native Arabic speaker. We then administered the survey to two different English-speaking intro physics courses and analyzed the results for consistency. The objective of the second part in my thesis is to expand my knowledge in an area of physics that I have interest in, and getting involved in a scientific research to develop skills I need as a teacher. My research is in optical physics, in particular, I am working on one of the challenges in implementing two photon excitation luorescence (TPEF) microscopy in imaging living systems. (TPEF) microscopy has been shown to be an invaluable tool for investigating biological structure and function in living organisms. The utility of (TPEF) imaging for this application arises from several important factors including it's ability to image deep within tissue, and to do so without harming the organism. Both of these advantages arise from the fact that (TPEF) imaging is done with

  7. Evaluation of Injured Axons Using Two-Photon Excited Fluorescence Microscopy after Spinal Cord Contusion Injury in YFP-H Line Mice.

    Science.gov (United States)

    Horiuchi, Hideki; Oshima, Yusuke; Ogata, Tadanori; Morino, Tadao; Matsuda, Seiji; Miura, Hiromasa; Imamura, Takeshi

    2015-07-13

    Elucidation of the process of degeneration of injured axons is important for the development of therapeutic modules for the treatment of spinal cord injuries. The aim of this study was to establish a method for time-lapse observation of injured axons in living animals after spinal cord contusion injury. YFP (yellow fluorescent protein)-H transgenic mice, which we used in this study, express fluorescence in their nerve fibers. Contusion damage to the spinal cord at the 11th vertebra was performed by IH (Infinite Horizon) impactor, which applied a pressure of 50 kdyn. The damaged spinal cords were re-exposed during the observation period under anesthesia, and then observed by two-photon excited fluorescence microscopy, which can observe deep regions of tissues including spinal cord axons. No significant morphological change of injured axons was observed immediately after injury. Three days after injury, the number of axons decreased, and residual axons were fragmented. Seven days after injury, only fragments were present in the damaged tissue. No hind-limb movement was observed during the observation period after injury. Despite the immediate paresis of hind-limbs following the contusion injury, the morphological degeneration of injured axons was delayed. This method may help clarification of pathophysiology of axon degeneration and development of therapeutic modules for the treatment of spinal cord injury.

  8. Saturable absorption and two-photon absorption of 1,2,5-thiadiazolo[3,4-g]quinoxaline based derivatives with near-infrared fluorescence

    Science.gov (United States)

    Du, Yabing; Lin, Xiaodong; Jia, Tingjian; Dong, Jun

    2015-03-01

    Organic molecules with near-infrared (NIR) fluorescence are extremely interesting for the applications in nonlinear optical devices and bioimaging. However, such kind of materials have been relatively rarely studied. In this work, the nonlinear optical properties of 1,2,5-thiadiazolo[3,4-g]quinoxaline based derivatives with NIR fluorescence emission have been investigated for the first time. Under the excitation of femtosecond pulses at 532 nm, the chromophore with dithienyl as donor (TQ2) presents saturable absorption (SA) behavior, while no SA has been observed in the derivative with biphenyl (TQ1) as donor. Moreover, TQ2 exhibits much larger two-photon absorption (TPA) cross-sections with strong NIR fluorescence in the second biological window. The larger nonlinear optical properties of TQ2 is due to the introduction of stronger electron-donating group (dithienyl) and the resultant enhanced intramolecular charge transfer properties. At the end, TPA based optical limiting behaviors of the molecules are demonstrated in THF solutions, thanks to their large solubility and strong TPA.

  9. Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy

    DEFF Research Database (Denmark)

    Norlén, Lars; Plasencia Gil, Maria Inés; Bagatolli, Luis

    2008-01-01

    Skin moisturization is largely a function of stratum corneum barrier capacity, which in turn is a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix [ J. Invest. Dermatol.18, 433 (1952); AIChE J. 21, 985 (1975); Acta Derm. Venereol.74, 1...... into co-existing microscopic domains below pH 6 [ Biophys. J.93, 3142 (2007) ]. It was further shown that the role of cholesterol is related to dispersion of ceramide-enriched domains. This effect is counteracted by the presence of free fatty acids, which mix with skin ceramides but not with cholesterol...... (1994); J. Invest. Dermatol.117, 830 (2001) ]. Three unsolved key questions with respect to this lipid matrix' structural organization [ Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001); J. Invest. Dermatol.118, 897 (2002); J. Invest. Dermatol.118, 899 (2002) ] are: i) whether...

  10. Two-photon physics

    Energy Technology Data Exchange (ETDEWEB)

    Bardeen, W.A.

    1981-10-01

    A new experimental frontier has recently been opened to the study of two photon processes. The first results of many aspects of these reactions are being presented at this conference. In contrast, the theoretical development of research ito two photon processes has a much longer history. This talk reviews the many different theoretical ideas which provide a detailed framework for our understanding of two photon processes.

  11. Identification of the boundary between normal brain tissue and ischemia region using two-photon excitation fluorescence microscopy

    Science.gov (United States)

    Du, Huiping; Wang, Shu; Wang, Xingfu; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Jianxin

    2016-10-01

    Ischemic stroke is one of the common neurological diseases, and it is becoming the leading causes of death and permanent disability around the world. Early and accurate identification of the potentially salvageable boundary region of ischemia brain tissues may enable selection of the most appropriate candidates for early stroke therapies. In this work, TPEF microscopy was used to image the microstructures of normal brain tissues, ischemia regions and the boundary region between normal and ischemia brain tissues. The ischemia brain tissues from Sprague-Dawley (SD) rats were subjected to 6 hours of middle cerebral artery occlusion (MCAO). Our study demonstrates that TPEF microscopy has the ability to not only reveal the morphological changes of the neurons but also identify the boundary between normal brain tissue and ischemia region, which correspond well to the hematoxylin and eosin (H and E) stained images. With the development of miniaturized TPEF microscope imaging devices, TPEF microscopy can be developed into an effectively diagnostic and monitoring tool for cerebral ischemia.

  12. Femtosecond, two-photon-absorption, laser-induced-fluorescence (fs-TALIF) imaging of atomic hydrogen and oxygen in non-equilibrium plasmas

    Science.gov (United States)

    Schmidt, Jacob B.; Roy, Sukesh; Kulatilaka, Waruna D.; Shkurenkov, Ivan; Adamovich, Igor V.; Lempert, Walter R.; Gord, James R.

    2017-01-01

    Femtosecond, two-photon-absorption laser-induced fluorescence (fs-TALIF) is employed to measure space- and time-resolved distributions of atomic hydrogen and oxygen in moderate-pressure, non-equilibrium, nanosecond-duration pulsed-discharge plasmas. Temporally and spatially resolved hydrogen and oxygen TALIF images are obtained over a range of low-temperature plasmas in mixtures of helium and argon at 100 Torr total pressure. The high-peak-intensity, low-average-energy fs pulses combined with the increased spectral bandwidth compared to traditional ns-duration laser pulses provide a large number of photon pairs that are responsible for the two-photon excitation, which results in an enhanced TALIF signal. Krypton and xenon TALIF are used for quantitative calibration of the hydrogen and oxygen concentrations, respectively, with similar excitation schemes being employed. This enables 2D collection of atomic-hydrogen and -oxygen TALIF signals with absolute number densities ranging from 2  ×  1012 cm-3 to 6  ×  1015 cm-3 and 1  ×  1013 cm-3 to 3  ×  1016 cm-3, respectively. These 2D images are the first application of TALIF imaging in moderate-pressure plasma discharges. 1D self-consistent modeling predictions show agreement with experimental results within the estimated experimental error of 25%. The present results can be used to further the development of higher fidelity kinetic models while quantifying plasma-source characteristics.

  13. Development of a DMD-based fluorescence microscope

    NARCIS (Netherlands)

    Chakrova, N.; Rieger, B.; Stallinga, S.

    2015-01-01

    We present a versatile fluorescence microscope, built by complementing a conventional fluorescence microscope with a digital micro-mirror device (DMD) in the illumination path. Arbitrary patterns can be created on the DMD and projected onto the sample. This patterned illumination can be used to impr

  14. A multifunctional magnetic nanocarrier bearing fluorescent dye for targeted drug delivery by enhanced two-photon triggered release.

    Science.gov (United States)

    Banerjee, Shashwat S; Chen, Dong-Hwang

    2009-05-06

    We report a novel nanoformulation for targeted drug delivery which utilizes nanophotonics through the fusion of nanotechnology with biomedical application. The approach involves an energy-transferring magnetic nanoscopic co-assembly fabricated of rhodamine B (RDB) fluorescent dye grafted gum arabic modified Fe(3)O(4) magnetic nanoparticle and photosensitive linker by which dexamethasone drug is conjugated to the magnetic nano-assembly. The advantage offered by this nanoformulation is the indirect photo-triggered-on-demand drug release by efficient up-converting energy of the near-IR (NIR) light to higher energy and intraparticle energy transfer from the dye grafted magnetic nanoparticle to the linker for drug release by cleavage. The synthesized nanoparticles were found to be of ultra-small size (13.33 nm) and are monodispersed in an aqueous suspension. Dexamethasone (Dexa) drug conjugated to RDB-GAMNP by photosensitive linker showed appreciable release of Dexa by photo-triggered response on exposure to radiation having a wavelength in the NIR region whereas no detectable release was observed in the dark. Photo-triggered response for the nanoformulation not bearing the rhodamine B dye was drastically less as less Dexa was released on exposure to NIR radiation which suggest that the photo-cleavage of linker and release of Dexa mainly originated from the indirect excitation through the uphill energy conversions based on donor-acceptor model FRET. The promising pathway of nanophotonics for the on-demand release of the drug makes this nanocarrier very promising for applications in nanomedicine.

  15. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    Science.gov (United States)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  16. Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

    Science.gov (United States)

    Mely, Yves; Azoulay, Joel; Beltz, Herve; Clamme, Jean-Pierre; Bernacchi, Serena; Ficheux, Damien; Roques, Bernard P.; Darlix, Jean-Luc

    2004-09-01

    The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3" terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

  17. Two-photon microscopy using fiber-based nanosecond excitation.

    Science.gov (United States)

    Karpf, Sebastian; Eibl, Matthias; Sauer, Benjamin; Reinholz, Fred; Hüttmann, Gereon; Huber, Robert

    2016-07-01

    Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.

  18. Polymerized LB Films Imaged with a Combined Atomic Force Microscope-Fluorescence Microscope

    NARCIS (Netherlands)

    Putman, C.A.J.; Putman, Constant A.J.; Hansma, Helen G.; Gaub, Hermann E.; Hansma, Paul K.

    1992-01-01

    The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined

  19. Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

    NARCIS (Netherlands)

    Putman, Constant A.J.; Hansma, Helen G.; Gaub, Hermann E.; Hansma, Paul K.

    1992-01-01

    The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microsc

  20. Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

    Science.gov (United States)

    Geissler, David; Belder, Detlev

    2015-12-01

    One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (body polymer microfluidic devices. This was achieved by means of two-photon excitation in the visible range (λex = 532 nm). Issues associated with the low optical transmittance of plastics in the UV range were successfully circumvented in this way. The technique was investigated by application to microchip electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Absolute atomic oxygen density measurements for nanosecond-pulsed atmospheric-pressure plasma jets using two-photon absorption laser-induced fluorescence spectroscopy

    Science.gov (United States)

    Jiang, C.; Carter, C.

    2014-12-01

    Nanosecond-pulsed plasma jets that are generated under ambient air conditions and free from confinement of electrodes have become of great interest in recent years due to their promising applications in medicine and dentistry. Reactive oxygen species that are generated by nanosecond-pulsed, room-temperature non-equilibrium He-O2 plasma jets among others are believed to play an important role during the bactericidal or sterilization processes. We report here absolute measurements of atomic oxygen density in a 1 mm-diameter He/(1%)O2 plasma jet at atmospheric pressure using two-photon absorption laser-induced fluorescence spectroscopy. Oxygen number density on the order of 1013 cm-3 was obtained in a 150 ns, 6 kV single-pulsed plasma jet for an axial distance up to 5 mm above the device nozzle. Temporally resolved O density measurements showed that there are two maxima, separated in time by 60-70 µs, and a total pulse duration of 260-300 µs. Electrostatic modeling indicated that there are high-electric-field regions near the nozzle exit that may be responsible for the observed temporal behavior of the O production. Both the field-distribution-based estimation of the time interval for the O number density profile and a pulse-energy-dependence study confirmed that electric-field-dependent, direct and indirect electron-induced processes play important roles for O production.

  2. Production mechanism of atomic nitrogen in atmospheric pressure pulsed corona discharge measured using two-photon absorption laser-induced fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Teramoto, Yoshiyuki; Ono, Ryo [Department of Advanced Energy, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 227-8568 (Japan); Oda, Tetsuji [Department of Electrical Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)

    2012-06-01

    To study the production mechanism of atomic nitrogen, the temporal profile and spatial distribution of atomic nitrogen are measured in atmospheric pressure pulsed positive corona discharge using two-photon absorption laser-induced fluorescence. The absolute atomic nitrogen density in the streamer filaments is estimated from decay rate of atomic nitrogen in N{sub 2} discharge. The results indicate that the absolute atomic nitrogen density is approximately constant against discharge energy. When the discharge voltage is 21.5 kV, production yield of atomic nitrogen produced by an N{sub 2} discharge pulse is estimated to be 2.9 - 9.8 Multiplication-Sign 10{sup 13} atoms and the energy efficiency of atomic nitrogen production is estimated to be about 1.8 - 6.1 Multiplication-Sign 10{sup 16} atoms/J. The energy efficiency of atomic nitrogen production in N{sub 2} discharge is constant against the discharge energy, while that in N{sub 2}/O{sub 2} discharge increases with discharge energy. In the N{sub 2}/O{sub 2} discharge, two-step process of N{sub 2} dissociation plays significant role for atomic nitrogen production.

  3. The effect of polyunsaturated fatty acids on the homeostasis of yolk lipoprotein in C. elegans examined by CARS and two-photon excitation fluorescence (TPE-F) microscopy

    Science.gov (United States)

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Lin, Yi-Chun; Ma, Tian-Hsiang; Lo, Szecheng J.; Chang, Ta-Chau

    2016-03-01

    Yolk lipoprotein constitutes the major source of energy and the materials for synthesizing signaling factors for the development of oocytes and embryos in C. elegans. Polyunsaturated fatty acids (PUFAs) packed in yolk lipoprotein have been recently recognized as critical molecules for fertilization and reproduction.1 However, the relation between PUFAs and the homeostasis of yolk lipoprotein is not clear. Here we use coherent anti-Stokes Raman scattering (CARS) microscopy and two-photon excitation fluorescence (TPE-F) microscopy to examine the transportation of yolk lipoprotein. We demonstrate that CARS microscopy is a more sensitive method than the traditional Nile Red staining method in probing the abnormal accumulation of yolk lipoprotein in the body cavity of C. elegans. It is found that the accumulation of yolk lipoprotein is a time-dependent process. In addition, a negative correlation (r = -0.955) between reproductive aging and abnormal accumulation of yolk lipoprotein is established. We further examine wild-type, fat-1, and fat-2 worms with or without the expression of GFP-tagged yolk lipoprotein (VIT-2-GFP). Our data reveal that PUFAs have a positive effect on the synthesis and endocytosis of yolk lipoprotein, confirming the model proposed by Edmonds et al.2

  4. Two-Photon Flow Cytometry

    Science.gov (United States)

    Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

    2004-01-01

    Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

  5. Control of excitation in the fluorescence microscope.

    Science.gov (United States)

    Lea, D J; Ward, D J

    1979-01-01

    In fluorescence microscopy image brightness and contrast and the rate of fading depend upon the intensity of illumination of the specimen. An iris diaphragm or neutral density filters may be used to reduce fluorescence excitation. Also the excitation bandwidth may be varied by using a broad band exciter filter with a set of interchangeable yellow glass filters at the lamphouse.

  6. Fluorescein sodium fluorescence microscope-integrated lymphangiography for lymphatic supermicrosurgery.

    Science.gov (United States)

    Ayestaray, Benoit; Bekara, Farid

    2015-07-01

    Microscope-integrated lymphangiography is a useful method in the field of lymphatic supermicrosurgery. Fluorescence based on indocyanine green (ICG) is the most commonly used. Fluorescein sodium is a fluorescent tracer used for retinal and neurosurgical angiography but not yet for lymphatic supermicrosurgery. In this report, we present a case in which the fluorescein sodium fluorescence microscope-integrated lymphangiography was used for assessment of lymphatic drainage pathway and patency in a patient treated for secondary lymphedema by lymphaticovenular anastomoses. Fluorescein sodium fluorescence microscope-integrated lymphangiography was evaluated in a 67-year-old female presented for a Campisi clinical stage IV lymphedema of the upper limb. Transcutaneous guidance and vascular fluorescence were assessed. A comparison with ICG fluorescence was made intraoperatively. Two lymphaticovenular anastomoses were performed and their patency were checked by lymphangiography. Transcutaneous signal was found higher with fluorescein sodium fluorescence. Intraluminal visualization was possible with fluorescein sodium coloration during lymphaticovenular anastomoses. No adverse reaction occurred. The circumferential differential reduction rate of affected limb was 8.1% 3 months after lymphaticovenular anastomoses. The use of fluorescence microscope-integrated lymphangiography with fluorescein sodium may be superior to ICG fluorescence in assistance of lymphaticovenular anastomoses. © 2015 Wiley Periodicals, Inc.

  7. Comparison of nanosecond and picosecond excitation for interference-free two-photon laser-induced fluorescence detection of atomic hydrogen in flames.

    Science.gov (United States)

    Kulatilaka, Waruna D; Patterson, Brian D; Frank, Jonathan H; Settersten, Thomas B

    2008-09-10

    Two-photon laser-induced fluorescence (TP-LIF) line imaging of atomic hydrogen was investigated in a series of premixed CH4/O2/N2, H2/O2, and H2/O2/N2 flames using excitation with either picosecond or nanosecond pulsed lasers operating at 205 nm. Radial TP-LIF profiles were measured for a range of pulse fluences to determine the maximum interference-free signal levels and the corresponding picosecond and nanosecond laser fluences in each of 12 flames. For an interference-free measurement, the shape of the TP-LIF profile is independent of laser fluence. For larger fluences, distortions in the profile are attributed to photodissociation of H2O, CH3, and/or other combustion intermediates, and stimulated emission. In comparison with the nanosecond laser, excitation with the picosecond laser can effectively reduce the photolytic interference and produces approximately an order of magnitude larger interference-free signal in CH4/O2/N2 flames with equivalence ratios in the range of 0.5laser fluence in all flames, stimulated emission, occurring between the laser-excited level, H(n=3), and H(n=2), is the limiting factor for picosecond excitation in the flames with the highest H atom concentration. Nanosecond excitation is advantageous in the richest (Phi=1.64) CH4/O2/N2 flame and in H2/O2/N2 flames. The optimal excitation pulse width for interference-free H atom detection depends on the relative concentrations of hydrogen atoms and photolytic precursors, the flame temperature, and the laser path length within the flame.

  8. Fano interference in two-photon transport

    Science.gov (United States)

    Xu, Shanshan; Fan, Shanhui

    2016-10-01

    We present a general input-output formalism for the few-photon transport in multiple waveguide channels coupled to a local cavity. Using this formalism, we study the effect of Fano interference in two-photon quantum transport. We show that the physics of Fano interference can manifest as an asymmetric spectral line shape in the frequency dependence of the two-photon correlation function. The two-photon fluorescence spectrum, on the other hand, does not exhibit the physics of Fano interference.

  9. Two-dimensional imaging of molecular hydrogen in H2-air diffusion flames using two-photon laser-induced fluorescence

    Science.gov (United States)

    Lempert, W.; Kumar, V.; Glesk, I.; Miles, R.; Diskin, G.

    1991-01-01

    The use of a tunable ArF laser at 193.26 nm to record simultaneous single-laser-shot, planar images of molecular hydrogen and hot oxygen in a turbulent H2-air diffusion flame. Excitation spectra of fuel and oxidant-rich flame zones confirm a partial overlap of the two-photon H2 and single-photon O2 Schumann-Runge absorption bands. UV Rayleigh scattering images of flame structure and estimated detection limits for the H2 two-photon imaging are also presented.

  10. A portable fluorescence microscopic imaging system for cholecystectomy

    Science.gov (United States)

    Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

    2016-03-01

    In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

  11. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

    Science.gov (United States)

    Hayashi, Shinichi; Okada, Yasushi

    2015-05-01

    Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  12. Technical principles and neurosurgical applications of fluorescein fluorescence using a microscope-integrated fluorescence module.

    Science.gov (United States)

    Rey-Dios, Roberto; Cohen-Gadol, Aaron A

    2013-04-01

    Fluorescent technology has recently become a valuable tool in the surgical management of neoplastic and vascular lesions. The availability of microscope-integrated fluorescent modules has facilitated incorporation of this technology within the microsurgical workflow. The currently available microscope integrated modules use 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) as fluorophores. Fluorescein sodium is a fluorescent molecule that has been used specifically in ophthalmology for the treatment of retinal angiography. A new microscope-integrated fluorescent module has been recently developed for fluorescein. We employed this technology to maximize resection of tumors and perform intraoperative angiography to guide microsurgical management of aneurysms and arteriovenous malformations. Fluorescein fluorescence allows the surgeon to appreciate fluorescent structures through the oculars while visualizing non-fluorescent tissues in near natural colors. Therefore, the operator can proceed with microsurgery under the fluorescent mode. We present three representative cases in which the use of fluorescein fluorescence was found useful in the surgeon's decision making during surgery. The applications of this new microscope-integrated fluorescent module are multiple, and include vascular and oncologic neurosurgery. Further clinical investigations with large patient cohorts are needed to fully establish the role of this new technology.

  13. Responsive mechanism of 2-(2’-hydroxyphenyl)benzoxazole-based two-photon fluorescent probes for zinc and hydroxide ions

    Institute of Scientific and Technical Information of China (English)

    张玉瑾; 张秋月; 丁红娟; 宋秀能; 王传奎

    2015-01-01

    The response theory is used to investigate one-and two-photon absorption (TPA) as well as emission properties of a series of potential zinc ion and pH sensitive materials containing 2-(2’-hydroxyphenyl)benzoxazole (HPBO) end groups. Special emphasis is placed on the evolution of their optical properties upon combining with zinc ions or deprotonation. Calculated results indicate that upon combining with zinc ions or deprotonation, these HPBO derivatives show drastic changes in their one-photon absorption (OPA), emission, and TPA properties. Moreover, mechanisms of the probes are analyzed to be intramolecular charge transfer. These compounds are thus proved to be excellent candidates for two-photon fl uorescent zinc and pH probes.

  14. Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Jeshtadi, Ananya; Burgos, Pierre; Stubbs, Christopher D; Parker, Anthony W; King, Linda A; Skinner, Michael A; Botchway, Stanley W

    2010-12-01

    Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

  15. Visualizing Fluorescence: Using a Homemade Fluorescence "Microscope" to View Latent Fingerprints on Paper

    Science.gov (United States)

    LaFratta, Christopher N.; Huh, Sun Phill; Mallillin, Allistair C.; Riviello, Peter J.; Walt, David R.

    2010-01-01

    We describe an inexpensive hand-held fluorescence imager (low-magnification microscope), constructed from poly(vinyl chloride) pipe and other inexpensive components for use as a teaching tool to understand the principles of fluorescence detection. Optical filters are used to select the excitation and emission wavelengths and can be easily…

  16. Pulp tissue in sex determination: A fluorescent microscopic study

    Directory of Open Access Journals (Sweden)

    Amit Nayar

    2014-01-01

    Full Text Available Aims: To determine and compare the reliability of pulp tissue in determination of sex and to analyze whether caries have any effect on fluorescent body test. Materials and Methods: This study was carried on 50 maxillary and mandibular teeth (25 male teeth and 25 female teeth, which were indicated for extraction. The teeth are categorized into 5 groups, 10 each (5 from males and 5 from females on the basis of caries progression. The pulp cells are stained with quinacrine hydrochloride and observed with fluorescent microscope for fluorescent body. Gender is determined by identification of Y chromosome fluorescence in dental pulp. Results: Fluorescent bodies were found to be more in sound teeth in males as the caries increase the mean percentage of fluorescent bodies observed decreases in males. We also observed the fluorescent spots in females, and the value of the spot increases in female as the caries progresses, thereby giving false positive results in females. Conclusion: Sex determination by fluorescent staining of the Y chromosome is a reliable technique in teeth with healthy pulps or caries with enamel or up to half way of dentin. Teeth with caries involving pulp cannot be used for sex determination.

  17. Two-photon imaging and analysis of neural network dynamics

    Science.gov (United States)

    Lütcke, Henry; Helmchen, Fritjof

    2011-08-01

    The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to measure and analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behavior. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so-called 'microcircuits') remains comparably poor. Predominantly, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near-millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.

  18. Two-photon imaging and analysis of neural network dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Luetcke, Henry; Helmchen, Fritjof [Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland)

    2011-08-15

    The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to measure and analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behavior. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so-called 'microcircuits') remains comparably poor. Predominantly, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near-millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.

  19. The Gray Institute 'open' high-content, fluorescence lifetime microscopes.

    Science.gov (United States)

    Barber, P R; Tullis, I D C; Pierce, G P; Newman, R G; Prentice, J; Rowley, M I; Matthews, D R; Ameer-Beg, S M; Vojnovic, B

    2013-08-01

    We describe a microscopy design methodology and details of microscopes built to this 'open' design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off-the-shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup. Several automated high-content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time-domain FLIM via time-correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide-field and laser scanning capabilities designed for high-content microscopy. Devices using these designs also form radiation-beam 'end-stations' at Oxford and Surrey Universities, showing the versatility and extendibility of this approach. © 2013 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

  20. Trends in environmental science using microscopic X-ray fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Fittschen, Ursula Elisabeth Adriane, E-mail: ursula.fittschen@chemie.uni-hamburg.de [Department of Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg (Germany); Falkenberg, Gerald [Deutsches Elektronen-Synchrotron, Notkestr. 85, 22603 Hamburg (Germany)

    2011-08-15

    Microscopic X-ray fluorescence (micro-XRF) is a versatile tool in environmental analysis. We review work done in this field from 2008 to 2010 and highlight new aspects. Overall, there is a strong trend to combine fluorescence data with other data like diffraction or absorption spectroscopy. Also, the use of laboratory based instrumentation has become wide spread as more commercial instruments are available. At laboratories and synchrotron sites the trend towards higher spatial resolution is still persistent hitting sub micrometer values in case of synchrotron set ups.

  1. Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination.

    Science.gov (United States)

    Chu, Kengyeh K; Lim, Daryl; Mertz, Jerome

    2007-10-01

    We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

  2. Quantitative imaging of fibrotic and morphological changes in liver of non-alcoholic steatohepatitis (NASH) model mice by second harmonic generation (SHG) and auto-fluorescence (AF) imaging using two-photon excitation microscopy (TPEM).

    Science.gov (United States)

    Yamamoto, Shin; Oshima, Yusuke; Saitou, Takashi; Watanabe, Takao; Miyake, Teruki; Yoshida, Osamu; Tokumoto, Yoshio; Abe, Masanori; Matsuura, Bunzo; Hiasa, Yoichi; Imamura, Takeshi

    2016-12-01

    Non-alcoholic steatohepatitis (NASH) is a common liver disorder caused by fatty liver. Because NASH is associated with fibrotic and morphological changes in liver tissue, a direct imaging technique is required for accurate staging of liver tissue. For this purpose, in this study we took advantage of two label-free optical imaging techniques, second harmonic generation (SHG) and auto-fluorescence (AF), using two-photon excitation microscopy (TPEM). Three-dimensional ex vivo imaging of tissues from NASH model mice, followed by image processing, revealed that SHG and AF are sufficient to quantitatively characterize the hepatic capsule at an early stage and parenchymal morphologies associated with liver disease progression, respectively.

  3. Wide-field two-photon microscopy: features and advantages for biomedical applications

    Science.gov (United States)

    Wachsmann-Hogiu, S.; Hwang, J. Y.; Lindsley, E.; Farkas, D. L.

    2007-02-01

    We describe a simple fluorescence microscope based on wide-field two-photon excitation. While still taking advantage of some inherent properties of non-linear (two-photon) microscopy, such as increased penetration depth through tissue and reduced phototoxicity, this approach provides video frame rate imaging, can be easily coupled to fluorescence spectral and lifetime detection modules, and makes efficient use of the high average power currently available from ultrashort pulsed lasers. For a standard histopathology specimen, we were able to identify different structures based on spectral and fluorescence lifetime detection and analysis. We examined the use of 200fs and 2ps pulses from Spectra Physics MaiTai and Tsunami lasers, respectively, with average power ranging from 50mW to 500mW.

  4. Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope

    CERN Document Server

    McHale, Kevin

    2009-01-01

    We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system, and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity and long time-scale decays in comparison to traditional fluorescence correlation spectroscopy. We demonstrate our approach by using a three-dimensional tracking microscope to study genomic $\\lambda$-phage DNA molecules with various fluorescence label configurations. We conclude with a discussion of related techniques, including computation of the relevant statistics for camera-based intramolecular correlation measurements.

  5. Fluorescence labeling of carbon nanotubes and visualization of a nanotube-protein hybrid under fluorescence microscope.

    Science.gov (United States)

    Yoshimura, Shige H; Khan, Shahbaz; Maruyama, Hiroyuki; Nakayama, Yoshikazu; Takeyasu, Kunio

    2011-04-11

    Biological applications of carbon nanotubes have been hampered by the inability to visualize them using conventional optical microscope, which is the most common tool for the observation and measurement of biological processes. Recently, a number of fluorescence labeling methods for biomolecules and various fluorescence probes have been developed and widely utilized in biological fields. Therefore, labeling carbon nanotubes with such fluorophores under physiological conditions will be highly useful in their biological applications. In this Article, we present a method to fluorescently label nanotubes by combining a detergent and a fluorophore commonly used in biological experiments. Fluorophores carrying an amino group (Texas Red hydrazide or BODIPY FL-hydrazide) were covalently attached to the hydroxyl groups of Tween 20 using carbonyldiimidazole. Fluorescence microscopy demonstrated that nanotubes were efficiently solubilized and labeled by this fluorescently labeled detergent. By using this technique, we also demonstrated multicolor fluorescence imaging of a nanotube-protein hybrid.

  6. Two photon physics. Personal recollection

    CERN Document Server

    Ginzburg, Ilya F

    2015-01-01

    The term two--photon processes is used for the reactions in which some system of particles is produced in collision of two photons, either real or virtual. In the study of these processes our main goal was to suggest approach, allowing to extract from the data information on proper two--photon process separating it from mechanism which responsible for the production of photons. Here I present my view for history of two--photon physics. I don't try to give complete review, concentrating mainly on works of our team (which cover essential part of the topic) and some colleagues. My citation is strongly incomplete. I cite here only papers which were essential in our understanding of the problems. The choice of presented details is the result of my discussions with Gleb Kotkin and Valery Serbo. 1. Prehistory. 2. Two photon processes at e^+e^- colliders. 3. Photon colliders. 4. Notes on physical program.

  7. Two-photon ionization of colliding atoms

    Energy Technology Data Exchange (ETDEWEB)

    Nayfeh, M.H.

    1977-09-01

    Semiclassical expressions of two-photon ionization of two colliding atoms are derived for a wide range of electromagnetic field intensity and detunings from the isolated atom line. The dependence of the ionization yield on the details of the interaction potential of the system is derived. This process promises an extremely sensitive method for studying line broadening on the far wing, especially when absorption or fluorescence becomes very weak.

  8. An automated protocol for performance benchmarking a widefield fluorescence microscope.

    Science.gov (United States)

    Halter, Michael; Bier, Elianna; DeRose, Paul C; Cooksey, Gregory A; Choquette, Steven J; Plant, Anne L; Elliott, John T

    2014-11-01

    Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other imaging applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy-to-use benchmarking methods would facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and would aid the determination of instrumental method validation for commercial product development applications. We describe and evaluate an automated method to characterize a fluorescence imaging system's performance by benchmarking the detection threshold, saturation, and linear dynamic range to a reference material. The benchmarking procedure is demonstrated using two different materials as the reference material, uranyl-ion-doped glass and Schott 475 GG filter glass. Both are suitable candidate reference materials that are homogeneously fluorescent and highly photostable, and the Schott 475 GG filter glass is currently commercially available. In addition to benchmarking the analytical performance, we also demonstrate that the reference materials provide for accurate day to day intensity calibration. Published 2014 Wiley Periodicals Inc.

  9. Lagragian 3D tracking of fluorescent microscopic objects under flow

    CERN Document Server

    Darnige, T; Bohec, P; Lindner, A; Clément, E

    2016-01-01

    We detail the elaboration of a tracking device mounted on an epifluorescent inverted microscope and suited to obtain time resolved 3D Lagrangian tracks of fluorescent micro-objects. The system is based on a real-time image processing driving a mechanical X-Y stage displacement and a Z refocusing piezo mover such as to keep the designed object at a fixed position in a moving frame. Track coordinates with respect to the microfluidic device, as well as images of the object in the laboratory reference frame are thus obtained at a frequency of several tenths of Hertz. This device is particularly adapted to follow the trajectory of motile micro-organisms in microfluidic devices with or without flow.

  10. Multimodal optical setup for nonlinear and fluorescence lifetime imaging microscopies: improvement on a commercial confocal inverted microscope

    Science.gov (United States)

    Pelegati, V. B.; Adur, J.; de Thomaz, A. A.; Almeida, D. B.; Baratti, M. O.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    In this work we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus FV300) to include nonlinear optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). The NLO microscopies included two-photon fluorescence (TPFE), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG). The whole system, including FLIM, used only one laser source composed of an 80 MHz femtosecond laser. The commercial Ti:sapphire lasers can be tuned up to 690-1040 nm bringing the THG signal to the 350 nm region where most microscope optics do not work. However, the third harmonic is only generated at the sample, meaning that we only have to take care of the collection optics. To do that we used a remote photomultiplier to acquire the THG signal at the 310-350 nm wavelength window. After performing the tests to guarantee that we are observing actually SHG/THG signals we than used this system to acquire multimodal images of several biological samples, from epithelial cancer to vegetables. The ability to see the collagen network together with the cell nuclei proved to be important for cancer tissues diagnosis. Moreover, FLIM provides information about the cell metabolism, also very important for cancer cell processes.

  11. Excited-state dynamics and two-photon absorption cross sections of fluorescent diphenyl-tin(IV) derivatives with schiff bases: a comparative study of the effect of chelation from the ultrafast to the steady-state time scale.

    Science.gov (United States)

    Zugazagoitia, Jimena S; Maya, Mauricio; Damián-Zea, Carlos; Navarro, Pedro; Beltrán, Hiram I; Peon, Jorge

    2010-01-21

    Schiff bases bearing an intramolecular hydrogen bond are known to undergo excited-state intramolecular proton transfer and E-Z isomerization, which are related to their thermochromism and solvatochromism properties. In this study, we explored these ultrafast photoinduced processes for two doubly hydroxylated Schiff bases, salicylidene-2-aminophenol and 2-hydroxynaphthylmethylidene-2-aminophenol. From comparisons with our previously reported results for the parent monohidroxylated Schiff base salicylideneaniline, we were able to establish the lack of an effect of a second intramolecular hydrogen bond in the excited-state intramolecular proton-transfer process. Moreover, we synthesized and studied the photophysics of 14 diphenyl-tin(IV) derivatives with Schiff bases with the same framework as the former two. In these organometallic compounds, we observed an increase of more than 50 times in the excited-state decay times in comparison with those of the free ligands. This finding is attributed to the coordination with the metallic center, which restricts the fluctuations of the geometry of the organic Schiff base skeleton. The emission bands of these complexes can be easily tuned through substitutions at the Schiff base ligand and can be made to be centered well above 600 nm. The much enhanced emissive behavior of all diphenyl-tin(IV) derivatives allowed the study of several properties of their electronically excited states, including the effects of different substituents on their femtosecond and picosecond dynamics. Considering potential applications, we also performed transient absorption experiments to assess the wavelength interval for stimulated emission of this type of compound. Finally, we determined their two-photon absorption cross sections in the 760-820-nm range by measuring their two-photon induced fluorescence excitation spectra. Mainly, our results illustrate that the diphenyl-tin(IV) moiety, thanks to its size and its coordination mode with a single

  12. Higgs Decay to Two Photons

    OpenAIRE

    Marciano, William J.; Zhang, Cen; Willenbrock, Scott

    2011-01-01

    The amplitude for Higgs decay to two photons is calculated in renormalizable and unitary gauges using dimensional regularization at intermediate steps. The result is finite, gauge independent, and in agreement with previously published results. The large Higgs mass limit is examined using the Goldstone-boson equivalence theorem as a check on the use of dimensional regularization and to explain the absence of decoupling.

  13. A novel multimodal CARS miniaturized microscope

    Science.gov (United States)

    Smith, Brett; Naji, Majid; Murugkar, Sangeeta; Brideau, Craig; Stys, Peter; Anis, Hanan

    2012-03-01

    We demonstrate the operation of a novel portable, fibre delivery miniaturized multimodal microscope (exoscope) for coherent anti-Stokes Raman scattering and two-photon excitation fluorescence imaging using a single Ti:sapphire femtosecond pulsed laser. This microscope features a large mode area photonic crystal fibre for light delivery, as well as biaxial scanning microelectromechanical system mirrors and custom miniaturized optics corrected for chromatic aberration. We demonstrate imaging of polystyrene beads, two photon excitation fluorescence beads in both forward and backward (epi) directions. This miniaturized exoscope will enable in-vivo imaging of rat spinal cord.

  14. Two-photon imaging of stem cells

    Science.gov (United States)

    Uchugonova, A.; Gorjup, E.; Riemann, I.; Sauer, D.; König, K.

    2008-02-01

    A variety of human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, dental pulpa stem cells) have been investigated by femtosecond laser 5D two-photon microscopy. Autofluorescence and second harmonic generation have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, NADH and flavoprotein fluorescence was detected in stem cells. Major emission peaks at 460nm and 530nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured using time-correlated single photon counting and spectral imaging. Differentiated stem cells produced the extracellular matrix protein collagen which was detected by SHG signals at 435 nm.

  15. Pulse-shaping based two-photon FRET stoichiometry.

    Science.gov (United States)

    Flynn, Daniel C; Bhagwat, Amar R; Brenner, Meredith H; Núñez, Marcos F; Mork, Briana E; Cai, Dawen; Swanson, Joel A; Ogilvie, Jennifer P

    2015-02-09

    Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

  16. Denoising two-photon calcium imaging data.

    Science.gov (United States)

    Malik, Wasim Q; Schummers, James; Sur, Mriganka; Brown, Emery N

    2011-01-01

    Two-photon calcium imaging is now an important tool for in vivo imaging of biological systems. By enabling neuronal population imaging with subcellular resolution, this modality offers an approach for gaining a fundamental understanding of brain anatomy and physiology. Proper analysis of calcium imaging data requires denoising, that is separating the signal from complex physiological noise. To analyze two-photon brain imaging data, we present a signal plus colored noise model in which the signal is represented as harmonic regression and the correlated noise is represented as an order autoregressive process. We provide an efficient cyclic descent algorithm to compute approximate maximum likelihood parameter estimates by combing a weighted least-squares procedure with the Burg algorithm. We use Akaike information criterion to guide selection of the harmonic regression and the autoregressive model orders. Our flexible yet parsimonious modeling approach reliably separates stimulus-evoked fluorescence response from background activity and noise, assesses goodness of fit, and estimates confidence intervals and signal-to-noise ratio. This refined separation leads to appreciably enhanced image contrast for individual cells including clear delineation of subcellular details and network activity. The application of our approach to in vivo imaging data recorded in the ferret primary visual cortex demonstrates that our method yields substantially denoised signal estimates. We also provide a general Volterra series framework for deriving this and other signal plus correlated noise models for imaging. This approach to analyzing two-photon calcium imaging data may be readily adapted to other computational biology problems which apply correlated noise models.

  17. Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

    Directory of Open Access Journals (Sweden)

    Rumelo Amor

    Full Text Available We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca(2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca(2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

  18. Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

    CERN Document Server

    Amor, Rumelo; Robb, Gillian; Wilson, Louise; Rahman, Nor Zaihana Abdul; Dempster, John; Amos, William Bradshaw; Bushell, Trevor J; McConnell, Gail

    2015-01-01

    We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca$^{2+}$ events in primary rat neurone cultures loaded with the fluorescent Ca$^{2+}$ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

  19. Fluorescence behaviour and dural infiltration of meningioma analyzed by 5-ALA based fluorescence: Operating microscope versus mini-spectrometer.

    Science.gov (United States)

    Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

    2017-08-30

    To compare fluorescence intensity of tumor specimens as measured by a FGS-microscope and a spectrometer, 2.) to evaluate tumor infiltration of dura mater around meningiomas with help of these two different 5-ALA based fluorescence tools and 3.) to correlate fluorescence intensity with histopathological data. In a clinical series menigiomas were resected by 5-ALA fluorescence guided surgery. Fluorescence intensity was semi-quantitatively rated by the surgeon at pre-defined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analysed. Sampling was realized at the level of the dura in a centrifugal direction. 104 biopsies (n= 13 tumors) were analysed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Confocal and Two-Photon Microscopy: Foundations, Applications and Advances

    Science.gov (United States)

    Diaspro, Alberto

    2001-11-01

    Confocal and Two-Photon Microscopy Foundations, Applications, and Advances Edited by Alberto Diaspro Confocal and two-photon fluorescence microscopy has provided researchers with unique possibilities of three-dimensional imaging of biological cells and tissues and of other structures such as semiconductor integrated circuits. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances provides clear, comprehensive coverage of basic foundations, modern applications, and groundbreaking new research developments made in this important area of microscopy. Opening with a foreword by G. J. Brakenhoff, this reference gathers the work of an international group of renowned experts in chapters that are logically divided into balanced sections covering theory, techniques, applications, and advances, featuring: In-depth discussion of applications for biology, medicine, physics, engineering, and chemistry, including industrial applications Guidance on new and emerging imaging technology, developmental trends, and fluorescent molecules Uniform organization and review-style presentation of chapters, with an introduction, historical overview, methodology, practical tips, applications, future directions, chapter summary, and bibliographical references Companion FTP site with full-color photographs The significant experience of pioneers, leaders, and emerging scientists in the field of confocal and two-photon excitation microscopy Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances is invaluable to researchers in the biological sciences, tissue and cellular engineering, biophysics, bioengineering, physics of matter, and medicine, who use these techniques or are involved in developing new commercial instruments.

  1. Synthesis of two carbazole-based dyes and application of two-photon initiating polymerization

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Two carbazole-based polymerization initiators possessing blue fluorescence emission have been synthesized via Wittig reaction in the solid phase at room temperature.Two-photon excited fluorescence(TPEF) spectra for them were investigated under 800 nm fs laser pulse and two-photon absorption cross sections were determined by the Z-scan technique.Then two-photon initiating polymerization(TPIP) microfabrication experiments were successfully carried out.Three-dimensional lattice and artificial defects were gained,indicating that they were viable candidates for the two-photon polymerization initiator in practical application of microfabrication.

  2. Synthesis of two carbazole-based dyes and application of two-photon initiating polymerization

    Institute of Scientific and Technical Information of China (English)

    HU RenTao; LU LiangFei; RUAN BanFeng; WANG Peng; ZHANG MingLiang; ZHOU HongPing; LI ShengLi; WU JieYing; TIAN YuPeng

    2009-01-01

    Two carbazole-based polymerization initiators possessing blue fluorescence emission have been synthesized via Wittig reaction in the solid phase at room temperature.Two-photon excited fluorescence (TPEF) spectra for them were investigated under 800 nm fs laser pulse and two-photon absorption cross sections were determined by the Z-scan technique.Then two-photon initiating polymerization (TPIP) microfabrication experiments were successfully carried out.Three-dimensional lattice and artificial defects were gained,indicating that they were viable candidates for the two-photon polymerization initiator in practical application of microfabrication.

  3. Parameter estimation method for blurred cell images from fluorescence microscope

    Science.gov (United States)

    He, Fuyun; Zhang, Zhisheng; Luo, Xiaoshu; Zhao, Shulin

    2016-10-01

    Microscopic cell image analysis is indispensable to cell biology. Images of cells can easily degrade due to optical diffraction or focus shift, as this results in low signal-to-noise ratio (SNR) and poor image quality, hence affecting the accuracy of cell analysis and identification. For a quantitative analysis of cell images, restoring blurred images to improve the SNR is the first step. A parameter estimation method for defocused microscopic cell images based on the power law properties of the power spectrum of cell images is proposed. The circular radon transform (CRT) is used to identify the zero-mode of the power spectrum. The parameter of the CRT curve is initially estimated by an improved differential evolution algorithm. Following this, the parameters are optimized through the gradient descent method. Using synthetic experiments, it was confirmed that the proposed method effectively increased the peak SNR (PSNR) of the recovered images with high accuracy. Furthermore, experimental results involving actual microscopic cell images verified that the superiority of the proposed parameter estimation method for blurred microscopic cell images other method in terms of qualitative visual sense as well as quantitative gradient and PSNR.

  4. In Vivo Non Linear Optical (NLO) Imaging in Live Rabbit Eyes Using the Heidelberg Two-Photon Laser Ophthalmoscope

    Science.gov (United States)

    Hao, Ming; Flynn, Kevin; Nien-Shy, Chyong; Jester, Bryan E.; Winkler, Moritz; Brown, Donald J.; La Schiazza, Olivier; Bille, Josef; Jester, James V.

    2010-01-01

    Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW femtosecond laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 μm diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 hours after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas. PMID:20558159

  5. Enhanced two-photon absorption using true thermal light

    CERN Document Server

    Jechow, Andreas; Kurzke, Henning; Heuer, Axel; Menzel, Ralf

    2013-01-01

    Two-photon excited fluorescence (TPEF) is a standard technique in modern microscopy but still affected by photo-damage of the probe. It was proposed that TPEF can be enhanced by using entangled photons, but has proven to be challenging. Recently it was shown that some features of entangled photons can be mimicked with thermal light, which finds application in ghost imaging, sub-wavelength lithography and metrology. Here, we utilize true thermal light from a super-luminescence diode to demonstrate enhanced TPEF compared to coherent light using two common fluorophores and luminescent quantum dots. We find that the two-photon absorption rate is directly proportional to the measured degree of second-order coherence, as predicted by theory. Our results show that photon bunching can be exploited in two-photon microscopy with the photon statistic providing a new degree of freedom.

  6. All-optical histology using two photon laser scanning microscopy and ablation with ultrashort pulses

    Science.gov (United States)

    Tsai, Philbert S.

    This dissertation discusses the use of ultrashort laser pulses to image and manipulate tissue for the purpose of three-dimensional histological reconstruction of extended brain structures. Two photon laser scanning microscopy (TPLSM) and ultrashort pulsed laser ablation are used to provide in situ three-dimensional imaging through thick preparations of fixed tissue. Surface regions of fixed tissue are first imaged using TPLSM. The imaged regions are then removed by ablation with amplified, ultrashort laser pulses, thereby exposing a previously underlying tissue region for imaging. This process of imaging and ablation proceeds iteratively until the desired tissue volume has been processed. First, the principles, design, and construction of a two photon laser scanning microscope are discussed, followed by a discussion of the physical mechanisms of tissue ablation with ultrashort laser pulses. The compatibility of tissue ablation using ultrashort pulses with subsequent histological analysis, particularly with fluorescent microscopy, is evaluated. Tissue ablation with ultrashort laser pulses is found to produce ablated tissue surfaces that are smooth to within a micrometer. Intrinsic fluorescence as well as immunoreactivity are found to be resilient to the ablation process. The all-optical histological technique is demonstrated on brain tissue from rats and mice, including tissue from embryonic mouse as early at E15. The ablation process is shown to preserve both macroscopic and microscopic structures within tissue. To facilitate the all-optical histological analysis of neuronal vasculature and its relative distribution to surrounding neuronal tissue, a fluorescent gel perfusion technique is developed that provides a temperature-stabilized fluorescent label of the neuronal vasculature. The use of immunohistochemistry to label specific cell populations throughout an 800 micrometer-thick tissue section is demonstrated. Additionally, the immersion of fixed tissue in high

  7. Integrated single- and two-photon light sheet microscopy using accelerating beams

    DEFF Research Database (Denmark)

    Piksarv, Peeter; Marti, Dominik; Le, Tuan

    2017-01-01

    We demonstrate the first light sheet microscope using propagation invariant, accelerating Airy beams that operates both in single- and two-photon modes. The use of the Airy beam permits us to develop an ultra compact, high resolution light sheet system without beam scanning. In two-photon mode, a...

  8. Two-photon flow cytometer with laser scanning Bessel beams

    Science.gov (United States)

    Wang, Yongdong; Ding, Yu; Ray, Supriyo; Paez, Aurelio; Xiao, Chuan; Li, Chunqiang

    2016-03-01

    Flow cytometry is an important technique in biomedical discovery for cell counting, cell sorting and biomarker detection. In vivo flow cytometers, based on one-photon or two-photon excited fluorescence, have been developed for more than a decade. One drawback of laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is fairly small due to the short Rayleigh range of a focused Gaussian beam. Hence, the sampling volume is much smaller than one-photon flow cytometry, which makes it challenging to count or detect rare circulating cells in vivo. Bessel beams have narrow intensity profiles with an effective spot size (FWHM) as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making it an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Bessel beams rather than a Gaussian beam is the fact that Bessel beams have small concentric side rings that contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using scanned Bessel beams to form a light sheet that intersects the micro fluidic channel.

  9. Holographic Two-Photon Induced Photopolymerization

    Data.gov (United States)

    Federal Laboratory Consortium — Holographic two-photon-induced photopolymerization (HTPIP) offers distinct advantages over conventional one-photon-induced photopolymerization and current techniques...

  10. Two-Photon Microscopy Allows Imaging and Characterization of Cochlear Microvasculature In Vivo

    Directory of Open Access Journals (Sweden)

    Friedrich Ihler

    2015-01-01

    Full Text Available Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanate- or Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of 90.0±22.7 μm. Imaging yielded a mean contrast-to-noise ratio (CNR of 3.3±1.7. Mean diameter in vivo was 16.5±6.0 μm for arterioles and 8.0±2.4 μm for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with 12.2±1.6 μm and 6.6±1.0 μm, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P<0.001 and P=0.022, two-way ANOVA. Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research.

  11. Temporal dynamics of two-photon-pumped amplified spontaneous emission in slab organic crystals

    NARCIS (Netherlands)

    Fang, Hong-Hua; Chen, Qi-Dai; Ding, Ran; Yang, Jie; Ma, Yu-Guang; Wang, Hai-Yu; Gao, Bing-Rong; Feng, Jing; Sun, Hong-Bo; Fang, Honghua

    2010-01-01

    We have studied the ultrafast dynamics of two-photon-pumped amplified spontaneous emission (ASE) from a single crystal by the time-resolved fluorescence upconversion technique. With the increase of two-photon pump intensities, the emission decay time is dramatically shortened by 30 times (from 3 ns

  12. Three-dimensional microfabrication using two-photon polymerization

    Science.gov (United States)

    Cumpston, Brian H.; Ehrlich, Jeffrey E.; Kuebler, Stephen M.; Lipson, Matthew; Marder, Seth R.; McCord-Maughon, D.; Perry, Joseph W.; Roeckel, Harold; Rumi, Maria Cristina

    1998-09-01

    Photopolymerization initiated by the simultaneous absorption of two photons is unique in its ability to produce complex three-dimensional (3D) structures from a single, thick photopolymer film. Strong 3D confinement of the polymerization process is not possible in other polymer microfabrication techniques such as LIGA, rapid prototyping, and conventional photoresist technology. Two-photon polymerization also permits the fabrication of 3D structures and the definition of lithographic features on non-planar surfaces. We have developed a wide array of chromophores which hold great promise for 3D microfabrication, as well as other applications, such as two-photon fluorescence imaging and 3D optical data storage. These materials are based on a donor- (pi) -donor, donor-acceptor-donor, or acceptor-donor-acceptor structural motif. The magnitude of the two-photon absorption cross-section, (delta) , and the position of the two-photon absorption maximum, (lambda) (2)max, can be controlled by varying the length of the conjugated bridge and by varying the strength of the donor/acceptor groups. In this way, chromophores have been developed which exhibit strong two- photon absorption in the range of 500 - 975 nm, in some cases as high as 4400 X 10-50 cm4 s/photon-molecule. In the case of donor-(pi) -donor structures, quantum-chemical calculations show that the large absorption cross-sections arise from the symmetric re-distribution of charge from the donor end-groups to the conjugated bridge, resulting in an electronic excited-state which is more delocalized than the ground state. For many of these molecules, two-photon excitation populates a state which is sufficiently reducing that a charge transfer reaction can occur with acrylate monomers. The efficiency of these processes can be described using Marcus theory. Under suitable conditions, such reactions can induce radical polymerization of acrylate resins. Polymerization rates have been measured, and we show that these two-photon

  13. Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up.

    Science.gov (United States)

    Cortesi, Marilisa; Bandiera, Lucia; Pasini, Alice; Bevilacqua, Alessandro; Gherardi, Alessandro; Furini, Simone; Giordano, Emanuele

    2017-01-01

    Quantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system's functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories. A protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry. The high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope- when coupled with appropriate software for image processing- might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described. The experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).

  14. Fluorescence microscopic spectral study of fluorescein and some amino acids

    Science.gov (United States)

    Joshi, Narahari V.; Otero de Joshi, Virginia; Rodrigues, Nestor; Hernandes, Luis

    1995-04-01

    A conventional collinear laser induced fluorescence detection system for capillary electrophoresis was modified by replacing a GaAs photomultiplier by a Charge coupled Device which was operated at liquid nitrogen temperature. This permits longer exposure time to collect the weak radiation and hence extends the limit of detection and spectral analysis of lower concentration of biological active molecules. Using the above technique, and passing the solution through a capillary of 20 micrometers inside diameter, we have recorded fluorescence spectra of biological important molecules such as Arginine, Glutamate and Glutamine; all of them were derivatized with Fluorescein Isothiocyanate (FITC) isomer I. Spectra of FITC as a function of the pH were examined to lower the detection limit. The concentration of the molecules was as low as 10-14 M. All the spectra recorded lied in the expected region (510 nm to 550 nm) and showed considerable similarity. The spectrum of low concentration of molecules provides significant information as the intensity of intra-molecular interaction is reduced. The recorded spectra gave information about the distribution of vibrational states near S0 and S1 levels.

  15. Fast two-photon neuronal imaging and control using a spatial light modulator and ruthenium compounds

    Science.gov (United States)

    Peterka, Darcy S.; Nikolenko, Volodymyr; Fino, Elodie; Araya, Roberto; Etchenique, Roberto; Yuste, Rafael

    2010-02-01

    We have developed a spatial light modulator (SLM) based microscope that uses diffraction to shape the incoming two-photon laser source to any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision at high frame rates. Additionally, we have combined this microscope with a new class of two photon active neuromodulators with Ruthenium BiPyridine (RuBi) based cages that offer great flexibility for neuronal control.

  16. Two-photon imaging through a multimode fiber

    CERN Document Server

    Morales-Delgado, Edgar E; Moser, Christophe

    2015-01-01

    In this work we demonstrate 3D imaging using two-photon excitation through a 20 cm long multimode optical fiber (MMF) of 350 micrometers diameter. The imaging principle is similar to single photon fluorescence through a MMF, except that a focused femtosecond pulse is delivered and scanned over the sample. In our approach, focusing and scanning through the fiber is accomplished by digital phase conjugation using mode selection by time gating with an ultra-fast reference pulse. The excited two-photon emission is collected through the same fiber. We demonstrate depth sectioning by scanning the focused pulse in a 3D volume over a sample consisting of fluorescent beads suspended in a polymer. The achieved resolution is 1 micrometer laterally and 15 micrometers axially. Scanning is performed over an 80x80 micrometers field of view. To our knowledge, this is the first demonstration of high-resolution three-dimensional imaging using two-photon fluorescence through a multimode fiber.

  17. Imaging theory and resolution improvement of two-photon confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    唐志列; 杨初平; 裴红津; 梁瑞生; 刘颂豪

    2002-01-01

    The nonlinear effect of two-photon excitation on the imaging property of two-photonconfocal microscopy has been analyzed by the two-photon fluorescence intensity transfer functionderived in this paper. The two-photon fluorescence intensity transfer function in a confocal micros-copy is given. Furthermore the three-dimensional point spread function (3D-PSF) and thethree-dimensional optical transfer function (3D-OTF) of two-photon confocal microscopy are de-rived based on the nonlinear effect of two-photon excitation. The imaging property of two-photonconfocal microscopy is discussed in detail based on 3D-OTF. Finally the spatial resolution limit oftwo-photon confocal microscopy is discussed according to the uncertainty principle.

  18. Development of in vivo confocal microscope for reflection and fluorescence imaging simultaneously

    Science.gov (United States)

    Ahn, MyoungKi; Chun, ByungSeon; Song, Cheol; Gweon, DaeGab

    2010-02-01

    In-vivo confocal microscope technology can be applied to the medical imaging diagnosis and new drug development. We present an in-vivo confocal microscope that can acquire a reflection image and a fluorescence image simultaneously and independently. To obtain reflection confocal images, we used a linearly polarized diode laser with the wavelength of 830 nm. To acquire fluorescence confocal images, we used two diode lasers with the wavelength of 488 nm and 660 nm, respectively. Because of a broad wavelength bandwidth from visible (488 nm) to near-IR (830 nm), we designed and optimized the optical system to reduce various optical aberrations. With the developed in-vivo confocal microscope, we performed ex-vivo cell imaging and in-vivo imaging of the human skin.

  19. Evaluation of a miniature microscope objective designed for fluorescence array microscopy detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    McCall, Brian; Olsen, Randall J; Nelles, Nicole J; Williams, Dawn L; Jackson, Kevin; Richards-Kortum, Rebecca; Graviss, Edward A; Tkaczyk, Tomasz S

    2014-03-01

    A prototype miniature objective that was designed for a point-of-care diagnostic array microscope for detection of Mycobacterium tuberculosis and previously fabricated and presented in a proof of concept is evaluated for its effectiveness in detecting acid-fast bacteria. To evaluate the ability of the microscope to resolve submicron features and details in the image of acid-fast microorganisms stained with a fluorescent dye, and to evaluate the accuracy of clinical diagnoses made with digital images acquired with the objective. The lens prescription data for the microscope design are presented. A test platform is built by combining parts of a standard microscope, a prototype objective, and a digital single-lens reflex camera. Counts of acid-fast bacteria made with the prototype objective are compared to counts obtained with a standard microscope over matched fields of view. Two sets of 20 smears, positive and negative, are diagnosed by 2 pathologists as sputum smear positive or sputum smear negative, using both a standard clinical microscope and the prototype objective under evaluation. The results are compared to a reference diagnosis of the same sample. More bacteria are counted in matched fields of view in digital images taken with the prototype objective than with the standard clinical microscope. All diagnostic results are found to be highly concordant. An array microscope built with this miniature lens design will be able to detect M tuberculosis with high sensitivity and specificity.

  20. Adiabatic following in two-photon transition

    Energy Technology Data Exchange (ETDEWEB)

    Nayfeh, M.H.; Nayfeh, A.H.

    1977-01-01

    There has been much interest recently in coherent multiphoton transitions in many-level systems. The present work considers the effect of relaxation in the response of a three-level system to a smoothly varying, near-resonant, two-photon field. The relaxation-dependent contributions to the nonlinear refractive index are calculated. It is shown that the coherent interaction of two smoothly varying, near-resonant, two-photon pulses with a three-level system can be described by ''two-photon damped Bloch equations'' which are analogous to those for a one-photon transition in a two-level system except for the presence of a two-photon coupling and a frequency shift. 1 figure. (RWR)

  1. Two-Photon Physics in Hadronic Processes

    Energy Technology Data Exchange (ETDEWEB)

    Carl Carlson; Marc Vanderhaeghen

    2007-11-01

    Two-photon exchange contributions to elastic electron-scattering are reviewed. The apparent discrepancy in the extraction of elastic nucleon form factors between unpolarized Rosenbluth and polarization transfer experiments is discussed, as well as the understanding of this puzzle in terms of two-photon exchange corrections. Calculations of such corrections both within partonic and hadronic frameworks are reviewed. In view of recent spin-dependent electron scattering data, the relation of the two-photon exchange process to the hyperfine splitting in hydrogen is critically examined. The imaginary part of the two-photon exchange amplitude as can be accessed from the beam normal spin asymmetry in elastic electron-nucleon scattering is reviewed. Further extensions and open issues in this field are outlined.

  2. Sideband-Induced Two-Photon Transparency

    Institute of Scientific and Technical Information of China (English)

    CHENG Guang-Ling; HU Xiang-Ming

    2006-01-01

    @@ We show that it is possible to use a single sideband to induce two-photon transparency in a three-level cascade medium. The medium simultaneously absorbs two photons as a one-step process when the middle level is far off one-photon resonance. A resonant sideband coupling on the upper transition and the two-photon one-step process drive the medium into a trapped state, and the dominant component is the ground state. Thus almost all population is trapped in the ground state and the two-photon absorption is dramatically suppressed. We present a numerical calculation for arbitrary values of the atomic and field parameters and also provide an analytic description for the required conditions.

  3. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    Science.gov (United States)

    Yi, Dingrong; Xie, Shaochuan

    2013-12-01

    Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

  4. Correlations of two photons at hadron colliders

    OpenAIRE

    Kozlov, G. A.

    2011-01-01

    We study the Bose-Einstein correlations of two photons and their coherent properties that can provide the information about the space-time structure of the emitting source through the Higgs-boson decays into two photons. We argue that such an investigation could probe the Higgs-boson mass. The model is rather sensitive to the temperature of the environment and to the external distortion effect in medium.

  5. Platinum Acetylide Two-Photon Chromophores (Preprint)

    Science.gov (United States)

    2007-04-01

    the higher energy range that lead to its photodegradation . Secondly, because there is a quadratic dependence of two-photon absorption (2PA) on the...to either an electron donating amino- fluorenyl or electron withdrawing benzothiazolyl-fluorene that are themselves known as two-photon absorbing dyes ...groups in place of phenyl groups have shown a doubling of the intrinsic cr2value at 740 nm.40,41In this paper we describe novel platinum dyes that

  6. Two-photon autofluorescence lifetime and SHG imaging of healthy and diseased human corneas

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Seitz, Berthold; Morgado, António Miguel; König, Karsten

    2015-03-01

    Corneal function can be drastically affected by several degenerations and dystrophies, leading to blindness. Early diagnosis of corneal disease is of major importance and it may be accomplished by monitoring changes of the metabolic state and structural organization, the first detectable pathological signs, by two-photon excitation autofluorescence lifetime and second-harmonic generation imaging. In this study, we propose to use these imaging techniques to differentiate between healthy and pathological corneas. Images were acquired using a laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed laser and a 16-channel photomultiplier tube detector for signal collection. This setup allows the simultaneous excitation of metabolic co-factors and to identify them based on their fluorescence spectra. We were able to discriminate between healthy and pathological corneas using two-photon excitation autofluorescence lifetime and second-harmonic generation imaging from corneal epithelium and stroma. Furthermore, differences between different pathologies were observed. Alterations in the metabolic state of corneal epithelial cells were observed using the autofluorescence lifetime of the metabolic co-factors. In the corneal stroma, we observed not only alterations in the collagen fibril structural organization but also alterations in the autofluorescence lifetime. Further tests are required as the number of pathological samples must be increased. In the future, we intend to establish a correlation between the metabolic and structural changes and the disease stage. This can be a step forward in achieving early diagnosis.

  7. Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope.

    Science.gov (United States)

    Kim, Dong Uk; Moon, Sucbei; Song, Hoseong; Kwon, Hyuk-Sang; Kim, Dug Young

    2011-01-01

    High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.

  8. A cost-effective fluorescence mini-microscope for biomedical applications.

    Science.gov (United States)

    Zhang, Yu Shrike; Ribas, João; Nadhman, Akhtar; Aleman, Julio; Selimović, Šeila; Lesher-Perez, Sasha Cai; Wang, Ting; Manoharan, Vijayan; Shin, Su-Ryon; Damilano, Alessia; Annabi, Nasim; Dokmeci, Mehmet Remzi; Takayama, Shuichi; Khademhosseini, Ali

    2015-01-01

    We have designed and fabricated a miniature microscope from off-the-shelf components and a webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters, such as cell/tissue viability (e.g. live/dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60×, achieves a resolution as high as microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including, but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread application in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required.

  9. A Cost-Effective Fluorescence Mini-Microscope with Adjustable Magnifications for Biomedical Applications

    Science.gov (United States)

    Zhang, Yu Shrike; Ribas, João; Nadhman, Akhtar; Aleman, Julio; Selimović, Šeila; Lesher-Perez, Sasha Cai; Wang, Ting; Manoharan, Vijayan; Shin, Su-Ryon; Damilano, Alessia; Annabi, Nasim; Dokmeci, Mehmet Remzi; Takayama, Shuichi; Khademhosseini, Ali

    2015-01-01

    We have designed and fabricated a miniature microscope from off-the-shelf components and webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters such as cell/tissue viability (e.g. Live/Dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60X, achieves a resolution as high as microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread applications in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required. PMID:26282117

  10. Testing the sensitivity and specificity of the fluorescence microscope (Cyscope® for malaria diagnosis

    Directory of Open Access Journals (Sweden)

    Mudathir Mahmoud A

    2010-03-01

    Full Text Available Abstract Background Early diagnosis and treatment of malaria are necessary components in the control of malaria. The gold standard light microscopy technique has high sensitivity, but is a relatively time-consuming procedure especially during epidemics and in areas of high endemicity. This study attempted to test the sensitivity and specificity of a new diagnostic tool - the Cyscope® fluorescence microscope, which is based on the use of Plasmodium nucleic acid-specific fluorescent dyes to facilitate detection of the parasites even in low parasitaemia conditions due to the contrast with the background. Methods In this study, 293 febrile patients above the age of 18 years attending the malaria treatment centre in Sinnar State (Sudan were interviewed using a structured questionnaire. Finger-prick blood samples were also collected from the participants to be tested for malaria using the hospital's microscope, the reference laboratory microscope, as well as the Cyscope® microscope. The results of the investigations were then used to calculate the sensitivity, specificity, and positive and negative predictive values of the Cyscope® microscope in reference to gold standard light microscopy. Results The sensitivity was found to be 98.2% (95% CI: 90.6%-100%; specificity = 98.3% (95% CI: 95.7% - 99.5%; positive predictive value = 93.3% (95% CI: 83.8% - 98.2%; and negative predictive value = 99.6% (95% CI: 97.6% - 100%. Conclusions In conclusion, the Cyscope® microscope was found to be sensitive, specific and provide rapid, reliable results in a matter of less than 10 minutes. The Cyscope® microscope should be considered as a viable, cheaper and time-saving option for malaria diagnosis, especially in areas where Plasmodium falciparum is the predominant parasite.

  11. Synthesis of Fluorescent Gelators and Direct Observation of Gelation with a Fluorescence Microscope.

    Science.gov (United States)

    Hanabusa, Kenji; Ueda, Takuya; Takata, Shingo; Suzuki, Masahiro

    2016-11-14

    Fluorescein-, benzothiazole-, quinoline-, stilbene-, and carbazole-containing fluorescent gelators have been synthesized by connecting gelation-driving segments, including l-isoleucine, l-valine, l-phenylalanine, l-leucine residue, cyclo(l-asparaginyl-l-phenylalanyl), and trans-(1R,2R)-diaminocyclohexane. The emission behaviors of the gelators were investigated, and their gelation abilities studied against 15 solvents. The minimum gel concentration, variable-temperature spectroscopy, transmission electron microscopy, scanning electron microscopy, fluorescence microscopy (FM), and confocal laser scanning microscopy (CLSM) were used to characterize gelation. The intermolecular hydrogen bonding between the N-H and C=O of amide, van der Waals interactions and π-π stacking play important roles in gelation. The colors of emission are related to the fluorescence structures of gelators. Fibrous aggregates characterized by the color of their emission were observed by FM. 3D images are produced by the superposition of images captured by CLSM every 0.1 μm to a settled depth. The 3D images show that the large micrometer-sized aggregates spread out three dimensionally. FM observations of mixed gelators are studied. In the case of gelation, two structurally related gelators with the same gelation-driving segment lead to the gelators build up of the same aggregates through similar hydrogen-bonding patterns. When two gelators with structurally different gelation-driving segments induce gelation, the gelators build up each aggregate through individual hydrogen-bonding patterns. A fluorescent reagent that was incorporated into the aggregates of gels through van der Waals interactions was developed. The addition of this fluorescent reagent enables the successful observation of nonfluorescent gelators' aggregates by FM.

  12. In situ imaging of the mouse cochlea using two-photon microscopy

    Science.gov (United States)

    Yang, Xin; Pu, Ye; Psaltis, Demetri; Stankovic, Konstantina M.

    2013-04-01

    Intracochlear imaging is of great interest clinically because cochlea is the central organ of hearing. However, intracochlear imaging is technologically challenging due to the cochlea's small size and encasement in bone. The state-of- the-art imaging techniques are not adequate for high resolution cellular imaging to establish diagnosis without destroying the cochlea. We report in situ imaging of intact mouse cochlea using endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. TPEF eliminates the need for exogenous labeling and eradicating the staining-induced artifacts. We used a natural, membranous opening into the cochlea, the round window, as the optical access to reach the organ of Corti, requiring no additional slicing or opening. Our approach provides the maximum non-invasiveness in the imaging process. TPEF exhibits strong contrast allowing deep imaging of mouse cochlea with cellular and even subcellular resolution. Inner hair cell, outer hair cell and supporting cell are clearly identifiable in TPEF images. Distinct morphological differences are observed between healthy and noise-exposed cochleae, allowing detection of specific, noise-induced pathologic changes. The TPEF images taken through the round window are correlated with the whole mount sections, verifying their reliability. Compared with one-photon excitation fluorescence (OPEF) confocal microscope and wide-field transmission microscope images taken under the same magnification and resolution, TPEF images demonstrate clear advantages in terms of sharpness, signal to noise ratio and contrast. These capabilities provide a working foundation for microendoscopy-based clinical diagnostics of sensorineural hearing loss.

  13. The Gray Institute ‘open’ high-content, fluorescence lifetime microscopes

    OpenAIRE

    Barber, PR; TULLIS, IDC; PIERCE, GP; Newman, RG; PRENTICE, J; Rowley, MI; Matthews, DR; AMEER-BEG, SM; Vojnovic, B

    2013-01-01

    Summary We describe a microscopy design methodology and details of microscopes built to this ‘open’ design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a c...

  14. Fluorescence intensity and bright spot analyses using a confocal microscope for photodynamic diagnosis of brain tumors.

    Science.gov (United States)

    Yoneyama, Takeshi; Watanabe, Tetsuyo; Kagawa, Hiroyuki; Hayashi, Yutaka; Nakada, Mitsutoshi

    2017-03-01

    In photodynamic diagnosis using 5-aminolevulinic acid (5-ALA), discrimination between the tumor and normal tissue is very important for a precise resection. However, it is difficult to distinguish between infiltrating tumor and normal regions in the boundary area. In this study, fluorescent intensity and bright spot analyses using a confocal microscope is proposed for the precise discrimination between infiltrating tumor and normal regions. From the 5-ALA-resected brain tumor tissue, the red fluorescent and marginal regions were sliced for observation under a confocal microscope. Hematoxylin and eosin (H&E) staining were performed on serial slices of the same tissue. According to the pathological inspection of the H&E slides, the tumor and infiltrating and normal regions on confocal microscopy images were investigated. From the fluorescent intensity of the image pixels, a histogram of pixel number with the same fluorescent intensity was obtained. The fluorescent bright spot sizes and total number were compared between the marginal and normal regions. The fluorescence intensity distribution and average intensity in the tumor were different from those in the normal region. The probability of a difference from the dark enhanced the difference between the tumor and the normal region. The bright spot size and number in the infiltrating tumor were different from those in the normal region. Fluorescence intensity analysis is useful to distinguish a tumor region, and a bright spot analysis is useful to distinguish between infiltrating tumor and normal regions. These methods will be important for the precise resection or photodynamic therapy of brain tumors. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. The Gray Institute ‘open’ high-content, fluorescence lifetime microscopes

    Science.gov (United States)

    BARBER, PR; TULLIS, IDC; PIERCE, GP; NEWMAN, RG; PRENTICE, J; ROWLEY, MI; MATTHEWS, DR; AMEER-BEG, SM; VOJNOVIC, B

    2013-01-01

    Summary We describe a microscopy design methodology and details of microscopes built to this ‘open’ design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off-the-shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup. Several automated high-content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time-domain FLIM via time-correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide-field and laser scanning capabilities designed for high-content microscopy. Devices using these designs also form radiation-beam ‘end-stations’ at Oxford and Surrey Universities, showing the versatility and extendibility of this approach. PMID:23772985

  16. Single particle tracking through highly scattering media with multiplexed two-photon excitation

    Science.gov (United States)

    Perillo, Evan; Liu, Yen-Liang; Liu, Cong; Yeh, Hsin-Chih; Dunn, Andrew K.

    2015-03-01

    3D single-particle tracking (SPT) has been a pivotal tool to furthering our understanding of dynamic cellular processes in complex biological systems, with a molecular localization accuracy (10-100 nm) often better than the diffraction limit of light. However, current SPT techniques utilize either CCDs or a confocal detection scheme which not only suffer from poor temporal resolution but also limit tracking to a depth less than one scattering mean free path in the sample (typically validated our microscope by tracking (1) fluorescent nanoparticles in a prescribed motion inside gelatin gel (with 1% intralipid) and (2) labeled single EGFR complexes inside skin cancer spheroids (at least 8 layers of cells thick) for ~10 minutes. Furthermore we discuss future capabilities of our multiplexed two-photon microscope design, specifically to the extension of (1) simultaneous multicolor tracking (i.e. spatiotemporal co-localization analysis) and (2) FRET studies (i.e. lifetime analysis). The high resolution, high depth penetration, and multicolor features of this microscope make it well poised to study a variety of molecular scale dynamics in the cell, especially related to cellular trafficking studies with in vitro tumor models and in vivo.

  17. Medical prototyping using two photon polymerization

    Directory of Open Access Journals (Sweden)

    Roger J Narayan

    2010-12-01

    Full Text Available Two photon polymerization involves nearly simultaneous absorption of ultrashort laser pulses for selective curing of photosensitive material. This process has recently been used to create small-scale medical devices out of several classes of photosensitive materials, such as acrylate-based polymers, organically-modified ceramic materials, zirconium sol-gels, and titanium-containing hybrid materials. In this review, the use of two photon polymerization for fabrication of several types of small-scale medical devices, including microneedles, artificial tissues, microfluidic devices, pumps, sensors, and valves, from computer models is described. Necessary steps in the development of two photon polymerization as a commercially viable medical device manufacturing method are also considered.

  18. Two Photon Couplings of Hybrid Mesons

    CERN Document Server

    Page, P R

    1996-01-01

    A new formalism is developed for the two photon production of hybrid mesons via intermediate hadronic decays. In an adiabatic and non--relativistic context with spin 1 pair creation we obtain the first absolute estimates of unmixed hybrid production strengths to be small (0.03 - 3 eV) in relation to experimental meson widths (0.1 - 5 keV). Within this context, two photon collisions therefore strongly discriminate between hybrid and conventional meson wave function components at BaBar, Cleo II, LEP2 and LHC, filtering out non--gluonic components. Decay widths of unmixed hybrids are tiny. The formalism also induces conventional meson two photon widths roughly in agreement with experiment.

  19. Fluorescence liftime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes

    Science.gov (United States)

    Ruck, Angelika C.; Dolp, Frank; Happ, Claudia; Steiner, Rudolf; Beil, Michael

    2003-06-01

    A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter liftime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.

  20. Boosting accuracy of automated classification of fluorescence microscope images for location proteomics

    Directory of Open Access Journals (Sweden)

    Huang Kai

    2004-06-01

    Full Text Available Abstract Background Detailed knowledge of the subcellular location of each expressed protein is critical to a full understanding of its function. Fluorescence microscopy, in combination with methods for fluorescent tagging, is the most suitable current method for proteome-wide determination of subcellular location. Previous work has shown that neural network classifiers can distinguish all major protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Building on these results, we evaluate here new classifiers and features to improve the recognition of protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Results We report here a thorough comparison of the performance on this problem of eight different state-of-the-art classification methods, including neural networks, support vector machines with linear, polynomial, radial basis, and exponential radial basis kernel functions, and ensemble methods such as AdaBoost, Bagging, and Mixtures-of-Experts. Ten-fold cross validation was used to evaluate each classifier with various parameters on different Subcellular Location Feature sets representing both 2D and 3D fluorescence microscope images, including new feature sets incorporating features derived from Gabor and Daubechies wavelet transforms. After optimal parameters were chosen for each of the eight classifiers, optimal majority-voting ensemble classifiers were formed for each feature set. Comparison of results for each image for all eight classifiers permits estimation of the lower bound classification error rate for each subcellular pattern, which we interpret to reflect the fraction of cells whose patterns are distorted by mitosis, cell death or acquisition errors. Overall, we obtained statistically significant improvements in classification accuracy over the best previously published results, with the overall error rate being reduced by one-third to one-half and with the average

  1. High contrast two-photon imaging of fingermarks

    Science.gov (United States)

    Stoltzfus, Caleb R.; Rebane, Aleksander

    2016-04-01

    Optically-acquired fingermarks are widely used as evidence across law enforcement agencies as well as in the courts of law. A common technique for visualizing latent fingermarks on nonporous surfaces consists of cyanoacrylate fuming of the fingerprint material, followed by impregnation with a fluorescent dye, which under ultra violet (UV) illumination makes the fingermarks visible and thus accessible for digital recording. However, there exist critical circumstances, when the image quality is compromised due to high background scattering, high auto-fluorescence of the substrate material, or other detrimental photo-physical and photo-chemical effects such as light-induced damage to the sample. Here we present a novel near-infrared (NIR), two-photon induced fluorescence imaging modality, which significantly enhances the quality of the fingermark images, especially when obtained from highly reflective and/or scattering surfaces, while at the same time reducing photo-damage to sensitive forensic samples.

  2. Spectral characterization and unmixing of intrinsic contrast in intact normal and diseased gastric tissues using hyperspectral two-photon microscopy.

    Directory of Open Access Journals (Sweden)

    Lauren E Grosberg

    Full Text Available BACKGROUND: Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition. CONCLUSIONS/SIGNIFICANCE: These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative 'instant histology' of fresh tissue

  3. Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color Two-Photon Uncaging.

    Science.gov (United States)

    Fichte, Manuela A H; Weyel, Xenia M M; Junek, Stephan; Schäfer, Florian; Herbivo, Cyril; Goeldner, Maurice; Specht, Alexandre; Wachtveitl, Josef; Heckel, Alexander

    2016-07-25

    We successfully introduced two-photon-sensitive photolabile groups ([7-(diethylamino)coumarin-4-yl]methyl and p-dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three-dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double-strand displacement in a hydrogel and in neurons. Orthogonal two-photon uncaging of the two cages is possible, thus enabling complex scenarios of three-dimensional control of hybridization with light.

  4. On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope

    Energy Technology Data Exchange (ETDEWEB)

    Mazouchi, Amir; Liu Baoxu; Bahram, Abdullah [Department of Physics, Institute for Optical Sciences, University of Toronto, Toronto (Canada); Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, ON, L5L 1C6 (Canada); Gradinaru, Claudiu C., E-mail: claudiu.gradinaru@utoronto.ca [Department of Physics, Institute for Optical Sciences, University of Toronto, Toronto (Canada); Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, ON, L5L 1C6 (Canada)

    2011-02-28

    Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to {mu}M range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.

  5. Enhanced energy transfer in respiratory-deficient endothelial cells probed by microscopic fluorescence excitation spectroscopy

    Science.gov (United States)

    Schneckenburger, Herbert; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.; Steiner, Rudolf W.

    1996-12-01

    Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

  6. Two-photon physics at LEP2

    Energy Technology Data Exchange (ETDEWEB)

    Cartwright, Susan; Lehto, Mark [University of Sheffield Department of Physics, Sheffield S3 7RH (United Kingdom); Seymour, Michael H.; Close, Frank; Wright, Alison [Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX (United Kingdom); Affholderbach, Klaus; Cowan, Glen [Universitaet Siegen, Fachbereich Physik, D-57068 Siegen (Germany); Finch, Alex [University of Lancaster, Lancaster LA1 4YB (United Kingdom); Lauber, Jan [University College London, Gower Street, London WC1E 6BT (United Kingdom)

    1998-02-01

    The working group on two-photon physics concentrated on three main subtopics: modelling the hadronic final state of deep inelastic scattering on a photon; unfolding the deep inelastic scattering data to obtain the photon structure function; and resonant production of exclusive final states, particularly of glueball candidates. In all three areas, new results were presented. (author)

  7. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

    Science.gov (United States)

    Zhao, Qiaole; Schelen, Ben; Schouten, Raymond; van den Oever, Rein; Leenen, René; van Kuijk, Harry; Peters, Inge; Polderdijk, Frank; Bosiers, Jan; Raspe, Marcel; Jalink, Kees; Geert Sander de Jong, Jan; van Geest, Bert; Stoop, Karel; Young, Ian Ted

    2012-12-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

  8. Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.

    Science.gov (United States)

    Winter, Peter W; York, Andrew G; Nogare, Damian Dalle; Ingaramo, Maria; Christensen, Ryan; Chitnis, Ajay; Patterson, George H; Shroff, Hari

    2014-09-20

    Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 μm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

  9. Aggregation induced enhanced emission of conjugated dendrimers with a large intrinsic two-photon absorption cross-section

    NARCIS (Netherlands)

    Xu, Bin; Zhang, Jibo; Fang, Honghua; Ma, Suqian; Chen, Qidai; Sun, Hongbo; Im, Chan; Tian, Wenjing

    2014-01-01

    Organic nonlinear optical materials combining high luminescence quantum yields and large two-photon absorption cross-sections are attractive for both fundamental research and practical applications, such as up-converted lasers and two-photon fluorescence microscopy. Herein, we reported a series of

  10. In-trap fluorescence detection of atoms in a microscopic dipole trap

    CERN Document Server

    Hilliard, A J; Sompet, P; Carpentier, A V; Andersen, M F

    2015-01-01

    We investigate fluorescence detection using a standing wave of blue-detuned light of one or more atoms held in a deep, microscopic dipole trap. The blue-detuned standing wave realizes a Sisyphus laser cooling mechanism so that an atom can scatter many photons while remaining trapped. When imaging more than one atom, the blue detuning limits loss due to inelastic light-assisted collisions. Using this standing wave probe beam, we demonstrate that we can count from one to the order of 100 atoms in the microtrap with sub-poissonian precision.

  11. The Pocketscope: a spatial light modulator based epi-fluorescence microscope for optogenetics

    Science.gov (United States)

    Linnenberger, Anna; Peterka, Darcy S.; Quirin, Sean; Yuste, Rafael

    2014-09-01

    Microscopy incorporating spatial light modulators (SLMs) enables three dimensional (3D) excitation and monitoring of the activity of neuronal ensembles, enabling studies of neuronal circuit activity both in vitro and in vivo. In this paper we present a portable (22 cm x 42.5 cm x 30 cm), SLM-based epi-fluorescence upright microscope ("Pocketscope") that enables 3D calcium imaging and photoactivation of neurons in brain slices. Here we describe the implementation of the instrument; quantify the volume over which neural activity can be excited; and demonstrate the use of the system for mapping neural circuits in brain slices.

  12. Rapid imaging of mammalian brain slices with a compact light sheet fluorescent microscope

    Science.gov (United States)

    Yang, Zhengyi; Haslehurst, Peter; Scott, Suzanne; Emptage, Nigel; Dholakia, Kishan

    2017-02-01

    Light sheet fluorescent microscopy is able to provide high acquisition speed and high contrast images, as well as the low photo-bleaching and photo-damage brought to the sample. Here we describe a compact setup design optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We report this prototype instrument is capable of rapid imaging wide area of the dendritic or axonal arbor of a dye-filled neuron in hippocampal slice. We also show several applications of this compact light sheet fluorescent microscope, to demonstrate that our approach offers a powerful functionality to the neuroscience community that is not achievable with traditional imaging methods.

  13. Transparency induced by two photon interference in a beam splitter

    Institute of Scientific and Technical Information of China (English)

    Wang Kai-Ge; Yang Guo-Jian

    2004-01-01

    We propose a special two-photon state which is completely transparent in a 50/50 beam splitter. This effect is caused by the destructive two-photon interference and shows the signature of photon entanglement. We find that the symmetry of the two-photon spectrum plays the key role for the properties of two-photon interference.

  14. A new self-made digital slide scanner and microscope for imaging and quantification of fluorescent microspheres

    DEFF Research Database (Denmark)

    Henning, William; Bjerglund Andersen, Julie; Højgaard, Liselotte;

    2015-01-01

    Objective: A low-cost microscope slide scanner was constructed for the purpose of digital imaging of newborn piglet brain tissue and to quantify fluorescent microspheres in tissue. Methods: Using a standard digital single-lens reflex (DSLR) camera, fluorescent imaging of newborn piglet brain tissue...

  15. Two-photon luminescence microscopy of field enhancement at gold nanoparticles

    DEFF Research Database (Denmark)

    Beermann, Jonas; Bozhevolnyi, Sergey I.

    2005-01-01

    Using a reflection scanning optical microscope detecting two-photon luminescence (TPL) we have imaged square gold bumps positioned in a periodic array either on a smooth gold film or directly on a glass substrate. The second-harmonic (SH) and TPL response from these structures show both polarizat......Using a reflection scanning optical microscope detecting two-photon luminescence (TPL) we have imaged square gold bumps positioned in a periodic array either on a smooth gold film or directly on a glass substrate. The second-harmonic (SH) and TPL response from these structures show both...

  16. Two-photon cooling of magnesium atoms

    DEFF Research Database (Denmark)

    Malossi, N.; Damkjær, S.; Hansen, P. L.

    2005-01-01

    A two-photon mechanism for cooling atoms below the Doppler temperature is analyzed. We consider the magnesium ladder system (3s2)S01¿(3s3p)P11 at 285.2nm followed by the (3s3p)P11¿(3s3d)D21 transition at 880.7nm . For the ladder system quantum coherence effects may become important. Combined...... with the basic two-level Doppler cooling process this allows for reduction of the atomic sample temperature by more than a factor of 10 over a broad frequency range. First experimental evidence for the two-photon cooling process is presented and compared to model calculations. Agreement between theory...... and experiment is excellent. In addition, by properly choosing the Rabi frequencies of the two optical transitions a velocity independent atomic dark state is observed....

  17. Magnetic two-photon scattering and two-photon emission - Cross sections and redistribution functions

    Science.gov (United States)

    Alexander, S. G.; Meszaros, P.

    1991-01-01

    The magnetic two-photon scattering cross section is discussed within the framework of QED, and the corresponding scattering redistribution function for this process and its inverse, as well as the scattering source function are calculated explicitly. In a similar way, the magnetic two-photon emission process which follows the radiative excitation of Landau levels above ground is calculated. The two-photon scattering and two-photon emission are of the same order as the single-photon magnetic scattering. All three of these processes, and in optically thick cases also their inverses, are included in radiative transport calculations modeling accreting pulsars and gamma-ray bursters. These processes play a prominent role in determining the relative strength of the first two cyclotron harmonics, and their effects extend also to the higher harmonics.

  18. Observing Fluorescent Probes in Living Cells using a Low-Cost LED Flashlight Retrofitted to a Common Vintage Light Microscope

    Directory of Open Access Journals (Sweden)

    G. A. Babbitt

    2013-03-01

    Full Text Available While the application of molecular biological techniques based upon fluorescent probes has rapidly expanded over recent decades, the equipment cost of fluorescent microscopy has largely prevented its adoption in the college and high school classroom. We offer a simple solution to this problem by describing in detail how to build with simple tools, a fluorescent microscope using a common brand of colored LED flashlights and second-hand components of vintage Nikon microscopes. This extremely low cost solution is qualitatively compared to an expensive modern Zeiss system.

  19. Two-photon cooling of magnesium atoms

    DEFF Research Database (Denmark)

    Malossi, N.; Damkjær, S.; Hansen, P. L.;

    2005-01-01

    A two-photon mechanism for cooling atoms below the Doppler temperature is analyzed. We consider the magnesium ladder system (3s2)S01¿(3s3p)P11 at 285.2nm followed by the (3s3p)P11¿(3s3d)D21 transition at 880.7nm . For the ladder system quantum coherence effects may become important. Combined...

  20. Two-Photon Collective Atomic Recoil Lasing

    Directory of Open Access Journals (Sweden)

    James A. McKelvie

    2015-11-01

    Full Text Available We present a theoretical study of the interaction between light and a cold gasof three-level, ladder configuration atoms close to two-photon resonance. In particular, weinvestigate the existence of collective atomic recoil lasing (CARL instabilities in differentregimes of internal atomic excitation and compare to previous studies of the CARL instabilityinvolving two-level atoms. In the case of two-level atoms, the CARL instability is quenchedat high pump rates with significant atomic excitation by saturation of the (one-photoncoherence, which produces the optical forces responsible for the instability and rapid heatingdue to high spontaneous emission rates. We show that in the two-photon CARL schemestudied here involving three-level atoms, CARL instabilities can survive at high pump rateswhen the atoms have significant excitation, due to the contributions to the optical forces frommultiple coherences and the reduction of spontaneous emission due to transitions betweenthe populated states being dipole forbidden. This two-photon CARL scheme may form thebasis of methods to increase the effective nonlinear optical response of cold atomic gases.

  1. Two-photon super bunching of thermal light via multiple two-photon-path interference

    CERN Document Server

    Hong, Peilong; Zhang, Guoquan

    2012-01-01

    We propose a novel scheme to achieve two-photon super bunching of thermal light through multiple two-photon-path interference, in which two mutually first-order incoherent optical channels are introduced by inserting a modified Michelson interferometer into a traditional two-photon HBT interferometer, and the bunching peak-to-background ratio can reach 3 theoretically. Experimentally, the super bunching peak-to-background ratio was measured to be 2.4, much larger than the ratio 1.7 measured with the same thermal source in a traditional HBT interferometer. The peak-to-background ratio of two-photon super bunching of thermal light can be increased up to $2\\times1.5^n$ by inserting cascadingly $n$ pairs of mutually first-order incoherent optical channels into the traditional two-photon HBT interferometer. The two-photon super bunching of thermal light should be of great significance in improving the visibility of classical ghost imaging.

  2. Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.

    Science.gov (United States)

    Larson, Joshua; Kirk, Matt; Drier, Eric A; O'Brien, William; MacKay, James F; Friedman, Larry J; Hoskins, Aaron A

    2014-10-01

    Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.

  3. Two-Photon Absorption-Induced Emission Properties of Dye HMASPS Doped Polymer

    Institute of Scientific and Technical Information of China (English)

    王东; 周广勇; 任燕; 杨胜军; 许心光; 邵宗书; 蒋民华

    2002-01-01

    The 0.01M two-photon absorption dye trans-4-[p-(N-hydroxyethyl-N-methylamino)styryl]-N-methyl-pyridinium p-toluene sulfonate (HMASPS) doped polymer has been prepared. When pumped by the picosecond pulse from the pulsed mode-locked Nd: YAG laser, the polymer emits more intense upconverted fluorescence and superradiance compared to the solution sample of the dye. The two-photon pumped lasing with oscillating pulses has also been obtained. Compared to the dye in its solution state, the emission spectra of the polymer are all blueshifted.The polymer has a long upconverted fluorescent lifetime of about 4.041 ± 0.04 ns.

  4. Estimating pH at the Air/Water Interface with a Confocal Fluorescence Microscope.

    Science.gov (United States)

    Yang, Haiya; Imanishi, Yasushi; Harata, Akira

    2015-01-01

    One way to determine the pH at the air/water interface with a confocal fluorescence microscope has been proposed. The relation between the pH at the air/water interface and that in a bulk solution has been formulated in connection with the adsorption equilibrium and the dissociation equilibrium of the dye adsorbed. Rhodamine B (RhB) is used as a surface-active fluorescent pH probe. The corrected fluorescence spectrum of RhB molecules at the air/water interface with the surface density of 1.0 nmol m(-2) level shows pH-dependent shifts representing an acid-base equilibrium. Two ways to determine the unknown acid-base equilibrium constant of RhB molecules at the air/water interface have been discussed. With surface-tension measurements, the adsorption properties, maximum surface density, and adsorption equilibrium constants were estimated for both cationic and zwitterionic forms of RhB molecules at the air/water interface.

  5. Adiabatic following in two-photon transition

    Energy Technology Data Exchange (ETDEWEB)

    Nayfeh, M.H.; Nayfeh, A.H.

    1977-03-01

    The coherent interaction of two smoothly varying, near-resonant, two-photon pulses with a three-level system can be described by ''two-photon damped Bloch equations'' which are analogous to those for a one-photon transition in a two-level system except for the presence of a two-photon coupling and a frequency shift. These equations are solved for the cases ..gamma../sub 1/, ..gamma../sub 2/ very-much-less-than ..cap omega.., ..gamma../sub 1/ = ..gamma../sub 2/, and ..gamma../sub 2/k/sup 2/epsilon/sup 4//..cap omega../sup 2/, ..gamma../sub 1/ very-much-less-than ..cap omega.., where ..gamma../sub 1/ and ..gamma../sub 2/ are the atomic energy and phase relaxation widths, respectively, and ..cap omega.. is the Rabi frequency. The leading contribution to the refractive index is intensity dependent, caused by the level shifts inherent in multiphoton processes; it includes a relaxation dependent part which is important at times shorter than ..gamma../sup -1//sub 1/. The second-order contributions depend on the square of the intensity and the time-integrated square of the intensity. The latter contribution, which is relaxation dependent, causes line asymmetry at the long-wavelength wing; it consists of a term proportional to ..gamma../sub 2/-..gamma../sub 1/ and only important at early times and a term proportional to 2..gamma../sub 2/-..gamma../sub 1/.

  6. Reduction of the pulse duration of the ultrafast laser pulses of the Two-Photon Laser Scanning Microscopy (2PLSM

    Directory of Open Access Journals (Sweden)

    Reshak Ali

    2008-07-01

    Full Text Available Abstract Background We provide an update of our two-photon laser scanning microscope by compressing or reducing the broadening of the pulse width of ultrafast laser pulses for dispersion precompensation, to enable the pulses to penetrate deeply inside the sample. Findings The broadening comes as the pulses pass through the optical elements. We enhanced and modified the quality and the sharpness of images by enhancing the resolution using special polarizer namely Glan Laser polarizer GL10. This polarizer consists of two prisms separated by air space. This air separation between the two prisms uses to delay the red wavelength when the light leaves the first prism to the air then to second prism. We note a considerable enhancing with using the GL polarizer, and we can see the details of the leaf structure in early stages when we trying to get focus through z-stacks of images in comparison to exactly the same measurements without using GL polarizer. Hence, with this modification we able to reduce the time of exposure the sample to the laser radiation thereby we will reduce the probability of photobleaching and phototoxicity. When the pulse width reduced, the average power of the laser pulses maintained at a constant level. Significant enhancement is found between the two kinds of images of the Two-Photon Excitation Fluorescence (TPEF. Conclusion In summary reduction the laser pulse width allowed to collect more diffraction orders which will used to form the images. The more diffraction orders the higher resolution images.

  7. Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research

    Science.gov (United States)

    PERRY, SETH W.; BURKE, RYAN M.; BROWN, EDWARD B.

    2012-01-01

    Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality. PMID:22258888

  8. Two-photon laser confocal microscopy of micropermeability of resin-dentin bonds made with water or ethanol wet bonding.

    Science.gov (United States)

    Sauro, Salvatore; Watson, Timothy F; Mannocci, Francesco; Miyake, Katsuya; Huffman, Bradford P; Tay, Franklin R; Pashley, David H

    2009-07-01

    This study evaluated the micropermeability of six etch-and-rinse adhesives bonded to dentin. There were two principal groups: wet bonding with water or wet bonding with absolute ethyl alcohol. After bonding and the creation of composite build-ups, the pulp chambers were filled with 0.1% lucifer yellow. The contents of the pulp chamber were kept under 20 cm H(2)O pressure to simulate pulpal pressure for 3 h. The specimens were vertically sectioned into multiple 0.5-mm thick slabs that were polished and then examined using a two-photon confocal laser scanning microscope (TPCLSM). The results showed that specimens bonded with adhesives using the water wet-bonding condition all showed tracer taken up uniformly by the hybrid layer. This uptake of fluorescent tracer into the hybrid layer was quantified by computer software. The most hydrophobic experimental resins showed the highest fluorescent tracer uptake (ca. 1800 +/- 160 arbitrary fluorescent units/std. surface area). The most hydrophilic experimental resins showed the lowest tracer uptake into water-saturated hybrid layers. When ethanol wet-bonding was used, significantly less fluorescent tracer was seen in hybrid layers. The most hydrophilic experimental resins and Single Bond Plus showed little micropermeability. Clearly, ethanol wet-bonding seals dentin significantly better than water-wet dentin regardless of the adhesive in etch-and-rinse systems.

  9. Anomalous two-photon spectral features in warm rubidium vapor

    Science.gov (United States)

    Perrella, C.; Light, P. S.; Milburn, T. J.; Kielpinski, D.; Stace, T. M.; Luiten, A. N.

    2016-09-01

    We report observation of anomalous fluorescence spectral features in the environs of a two-photon transition in a rubidium vapor when excited with two different wavelength lasers that are both counterpropagating through the vapor. These features are characterized by an unusual trade-off between the detunings of the driving fields. Three different hypothetical processes are presented to explain the observed spectra: a simultaneous three-atom and four-photon collision, a four-photon excitation involving a light field produced via amplified spontaneous emission, and population pumping perturbing the expected steady-state spectra. Numerical modeling of each hypothetical process is presented, supporting the population pumping process as the most plausible mechanism.

  10. Analysis of mitochondrial mechanical dynamics using a confocal fluorescence microscope with a bent optical fibre.

    Science.gov (United States)

    Li, Yongbo; Honda, Satoshi; Iwami, Kentaro; Ohta, Yoshihiro; Umeda, Norihiro

    2015-11-01

    The cells in the cardiovascular system are constantly subjected to mechanical forces created by blood flow and the beating heart. The effect of forces on cells has been extensively investigated, but their effect on cellular organelles such as mitochondria remains unclear. We examined the impact of nano-Newton forces on mitochondria using a bent optical fibre (BOF) with a flat-ended tip (diameter exceeding 2 μm) and a confocal fluorescence microscope. By indenting a single mitochondrion with the BOF tip, we found that the mitochondrial elastic modulus was proportional to the (-1/2) power of the mitochondrial radius in the 9.6-115 kPa range. We stained the mitochondria with a potential-metric dye (TMRE) and measured the changes in TMRE fluorescence intensity. We confirmed that more active mitochondria exhibit a higher frequency of repetitive transient depolarization. The same trend was observed at forces lower than 50 nN. We further showed that the depolarization frequency of mitochondria decreases under an extremely large force (nearly 100 nN). We conclude that mitochondrial function is affected by physical environmental factors, such as external forces at the nano-Newton level.

  11. Two-photon microscopy with double-circle trajectories for in vivo cerebral blood flow measurements

    Science.gov (United States)

    Landolt, Andrin; Obrist, Dominik; Wyss, Matthias; Barrett, Matthew; Langer, Dominik; Jolivet, Renaud; Soltysinski, Tomasz; Roesgen, Thomas; Weber, Bruno

    2013-05-01

    Scanning microscopes normally use trajectories which produce full-frame images of an object at a low frame rate. Time-resolved measurements are possible if scans along a single line are repeated at a high rate. In conjunction with fluorescence labeling techniques, in vivo recording of blood flow in single capillaries is possible. The present work investigates scanning with double-circle trajectories to measure blood flow simultaneously in several vessels of a capillary network. With the trajectory centered near a bifurcation, a double circle crosses each vessel twice, creating a sensing gate for passing dark red blood cells in fluorescently labeled plasma. From the stack of scans repeated at 1,300 Hz, the time-resolved velocity is retrieved using an image correlation approach. Single bifurcation events can be identified from a few fluorescently labeled red blood cells. The applicability of the method for in vivo measurements is illustrated on the basis of two-photon laser scanning microscopy of the cerebral capillary network of mice. Its performance is assessed with synthetic data generated from a two-phase model for the perfusion in a capillary network. The calculation of velocities is found to be sufficiently robust for a wide range of conditions. The achievable limits depend significantly on the experimental conditions and are estimated to be in the 1 μm/s (velocity) and 0.1 s (time resolution) ranges, respectively. Some manual fine-tuning is required for optimal performance in terms of accuracy and time resolution. Further work may lead to improved reliability with which bifurcation events are identified in the algorithm and to include red blood cell flux and hematocrit measurements. With the capability for time-resolved measurements in all vessels of a bifurcation, double-circle scanning trajectories allow a detailed study of the dynamics in vascular networks.

  12. Planetary Surface Analysis Using Fast Laser Spectroscopic Techniques: Combined Microscopic Raman, LIBS, and Fluorescence Spectroscopy

    Science.gov (United States)

    Blacksberg, J.; Rossman, G. R.; Maruyama, Y.; Charbon, E.

    2011-12-01

    In situ exploration of planetary surfaces has to date required multiple techniques that, when used together, yield important information about their formation histories and evolution. We present a time-resolved laser spectroscopic technique that could potentially collect complementary sets of data providing information on mineral structure, composition, and hydration state. Using a picosecond-scale pulsed laser and a fast time-resolved detector we can simultaneously collect spectra from Raman, Laser Induced Breakdown Spectroscopy (LIBS), and fluorescence emissions that are separated in time due to the unique decay times of each process. The use of a laser with high rep rate (40 KHz) and low pulse energy (1 μJ/pulse) allows us to rapidly collect high signal to noise Raman spectra while minimizing sample damage. Increasing the pulse energy by about an order of magnitude creates a microscopic plasma near the surface and enables the collection of LIBS spectra at an unusually high rep rate and low pulse energy. Simultaneously, broader fluorescence peaks can be detected with lifetimes varying from nanosecond to microsecond. We will present Raman, LIBS, and fluorescence spectra obtained on natural mineral samples such as sulfates, clays, pyroxenes and carbonates that are of interest for Mars mineralogy. We demonstrate this technique using a photocathode-based streak camera detector as well as a newly-developed solid state Single Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. We will discuss the impact of system design and detector choice on science return of a potential planetary surface mission, with a specific focus on size, weight, power, and complexity. The research described here was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (NASA).

  13. Threshold Property of Photoresist Film for Two-photon Optical Memory

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jiangying; MING Hai; LIANG Zhongcheng; WANG Pei; XIE Jianping; XIE Aifang; ZHANG Zebo

    2001-01-01

    Two-photon threshold property of photoresist films have been studied by changing exposure energy. When photoresist film is irradiated by Ti∶Sapphire laser with wavelength 770 nm, pulse width 130 fs, repetition rate 82 MHz, the damage and recording thresholds of the material are 9.15×105 J/cm2 and below 5.57×105 J/cm2, respectively. The principle experiments of two-photon optical memory are demonstrated in photoresist film. The patterns of optical bit data storage are realized at different input power density. The corresponding 3-D tomographies of these recorded spots are scanned under near-field optical microscope.

  14. Simultaneous two-photon imaging and photo-stimulation with structured light illumination.

    Science.gov (United States)

    Dal Maschio, Marco; Difato, Francesco; Beltramo, Riccardo; Blau, Axel; Benfenati, Fabio; Fellin, Tommaso

    2010-08-30

    Holographic microscopy is increasingly recognized as a promising tool for the study of the central nervous system. Here we present a "holographic module", a simple optical path that can be combined with commercial scanheads for simultaneous imaging and uncaging with structured two-photon light. The present microscope is coupled to two independently tunable lasers and has two principal configurations: holographic imaging combined with galvo-steered uncaging and holographic uncaging combined with conventional scanning imaging. We applied this flexible system for simultaneous two-photon imaging and photostimulation of neuronal cells with complex light patterns, opening new perspectives for the study of brain function in situ and in vivo.

  15. Two-photon interference : spatial aspects of two-photon entanglement, diffraction, and scattering

    NARCIS (Netherlands)

    Peeters, Wouter Herman

    2010-01-01

    This dissertation contains scientific research within the realm of quantum optics, which is a branch of physics. An experimental and theoretical study is made of two-photon interference phenomena in various optical systems. Spatially entangled photon pairs are produced via the nonlinear optical proc

  16. Two-photon Interference with Non-identical Photons

    CERN Document Server

    Liu, Jianbin; Zheng, Huaibin; Chen, Hui; Li, Fu-Li; Xu, Zhuo

    2014-01-01

    The indistinguishability of non-identical photons is dependent on detection system in quantum physics. If two photons with different wavelengths are indistinguishable for a detection system, there can be two-photon interference when these two photons are incident to two input ports of a Hong-Ou-Mandel interferometer, respectively. The reason why two-photon interference phenomena are different for classical and nonclassical light is not due to interference, but due to the properties of light and detection system. These conclusions are helpful to understand the physics and applications of two-photon interference.

  17. A three-photon microscope with adaptive optics for deep-tissue in vivo structural and functional brain imaging

    Science.gov (United States)

    Tao, Xiaodong; Lu, Ju; Lam, Tuwin; Rodriguez, Ramiro; Zuo, Yi; Kubby, Joel

    2017-02-01

    We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.

  18. Two-Photon Excited Fluorescence from Biological Aerosol Particles

    Science.gov (United States)

    2010-09-29

    previously observed from serotonin (5-HT) and its precursor hyrdroxytryptophan (5- HTP ) using multi-photon excitation [17-19]. Visible emission from...Sivaprakasam, A. Huston, H.B. Lin, J.D. Eversole, P. Falkenstein and A. Schultz, “Field test results and ambient aerosol measurements using dual

  19. Insights into esophagus tissue architecture using two-photon confocal microscopy

    Science.gov (United States)

    Liu, Nenrong; Wang, Yue; Feng, Shangyuan; Chen, Rong

    2013-08-01

    In this paper, microstructures of human esophageal mucosa were evaluated using the two-photon laser scanning confocal microscopy (TPLSCM), based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). The distribution of epithelial cells, muscle fibers of muscularis mucosae has been distinctly obtained. Furthermore, esophageal submucosa characteristics with cancer cells invading into were detected. The variation of collagen, elastin and cancer cells is very relevant to the pathology in esophagus, especially early esophageal cancer. Our experimental results indicate that the MPM technique has the much more advantages for label-free imaging, and has the potential application in vivo in the clinical diagnosis and monitoring of early esophageal cancer.

  20. Arduino Due based tool to facilitate in vivo two-photon excitation microscopy.

    Science.gov (United States)

    Artoni, Pietro; Landi, Silvia; Sato, Sebastian Sulis; Luin, Stefano; Ratto, Gian Michele

    2016-04-01

    Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra. This tool is portable, fast, affordable and precise. It allowed studying the impact of scattering and of blood absorption on two-photon excitation light. In this way, we determined the wavelength-dependent deformation of excitation spectra occurring in deep tissues in vivo.

  1. Synthesis,structure and nonlinear optical properties of two novel two-photon absorption chromophores

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Two triphenylamine-based derivatives that can be used as two-photon absorption chromophore,tris{4-[4-(3-trifluoromethyl-3-oxopanoyl)]phenyl}amine (1) and tris{4-[4-(3-phenyl-3-oxopanoyl)] phenyl} amine (2) were successfully synthesized and fully characterized by elemental analysis,IR,1H NMR and MS. The single crystal X-ray diffraction analysis showed that the molecules possess D-(π-A)3 structures. One-and two-photon absorption and fluorescence in various solvents were experimentally investigated. A data recording experiment proved the potential application of these chromophores.

  2. Two-photon calcium imaging in mice navigating a virtual reality environment.

    Science.gov (United States)

    Leinweber, Marcus; Zmarz, Pawel; Buchmann, Peter; Argast, Paul; Hübener, Mark; Bonhoeffer, Tobias; Keller, Georg B

    2014-02-20

    In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior(1-6). Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

  3. Scanless functional imaging of hippocampal networks using patterned two-photon illumination through GRIN lenses

    KAUST Repository

    Moretti, Claudio

    2016-09-12

    Patterned illumination through the phase modulation of light is increasingly recognized as a powerful tool to investigate biological tissues in combination with two-photon excitation and light-sensitive molecules. However, to date two-photon patterned illumination has only been coupled to traditional microscope objectives, thus limiting the applicability of these methods to superficial biological structures. Here, we show that phase modulation can be used to efficiently project complex two-photon light patterns, including arrays of points and large shapes, in the focal plane of graded index (GRIN) lenses. Moreover, using this approach in combination with the genetically encoded calcium indicator GCaMP6, we validate our system performing scanless functional imaging in rodent hippocampal networks in vivo ~1.2 mm below the brain surface. Our results open the way to the application of patterned illumination approaches to deep regions of highly scattering biological tissues, such as the mammalian brain.

  4. Two-photon imaging and spectroscopy of fresh human colon biopsies

    Science.gov (United States)

    Cicchi, R.; Sturiale, A.; Nesi, G.; Tonelli, F.; Pavone, F. S.

    2012-03-01

    Two-photon fluorescence (TPEF) microscopy is a powerful tool to image human tissues up to 200 microns depth without any exogenously added probe. TPEF can take advantage of the autofluorescence of molecules intrinsically contained in a biological tissue, as such NADH, elastin, collagen, and flavins. Two-photon microscopy has been already successfully used to image several types of tissues, including skin, muscles, tendons, bladder. Nevertheless, its usefulness in imaging colon tissue has not been deeply investigated yet. In this work we have used combined two-photon excited fluorescence (TPEF), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two-photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of colon. Morphological and spectroscopic analyses allowed to characterize both healthy mucosa, polyp, and colon samples in a good agreement with common routine histology. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low, mild, and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or colonoscope to be used in in-vivo imaging applications.

  5. Highly Efficient and Excitation Tunable Two-Photon Luminescence Platform For Targeted Multi-Color MDRB Imaging Using Graphene Oxide

    Science.gov (United States)

    Pramanik, Avijit; Fan, Zhen; Chavva, Suhash Reddy; Sinha, Sudarson Sekhar; Ray, Paresh Chandra

    2014-08-01

    Multiple drug-resistance bacteria (MDRB) infection is one of the top three threats to human health according to the World Health Organization (WHO). Due to the large penetration depth and reduced photodamage, two-photon imaging is an highly promising technique for clinical MDRB diagnostics. Since most commercially available water-soluble organic dyes have low two-photon absorption cross-section and rapid photobleaching tendency, their applications in two-photon imaging is highly limited. Driven by the need, in this article we report extremely high two-photon absorption from aptamer conjugated graphene oxide (σ2PA = 50800 GM) which can be used for highly efficient two-photon fluorescent probe for MDRB imaging. Reported experimental data show that two-photon photoluminescence imaging color, as well as luminescence peak position can be tuned from deep blue to red, just by varying the excitation wavelength without changing its chemical composition and size. We have demonstrated that graphene oxide (GO) based two-photon fluorescence probe is capable of imaging of multiple antibiotics resistance MRSA in the first and second biological transparency windows using 760-1120 nm wavelength range.

  6. Two-photon excitation laser scanning microscopy of rabbit nasal septal cartilage following Nd:YAG-laser-mediated stress relaxation

    Science.gov (United States)

    Kim, Charlton C.; Wallace, Vincent P.; Coleno, Mariah L.; Dao, Xavier; Tromberg, Bruce J.; Wong, Brian J.

    2000-04-01

    Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within rabbit nasal septal cartilage tissue over a 12-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation. During laser irradiation surface temperature, stress relaxation, and diffuse reflectance, were measured dynamically. Each specimen received one or two sequential laser exposures. The cartilage reached a peak surface temperature of about 61 degrees C during irradiation. Cartilage denatured in 50 percent EtOH was used as a positive control. TPM was performed to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns, immediately following laser exposure, and also following 12 days in culture. Few differences in the pattern or intensity of fluorescence was observed between controls and irradiated specimens imaged immediately following exposure, regardless of the number of laser pulses. However, following twelve days in tissue culture, the irradiated specimens increase, whereas the native tissue diminishes, in intensity and distribution of fluorescence in the cytoplasm. In contrast, the positive control shows only extracellular matrices and empty lacuna, feature consistent with cell membrane lysis.

  7. Design, synthesis, and characterization of photoinitiators for two-photon polymerization

    Science.gov (United States)

    Whitby, Reece; MacMillan, Ryan; Janssens, Stefaan; Raymond, Sebastiampillai; Clarke, Dave; Kay, Andrew; Jin, Jianyong; Simpson, Cather M.

    2016-09-01

    A series of dipolar and quadrupolar two-photon absorption (2PA) photoinitiators (PIs) based around the well-known triphenylamine (TPA) core and tricyanofuran (TCF) acceptors have been prepared for use in two-photon polymerisation (TPP). The synthesised dipolar species are designated as 5 and 7, and the remaining quadrupolar species are 6, 8, 9 and 10. Large two-photon absorption cross-sections (δ2PA) ranging between 333 - 507 GM were measured at 780 nm using the z-scan technique. Fluorescence quantum yields (ΦF) were below 3% across the series when compared to Rhodamine 6G as a reference standard. Finally, TPP tests were conducted on PIs 7 and 8 to assess their ability to initiate the polymerisation of acrylate monomers using an 800 nm femtosecond Ti:Sapphire laser system.

  8. Two-Photon Absorption Properties of Mn-Doped ZnS Quantum Dots

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jia-Jin; ZHANG Gui-Lan; GUO Yang-Xue; WANG Xiao-Yan; CHEN Wen-Ju; ZHANG Xiao-Song; HUA Yu-Lin

    2006-01-01

    @@ We investigate the two-photon absorption and nonlinear refractive index properties of a quantum dot material based on ZnS nanocrystals doped with Mn isoelectronic impurities, using the Z-scan technique with 532nm picosecond laser pulses. The Mn-doped ZnS quantum dots have an average two-photon absorption cross section as high as 13600 Goeppert-Mayer units, which turn it into a very promising material for fluorescent label and imaging in biological samples. In addition, we also found that the two-photon absorption coeflicient initially increases and then decreases with increasing pulse irradiance, which demonstrates the presence of the higherorder nonlinearity under the strong excitation.

  9. Synchrotron radiation micro-X-ray fluorescence analysis: A tool to increase accuracy in microscopic analysis

    CERN Document Server

    Adams, F

    2003-01-01

    Microscopic X-ray fluorescence (XRF) analysis has potential for development as a certification method and as a calibration tool for other microanalytical techniques. The interaction of X-rays with matter is well understood and modelling studies show excellent agreement between experimental data and calculations using Monte Carlo simulation. The method can be used for a direct iterative calculation of concentrations using available high accuracy physical constants. Average accuracy is in the range of 3-5% for micron sized objects at concentration levels of less than 1 ppm with focused radiation from SR sources. The end-station ID18F of the ESRF is dedicated to accurate quantitative micro-XRF analysis including fast 2D scanning with collection of full X-ray spectra. Important aspects of the beamline are the precise monitoring of the intensity of the polarized, variable energy beam and the high reproducibility of the set-up measurement geometry, instrumental parameters and long-term stability.

  10. Two-photon excited photoconversion of cyanine-based dyes

    Science.gov (United States)

    Kwok, Sheldon J. J.; Choi, Myunghwan; Bhayana, Brijesh; Zhang, Xueli; Ran, Chongzhao; Yun, Seok-Hyun

    2016-03-01

    The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue.

  11. Conjugated polymers with pyrrole as the conjugated bridge: synthesis, characterization, and two-photon absorption properties.

    Science.gov (United States)

    Li, Qianqian; Zhong, Cheng; Huang, Jing; Huang, Zhenli; Pei, Zhiguo; Liu, Jun; Qin, Jingui; Li, Zhen

    2011-07-14

    The synthesis, one- and two-photon absorption (2PA) and emission properties of two novel pyrrole-based conjugated polymers (P1 and P2) are reported. They emitted strong yellow-green and orange fluorescence with fluorescent quantum yields (Φ) of 46 and 33%, respectively. Their maximal 2PA cross sections (δ) measured by the two-photon-induced fluorescence method using femtosecond laser pulses in THF were 2392 and 1938 GM per repeating unit, respectively, indicating that the 2PA chromophores consisting of the triphenylamine with nonplanar structure as the donor and electron-rich pyrrole as the conjugated bridge could be the effective repeating units to enhance the δ values.

  12. Near infrared two-photon excitation cross-sections of voltage-sensitive dyes.

    Science.gov (United States)

    Fisher, Jonathan A N; Salzberg, Brian M; Yodh, Arjun G

    2005-10-15

    Microscopy based on voltage-sensitive dyes has proven effective for revealing spatio-temporal patterns of neuronal activity in vivo and in vitro. Two-photon microscopy using voltage-sensitive dyes offers the possibility of wide-field visualization of membrane potential on sub-cellular length scales, hundreds of microns below the tissue surface. Very little information is available, however, about the utility of voltage-sensitive dyes for two-photon imaging purposes. Here we report on measurements of two-photon fluorescence excitation cross-sections for nine voltage-sensitive dyes in a solvent, octanol, intended to simulate the membrane environment. Ultrashort light pulses from a Ti:sapphire laser were used for excitation from 790 to 960 nm, and fluorescein dye was used as a calibration standard. Overall, dyes RH795, RH421, RH414, di-8-ANEPPS, and di-8-ANEPPDHQ had the largest two-photon excitation cross-sections ( approximately 15 x 10(-50)cm4 s photon(-1)) in this wavelength region and are therefore potentially useful for two-photon microscopy. Interestingly, di-8-ANEPPDHQ, a chimera constructed from the potentiometric dyes RH795 and di-8-ANEPPS, exhibited larger cross-sections than either of its constituents.

  13. A portable confocal hyperspectral microscope without any scan or tube lens and its application in fluorescence and Raman spectral imaging

    Science.gov (United States)

    Li, Jingwei; Cai, Fuhong; Dong, Yongjiang; Zhu, Zhenfeng; Sun, Xianhe; Zhang, Hequn; He, Sailing

    2017-06-01

    In this study, a portable confocal hyperspectral microscope is developed. In traditional confocal laser scanning microscopes, scan lens and tube lens are utilized to achieve a conjugate relationship between the galvanometer and the back focal plane of the objective, in order to achieve a better resolution. However, these lenses make it difficult to scale down the volume of the system. In our portable confocal hyperspectral microscope (PCHM), the objective is placed directly next to the galvomirror. Thus, scan lens and tube lens are not included in our system and the size of this system is greatly reduced. Furthermore, the resolution is also acceptable in many biomedical and food-safety applications. Through reducing the optical length of the system, the signal detection efficiency is enhanced. This is conducive to realizing both the fluorescence and Raman hyperspectral imaging. With a multimode fiber as a pinhole, an improved image contrast is also achieved. Fluorescent spectral images for HeLa cells/fingers and Raman spectral images of kumquat pericarp are present. The spectral resolution and spatial resolutions are about 0.4 nm and 2.19 μm, respectively. These results demonstrate that this portable hyperspectral microscope can be used in in-vivo fluorescence imaging and in situ Raman spectral imaging.

  14. Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source.

    Science.gov (United States)

    Yokoyama, Hiroyuki; Tsubokawa, Hiroshi; Guo, Hengchang; Shikata, Jun-ichi; Sato, Ki-ichi; Takashima, Keijiro; Kashiwagi, Kaori; Saito, Naoaki; Taniguchi, Hirokazu; Ito, Hiromasa

    2007-01-01

    We developed a novel scheme for two-photon fluorescence bioimaging. We generated supercontinuum (SC) light at wavelengths of 600 to 1200 nm with 774-nm light pulses from a compact turn-key semiconductor laser picosecond light pulse source that we developed. The supercontinuum light was sliced at around 1030- and 920-nm wavelengths and was amplified to kW-peak-power level using laboratory-made low-nonlinear-effects optical fiber amplifiers. We successfully demonstrated two-photon fluorescence bioimaging of mouse brain neurons containing green fluorescent protein (GFP).

  15. A new self-made digital slide scanner and microscope for imaging and quantification of fluorescent microspheres

    DEFF Research Database (Denmark)

    Henning, William; Bjerglund Andersen, Julie; Højgaard, Liselotte

    2015-01-01

    Objective: A low-cost microscope slide scanner was constructed for the purpose of digital imaging of newborn piglet brain tissue and to quantify fluorescent microspheres in tissue. Methods: Using a standard digital single-lens reflex (DSLR) camera, fluorescent imaging of newborn piglet brain tissue...... was performed. A computer algorithm available for download was created to detect fluorescent microspheres in the brain tissue slides and to calculate regional cerebral blood flow (rCBF). The precision of the algorithm was tested by comparing with manual counting of the fluorescent microspheres. Finally, bright...... with a resolution of 2.9 µm. The mean difference (SD) between manual and automatic counts was in absolute numbers 0.32 (1.5) microspheres ranging from -5 to 5 microspheres per slide. The relative total difference between automatic and manual counts was -3.1%. Conclusions: A slide scanner was constructed...

  16. Nonlinear fluorescence probe using photoinduced charge separation (Presentation Recording)

    Science.gov (United States)

    Mochizuki, Kentaro; Shi, Lanting; Mizukami, Shin; Yamanaka, Masahito; Tanabe, Mamoru; Gong, Wei-Tao; Palonpon, Almar F.; Kawano, Shogo; Kawata, Satoshi; Kikuchi, Kazuya; Fujita, Katsumasa

    2015-08-01

    Two-photon excitation microscopy (TPEM) provides spatial resolution beyond the optical diffraction limit using the nonlinear response of fluorescent molecules. One of the strong advantages of TPEM is that it can be performed using a laser-scanning microscope without a complicated excitation method or computational post-processing. However, TPEM has not been recognized as a super-resolution microscopy due to the use of near-infrared light as excitation source, which provides lower resolution than visible light. In our research, we aimed for the realization of nonlinear fluorescence response with visible light excitation to perform super-resolution imaging using a laser-scanning microscope. The nonlinear fluorescence response with visible light excitation is achieved by developing a probe which provides stepwise two-photon excitation through photoinduced charge separation. The probe named nitro-bisBODIPY consists of two fluorescent molecules (electron donor: D) and one electron acceptor (A), resulting to the structure of D-A-D. Excited by an incident photon, nitro-bisBODIPY generates a charge-separated pair between one of the fluorescent molecules and the acceptor. Fluorescence emission is obtained only when one more incident photon is used to excite the other fluorescent molecule of the probe in the charge-separated state. This stepwise two-photon excitation by nitro-bisBODIPY was confirmed by detection of the 2nd order nonlinear fluorescence response using a confocal microscope with 488 nm CW excitation. The physical model of the stepwise two-photon excitation was investigated by building the energy diagram of nitro-bisBODIPY. Finally, we obtained the improvement of spatial resolution in fluorescence imaging of HeLa cells using nitro-bisBODIPY.

  17. Mitigating thermal mechanical damage potential during two-photon dermal imaging.

    Science.gov (United States)

    Masters, Barry R; So, Peter T C; Buehler, Christof; Barry, Nicholas; Sutin, Jason D; Mantulin, William W; Gratton, Enrico

    2004-01-01

    Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.

  18. Efficient two-photon sensitized luminescence of europium (Ⅲ) complex based on hypersensitive transitions

    Institute of Scientific and Technical Information of China (English)

    Meng Shi; Hua Li; Mei Pan; Fufang Su; Lili Ma; Peigao Han; Hezhou Wang

    2011-01-01

    Red frequency-upconversion fluorescence emission is observed in europium(Ⅲ) complex with encapsulating polybenzimidazole tripodal ligands, pumped with 930- and 1070-nm picosecond laser pulses. The luminescence of transition 5D0 →7F2 (612 nm) is induced by two-photon absorption of hypersensitive transitions 7F0 →5D2 (465 nm) and 7F1 →5D1 (535 nm). Analysis results suggest that the two-photon excitation strength of these hypersensitive transitions is increased dramatically owing to the C3 symmetry of the coordination field.%@@ Red frequency-upconversion fluorescence emission is observed in europium(Ⅲ) complex with encapsulating polybenzimidazole tripodal ligands, pumped with 930- and 1070-nm picosecond laser pulses.The luminescence of transition 5D0 →7F2 (612 nm) is induced by two-photon absorption of hypersensitive transitions 7F0 →5D2 (465 nm) and 7F1 →5D1 (535 nm).Analysis results suggest that the two-photon excitation strength of these hypersensitive transitions is increased dramatically owing to the Ca symmetry of the coordination field.

  19. Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

    CERN Document Server

    Amor, Rumelo; Amos, William Bradshaw; McConnell, Gail

    2014-01-01

    Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report that the relative intensities in each plane of excitation depend on the Stokes shift of the fluorochrome. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ~90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.

  20. Development of a strain visualization system for microstructures using single fluorescent molecule tracking on a three-dimensional orientation microscope

    Science.gov (United States)

    Yoshida, Shintaro; Yoshiki, Keisuke; Namazu, Takahiro; Araki, Nozomu; Hashimoto, Mamoru; Kurihara, Makoto; Hashimoto, Nobuyuki; Inoue, Shozo

    2011-09-01

    We propose a technique that employs single fluorescent molecules for visualizing the distribution of strain induced in microstructures. We sprayed single-molecule tracers on microstructures by ultrasonic atomization and traced the position and orientation of the tracers by a single-molecule detection technique with a three-dimensional (3D) orientation microscope, which consists of a conventional fluorescent microscope and a polarization-mode converter. By using 3D spline interpolation, we visualized the surface geometry of a microelectromechanical (MEMS) device. We tracked the 3D position and orientation of tracers attached to a supporting beam of the MEMS mirror. The surface declination angles calculated from the orientation of the tracers were in agreement with the tilt angle obtained from the 3D position of the tracers.

  1. Two-photon autofluorescence spectroscopy of oral mucosa tissue

    Science.gov (United States)

    Edward, Kert; Shilagard, Tuya; Qiu, Suimin; Vargas, Gracie

    2011-03-01

    The survival rate for individuals diagnosed with oral cancer is correlated with the stage of detection. Thus the development of novel techniques for the earliest possible detection of malignancies is of critical importance. Single photon (1P) autofluorescence spectroscopy has proven to be a powerful diagnostic tool in this regard, but 2P (two photon) spectroscopy remains essentially unexplored. In this investigation, a spectroscopic system was incorporated into a custom-built 2P laser scanning microscope. Oral cancer was induced in the buccal pouch of Syrian Golden hamsters by tri-weekly topical application of 9,10-dimethyl-1,2-benzanthracene (DMBA).Three separated sites where investigated in each hamster at four excitation wavelengths from 780 nm to 890 nm. A Total of 8 hamsters were investigated (4 normal and 4 DMBA treated). All investigated sites were imaged via 2p imaging, marked for biopsy, processed for histology and H&E staining, and graded by a pathologist. The in vivo emission spectrum for normal, mild/high grade dysplasia and squamous cell carcinoma is presented. It is shown that the hamsters with various stages of dysplasia are characterized by spectral differences as a function of depth and excitation wavelength, compared to normal hamsters.

  2. Steady state anisotropy two-photon microscopy resolves multiple, spectrally similar fluorophores, enabling in vivo multilabel imaging.

    Science.gov (United States)

    Dubach, J Matthew; Vinegoni, Claudio; Weissleder, Ralph

    2014-08-01

    The use of spectrally distinguishable fluorescent dyes enables imaging of multiple targets. However, in two-photon microscopy, the number of fluorescent labels with distinct emission spectra that can be effectively excited and resolved is constrained by the confined tuning range of the excitation laser and the broad and overlapping nature of fluorophore two-photon absorption spectra. This limitation effectively reduces the number of available imaging channels. Here, we demonstrate that two-photon steady state anisotropy imaging (2PSSA) offers the capability to resolve otherwise unresolvable fluorescent tracers both in live cells and in mouse tumor models. This approach expands the number of biological targets that can be imaged simultaneously, increasing the total amount of information that can be obtained through imaging.

  3. Several Organic Salts with High Two-Photon Active

    Institute of Scientific and Technical Information of China (English)

    TIAN, Yu-Peng; JIANG, Min-Hua; WANG, He-Zhou; FANG, Qi

    2001-01-01

    Several organic salts with D-A molecular structure and different counterion have been prepared and experimentally investigated. The two-photon induced frequency-upconverted spectra and two-photon pumped lasing are measured for the organic salt solutions in various solvents. The results indicate that counterions have influence on their stability and lasing property.

  4. Two-photon absorption in arsenic sulfide glasses

    Science.gov (United States)

    Chunaev, D. S.; Snopatin, G. E.; Plotnichenko, V. G.; Karasik, A. Ya.

    2016-10-01

    The two-photon absorption coefficient of 1047-{\\text{nm}} light in {\\text{As}}35{\\text{S}}65 chalcogenide glass has been measured. CW probe radiation has been used to observe the linear absorption in glass induced by two-photon excitation. The induced absorption lifetime was found to be ∼ 2 {\\text{ms}}.

  5. Direct two-photon excitation of isomeric transition in thorium-229 nucleus

    CERN Document Server

    Romanenko, V I; Yatsenko, L P; Romanenko, A V; Litvinov, A N; Kazakov, G A

    2012-01-01

    A possibility of the two-photon excitation of an isomeric state in a nucleus of thorium-229 has been discussed. The fluorescence intensity of the excitation is demonstrated to be identical for the irradiation of nuclei with either monochromatic light or polychromatic radiation consisting of a sequence of short light pulses of the same intensity. The two-photon excitation of Th^{3+} ion in an electromagnetic trap with a focused laser beam with a wavelength of about 320 nm and power of 100 mW can lead to the absorption saturation, at which the fluorescence emission with the frequency of the transition in a nucleus is maximal. In crystals doped with Th^{4+} to a concentration of about 10^{18} cm^{-3} and irradiated with a laser radiation 10 W in power, the emission of several photons per second with a wavelength of about 160 nm becomes possible.

  6. The development of efficient two-photon singlet oxygen sensitizers

    DEFF Research Database (Denmark)

    Nielsen, Christian Benedikt

    The development of efficient two-photon singlet oxygen sensitizers is addressed focusing on organic synthesis. Photophysical measurements were carried out on new lipophilic molecules, where two-photon absorption cross sections and singlet oxygen quantumyields were measured. Design principles...... for making efficient two-photon singlet oxygen sensitizers were then constructed from these results. Charge-transfer in the excited state of the prepared molecules was shown to play a pivotal role in the generationof singlet oxygen. This was established through studies of substituent effects on both...... the singlet oxygen yield and the two-photon absorption cross section, where it was revealed that a careful balancing of the amount of charge transfer present in theexcited state of the sensitizer is necessary to obtain both a high singlet oxygen quantum yield and a high two-photon cross section. An increasing...

  7. Centimeter-deep tissue fluorescence microscopic imaging with high signal-to-noise ratio and picomole sensitivity

    CERN Document Server

    Cheng, Bingbing; Wei, Ming-Yuan; Pei, Yanbo; DSouza, Francis; Nguyen, Kytai T; Hong, Yi; Tang, Liping; Yuan, Baohong

    2015-01-01

    Fluorescence microscopic imaging in centimeter-deep tissue has been highly sought-after for many years because much interesting in vivo micro-information, such as microcirculation, tumor angiogenesis, and metastasis, may deeply locate in tissue. In this study, for the first time this goal has been achieved in 3-centimeter deep tissue with high signal-to-noise ratio (SNR) and picomole sensitivity under radiation safety thresholds. These results are demonstrated not only in tissue-mimic phantoms but also in actual tissues, such as porcine muscle, ex vivo mouse liver, ex vivo spleen, and in vivo mouse tissue. These results are achieved based on three unique technologies: excellent near infrared ultrasound-switchable fluorescence (USF) contrast agents, a sensitive USF imaging system, and an effective correlation method. Multiplex USF fluorescence imaging is also achieved. It is useful to simultaneously image multiple targets and observe their interactions. This work opens the door for future studies of centimeter...

  8. Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes.

    Science.gov (United States)

    Kress, Matthias; Meier, Thomas; Steiner, Rudolf; Dolp, Frank; Erdmann, Rainer; Ortmann, Uwe; Rück, Angelika

    2003-01-01

    This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging

  9. Two-photon absorption in mesoionic compounds pumped at the visible and at the infrared

    CERN Document Server

    Rakov, N; Da Rocha, G B; Simas, A M; Athayde-Filho, P A F; Miller, J

    2000-01-01

    Intensity dependent transmission and laser-induced fluorescence were observed in liquid solutions of mesoionic compounds (MIC) pumped with nanosecond lasers operating at 1064, 604, and 570 nm. The results indicate that two-photon absorption (TPA) is the dominant mechanism which causes the observed behavior. The TPA cross-sections measured have values from 0.33*10/sup -20/ cm/sup 4//GW to 0.43*10/sup -18/ cm /sup 4//GW. (20 refs).

  10. Functionalized 3D Architected Materials via Thiol-Michael Addition and Two-Photon Lithography.

    Science.gov (United States)

    Yee, Daryl W; Schulz, Michael D; Grubbs, Robert H; Greer, Julia R

    2017-04-01

    Fabrication of functionalized 3D architected materials is achieved by a facile method using functionalized acrylates synthesized via thiol-Michael addition, which are then polymerized using two-photon lithography. A wide variety of functional groups can be attached, from Boc-protected amines to fluoroalkanes. Modification of surface wetting properties and conjugation with fluorescent tags are demonstrated to highlight the potential applications of this technique. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Field Observations of Bioaerosols: What We've Learned from Fluorescence, Genetic, and Microscopic Techniques (Invited)

    Science.gov (United States)

    Huffman, J. A.; Fröhlich-Nowoisky, J.; Després, V. R.; Elbert, W.; Sinha, B.; Andreae, M. O.; Pöschl, U.

    2009-12-01

    Biogenic aerosols are ubiquitous in the Earth’s atmosphere, influencing atmospheric chemistry and physics, the biosphere, climate, and public health. They play an important role in the spread of biological organisms, and they can cause or enhance human, animal, and plant diseases. Moreover, they can initiate the formation of clouds and precipitation as cloud condensation and ice nuclei (CCN, IN). Primary biogenic aerosol particles (PBAP) such as pollen, fungal spores, and bacteria are emitted directly from the biosphere to the atmosphere. Microscopic investigations have shown that PBAP account for up to ~30% of fine and up to ~70% of coarse particulate matter in rural and rain forest air, and the estimates of PBA emissions range from ~60 Tg a-1 of fine particles up to ~1000 Tg a-1 of total particulate matter. Fungal spores account for a large proportion of PBA with typical number and mass concentrations of ~104 m-3 and ~1 μg m-3 in continental boundary layer air and estimated global emissions of the order of ~50 Tg a-1 and 200 m-2 s-1, respectively [1]. The actual abundance, variability and diversity of PBAP are still poorly understood and quantified, however. By measuring fluorescence at excitation and emission wavelengths specific to viable cells, online techniques with time resolution of minutes are able to detect fluorescent biological aerosol particles (FBAP), which represent a lower limit for the actual abundance of coarse (> 1 μm) PBAP [2]. Continuous sampling (1 - 4 months) was performed at various locations including pristine rain forest, rural and polluted urban sites. Each study exhibited a similar average particle number distribution dominated by a peak at ~3 μm, with coarse FBAP concentrations of the order of ~5x104 m-3 and ~1 μg m-3. Recent advances in the DNA analysis and molecular genetic characterization of aerosol filter samples yield new information about the sources and composition of PBA and provide new insight into regional and global

  12. Imaging marine virus CroV and its host Cafeteria roenbergensis with two-photon microscopy

    Science.gov (United States)

    Cao, Bin; Chakraborty, Sayan; Sun, Wenqing; Aghvami, Seyedmohammadali; Fischer, Matthias G.; Qian, Wei; Xiao, Chuan; Li, Chunqiang

    2014-02-01

    We use two-photon microscopy to monitor the infection process of marine zooplankton, Cafeteria roenbergensis (C.roenbergensis), by Cafeteria roenbergensis virus (CroV), a giant DNA virus named after its host. Here, we image C.roenbergensis in culture by two-photon excited NADH autofluorescence at video-rate (30 frame/s), and the movement of C.roenbergensis is recorded in live videos. Moreover, CroV is stained with DNA dye SYBR gold and recorded simultaneously with this two-photon microscope. We observed the initial infection moment with this method. The result demonstrates the potential use of two-photon microscopy to investigate the fast dynamic interaction between C.roenbergensis with virus CroV. After catching this initial moment, we will freeze the sample in liquid nitrogen for cryo-electron microscopy (EM) study to resolve the virus-host interaction at molecular level. The long-term goal is to study similar fast moving pathogen-host interaction process which could lead to important medical applications.

  13. Phosphorescent probes for two-photon microscopy of oxygen (Conference Presentation)

    Science.gov (United States)

    Vinogradov, Sergei A.; Esipova, Tatiana V.

    2016-03-01

    The ability to quantify oxygen in vivo in 3D with high spatial and temporal resolution is much needed in many areas of biological research. Our laboratory has been developing the phosphorescence quenching technique for biological oximetry - an optical method that possesses intrinsic microscopic capability. In the past we have developed dendritically protected oxygen probes for quantitative imaging of oxygen in tissue. More recently we expanded our design on special two-photon enhanced phosphorescent probes. These molecules brought about first demonstrations of the two-photon phosphorescence lifetime microscopy (2PLM) of oxygen in vivo, providing new information for neouroscience and stem cell biology. However, current two-photon oxygen probes suffer from a number of limitations, such as sub-optimal brightness and high cost of synthesis, which dramatically reduce imaging performance and limit usability of the method. In this paper we discuss principles of 2PLM and address the interplay between the probe chemistry, photophysics and spatial and temporal imaging resolution. We then present a new approach to brightly phosphorescent chromophores with internally enhanced two-photon absorption cross-sections, which pave a way to a new generation of 2PLM probes.

  14. Synthesis of Two-Photon Materials and Two-Photon Liquid Crystals

    Science.gov (United States)

    Subramaniam, Girija

    2001-01-01

    The duration of the grant was interrupted by two major accidents that the PI met with-- an auto accident in Pasadena, CA during her second summer at JPL which took almost eight months for recovery and a second accident during Fall 2000 that left her in crutches for the entire semester. Further, the time released agreed by the University was not given in a timely fashion. The candidate has been given post-grant expire time off. In spite of all these problems, the PI synthesized a number of new two-photon materials and studied the structure-activity correlation to arrive at the best-optimized structure. The PI's design proved to be one of the best in the sense that these materials has a hitherto unreported two-photon absorption cross section. Many materials based on PI's design was later made by the NASA colleague. This is Phase 1. Phase II of this grant is to orate liquid crystalline nature into this potentially useful materials and is currently in progress. Recent observations of nano- and pico-second response time of homeotropically aligned liquid crystals suggest their inherent potentials to act as laser hardening materials, i.e., as protective devices against short laser pulses. The objective of the current project is to exploit this potential by the synthesis of liquid crystals with high optical nonlinearity and optimizing their performance. The PI is trying structural variations to bring in liquid crystalline nature without losing the high two-photon cross section. Both Phase I and Phase II led to many invited presentations and publications in reputed journals like 'Science' and 'Molecular Crystals'. The list of presentations and reprints are enclosed. Another important and satisfying outcome of this grant is the opportunity that this grant offered to the budding undergraduate scientists to get involved in a visible research of international importance. All the students had a chance to learn a lot during research, had the opportunity to present their work at

  15. Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

    NARCIS (Netherlands)

    Zhao, Q.; Schelen, B.; Schouten, R., et al.

    2012-01-01

    We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device des

  16. Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy (Conference Presentation)

    Science.gov (United States)

    Kwok, Sheldon J. J.; Kuznetsov, Ivan A.; Kim, Moonseok; Choi, Myunghwan; Scarcelli, Giuliano; Yun, Seok-Hyun

    2017-02-01

    Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively. Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering.

  17. Two-photon processes in highly charged ions

    Energy Technology Data Exchange (ETDEWEB)

    Jahrsetz, Thorsten

    2015-03-05

    Two-photon processes are atomic processes in which an atom interacts simultaneously with two photons. Such processes describe a wide range of phenomena, such as two-photon decay and elastic or inelastic scattering of photons. In recent years two-photon processes involving highly charged heavy ions have become an active area of research. Such studies do not only consider the total transition or scattering rates but also their angular and polarization dependence. To support such examinations in this thesis I present a theoretical framework to describe these properties in all two-photon processes with bound initial and final states and involving heavy H-like or He-like ions. I demonstrate how this framework can be used in some detailed studies of different two-photon processes. Specifically a detailed analysis of two-photon decay of H-like and He-like ions in strong external electromagnetic fields shows the importance of considering the effect of such fields for the physics of such systems. Furthermore I studied the elastic Rayleigh as well as inelastic Raman scattering by heavy H-like ions. I found a number of previously unobserved phenomena in the angular and polarization dependence of the scattering cross-sections that do not only allow to study interesting details of the electronic structure of the ion but might also be useful for the measurement of weak physical effects in such systems.

  18. Two-photon interference of temporally separated photons

    Science.gov (United States)

    Kim, Heonoh; Lee, Sang Min; Moon, Han Seb

    2016-10-01

    We present experimental demonstrations of two-photon interference involving temporally separated photons within two types of interferometers: a Mach-Zehnder interferometer and a polarization-based Michelson interferometer. The two-photon states are probabilistically prepared in a symmetrically superposed state within the two interferometer arms by introducing a large time delay between two input photons; this state is composed of two temporally separated photons, which are in two different or the same spatial modes. We then observe two-photon interference fringes involving both the Hong-Ou-Mandel interference effect and the interference of path-entangled two-photon states simultaneously in a single interferometric setup. The observed two-photon interference fringes provide simultaneous observation of the interferometric properties of the single-photon and two-photon wavepackets. The observations can also facilitate a more comprehensive understanding of the origins of the interference phenomena arising from spatially bunched/anti-bunched two-photon states comprised of two temporally separated photons within the interferometer arms.

  19. Time-Lapse Observation of Electrolysis of Copper Sulfate with a Full-Field X-ray Fluorescence Imaging Microscope

    Science.gov (United States)

    Ohigashi, Takuji; Aota, Tatsuya; Watanabe, Norio; Takano, Hidekazu; Yokosuka, Hiroki; Aoki, Sadao

    2008-06-01

    The time-lapse observation of the electrodeposition of copper in copper sulfate solution was performed by imaging X-ray fluorescence from the copper deposition. The X-ray fluorescence was directly imaged with a full-field Wolter mirror microscope, which was constructed at the Photon Factory. Controlling the electric current in the solution from 0 to 71.7 µA, the deposition of copper on a Pt cathode was directly observed by imaging its X-ray fluorescence. One exposure time for obtaining an X-ray fluorescence image was 80 s. Then, it was 17 min later from the beginning of the electrolysis when the X-ray fluorescence image of the electrodeposition is observed for the first time. At this exposure time, the detection limit of the mass of copper was estimated to be 0.60 pg/image, which was calculated using test samples of 1.00×10-3-1.00 mol/l copper sulfate solutions.

  20. OPTIMIZATION OF LAMBLIASIS MICROSCOPIC DIAGNOSTICS BY THE METHOD OF POLARIZED FLUORESCENCE FOR PATIENTS WITH ROSACEA AND URTICARIAL

    Directory of Open Access Journals (Sweden)

    Maryana Kovalchuk

    2013-07-01

    Full Text Available Introduction: There is little information about diagnosis of concurrent lambliasis in patients with rosacea and urticaria. We used method of polarized fluorescence to diagnose liambliasis, taking into account belonging of macromolecular structures of unicellular parasites Giardia lamblia to the optically active substances with the properties of liquid crystals. Material and Methods: Lambliasis was diagnosed on the basis of feces parasitological research and duodenal contents by methods of light and optic microscopy and polarized fluorescence in 105 patients with rosacea and urticaria. Research results were processed by the method of variation statistics in the Statgraf program by using Student’s criterion. Results: Search results of lamblia in patients with rosacea and urticaria depended on the conditions of its holding, patients’ preparation and from the previously received basic therapy if it consisted absorbents. Due to the fact that the fluorescence polarization as a physical method does not require the use of any generally toxic, dye- fluorochromes, qualitative cyto fluorescent analysis of lamblia in greeting microdrugs enables to distinguish vegetative forms of cysts. Conclussions: Polarized fluorescence method allows optimize the microscopic diagnosis of lambliasis, increasing its sensitivity. Previous preparation for the laboratory examination of Giardia lamblia is needed for the best exposure of vermin for patients with rosacea and urticaria.

  1. The use of fluorescence enhancement to improve the microscopic diagnosis of falciparum malaria

    Directory of Open Access Journals (Sweden)

    Liu Paul

    2007-07-01

    Full Text Available Abstract Background Giemsa staining of thick blood smears remains the "gold standard" for detecting malaria. However, this method is not very good for diagnosing low-level infections. A method for the simultaneous staining of Plasmodium-parasitized culture and blood smears for both bright field and fluorescence was developed and its ability to improve detection efficiency tested. Methods A total of 22 nucleic acid-specific fluorescent dyes were tested for their ability to provide easily observable staining of Plasmodium falciparum-parasitized red blood cells following Giemsa staining. Results Of the 14 dyes that demonstrated intense fluorescence staining, only SYBR Green 1, YOYO-1 and ethidum homodimer-2 could be detected using fluorescent microscopy, when cells were first stained with Giemsa. Giemsa staining was not effective when applied after the fluorescent dyes. SYBR Green 1 provided the best staining in the presence of Giemsa, as a very high percentage of the parasitized cells were simultaneously stained. When blood films were screened using fluorescence microscopy the parasites were more readily detectable due to the sharp contrast between the dark background and the specific, bright fluorescence produced by the parasites. Conclusion The dual staining method reported here allows fluorescence staining, which enhances the reader's ability to detect parasites under low parasitaemia conditions, coupled with the ability to examine the same cell under bright field conditions to detect the characteristic morphology of Plasmodium species that is observed with Giemsa staining.

  2. Quantum homodyne tomography of a two-photon Fock state

    CERN Document Server

    Ourjoumtsev, A; Grangier, P; Ourjoumtsev, Alexei; Tualle-Brouri, Rosa; Grangier, Philippe

    2006-01-01

    We present a continuous-variable experimental analysis of a two-photon Fock state of free-propagating light. This state is obtained from a pulsed non-degenerate parametric amplifier, which produces two intensity-correlated twin beams. Counting two photons in one beam projects the other beam in the desired two-photon Fock state, which is analyzed by using a pulsed homodyne detection. The Wigner function of the measured state is clearly negative. We developed a detailed analytic model which allows a fast and efficient analysis of the experimental results.

  3. Quantum homodyne tomography of a two-photon Fock state.

    Science.gov (United States)

    Ourjoumtsev, Alexei; Tualle-Brouri, Rosa; Grangier, Philippe

    2006-06-02

    We present a continuous-variable experimental analysis of a two-photon Fock state of free-propagating light. This state is obtained from a pulsed nondegenerate parametric amplifier, which produces two intensity-correlated twin beams. Counting two photons in one beam projects the other beam in the desired two-photon Fock state, which is analyzed by using a pulsed homodyne detection. The Wigner function of the measured state is clearly negative. We developed a detailed analytic model which allows a fast and efficient analysis of the experimental results.

  4. Scattering of two photons from two distant qubits: exact solution

    Energy Technology Data Exchange (ETDEWEB)

    Laakso, Matti; Pletyukhov, Mikhail [Institute for Theory of Statistical Physics, RWTH Aachen, 52056 Aachen (Germany)

    2015-07-01

    We consider the inelastic scattering of two photons from two qubits separated by an arbitrary distance and coupled to a one-dimensional transmission line. We present an exact, analytical solution to the problem, and use it to explore a particular configuration of qubits which is transparent to single-photon scattering, thus highlighting non-Markovian effects of inelastic two-photon scattering: Strong two-photon interference and momentum dependent photon (anti)bunching. This latter effect can be seen as an inelastic generalization of the Hong-Ou-Mandel effect.

  5. Acute two-photon imaging of the neurovascular unit in the cortex of active mice

    Science.gov (United States)

    Tran, Cam Ha T.; Gordon, Grant R.

    2015-01-01

    In vivo two-photon scanning fluorescence imaging is a powerful technique to observe physiological processes from the millimeter to the micron scale in the intact animal. In neuroscience research, a common approach is to install an acute cranial window and head bar to explore neocortical function under anesthesia before inflammation peaks from the surgery. However, there are few detailed acute protocols for head-restrained and fully awake animal imaging of the neurovascular unit during activity. This is because acutely performed awake experiments are typically untenable when the animal is naïve to the imaging apparatus. Here we detail a method that achieves acute, deep-tissue two-photon imaging of neocortical astrocytes and microvasculature in behaving mice. A week prior to experimentation, implantation of the head bar alone allows mice to train for head-immobilization on an easy-to-learn air-supported ball treadmill. Following just two brief familiarization sessions to the treadmill on separate days, an acute cranial window can subsequently be installed for immediate imaging. We demonstrate how running and whisking data can be captured simultaneously with two-photon fluorescence signals with acceptable movement artifacts during active motion. We also show possible applications of this technique by (1) monitoring dynamic changes to microvascular diameter and red blood cells in response to vibrissa sensory stimulation, (2) examining responses of the cerebral microcirculation to the systemic delivery of pharmacological agents using a tail artery cannula during awake imaging, and (3) measuring Ca2+ signals from synthetic and genetically encoded Ca2+ indicators in astrocytes. This method will facilitate acute two-photon fluorescence imaging in awake, active mice and help link cellular events within the neurovascular unit to behavior. PMID:25698926

  6. Acute two-photon imaging of the neurovascular unit in the cortex of active mice

    Directory of Open Access Journals (Sweden)

    Cam Ha Thai Tran

    2015-02-01

    Full Text Available In vivo two-photon scanning fluorescence imaging is a powerful technique to observe physiological processes from the millimeter to the micron scale in the intact animal. In neuroscience research, a common approach is to install an acute cranial window and head bar to explore neocortical function under anesthesia before inflammation peaks from the surgery. However, there are few detailed acute protocols for head-restrained and fully awake animal imaging of the neurovascular unit during activity. This is because acutely performed awake experiments are typically untenable when the animal is naïve to the imaging apparatus. Here we detail a protocol that achieves acute, deep-tissue two-photon imaging of neocortical astrocytes and microvasculature in behaving mice. A week prior to experimentation, implantation of the head bar alone allows mice to train for head-immobilization on an easy-to-learn air-supported ball treadmill. Following just two brief familiarization sessions to the treadmill on separate days, an acute cranial window can subsequently be installed for immediate imaging. We demonstrate how running and whisking data can be captured simultaneously with two-photon fluorescence signals with acceptable movement artifacts during active motion. We also show possible applications of this technique by 1 monitoring dynamic changes to microvascular diameter and red blood cells movements in response to vibrissa sensory stimulation, 2 examining responses of the cerebral microcirculation to the systemic delivery of pharmacological agents using a tail artery cannula during awake imaging, and 3 measuring Ca2+ signals from synthetic and genetically encoded Ca2+ indicators in astrocytes. This method will facilitate acute two-photon fluorescence imaging in awake, active mice and help link cellular events within the neurovascular unit to behaviour.

  7. A femtosecond Raman generator for long wavelength two-photon and third harmonic generation imaging

    Science.gov (United States)

    Trägârdh, J.; Schniete, J.; Parsons, M.; McConnell, G.

    2016-12-01

    We demonstrate a femtosecond single pass Raman generator based on an YVO4 crystal pumped by a high energy fiber laser at a wavelength of 1064 nm and a repetition rate of 1 MHz. The Raman generator shifts the pump wavelength to 1175 nm, in a broadband spectrum, making it suitable for multi-photon microscopy. We use the Raman generator for third harmonic generation imaging of live plant specimens as well as for two-photon fluorescence imaging of red fluorescent protein expressing HeLa cells. We demonstrate that the photo-damage to a live specimen is low.

  8. Second harmonic imaging of plants tissues and cell implosion using two-photon process in ZnO nanoparticles.

    Science.gov (United States)

    Urban, Ben E; Neogi, Purnima B; Butler, Sween J; Fujita, Yasuhisa; Neogi, Arup

    2012-03-01

    The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto-fluorescence above 750 nm and minimal auto-fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near-infrared femtosecond laser source ranging from 750-980 nm. For laser energies exceeding the two-photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two-photon emission becomes the dominant signal. The heat generated due to two-photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710-750 nm. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. NLO Electroweak Corrections to Higgs Decay to Two Photons

    OpenAIRE

    Actis, Stefano

    2009-01-01

    The recent calculation of the next-to-leading order electroweak corrections to the decay of the Standard Model Higgs boson to two photons in the framework of the complex-mass scheme is briefly summarized.

  10. Standard Model Higgs decay for two Photons in CMS

    CERN Multimedia

    Daniel Denegri

    2000-01-01

    Simulated two-photon mass distribution for SM Higgs and expected background in the CMS PbW04 crystal calorimeter for an integrated luminosity of 10 . 5 pb-1, with detailed simulation of calorimeter response.

  11. Two-photon pumped lead halide perovskite nanowire lasers

    CERN Document Server

    Gu, Zhiyuan; Sun, Wenzhao; Li, Jinakai; Liu, Shuai; Song, Qinghai; Xiao, Shumin

    2015-01-01

    Solution-processed lead halide perovskites have shown very bright future in both solar cells and microlasers. Very recently, the nonlinearity of perovskites started to attract considerable research attention. Second harmonic generation and two-photon absorption have been successfully demonstrated. However, the nonlinearity based perovskite devices such as micro- & nano- lasers are still absent. Here we demonstrate the two-photon pumped nanolasers from perovskite nanowires. The CH3NH3PbBr3 perovskite nanowires were synthesized with one-step solution self-assembly method and dispersed on glass substrate. Under the optical excitation at 800 nm, two-photon pumped lasing actions with periodic peaks have been successfully observed at around 546 nm. The obtained quality (Q) factors of two-photon pumped nanolasers are around 960, and the corresponding thresholds are about 674?J=cm2. Both the Q factors and thresholds are comparable to conventional whispering gallery modes in two-dimensional polygon microplates. Ou...

  12. Synthesis of a Series of Novel Organic Compounds with Two-photon Absorption and Two-photon pumped Lasing

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A series of novel organic compounds named as CSPI, DPASPI, PSPI DEASPI and HEASPI respectively, with large two-photon absorption has been synthesized and their structures have been determined by 1HNMR and elemental analysis. The highest two-photon pumped (TPP) output /input efficiency is as high as 13.4% for PSPI in DMF with d0 = 0.03 mol/L and the effective two-photon absorption cross section is 8.8′10-48 cm4×s/photon for DPASPI in DMF with d0= 0.05mol/L.

  13. Mass distribution for the two-photon channel

    CERN Multimedia

    ATLAS, collaboration

    2012-01-01

    Mass distribution for the two-photon channel. The strongest evidence for this new particle comes from analysis of events containing two photons. The smooth dotted line traces the measured background from known processes. The solid line traces a statistical fit to the signal plus background. The new particle appears as the excess around 126.5 GeV. The full analysis concludes that the probability of such a peak is three chances in a million.

  14. Two-photon absorption and frequency-upconversion properties of a new organic dye HMASPS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two-photon absorption (TPA) and frequency- upconversion properties of a new upconversion laser dye trans-4-[p-(N-hydroxyethyl-N-methyl-amino)styryl]-N-meth- ylpyridinium toluene-p-sulfonate (abbreviated to HMASPS) were reported in this note. The linear absorption, TPA, single-photon induced fluorescence, TPA induced fluorescence and TPA induced upconverted lasing spectra of HMASPS solution in dimethyl formamide (abbreviated to DMF) were measured at room temperature. The red shift for the central wavelength of TPA induced fluorescence peak, which was compared with that of the single-photon induced fluorescen-ce peak, and the blue shift for that of TPA induced upcon-verted lasing compared with that of TPA induced fluores-cence, were explained by using re-absorption effect. TPA peak was at 930 nm. There is an 11 nm blue shift for two-photon energy of TPA peak compared with the linear ab-sorption peak. The molecular TPA cross-section at 1064 nm was measured to be 6.0′10-48 cm4 ·s/photon by using the open aperture Z-scanning system. The highest upconversion efficiency was measured to be 8.4% at 1064 nm.

  15. Evaluation of transdermal delivery of nanoemulsions in ex vivo porcine skin using two-photon microscopy and confocal laser-scanning microscopy

    Science.gov (United States)

    Choi, Sanghoon; Kim, Jin Woong; Lee, Yong Joong; Delmas, Thomas; Kim, Changhwan; Park, Soyeun; Lee, Ho

    2014-10-01

    This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.

  16. Automatic choroid cells segmentation and counting based on approximate convexity and concavity of chain code in fluorescence microscopic image

    Science.gov (United States)

    Lu, Weihua; Chen, Xinjian; Zhu, Weifang; Yang, Lei; Cao, Zhaoyuan; Chen, Haoyu

    2015-03-01

    In this paper, we proposed a method based on the Freeman chain code to segment and count rhesus choroid-retinal vascular endothelial cells (RF/6A) automatically for fluorescence microscopy images. The proposed method consists of four main steps. First, a threshold filter and morphological transform were applied to reduce the noise. Second, the boundary information was used to generate the Freeman chain codes. Third, the concave points were found based on the relationship between the difference of the chain code and the curvature. Finally, cells segmentation and counting were completed based on the characteristics of the number of the concave points, the area and shape of the cells. The proposed method was tested on 100 fluorescence microscopic cell images, and the average true positive rate (TPR) is 98.13% and the average false positive rate (FPR) is 4.47%, respectively. The preliminary results showed the feasibility and efficiency of the proposed method.

  17. Characterization of heterogeneous SiO{sub 2} materials by scanning electron microscope and micro fluorescence XAS techniques

    Energy Technology Data Exchange (ETDEWEB)

    Khouchaf, L. [Centre de Recherche de l' Ecole des Mines deDouai, 941, rue Charles Bourseul, BP. 10838, 59508 Douai (France)]. E-mail: khouchaf@ensm-douai.fr; Boinski, F. [Centre de Recherche de l' Ecole des Mines deDouai, 941, rue Charles Bourseul, BP. 10838, 59508 Douai (France); Tuilier, M.H. [GMP Equipe de recherche: MMPF, Universite de Haute-Alsace, 61 rue Albert Camus, F-68093, Mulhouse Cedex (France); Flank, A.M. [SOLEIL and Swiss Light Source SLS CH-5232 Villigen PSI (Switzerland)

    2006-11-15

    Micro X-ray absorption near edge structure XANES and micro fluorescence experiments have been carried out using X-ray microbeam from synchrotron radiation source with high brightness to investigate the local structural evolutions of heterogeneous and natural SiO{sub 2} submitted to alkali-silica reaction ASR process. Compared to elemental maps obtained by Environmental Scanning Electron Microscope ESEM, micro fluorescence X maps showed the diffusion of potassium cations inside the grains with higher accuracy. Si K-edge spectra show the disorder induced by the dissolution of the grain from the outside to the inside. Potassium K-edge spectra do not show significant changes around K cations. The breaking of Si-O-Si bonds and the disorder of the (SiO{sub 4}) {sub n} network may be affected to potassium cations.

  18. Comparison of calcium imaging in dorsal root ganglion neurons by using laser scanning confocal and two-photon microscopy

    Science.gov (United States)

    Huang, Yimei; Yang, Hongqin; Chen, Jiangxu; Shen, Xiuqiu; Zheng, Liqin; Wang, Yuhua; Xie, Shusen

    2012-03-01

    As one of the most important second messengers, calcium in nerve cells plays a critical role in neuronal processes, including excitability, neurotransmitter release, synaptic plasticity. Modulation of the calcium concentration is an important means of regulating diverse neuronal functions. To evaluate the role of calcium, quantitative measurement of cytosolic free calcium concentrations is necessary. There are several optical techniques that are available for measurement of calcium in live cells. Laser scanning confocal microscopy and two-photon microscopy are two prevalent techniques for their advantage in spatial resolution. In this paper, calcium in dorsal root ganglion neurons was imaged by laser scanning confocal microscopy and two-photon microscopy with Fluo-3, a calcium specific fluorescence probe. Both of spatial resolution and photobleaching, two common limitations of optical image modality, were compared between laser scanning confocal microscopy and two-photon microscopy, respectively. Three dimension images showed that laser scanning confocal microscopy and two-photon microscopy had not only similar lateral resolution but also parallel vertical resolution. However, Laser scanning confocal microscopy had an advantage over the two-photon microcopy in photobleaching. These results indicated that laser scanning confocal microscopy was more suitable than two-photon microscopy to be applied in imaging calcium in dorsal root ganglion neurons with Fluo-3.

  19. Laser scanning fluorescence microscopic measurement of the movement of cleaving egg surface of Rana Amurensis

    Institute of Scientific and Technical Information of China (English)

    GUGUOYAN; ChengtangXu; 等

    1995-01-01

    By laser scanning fluorescence microscopy for quantitative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate during cleavage furrow extending forward,it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape,indicating dark stripe appeared in that place.Then the fluorescence intensity increased at the place where the bottom of ∨ shape had located,and the scanning curve turned to ∧ shape,indicating single stripe was formed.While enhanced fluorescence appeared on the borders of ∧ shape,an M shape curve was found,showing double stripe occurred.During the distance between two borders of M shape incresing from 50μm to 100μm,a fluorescence peak came to sight in the middle of the M shape,which being the cleavge furrow bottom.The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane.At that time the curve became W shape.By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage,showing the stretching of the egg surface being increased.With our[31,33]and others[32] reports that polylysine could induce the appearance of nascent membrane and phytohemagglutinins could decrease or prevent the appearance of nascent membrane,we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stresslay to the outsides of cleavage furrow.

  20. Spectroscopic Study of ThCl+ by Two-Photon Ionization

    Science.gov (United States)

    Bartlett, Joshua; VanGundy, Robert A.; Heaven, Michael; Peterson, Kirk

    2016-06-01

    Despite the irreplaceable role experimental data plays for evaluating the performance of computational predictions, diatomic actinide species have not received much spectroscopic attention. As an early actinide element, thorium-containing species are ideal candidates for these types of studies. The electronic structure is expected to be relatively simple compared to later actinides, and therefore allows straightforward assessment of calculations. Here, we have studied ThCl+ for the first time via resonant two-photon ionization of jet-cooled ThCl produced by laser ablation of the metal reacted with dilute Cl2. Laser-induced Fluorescence (LIF) spectra have been recorded for the neutral molecule from 16000 - 23500 cm-1 in search of a suitable intermediate state for subsequent two-photon ionization experiments. Monochromator dispersion of the fluorescence has recovered the ground state vibration and anharmonic constants of ThCl. Resonant Two-Photon Ionization (R2PI) within a time-of-flight mass spectrometer was used to confirm ThCl production, and Pulsed Field Ionization Zero Kinetic Energy photoelectron spectroscopy (PFI-ZEKE) has been performed to identify the ionization energy as well as several of the low-lying states of the ThCl+ molecule. These constants have been predicted at the CASPT2 and CCSD(T) levels of theory, and a discussion of the calculations' performance will be presented alongside the recorded spectra.

  1. Calculation of the spatial resolution in two-photon absorption spectroscopy applied to plasma diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Lechuga, M. [Departamento de Física Teórica, Atómica y Óptica, Universidad de Valladolid, 47011-Valladolid (Spain); Laser Processing Group, Instituto de Óptica “Daza de Valdés,” CSIC, 28006-Madrid (Spain); Fuentes, L. M. [Departamento de Física Aplicada, Universidad de Valladolid, 47011-Valladolid (Spain); Grützmacher, K.; Pérez, C., E-mail: concha@opt.uva.es; Rosa, M. I. de la [Departamento de Física Teórica, Atómica y Óptica, Universidad de Valladolid, 47011-Valladolid (Spain)

    2014-10-07

    We report a detailed characterization of the spatial resolution provided by two-photon absorption spectroscopy suited for plasma diagnosis via the 1S-2S transition of atomic hydrogen for optogalvanic detection and laser induced fluorescence (LIF). A precise knowledge of the spatial resolution is crucial for a correct interpretation of measurements, if the plasma parameters to be analysed undergo strong spatial variations. The present study is based on a novel approach which provides a reliable and realistic determination of the spatial resolution. Measured irradiance distribution of laser beam waists in the overlap volume, provided by a high resolution UV camera, are employed to resolve coupled rate equations accounting for two-photon excitation, fluorescence decay and ionization. The resulting three-dimensional yield distributions reveal in detail the spatial resolution for optogalvanic and LIF detection and related saturation due to depletion. Two-photon absorption profiles broader than the Fourier transform-limited laser bandwidth are also incorporated in the calculations. The approach allows an accurate analysis of the spatial resolution present in recent and future measurements.

  2. Two-photon excited spectroscopies of ex vivo human skin endogenous species irradiated by femtosecond laser pulses

    Institute of Scientific and Technical Information of China (English)

    Jianxin Chen; Shuangmu Zhuo; Tianshu Luo; Jingjun Zhao

    2006-01-01

    Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin,NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.

  3. Two-photon excited spectroscopies of ex vivo human skin endogenous species irradiated by femtosecond laser pulses

    Science.gov (United States)

    Chen, Jianxin; Zhuo, Shuangmu; Luo, Tianshu; Zhao, Jingjun

    2006-10-01

    Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin, NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.

  4. Correction of cell-induced optical aberrations in a fluorescence fluctuation microscope

    CERN Document Server

    Leroux, Charles-Edouard; Wang, Irène; Delon, Antoine

    2014-01-01

    We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements. Our data show that AO is not only useful when imaging deep in tissues but also when performing FFM measurements through a single cellular layer. This work paves the way for the application of FFM to complex three-dimensional multicellular samples.

  5. Two-Photon Absorption of Metal-Assisted Chromophores.

    Science.gov (United States)

    Li, Xin; Rinkevicius, Zilvinas; Ågren, Hans

    2014-12-09

    Aiming to understand the effect of a metal surface on nonlinear optical properties and the combined effects of surface and solvent environments on such properties, we present a multiscale response theory study, integrated with dynamics of the two-photon absorption of 4-nitro-4'-amino-trans-stilbene physisorbed on noble metal surfaces, considering two such surfaces, Ag(111) and Au(111), and two solvents, cyclohexane and water, as cases for demonstration. A few conclusions of general character could be drawn: While the geometrical change of the chromophore induced by the environment was found to notably alter (diminish) the two-photon absorption cross section in the polar medium, the effects of the metal surface and solvent on the electronic structure of the chromophore surpasses the geometrical effects and leads to a considerably enhanced two-photon absorption cross section in the polar solvent. This enhancement of two-photon absorption arises essentially from the metal charge image induced enlargement of the difference between the dipole moment of the excited state and the ground state. The orientation-dependence of the two-photon absorption is found to connect with the lateral rotation of the chromophore, where the two-photon absorption reaches its maximum when the polarization of the incident light coincides with the long-axis of the chromophore. Our results demonstrate a distinct enhancement of the two-photon absorption by a metal surface and a polar medium and envisage the employment of metal-chromophore composite materials for future development of nonlinear optical materials with desirable properties.

  6. Molecular engineering of nanoscale quadrupolar chromophores for two-photon absorption

    Science.gov (United States)

    Porres, Laurent; Mongin, Olivier; Blanchard-Desce, Mireille H.; Ventelon, Lionel; Barzoukas, Marguerite; Moreaux, Laurent; Pons, Thomas; Mertz, Jerome

    2003-02-01

    Our aim has been the design of optimized NLO-phores with very high two-photon absorption (TPA) cross-sections (s2) in the red-NIR region, while maintaining high linear transparency and high fluorescence quantum yield. Our molecular engineering strategy is based on the push-push or pull-pull functionalization of semi-rigid nanoscale conjugated systems. The central building blocks were selected as rigid units that may assist quadrupolar intramolecular charge transfer by acting either as a (weak) donor or acceptor core. Quadrupolar molecules derived either from a phenyl unit, a rigidified biphenyl moiety or a fused bithiophene unit have been considered. Conjugated oligomers made of phenylene-vinylene and/or phenylene-ethynylene units were selected as connecting spacers between the core and the electroactive end groups to ensure effective electronic conjugation while maintaining suitable transparency/fluorescence. The TPA cross-sections were determined by investigating the two-photon-excited fluorescence properties using a Ti:sapphire laser delivering fs pulses. Both the nature of the end groups and of the core moiety play an important role in determining the TPA spectra. In addition, by adjusting the length and nature of the conjugated extensor, both amplification and spectral tuning of TPA cross-sections can be achieved. As a result, push-push fluorophores which demonstrate giant TPA cross-sections (up to 3000 GM) in the visible red, high fluorescence quantum yields and good transparency in the visible range have been obtained.

  7. A fiber-optic fluorescence microscope using a consumer-grade digital camera for in vivo cellular imaging.

    Directory of Open Access Journals (Sweden)

    Dongsuk Shin

    Full Text Available BACKGROUND: Early detection is an essential component of cancer management. Unfortunately, visual examination can often be unreliable, and many settings lack the financial capital and infrastructure to operate PET, CT, and MRI systems. Moreover, the infrastructure and expense associated with surgical biopsy and microscopy are a challenge to establishing cancer screening/early detection programs in low-resource settings. Improvements in performance and declining costs have led to the availability of optoelectronic components, which can be used to develop low-cost diagnostic imaging devices for use at the point-of-care. Here, we demonstrate a fiber-optic fluorescence microscope using a consumer-grade camera for in vivo cellular imaging. METHODS: The fiber-optic fluorescence microscope includes an LED light, an objective lens, a fiber-optic bundle, and a consumer-grade digital camera. The system was used to image an oral cancer cell line labeled with 0.01% proflavine. A human tissue specimen was imaged following surgical resection, enabling dysplastic and cancerous regions to be evaluated. The oral mucosa of a healthy human subject was imaged in vivo, following topical application of 0.01% proflavine. FINDINGS: The fiber-optic microscope resolved individual nuclei in all specimens and tissues imaged. This capability allowed qualitative and quantitative differences between normal and precancerous or cancerous tissues to be identified. The optical efficiency of the system permitted imaging of the human oral mucosa in real time. CONCLUSION: Our results indicate this device as a useful tool to assist in the identification of early neoplastic changes in epithelial tissues. This portable, inexpensive unit may be particularly appropriate for use at the point-of-care in low-resource settings.

  8. A fiber-optic fluorescence microscope using a consumer-grade digital camera for in vivo cellular imaging.

    Science.gov (United States)

    Shin, Dongsuk; Pierce, Mark C; Gillenwater, Ann M; Williams, Michelle D; Richards-Kortum, Rebecca R

    2010-06-23

    Early detection is an essential component of cancer management. Unfortunately, visual examination can often be unreliable, and many settings lack the financial capital and infrastructure to operate PET, CT, and MRI systems. Moreover, the infrastructure and expense associated with surgical biopsy and microscopy are a challenge to establishing cancer screening/early detection programs in low-resource settings. Improvements in performance and declining costs have led to the availability of optoelectronic components, which can be used to develop low-cost diagnostic imaging devices for use at the point-of-care. Here, we demonstrate a fiber-optic fluorescence microscope using a consumer-grade camera for in vivo cellular imaging. The fiber-optic fluorescence microscope includes an LED light, an objective lens, a fiber-optic bundle, and a consumer-grade digital camera. The system was used to image an oral cancer cell line labeled with 0.01% proflavine. A human tissue specimen was imaged following surgical resection, enabling dysplastic and cancerous regions to be evaluated. The oral mucosa of a healthy human subject was imaged in vivo, following topical application of 0.01% proflavine. The fiber-optic microscope resolved individual nuclei in all specimens and tissues imaged. This capability allowed qualitative and quantitative differences between normal and precancerous or cancerous tissues to be identified. The optical efficiency of the system permitted imaging of the human oral mucosa in real time. Our results indicate this device as a useful tool to assist in the identification of early neoplastic changes in epithelial tissues. This portable, inexpensive unit may be particularly appropriate for use at the point-of-care in low-resource settings.

  9. LAURDAN fluorescence properties in membranes: a journey from the fluorometer to the microscope

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2012-01-01

    After 32 years since its introduction, the particular fluorescence properties of 6-lauroyl-2-(dimethylamino)-naphtalene (LAURDAN) in model and biological membranes are revisited. This review includes a historical perspective about the design, synthesis and initial description of the probe’s fluor...

  10. In situ, dual-mode monitoring of organ-on-a-chip with smartphone-based fluorescence microscope.

    Science.gov (United States)

    Cho, Soohee; Islas-Robles, Argel; Nicolini, Ariana M; Monks, Terrence J; Yoon, Jeong-Yeol

    2016-12-15

    The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Two photon dissociation of benzene, phenylacetylene, and benzaldehyde at 243 nm: translational energy releases in the H atom channel

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Seung Keun; Kim, Hong Lae [Kangwon National Univ., Chuncheon (Korea, Republic of); Park, Chan Ryang [Kookmin Univ., Seoul (Korea, Republic of)

    2002-02-01

    Hydrogen atom production channels from photodissociation of benzene, phenylacetylene, and benzaldehyde at 243 nm have been investigated by detecting H atoms using two photon absorption at 243.2 nm and induced fluorescence at 121.6 nm. Translational energies of the H atoms were measured by Doppler broadened H atom spectra. By absorption of two photons at 243 nm, the H atoms are statistically produced from benzene and phenylacetylene whereas the H atoms from the aldehyde group in benzaldehyde are produced from different pathways. The possible dissociation mechanisms are discussed from the measured translational energy releases.

  12. Ultra-thin rigid endoscope: Two-photon imaging through a graded-index multi-mode fiber

    CERN Document Server

    Sivankutty, Siddharth; Cossart, Rosa; Bouwmans, Géraud; Monneret, Serge; Rigneault, Hervé

    2015-01-01

    Rigid endoscopes like graded-index (GRIN) lenses are known tools in biological imaging, but it is conceptually difficult to miniaturize them. In this letter, we demonstrate an ultra-thin rigid endoscope with a diameter of only 125 microns. In addition, we identify a domain where two-photon endoscopic imaging with fs-pulse excitation is possible. We validate the ultra-thin rigid endoscope consisting of a few cm of graded-index multi-mode fiber by using it to acquire optically sectioned two-photon fluorescence endoscopic images of three-dimensional samples.

  13. Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

    Science.gov (United States)

    Podgorski, Kaspar; Terpetschnig, Ewald; Klochko, Oleksii P; Obukhova, Olena M; Haas, Kurt

    2012-01-01

    Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM) at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647) by imaging dye-filled neurons in vivo over 5 days, and utility for sensitive subcellular imaging by synthesizing a specific peptide-conjugate label for the synaptic protein PSD-95.

  14. Ultra-bright and -stable red and near-infrared squaraine fluorophores for in vivo two-photon imaging.

    Directory of Open Access Journals (Sweden)

    Kaspar Podgorski

    Full Text Available Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647 by imaging dye-filled neurons in vivo over 5 days, and utility for sensitive subcellular imaging by synthesizing a specific peptide-conjugate label for the synaptic protein PSD-95.

  15. Two-photon luminescence microscopy of field enhancement at gold nanoparticles

    DEFF Research Database (Denmark)

    Beermann, Jonas; Bozhevolnyi, Sergey I.

    2005-01-01

    Using a reflection scanning optical microscope detecting two-photon luminescence (TPL) we have imaged square gold bumps positioned in a periodic array either on a smooth gold film or directly on a glass substrate. The second-harmonic (SH) and TPL response from these structures show both...... polarization and wavelength dependence. The gold bumps on gold film showed extremely high sensitivity to the incident field, with the strongest TPL response from the gold bumps being enhanced nearly 103 times compared to the TPL response from the smooth gold surface. For gold bumps directly on glass...

  16. Nonlinear quantitative photoacoustic tomography with two-photon absorption

    CERN Document Server

    Ren, Kui

    2016-01-01

    Two-photon photoacoustic tomography (TP-PAT) is a non-invasive optical molecular imaging modality that aims at inferring two-photon absorption property of heterogeneous media from photoacoustic measurements. In this work, we analyze an inverse problem in quantitative TP-PAT where we intend to reconstruct optical coefficients in a semilinear elliptic PDE, the mathematical model for the propagation of near infra-red photons in tissue-like optical media with two-photon absorption, from the internal absorbed energy data. We derive uniqueness and stability results on the reconstructions of single and multiple optical coefficients, and present some numerical reconstruction results based on synthetic data to complement the theoretical analysis.

  17. Two-photon interference between disparate sources for quantum networking

    Science.gov (United States)

    McMillan, A. R.; Labonté, L.; Clark, A. S.; Bell, B.; Alibart, O.; Martin, A.; Wadsworth, W. J.; Tanzilli, S.; Rarity, J. G.

    2013-06-01

    Quantum networks involve entanglement sharing between multiple users. Ideally, any two users would be able to connect regardless of the type of photon source they employ, provided they fulfill the requirements for two-photon interference. From a theoretical perspective, photons coming from different origins can interfere with a perfect visibility, provided they are made indistinguishable in all degrees of freedom. Previous experimental demonstrations of such a scenario have been limited to photon wavelengths below 900 nm, unsuitable for long distance communication, and suffered from low interference visibility. We report two-photon interference using two disparate heralded single photon sources, which involve different nonlinear effects, operating in the telecom wavelength range. The measured visibility of the two-photon interference is 80 +/- 4%, which paves the way to hybrid universal quantum networks.

  18. Two-photon interference with non-identical photons

    Science.gov (United States)

    Liu, Jianbin; Zhou, Yu; Zheng, Huaibin; Chen, Hui; Li, Fu-li; Xu, Zhuo

    2015-11-01

    Two-photon interference with non-identical photons is studied based on the superposition principle in Feynman's path integral theory. The second-order temporal interference pattern is observed by superposing laser and pseudothermal light beams with different spectra. The reason why there is two-photon interference for photons of different spectra is that non-identical photons can be indistinguishable for the detection system when Heisenberg's uncertainty principle is taken into account. These studies are helpful to understand the second-order interference of light in the language of photons.

  19. Two-Photon Total Annihilation of Molecular Positronium

    CERN Document Server

    Pérez-Ríos, Jesús; Greene, Chris H

    2014-01-01

    The rate for complete two-photon annihilation of molecular positronium Ps$_{2}$ is reported. This decay channel involves a four-body collision among the fermions forming Ps$_{2}$, and two photons of 1.022 MeV, each, as the final state. The quantum electrodynamics result for the rate of this process is found to be $\\Gamma_{Ps_{2} \\rightarrow \\gamma\\gamma}$ = 9.0 $\\times 10^{-12}$ s$^{-1}$. This decay channel completes the most comprehensive decay chart for Ps$_{2}$ up to date.

  20. Two-photon Compton process in pulsed intense laser fields

    CERN Document Server

    Seipt, D

    2012-01-01

    Based on strong-field QED in the Furry picture we use the Dirac-Volkov propagator to derive a compact expression for the differential emission probability of the two-photon Compton process in a pulsed intense laser field. The relation of real and virtual intermediate states is discussed, and the natural regularization of the on-shell contributions due to the finite laser pulse is highlighted. The inclusive two-photon spectrum is two orders of magnitude stronger than expected from a perturbative estimate.

  1. Precision two-photon spectroscopy of alkali elements

    Indian Academy of Sciences (India)

    P V Kiran Kumar; M V Suryanarayana

    2014-08-01

    In this paper, we have briefly reviewed the work on two-photon spectroscopy of alkali elements and its applications. The technique of Doppler-free two-photon spectroscopy is briefly summarized. A review of various techniques adopted for measuring absolute frequencies of the atomic transitions and precision measurements of isotope shifts and hyperfine structures (HFS) is presented. Some of the recent works on precision measurements of HFS constants of 6 ${}^2S_{1/2}$ level of ${}^{39}$K and ${}^{41}$K, 9 ${}^2S_{1/2}$ level and 7 ${}^2D_{3/2}$ level of 133Cs are also discussed.

  2. Modulation of attosecond beating by resonant two-photon transition

    CERN Document Server

    Galán, Álvaro Jiménez; Martín, Fernando

    2015-01-01

    We present an analytical model that characterizes two-photon transitions in the presence of autoionising states. We applied this model to interpret resonant RABITT spectra, and show that, as a harmonic traverses a resonance, the phase of the sideband beating significantly varies with photon energy. This phase variation is generally very different from the $\\pi$ jump observed in previous works, in which the direct path contribution was negligible. We illustrate the possible phase profiles arising in resonant two-photon transitions with an intuitive geometrical representation.

  3. New developments in two-photon analysis of human skin in vivo

    Science.gov (United States)

    Riemann, I.; Schwarz, M.; Stracke, F.; Ehlers, A.; Dimitrow, E.; Kaatz, M.; König, K.; Le Harzic, R.

    2009-02-01

    Two-photon imaging of human skin using ultra short laser pulses can be used to obtain information about the state of cells and tissues by means of their natural autofluorescence. Using this method, it is possible to determine whether the normal cell pattern is disturbed or the autofluorescence is influenced by internal or external stimuli. Two-photon fluorescence lifetime imaging (FLIM) can further enhance this providing information about physiological processes, fluorophores (like NAD(P)H, collagen, keratin, elastin, flavins, melanin,...) and external applied probes inside cells and tissue parts. For example the part of the cells metabolism and energy level can be determined by analyzing the NADH regarding its free / bound state and its oxidized / reduced state. The combination of two-photon imaging with FLIM may lead to a better understanding and diagnosis of skin reactions and disorders. We also present some results of in vivo simultaneous collagen and elastin measurements in skin dermis. Changes of dermal collagen and elastin content are characteristic for skin aging as well as for pathological skin conditions.

  4. Enhanced two-photon emission in coupled metal nanoparticles induced by conjugated polymers.

    Science.gov (United States)

    Guan, Zhenping; Polavarapu, Lakshminarayana; Xu, Qing-Hua

    2010-12-01

    Interactions between noble metal (Ag and Au) nanoparticles and conjugated polymers as well as their one- and two-photon emission have been investigated. Ag and Au nanoparticles exhibited extraordinary quenching effects on the fluorescence of cationic poly(fluorinephenylene). The quenching efficiency by 37-nm Ag nanoparticles is ∼19 times more efficient than that by 13-nm Au nanoparticles, and 9-10 orders of magnitude more efficient than typical small molecule dye-quencher pairs. On the other hand, the cationic conjugated polymers induce the aggregate formation and plasmonic coupling of the metal nanoparticles, as evidenced by transmission electron microscopy images and appearance of a new longitudinal plasmon band in the near-infrared region. The two-photon emissions of Ag and Au nanoparticles were found to be significantly enhanced upon addition of conjugated polymers, by a factor of 51-times and 9-times compared to the isolated nanoparticles for Ag and Au, respectively. These studies could be further extended to the applications of two-photon imaging and sensing of the analytes that can induce formation of metal nanoparticle aggregates, which have many advantages over the conventional one-photon counterparts.

  5. Multidimensional two-photon imaging and spectroscopy of fresh human bladder biopsies

    Science.gov (United States)

    Cicchi, Riccardo; Crisci, Alfonso; Cosci, Alessandro; Nesi, Gabriella; Giancane, Saverio; Carini, Marco; Pavone, Francesco S.

    2010-02-01

    Two-photon microscopy has been successfully used to image several types of tissues, including skin, muscles, tendons. Nevertheless, its usefulness in imaging bladder tissue has not been investigated yet. In this work we used combined twophoton excited fluorescence, second-harmonic generation microscopy, fluorescence lifetime imaging microscopy, and multispectral two-photon emission detection to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopic analyses allowed to characterize both healthy mucosa and carcinoma in-situ samples in a good agreement with common routine histology. Cancer cells showed different morphology with respect to the corresponding healthy cells: they appeared more elongated and with a larger nucleus to cytoplasm ratio. From the spectroscopic point of view, differences between the two tissue types in both spectral emission and fluorescence lifetime distribution were found. Even if further analysis, as well as a more significant statistics on a larger number of samples would be helpful to discriminate between low, mild, and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well to be implemented in a multi-photon endoscope or in a spectroscopic for in in-vivo imaging applications.

  6. Two-photon spectroscopic behaviors and photodynamic effect on the BEL-7402 cancer cells of the new chlorophyll photosensitizer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The spectroscopic properties of a new chlorophyll derivate photosensitizer(CDP) are studied under the excitation wavelengths at 800 and 400 nm using femtosecond pulses from a Ti:sapphire laser.The damaging effect of CDP on the BEL-7402 cancer cells is also investigated upon two-photon illumination at 800 nm.The normalized fluorescence spectra of CDP in tetrahydrofuran(THF) show that two-photon and one-photon spectra have the same distributions and the same emission bands(675 nm).The life-times of two-and one-photon induced fluorescence of this molecule are of the order of 5.0 ns.By comparing the data it is shown that there is some difference between the two lifetimes,but the differ-ence is less than one nanosecond.The two-photon absorption cross section of the molecule is also measured at 800 nm and estimated as about σ′2 ≈ 31.5×10-50 cm4·s·photon-1.The results of two-photon photodynamic therapy(TPPDT) tests show that CDP can kill all of the tested cancer cells according to the usual Eosine assessment.Our results indicate that the two-photon-induced photophysical,photo-chemical and photosensitizing processes of CDP may be basically similar to those of one-photon ex-citation.These behaviors of the sample suggest that one may find other possible methods to estimate some photosensitizers’ effects in details such as their distribution in cells and the reactive targets of the sub-cellular parts of some tumor cells via two-photon excitation techniques.

  7. Two-photon spectroscopic behaviors and photodynamic effect on the BEL-7402 cancer cells of the new chlorophyll photosensitizer

    Institute of Scientific and Technical Information of China (English)

    ZHAO PeiDe; ZHANG GuiLan; CHEN WenJu; CHEN Ping; TANG GuoQing; LIU JinWei; LIN Lie; GUO Peng; YU Qing; YAO JianZhong; MA DongMing

    2008-01-01

    The spectroscopic properties of a new chlorophyll derivate photosensitizer (CDP) are studied under the excitation wavelengths at 800 and 400 nm using femtosecond pulses from a Ti: sapphire laser. The damaging effect of CDP on the BEL-7402 cancer cells is also investigated upon two-photon illumination at 800 nm. The normalized fluorescence spectra of CDP in tetrahydrofuran (THF) show that two-photon and one-photon spectra have the same distributions and the same emission bands (675 nm). The life-times of two- and one-photon induced fluorescence of this molecule are of the order of 5.0 ns. By comparing the data it is shown that there is some difference between the two lifetimes, but the differ-ence is less than one nanosecond. The two-photon absorption cross section of the molecule is also measured at 800 nm and estimated as about σ'2≈31.5×10-50 cm4·s·photon-1. The results of two-photon photodynamic therapy (TPPDT) tests show that CDP can kill all of the tested cancer cells according to the usual Eosine assessment. Our results indicate that the two-photon-induced photophysical, photochemical and photosensitizing processes of CDP may be basically similar to those of one-photon excitation. These behaviors of the sample suggest that one may find other possible methods to estimate some photosensitizers' effects in details such as their distribution in cells and the reactive targets of the sub-cellular parts of some tumor cells via two-photon excitation techniques.

  8. Intravital two-photon imaging: a versatile tool for dissecting the immune system.

    Science.gov (United States)

    Ishii, Taeko; Ishii, Masaru

    2011-03-01

    During the past decade, multi-photon or 'two-photon' excitation microscopy has launched a new era in the field of biological imaging. The near-infrared excitation laser for two-photon microscopy can penetrate thicker specimens, enabling the visualisation of living cell behaviour deep within tissues and organs without thin sectioning. The minimised photobleaching and toxicity enables the visualisation of live and intact specimens for extended periods. In this brief review, recent findings in intravital two-photon imaging for the physiology and pathology of the immune system are discussed. The immune system configures highly dynamic networks, where many cell types actively travel throughout the body and interact with each other in specific areas. Hence, real-time intravital imaging may be a powerful tool for dissecting the mechanisms of this dynamic system. The most unique characteristic of the immune system is its highly dynamic nature. A variety of cell types, such as lymphocytes, macrophages and dendritic cells (DCs), are continuously circulating throughout the body, migrating through the peripheral tissues and interacting with each other in their respective niches. Conventional methodologies in immunology, such as flow cytometry, cell or tissue culture, biochemistry and histology, have brought tremendous achievement within this field, although the dynamics of immune cells in an entire animal remain less clear. Technological progress of fluorescence microscopy has enabled us to visualise the intact biological phenomenon that has been uninvestigated. Among the advancements, the recent emergence and prevalence of two-photon, excitation-based, laser microscopy has revolutionised the research field, such that the dynamic behaviour of cells deep inside living organs can be visualised and analysed.

  9. Electron energy dependence of characteristics of fluorescent plates for ultrahigh-voltage electron microscopes.

    Science.gov (United States)

    Nishi, R; Yoshida, K; Takaoka, A; Katsuta, T

    1996-03-01

    The characteristics of fluorescent plates for high energy electron beams (0.5-2.0 MeV) are examined. The thickness and the optical transparency of plates strongly affect the luminous broadening and intensity. The spatial luminous broadening in fluorescent plates is measured and is simply represented by the rise width of a knife edge image. When the thickness is much smaller than the range of incident electrons, the rise width is 1/4-1/5 of the thickness in the case of YAG single crystal plates that are transparent for light, while the rise width is nearly equal to the thickness for the packed P22 powder plates that are opaque for light. To suppress the luminous broadening under 50 microm, the thickness of YAG plates has to be thinner than 250 microm in the energy region around 2 MeV. Under the same condition of the rise width, the luminous intensity of YAG plates is twice as high as that of the P22 plates.

  10. Organic nanostructure-based probes for two-photon imaging of mitochondria and microbes with emission between 430 nm and 640 nm.

    Science.gov (United States)

    Yang, Xinglong; Wang, Nuoxin; Zhang, Lingmin; Dai, Luru; Shao, Huawu; Jiang, Xingyu

    2017-04-06

    Multi-photon excitation and versatile fluorescent probes are in high need for biological imaging, since one probe can satisfy many needs as a biosensor. Herein we synthesize a series of two-photon excited probes based on tetraphenylethene (TPE) structures (TPE-Acr, TPE-Py, and TPE-Quino), which can image both mammalian cells and bacteria based on aggregation-induced emission (AIE) without washing them. Because of cationic moieties, the fluorescent molecules can aggregate into nanoscale fluorescent organic nanoscale dots to image mitochondria and bacteria with tunable emissions using both one-photon and two-photon excitation. Our research demonstrates that these AIE-dots expand the functions of luminescent organic dots to construct efficient fluorescent sensors applicable to both one-photon and two-photon excitation for bio-imaging of bacteria and mammalian cells.

  11. An evaluation of the performance and acceptability of three LED fluorescent microscopes in Zambia: lessons learnt for scale-up.

    Science.gov (United States)

    Turnbull, Eleanor R; Kaunda, Kaunda; Harris, Jennifer B; Kapata, Nathan; Muvwimi, Mweemba W; Kruuner, Annika; Henostroza, German; Reid, Stewart E

    2011-01-01

    The World Health Organization recommends the roll-out of light-emitting diode (LED) fluorescent microscopes (FM) as an alternative to light microscopes in resource-limited settings. We evaluated the acceptability and performance of three LED FMs after a short orientation among laboratory technicians from government health centers in Zambia. Sixteen technicians with varied light microscopy experience were oriented to FMs and divided into groups; each group read a different set of 40 slides on each LED FM (Primo Star iLED™, Lumin™, FluoLED™) and on a reference mercury-vapor FM (Olympus BX41TF). Slide reading times were recorded. An experienced FM technician examined each slide on the Olympus BX41TF. Sensitivity and specificity compared to TB culture were calculated. Misclassification compared to the experienced technician and inter-rater reliability between trainees was assessed. Trainees rated microscopes on technical aspects. Primo Star iLED™, FluoLED™ and Olympus BX41TF had comparable sensitivities (67%, 65% and 65% respectively), with the Lumin™ significantly worse (56%; p<0.05). Specificity was low for trainees on all microscopes (75.9%) compared to the experienced technician on Olympus BX41TF (100%). Primo Star iLED™ had significantly less misclassification (21.1% p<0.05) than FluoLED™ (26.5%) and Lumin™ (26.8%) and significantly higher inter-rater reliability (0.611; p<0.05), compared to FluoLED™ (0.523) and Lumin™ (0.492). Slide reading times for LED FMs were slower than the reference, but not significantly different from each other. Primo Star iLED™ rated highest in acceptability measures, followed by FluoLED™ then Lumin™. Primo Star iLED™ was consistently better than FluoLED™ and Lumin™, and performed comparably to the Olympus BX41TF in all analyses, except reading times. The Lumin™ compared least favorably and was thought unacceptable for use. Specificity and inter-rater reliability were low for all microscopes

  12. An evaluation of the performance and acceptability of three LED fluorescent microscopes in Zambia: lessons learnt for scale-up.

    Directory of Open Access Journals (Sweden)

    Eleanor R Turnbull

    Full Text Available The World Health Organization recommends the roll-out of light-emitting diode (LED fluorescent microscopes (FM as an alternative to light microscopes in resource-limited settings. We evaluated the acceptability and performance of three LED FMs after a short orientation among laboratory technicians from government health centers in Zambia. Sixteen technicians with varied light microscopy experience were oriented to FMs and divided into groups; each group read a different set of 40 slides on each LED FM (Primo Star iLED™, Lumin™, FluoLED™ and on a reference mercury-vapor FM (Olympus BX41TF. Slide reading times were recorded. An experienced FM technician examined each slide on the Olympus BX41TF. Sensitivity and specificity compared to TB culture were calculated. Misclassification compared to the experienced technician and inter-rater reliability between trainees was assessed. Trainees rated microscopes on technical aspects. Primo Star iLED™, FluoLED™ and Olympus BX41TF had comparable sensitivities (67%, 65% and 65% respectively, with the Lumin™ significantly worse (56%; p<0.05. Specificity was low for trainees on all microscopes (75.9% compared to the experienced technician on Olympus BX41TF (100%. Primo Star iLED™ had significantly less misclassification (21.1% p<0.05 than FluoLED™ (26.5% and Lumin™ (26.8% and significantly higher inter-rater reliability (0.611; p<0.05, compared to FluoLED™ (0.523 and Lumin™ (0.492. Slide reading times for LED FMs were slower than the reference, but not significantly different from each other. Primo Star iLED™ rated highest in acceptability measures, followed by FluoLED™ then Lumin™. Primo Star iLED™ was consistently better than FluoLED™ and Lumin™, and performed comparably to the Olympus BX41TF in all analyses, except reading times. The Lumin™ compared least favorably and was thought unacceptable for use. Specificity and inter-rater reliability were low for all microscopes

  13. 双光子技术及其在生物医药分析中的应用%Two-photon techniques and their applications in biomedical and pharmaceutical analysis

    Institute of Scientific and Technical Information of China (English)

    许佳丽; 熊祖洪; 黄承志

    2011-01-01

    Two-photon techniques, which have undergone rapid development during the last two decades, have attracted a great deal of attention from scientists and researchers in many fields. This has led to a wide range of applications in three-dimensional data storage, medicine, defense, biology, and life sciences. At the same time, there has been increased integration and cooperation among various disciplines. Two-photon techniques have increasingly important roles in revealing the basic principles and origins of life activities, and provide a much more effective means for developing biological and medical imaging. Combining the history of the two-photon technique with its applications in the life sciences, this mini-review provides a simple introduction to these emerging technologies. It focuses on the development of two-photon techniques, two-photon materials used in scientific research and their applications in optical and medical research, and the imaging principles of the two-photon laser scanning fluorescent microscope.%作为近20年来迅速发展的一门新技术,双光子技术已被广泛应用于三维数据存储材料、医药、军事、生物以及生命科学等领域,并随学科交叉与融合,在揭示生命活动基本规律和起源的研究中发挥越来越重要的作用,为生物医学提供了更多、更为有效的手段.本文从常用的双光子材料以及双光子激光扫描荧光显微镜成像原理等方面,结合双光子技术在生物医学研究中的应用,探讨了这一新兴技术在生物医药分析领域的前景.

  14. Internal conversions in Higgs decays to two photons

    OpenAIRE

    Firan, Ana; Stroynowski, Ryszard

    2007-01-01

    We evaluate the partial widths for internal conversions in the Higgs decays to two photons. For the Higgs masses of interest at LHC in the range of 100-150 GeV, the conversions to pairs of fermions represent significant fraction of Higgs decays.

  15. Two-Photon-Pumped Perovskite Semiconductor Nanocrystal Lasers.

    Science.gov (United States)

    Xu, Yanqing; Chen, Qi; Zhang, Chunfeng; Wang, Rui; Wu, Hua; Zhang, Xiaoyu; Xing, Guichuan; Yu, William W; Wang, Xiaoyong; Zhang, Yu; Xiao, Min

    2016-03-23

    Two-photon-pumped lasers have been regarded as a promising strategy to achieve frequency up-conversion for situations where the condition of phase matching required by conventional approaches cannot be fulfilled. However, their practical applications have been hindered by the lack of materials holding both efficient two-photon absorption and ease of achieving population inversion. Here, we show that this challenge can be tackled by employing colloidal nanocrystals of perovskite semiconductors. We observe highly efficient two-photon absorption (with a cross section of 2.7 × 10(6) GM) in toluene solutions of CsPbBr3 nanocrystals that can excite large optical gain (>500 cm(-1)) in thin films. We have succeeded in demonstrating stable two-photon-pumped lasing at a remarkable low threshold by coupling CsPbBr3 nanocrystals with microtubule resonators. Our findings suggest perovskite nanocrystals can be used as excellent gain medium for high-performance frequency-up-conversion lasers toward practical applications.

  16. Two-photon absorbing porphyrins for oxygen microscopy (Conference Presentation)

    Science.gov (United States)

    Esipova, Tatiana V.; Vinogradov, Sergei A.

    2016-03-01

    The ability to quantify oxygen in vivo in 3D with high spatial and temporal resolution is invaluable for many areas of the biomedical science, including ophthalmology, neuroscience, cancer and stem biology. An optical method based on oxygen-dependent quenching of phosphorescence is being developed, that allows quantitative minimally invasive real-time imaging of partial pressure of oxygen (pO2) in tissue. In the past, dendritically protected phosphorescent oxygen probes with controllable quenching parameters and defined bio-distributions have been developed. More recently our probe strategy has extended to encompass two-photon excitable oxygen probes, which brought about first demonstrations of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen in vivo, providing new valuable information for neuroscience and stem cell biology. However, current two-photon oxygen probes suffer from a number of limitations, such as low brightness and high cost of synthesis, which dramatically reduce imaging performance and limit usability of the method. Here we present an approach to new bright phosphorescent chromophores with internally enhanced two-photon absorption cross-sections, which pave a way to novel proves for 2PLM. In addition to substantial increase in performance, the new probes can be synthesized by much more efficient methods, thereby greatly reducing the cost of the synthesis and making the technique accessible to a broader range of researchers across different fields.

  17. Direct Writing of Photonic Structures by Two-Photon Polymerization

    Directory of Open Access Journals (Sweden)

    Li Yan

    2013-11-01

    Full Text Available Single-mode dielectric-loaded surface plasmon-polariton nanowaveguides with strong mode confinement at excitation wavelength of 830 nm and high-Q polymer whispering gallery mode microcavities with surface roughness less than 12 nm have been directly written by two-photon polymerization, which pave the way to fabricate 3D plasmonic photonic structures by direct laser writing.

  18. Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization?

    Science.gov (United States)

    MacDonald, Laura; Baldini, Giulia; Storrie, Brian

    2015-01-01

    Conventional microscopy techniques, namely, the confocal microscope or deconvolution processes, are resolution limited to approximately 200-250 nm by the diffraction properties of light as developed by Ernst Abbe in 1873. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20-50 nm in diameter. In the mid-2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular co-localization questions. This "breaking" of the diffraction barrier, affording resolution beyond 200 nm, is termed super-resolution microscopy. More recent approaches include single-molecule localization (such as photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM)) and point spread function engineering (such as stimulated emission depletion (STED) microscopy). In this review, we explain basic principles behind currently commercialized super-resolution setups and address advantages and considerations in applying these techniques to protein co-localization in biological systems.

  19. Recruitment of HIV-1 envelope occurs subsequent to lipid mixing: a fluorescence microscopic evidence

    Directory of Open Access Journals (Sweden)

    Lin Chi-Hui

    2009-03-01

    Full Text Available Abstract Entry of the human immunodeficiency virus (HIV into the target cell is initiated by fusion with the cell membrane, mediated through the envelope glycoproteins gp120 and gp41, following engagement to CD4 and the co-receptor. Previous fusion kinetics studies on the HXB2 envelope protein (Env revealed that Env recruitment occurred at about 13 min concurrent with the lipid mixing. To resolve the temporal sequence of lipid mixing and recruitment, we employed an inhibitory assay monitored by fluorescence microscopy using a gp41 ectodomain (gp41e fragment, which blocked Env recruitment in stark contrast to the lack of gp41e effect on the lipid mixing. In addition, to demonstrate the mode of action for the inhibition of gp41e, our results strongly suggested that lipid mixing precedes the Env recruitment because lipid mixing can proceed with Env recruitment inhibited by exogeneous gp41e molecules. Importantly, it was found that the random clustering of Env molecules on the membrane surface occurred at ~1 minute whereas the Env recruitment was observed at 13 minutes after the attachment of Env-expressing cell to the target cell. This > 10-fold temporal discrepancy highlights that the productive assembly of Env molecules leading to fusion requires spatio-temporal coordination of several adjacent Env trimers aggregated via directed movement.

  20. Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2015-03-01

    High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.

  1. Two-Photon Absorption Spectroscopy of Rubidium with a Dual-Comb Tequnique

    Science.gov (United States)

    Nishiyama, Akiko; Yoshida, Satoru; Hariki, Takuya; Nakajima, Yoshiaki; Minoshima, Kaoru

    2017-06-01

    Dual-comb spectroscopies have great potential for high-resolution molecular and atomic spectroscopies, thanks to the broadband comb spectrum consisting of dense narrow modes. In this study, we apply the dual-comb system to Doppler-free two-photon absorption spectroscopy. The outputs of two frequency combs excite several two-photon transitions of rubidium, and we obtained broadband Doppler-free spectra from dual-comb fluorescence signals. The fluorescence detection scheme circumvents the sensitivity limit which is effectively determined by the dynamic range of photodetectors in absorption-based dual-comb spectroscopies. Our system realized high-sensitive, Doppler-free high-resolution and broadband atomic spectroscopy. A part of observed spectra of 5S_{1/2} - 5D_{5/2} transition is shown in the figure. The hyperfine structures of the F" = 1 - F' = 3,2,1 transitions are fully-resolved and the spectral widths are approximately 5 MHz. The absolute frequency axis is precisely calibrated from comb mode frequencies which were stabilized to a GPS-disciplined clock. This work was supported by JST through the ERATO MINOSHIMA Intelligent Optical Synthesizer Project and Grant-in-Aid for JSPS Fellows (16J02345). A. Nishiyama, S. Yoshida, Y. Nakajima, H. Sasada, K. Nakagawa, A. Onae, K. and Minoshima, Opt. Express 24, 25894 (2016). A. Hipke, S. A. Meek, T. Ideguchi, T.W. Hänsch, and N. Picqué, Phys. Rev. A 90, 011805(R) (2014).

  2. Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo

    Science.gov (United States)

    Song, Wei; Xu, Qiang; Zhang, Yang; Zhan, Yang; Zheng, Wei; Song, Liang

    2016-01-01

    The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling. PMID:27576922

  3. Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo

    Science.gov (United States)

    Song, Wei; Xu, Qiang; Zhang, Yang; Zhan, Yang; Zheng, Wei; Song, Liang

    2016-08-01

    The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling.

  4. Two-photon absorption of Zn(II) octupolar molecules.

    Science.gov (United States)

    Mazzucato, Simone; Fortunati, Ilaria; Scolaro, Sara; Zerbetto, Michele; Ferrante, Camilla; Signorini, Raffaella; Pedron, Danilo; Bozio, Renato; Locatelli, Danika; Righetto, Stefania; Roberto, Dominique; Ugo, Renato; Abbotto, Alessandro; Archetti, Graziano; Beverina, Luca; Ghezzi, Sergio

    2007-06-21

    In this work we present an investigation of the non-linear optical (NLO) properties of two octupolar chromophores: [Zn(4,4'-bis(dibutylaminostyryl)-[2,2']-bipyridine)(3)](2+) and [Zn(4,4'-bis((E)-2-(N-(TEG)pyrrol-2-yl)vinyl)-[2,2']-bipyridine)(3)](2+) with Zn(ii) as the coordination center, using two-photon emission technique (TPE) in fs-pulse temporal regime. Compared to the free ligands, our results do not show a net increase in the two-photon absorption (TPA) cross-section for the octupolar complexes, once normalized to the ligand unit. This is in partial disagreement with a previous theoretical study investigating the first molecule where a significant increase of the TPA cross-section was predicted (X. J. Liu, et al., J. Chem. Phys., 2004, 120, 11 493).

  5. Synthesizing arbitrary two-photon polarization mixed states

    CERN Document Server

    Wei, T C; Branning, D; Goldbart, P M; James, D F V; Jeffrey, E; Kwiat, P G; Mukhopadhyay, S; Peters, N A; Wei, Tzu-Chieh; Altepeter, Joseph B.; Branning, David; Goldbart, Paul M.; Jeffrey, Evan; Kwiat, Paul G.; Mukhopadhyay, Swagatam; Peters, Nicholas A.

    2005-01-01

    Two methods for creating arbitrary two-photon polarization pure states are introduced. Based on these, four schemes for creating two-photon polarization mixed states are proposed and analyzed. The first two schemes can synthesize completely arbitrary two-qubit mixed states, i.e., control all 15 free parameters: Scheme I requires several sets of crystals, while Scheme II requires only a single set, but relies on decohering the pump beam. Additionally, we describe two further schemes which are much easier to implement. Although the total capability of these is still being studied, we show that they can synthesize all two-qubit Werner states, maximally entangled mixed states, Collins-Gisin states, and arbitrary Bell-diagonal states.

  6. Direct frequency comb two-photon laser cooling and trapping

    Science.gov (United States)

    Jayich, Andrew; Long, Xueping; Campbell, Wesley C.

    2016-05-01

    Generating and manipulating high energy photons for spectroscopy on electric dipole transitions of atoms and molecules with deeply bound valence electrons is difficult. Further, laser cooling of such species is even more challenging for lack of laser power. A possible solution is to drive two-photon transitions. This may alleviate the photon energy problem and open the door to cold, trapped samples of highly desirable species with tightly bound electrons. We perform a proof of principle experiment with rubidium by driving a two-photon transition with an optical frequency comb. We perform optical cooling and extend this technique to trapping, where we are able to make a magneto-optical trap in one dimension. This work is supported by the National Science Foundation CAREER program.

  7. Modulation of attosecond beating in resonant two-photon ionization

    CERN Document Server

    Galán, Álvaro J; Martín, Fernando

    2014-01-01

    We present a theoretical study of the photoelectron attosecond beating at the basis of RABBIT (Reconstruction of Attosecond Beating By Interference of Two-photon transitions) in the presence of autoionizing states. We show that, as a harmonic traverses a resonance, its sidebands exhibit a peaked phase shift as well as a modulation of the beating frequency itself. Furthermore, the beating between two resonant paths persists even when the pump and the probe pulses do not overlap, thus providing a sensitive non-holographic interferometric means to reconstruct coherent metastable wave packets. We characterize these phenomena quantitatively with a general finite-pulse analytical model that accounts for the effect of both intermediate and final resonances on two-photon processes, at a negligible computational cost. The model predictions are in excellent agreement with those of accurate ab initio calculations for the helium atom in the region of the N=2 doubly excited states.

  8. Two-photon excited ultraviolet photoluminescence of zinc oxide nanorods.

    Science.gov (United States)

    Zhu, Guangping; Xu, Chunxiang; Zhu, Jing; Lu, Changgui; Cui, Yiping; Sun, Xiaowei

    2008-11-01

    High density zinc oxide nanorods with uniform size were synthesized on (100) silicon substrate by vapor-phase transport method. The scanning electron microscopy images reveal that the nanorods have an average diameter of about 400 nm. The X-ray diffraction pattern demonstrates the wurtzite crystalline structure of the ZnO nanorods growing along [0001] direction. The single-photon excited photoluminescence presents a strong ultraviolet emission band at 394 nm and a weak visible emission band at 600 nm. When the ZnO nanorods were respectively pumped by various wavelength lasers from 520 nm to 700 nm, two-photon excited ultraviolet photoluminescence was observed. The dependence of the two-photon excited photoluminescence intensity on the excitation wavelength and power was investigated in detail.

  9. High-order dispersion effects in two-photon interference

    CERN Document Server

    Mazzotta, Z; Cipriani, D; Olivares, S; Paris, M G A

    2016-01-01

    Two-photon interference and Hong-Ou-Mandel (HOM) effect are relevant tools for quantum metrology and quantum information processing. In optical coherence tomography, HOM effect is exploited to achieve high-resolution measurements with the width of the HOM dip being the main parameter. On the other hand, applications like dense coding require high-visibility performances. Here we address high-order dispersion effects in two-photon interference and study, theoretically and experimentally, the dependence of the visibility and the width of the HOM dip on both the pump spectrum and the downconverted photon spectrum. In particular, a spatial light modulator is exploited to experimentally introduce and manipulate a custom phase function to simulate the high-order dispersion effects.

  10. Two-photon interference from two blinking quantum emitters

    Science.gov (United States)

    Jöns, Klaus D.; Stensson, Katarina; Reindl, Marcus; Swillo, Marcin; Huo, Yongheng; Zwiller, Val; Rastelli, Armando; Trotta, Rinaldo; Björk, Gunnar

    2017-08-01

    We investigate the effect of blinking on the two-photon interference measurement from two independent quantum emitters. We find that blinking significantly alters the statistics in the Hong-Ou-Mandel second-order intensity correlation function g(2 )(τ ) and the outcome of two-photon interference measurements performed with independent quantum emitters. We theoretically demonstrate that the presence of blinking can be experimentally recognized by a deviation from the gD(2 )(0 ) =0.5 value when distinguishable photons from two emitters impinge on a beam splitter. Our findings explain the significant differences between linear losses and blinking for correlation measurements between independent sources and are experimentally verified using a parametric down-conversion photon-pair source. We show that blinking imposes a mandatory cross-check measurement to correctly estimate the degree of indistinguishability of photons emitted by independent quantum emitters.

  11. Two-photon interaction between trapped ions and cavity fields

    CERN Document Server

    Semião, F L

    2006-01-01

    In this paper, we generalize the ordinary two-photon Jaynes-Cummings model (TPJCM) by considering the atom (or ion) to be trapped in a simple harmonic well. A typical setup would be an optical cavity containing a single ion in a Paul trap. Due to the inclusion of atomic vibrational motion, the atom-field coupling becomes highly nonlinear what brings out quite different behaviors for the system dynamics when compared to the ordinary TPJCM. In particular, we derive an effective two-photon Hamiltonian with dependence on the number operator of the ion's center-of-mass motion. This dependence occurs both in the cavity induced Stark-shifs and in the ion-field coupling, and its role in the dynamics is illustrated by showing the time evolution of the probability of occupation of the electronic levels for simple initial preparations of the state of the system.

  12. Two-photon-induced cycloreversion reaction of chalcone photodimers

    Science.gov (United States)

    Träger, J.; Härtner, S.; Heinzer, J.; Kim, H.-C.; Hampp, N.

    2008-04-01

    The photocleavage reaction of chalcone photodimers has been studied using a two-photon process. For this purpose, a novel chalcone dimer has been synthesized as a low molecular weight model substance for polymer bound chalcones and its photochemistry triggered by two-photon-absorption (2PA) has been investigated using a pulsed frequency-doubled Nd:YAG-laser. The 2PA-induced cycloreversion reaction selectively leads to the cleavage of the chalcone photodimers resulting in the formation of monomeric chalcone molecules. Hence, as an application chalcones can be used as a photosensitive linker which can be cleaved beyond an UV-absorbing barrier. The 2PA cross section of the chalcone photodimer was determined to be of 1.1 × 10 -49 cm 4 s photon -1 (11 GM).

  13. Four-dimensional multi-site two-photon excitation

    CERN Document Server

    Daria, Vincent Ricardo; Bowman, Richard; Redman, Stephen; Bachor, Hans-A

    2009-01-01

    We report the first demonstration of dynamic and arbitrary multi-site two-photon excitation in three-dimensional (3D) space using the holographic projection method. Rapid temporal response (fourth dimension) is achieved through high-speed non-iterative and non-optimized calculation of the hologram using a video graphics accelerator board. We verify that the projected asymmetric spot configurations have sufficient spatiotemporal photon density for localized two-photon excitation. This system is a significant advance and ready for applications such as time-resolved 3D photolysis of complex biological cell and neuronal networks, 3D microscopy, non-linear micro-fabrication and volume holographic optical storage.

  14. Simultaneous two-photon excitation of photodynamic therapy agents

    Energy Technology Data Exchange (ETDEWEB)

    Wachter, E.A.; Fisher, W.G. [Oak Ridge National Lab., TN (United States)]|[Photogen, Inc., Knoxville, TN (United States); Partridge, W.P. [Oak Ridge National Lab., TN (United States); Dees, H.C. [Photogen, Inc., Knoxville, TN (United States); Petersen, M.G. [Univ. of Tennessee, Knoxville, TN (United States). College of Veterinary Medicine

    1998-01-01

    The spectroscopic and photochemical properties of several photosensitive compounds are compared using conventional single-photon excitation (SPE) and simultaneous two-photon excitation (TPE). TPE is achieved using a mode-locked titanium:sapphire laser, the near infrared output of which allows direct promotion of non-resonant TPE. Excitation spectra and excited state properties of both type 1 and type 2 photodynamic therapy (PDT) agents are examined.

  15. Two-photon production of charged pion and kaon pairs

    CERN Document Server

    Dominick, J; Sanghera, S; Shelkov, V; Skwarnicki, T; Stroynowski, R; Volobuev, I P; Wei, G; Zadorozhny, P; Artuso, M; Goldberg, M; He, D; Horwitz, N; Kennett, R; Mountain, R; Moneti, G C; Muheim, F; Mukhin, Y; Playfer, S; Rozen, Y; Stone, S; Thulasidas, M; Vasseur, G; Zhu, G; Bartelt, J; Csorna, S E; Egyed, Z; Jain, V; Kinoshita, K; Edwards, K W; Ogg, M; Britton, D I; Hyatt, E R F; MacFarlane, D B; Patel, P M; Akerib, D S; Barish, B C; Chadha, M; Chan, S; Cowen, D F; Eigen, G; Miller, J S; O'Grady, C; Urheim, J; Weinstein, A J; Acosta, D; Athanas, M; Masek, G E; Paar, H P; Sivertz, M; Gronberg, J B; Kutschke, R; Menary, S R; Morrison, R J; Nakanishi, S; Nelson, H N; Nelson, T K; Qiao, C; Richman, J D; Ryd, A; Tajima, H; Sperka, D; Witherell, M S; Procario, M; Balest, R; Cho, K; Daoudi, M; Ford, W T; Johnson, D R; Lingel, K; Lohner, M; Rankin, P; Smith, J G; Alexander, J P; Bebek, C; Berkelman, K; Bloom, K; Browder, T E; Cassel, David G; Cho, H A; Coffman, D M; Drell, P S; Ehrlich, R; Gaidarev, P B; Galik, R S; García-Sciveres, M; Geiser, B; Gittelman, B; Gray, S W; Hartill, D L; Heltsley, B K; Jones, C D; Jones, S L; Kandaswamy, J; Katayama, N; Kim, P C; Kreinick, D L; Ludwig, G S; Masui, J; Mevissen, J; Mistry, N B; Ng, C R; Nordberg, E; Patterson, J R; Peterson, D; Riley, D; Salman, S; Sapper, M; Würthwein, F; Avery, P; Freyberger, A P; Rodríguez, J; Stephens, R; Yang, S; Yelton, J; Cinabro, D; Henderson, S; Liu, T; Saulnier, M; Wilson, R; Yamamoto, H; Bergfeld, T; Eisenstein, B I; Gollin, G; Ong, B; Palmer, M; Selen, M; Thaler, J J; Sadoff, A J; Ammar, R; Ball, S; Baringer, P; Bean, A; Besson, D; Coppage, D; Copty, N K; Davis, R; Hancock, N; Kelly, M; Kwak, N; Lam, H; Kubota, Y; Lattery, M; Nelson, J K; Patton, S; Perticone, D; Poling, R A; Savinov, V; Schrenk, S; Wang, R; Alam, M S; Kim, I J; Nemati, B; O'Neill, J J; Severini, H; Sun, C R; Zoeller, M M; Crawford, G; Daubenmier, C M; Fulton, R; Fujino, D; Gan, K K; Honscheid, K; Kagan, H; Kass, R; Lee, J; Malchow, R L; Skovpen, Y; Sung, M; White, C; Butler, F; Fu, X; Kalbfleisch, G R; Ross, W R; Skubic, P L; Snow, J; Wang, P L; Wood, M; Brown, D N; Fast, J; McIlwain, R L; Miao, T; Miller, D H; Modesitt, M; Payne, D; Shibata, E I; Shipsey, I P J; Wang Pei Ning; Battle, M; Ernst, J; Kwon, Y; Roberts, S; Thorndike, E H; Wang, C H

    1994-01-01

    A measurement of the cross section for the combined two-photon production of charged pion and kaon pairs is performed using 1.2~\\rm fb^{-1} of data collected by the CLEO II detector at the Cornell Electron Storage Ring. The cross section is measured at invariant masses of the two-photon system between 1.5 and 5.0~GeV/c^2, and at scattering angles more than 53^\\circ away from the \\gamma\\gamma collision axis in the \\gamma\\gamma center-of-mass frame. The large background of leptonic events is suppressed by utilizing the CsI calorimeter in conjunction with the muon chamber system. The reported cross section is compared with leading order QCD models as well as previous experiments. In particular, although the functional dependence of the measured cross section disagrees with leading order QCD at small values of the two-photon invariant mass, the data show a transition to perturbative behavior at an invariant mass of approximately 2.5~GeV/c^2. hardcopies with figures can be obtained by writing to to: Pam Morehouse ...

  16. Two photon exchange in elastic electron-nucleon scattering

    Energy Technology Data Exchange (ETDEWEB)

    Peter Blunden; Wolodymyr Melnitchouk; John Tjon

    2005-06-01

    A detailed study of two-photon exchange in unpolarized and polarized elastic electron-nucleon scattering is presented, taking particular account of nucleon finite size effects. Contributions from nucleon elastic intermediate states are found to have a strong angular dependence, which leads to a partial resolution of the discrepancy between the Rosenbluth and polarization transfer measurements of the proton electric to magnetic form factor ratio. The two-photon exchange contribution to the longitudinal polarization transfer ratio P{sub L} is small, whereas the contribution to the transverse polarization transfer ratio P{sub T} is enhanced at backward angles by several percent, increasing with Q{sup 2}. This gives rise to a several percent enhancement of the polarization transfer ratio P{sub T}/P{sub l} at large Q{sup 2} and backward angles. We compare the two-photon exchange effects with data on the ratio of e{sup +p} to e{sup -p} cross sections, which is predicted to be enhanced at backward angles. Finally, we evaluate the corrections to the form factors of the neutron, and estimate the elastic intermediate state contribution to the {sup 3}He form factors.

  17. Recent two-photon physics results from ARGUS

    Science.gov (United States)

    Živko Representing Argus Collaboration, Tomi

    1995-07-01

    Two photon production of π+π+π0π-π-, K+K-π+π-, K+K-π+π0π-, π+π0π-, and π+π- has been studied using the ARGUS detector at the e+e- storage ring DORIS II at DESY. A partial wave analysis was performed on the five-pion and three-pion final states. In the reaction γγ→ωρ0 is showed that the partial-wave with spin and parity (JP,Jz)=(2+,±2) dominates. The cross section and angular distributions of the reaction γγ→φρ0→K+K-π+π- were measured for the first time. The production of the vector-meson pair φω is observed in the two-photon reaction γγ→K+K-π+π0π-. The two-photon width of the tensor meson a2(1320) was measured in the decay channel π+π0π-. An upper limit, significantly lower than indicated by previous experiments was set on the radiative width of the π2(1670) meson. An upper limit was set on the radiative width of the f0(975)in the decay channel π+π-.

  18. Two-Photon Absorption in Organometallic Bromide Perovskites

    KAUST Repository

    Walters, Grant

    2015-07-21

    Organometallic trihalide perovskites are solution processed semiconductors that have made great strides in third generation thin film light harvesting and light emitting optoelectronic devices. Recently it has been demonstrated that large, high purity single crystals of these perovskites can be synthesized from the solution phase. These crystals’ large dimensions, clean bandgap, and solid-state order, have provided us with a suitable medium to observe and quantify two-photon absorption in perovskites. When CH3NH3PbBr3 single crystals are pumped with intense 800 nm light, we observe band-to-band photoluminescence at 572 nm, indicative of two-photon absorption. We report the nonlinear absorption coefficient of CH3NH3PbBr3 perovskites to be 8.6 cm GW-1 at 800 nm, comparable to epitaxial single crystal semiconductors of similar bandgap. We have leveraged this nonlinear process to electrically autocorrelate a 100 fs pulsed laser using a two-photon perovskite photodetector. This work demonstrates the viability of organometallic trihalide perovskites as a convenient and low-cost nonlinear absorber for applications in ultrafast photonics.

  19. Exploring control parameters of two photon processes in solutions

    Indian Academy of Sciences (India)

    Debabrata Goswami; Amit Nag

    2012-01-01

    Two-photon microscopy depends extensively on the two-photon absorption cross-sections of biologically relevant chromophores. High repetition rate (HRR) lasers are essential in multiphoton microscopy for generating satisfactory signal to noise at low average powers. However, HRR lasers generate thermal distortions in samples even with the slightest single photon absorption. We use an optical chopper with HRR lasers to intermittently `blank’ irradiation and effectively minimize thermal effects to result in a femtosecond z-scan setup that precisely measures the two-photon absorption (TPA) cross-sections of chromophores. Though several experimental factors impact such TPA measurements, a systematic effort to modulate and influence TPA characteristics is yet to evolve. Here, we present the effect of several control parameters on the TPA process that are independent of chromophore characteristics for femtosecond laser pulse based measurements; and demonstrate how the femtosecond laser pulse repetition rate, chromophore environment and incident laser polarization can become effective control parameters for such nonlinear optical properties.

  20. Two-Photon Absorption in Organometallic Bromide Perovskites.

    Science.gov (United States)

    Walters, Grant; Sutherland, Brandon R; Hoogland, Sjoerd; Shi, Dong; Comin, Riccardo; Sellan, Daniel P; Bakr, Osman M; Sargent, Edward H

    2015-09-22

    Organometallic trihalide perovskites are solution-processed semiconductors that have made great strides in third-generation thin film light-harvesting and light-emitting optoelectronic devices. Recently, it has been demonstrated that large, high-purity single crystals of these perovskites can be synthesized from the solution phase. These crystals' large dimensions, clean bandgap, and solid-state order have provided us with a suitable medium to observe and quantify two-photon absorption in perovskites. When CH3NH3PbBr3 single crystals are pumped with intense 800 nm light, we observe band-to-band photoluminescence at 572 nm, indicative of two-photon absorption. We report the nonlinear absorption coefficient of CH3NH3PbBr3 perovskites to be 8.6 cm GW(-1) at 800 nm, comparable to epitaxial single-crystal semiconductors of similar bandgap. We have leveraged this nonlinear process to electrically autocorrelate a 100 fs pulsed laser using a two-photon perovskite photodetector. This work demonstrates the viability of organometallic trihalide perovskites as a convenient and low-cost nonlinear absorber for applications in ultrafast photonics.

  1. Solvent and branching effect on the two-photon absorption properties of push-pull triphenylamine derivatives

    OpenAIRE

    Cvejn, Daniel; Michail, E.; Seintis, M.; Klikar, M.; Pytela, Oldřich; Mikysek, Tomáš; Almonasy, Numan; Ludwig, Miroslav; Giannetas, V.; Fakis, M.; Bureš, Filip

    2016-01-01

    The photophysical and two-photon absorption (2PA) properties of two tri-podal molecules and of their quadrupolar and dipolar counterparts are reported for a series of solvents with varying polarity. The molecules possess a tri-phenylamine electron donating group and mono-cyano acceptors while olefinic and acetylenic π-linkers have been used. Branching led to an increase of the molar extinction coefficient and to a slight bathochromic shift of the absorption spectra while the fluorescence quan...

  2. (Un)determined finite regularization dependent quantum corrections: the Higgs decay into two photons and the two photon scattering examples

    CERN Document Server

    Cherchiglia, A L; Nemes, M C; Sampaio, Marcos

    2012-01-01

    We investigate the appearance of arbitrary, regularization dependent parameters introduced by divergent integrals in two a priori finite but superficially divergent amplitudes: the Higgs decay into two photons and the two photon scattering. We use a general parametrization of ultraviolet divergences which explicitates such ambiguities. Thus we separate in a consistent way using Implicit Regularization the divergent, finite and regularization dependent parts of the amplitudes which in turn are written as surface terms. We find that, although finite, these amplitudes are ambiguous before the imposition of physical conditions namely momentum routing invariance in the loops of Feynman diagrams. In the examples we study momentum routing invariance turns out to be equivalent to gauge invariance. We also discuss the results obtained by different regularizations and show how they can be reproduced within our framework allowing for a clear view on the origin of regularization ambiguities.

  3. A two-photon probe for Al(3+) in aqueous solution and its application in bioimaging.

    Science.gov (United States)

    Wang, Haihong; Wang, Bei; Shi, Zhaohua; Tang, Xiaoliang; Dou, Wei; Han, Qingxin; Zhang, Yange; Liu, Weisheng

    2015-03-15

    A salicylimine probe L with a simple structure has been researched more in-depth on fluorescence sensor properties based on two-photon (TP) absorption. L displays excellent selective turn-on fluorescence response for Al(3+) in hexamethylenetetramine-buffered (HMTA) aqueous solution (0.3M, pH=5.8) under one-photon (OP) excitation. With the help of OP fluorescence, TP fluorescence titration, UV-spectra titration and Job's plot, the stoichiometric ratio of L with Al(3+) was determined to be 1:1. The coordination sites and the coordination mechanism of L with Al(3+) were analyzed in detail through (1)H NMR data. Not only with a detection limit of 5.2×10(-9)M in vitro, but also the probe has been successfully used in the live cells and tissues for the imaging of Al(3+) with TP fluorescence microscopy due to the enlarged TP cross section, providing a novel testing method for measuring Al(3+) in solution or cell tissue with low autofluorescence and cytotoxicity.

  4. Development of an automated two-photon absorption cross section spectrometer%双光子吸收截面自动化测量系统研究

    Institute of Scientific and Technical Information of China (English)

    屈军乐; 周藩; 邵永红; 张新富; 仉华; 姜娜; 彭孝军; 肖义

    2013-01-01

    为快速精确测量双光子材料的吸收截面,研究制作了一套基于双光子诱导荧光法的自动化双光子吸收截面谱仪.该系统基于虚拟仪器平台,实现了功率实时反馈、步进电机同步控制、荧光光谱快速采集、线性分析和双光子吸收光谱分析等功能,是集功率反馈控制到光谱采集、处理为一体的软件自动化操作平台,是研究双光子吸收截面的实用工具.%Two-photon absorption cross section is an important property of organic two-photon fluorophores and is critical to the study of two-photon materials. In order to measure two-photon absorption cross section quickly and accurately, we developed an automated two-photon absorption spectrometer that is based on two-photon induced fluorescence method. The system can perform the functions of real-time feedback of power, stepper motor synchronous control, fast acquisition of fluorescence spectra, linear analysis and two-photon absorption spectroscopy analysis using a virtual instrument platform. The system has an integrated and automated software platform for power feedback control, spectra acquisition and data processing. It can function as an important tool in the study of two-photon absorption cross section of fluorophores

  5. Two-photon excitation laser scanning microscopy of porcine nasal septal cartilage following Nd:YAG laser-mediated stress relaxation

    Science.gov (United States)

    Kim, Charlton C.; Wallace, Vincent P.; Rasouli, Alexandre; Coleno, Mariah L.; Dao, Xavier; Tromberg, Bruce J.; Wong, Brian J.

    2000-05-01

    Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within porcine nasal septal cartilage tissue over a 4-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation (lambda equals 1.32 micrometer) using parameters that result in mechanical stress relaxation (6.0 W, 5.4 mm spot diameter). TPM excitation (780 nm) result in induction of fluorescence from endogenous agents such as NADH, NADPH, and flavoproteins in the 400 - 500 nm spectral region. During laser irradiation diffuse reflectance (from a probe HeNe laser, (lambda) equals 632.8 nm), surface temperature, and stress relaxation were measured dynamically. Each specimen received one, two, or three sequential laser exposures (average irradiation times of 5, 6, and 8 seconds). The cartilage reached a peak surface temperature of about 70 degrees Celsius during irradiation. Cartilage denatured in 50% EtOH (20 minutes) was used as a positive control. TPM was performed using a mode-locked 780 nm Titanium:Sapphire (Ti:Al203) beam with a, 63X, 1.2 N.A. water immersion objective (working distance of 200 mm) to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns (lateral resolution equals 35 micrometer X 35 micrometer). Images were obtained immediately following laser exposure, and also after 4 days in culture. In both cases, the irradiated and non-irradiated specimens do not show any discernible difference in general shape or auto fluorescence. In contrast, positive controls (immersed in 50% ethanol), show markedly increased fluorescence relative to both the native and irradiated specimens, in the cytoplasmic region.

  6. Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels.

    Directory of Open Access Journals (Sweden)

    Philipp Steven

    Full Text Available BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM. Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible

  7. Confinement of pyridinium hemicyanine dye within an anionic metal-organic framework for two-photon-pumped lasing

    Science.gov (United States)

    Yu, Jiancan; Cui, Yuanjing; Xu, Hui; Yang, Yu; Wang, Zhiyu; Chen, Banglin; Qian, Guodong

    2013-10-01

    Two-photon-pumped dye lasers are very important because of their applications in wavelength up-conversion, optical data storage, biological imaging and photodynamic therapy. Such lasers are very difficult to realize in the solid state because of the aggregation-caused quenching. Here we demonstrate a new two-photon-pumped micro-laser by encapsulating the cationic pyridinium hemicyanine dye into an anionic metal-organic framework (MOF). The resultant MOF⊃dye composite exhibits significant two-photon fluorescence because of the large absorption cross-section and the encapsulation-enhanced luminescent efficiency of the dye. Furthermore, the well-faceted MOF crystal serves as a natural Fabry-Perot resonance cavity, leading to lasing around 640 nm when pumped with a 1064-nm pulse laser. This strategy not only combines the crystalline benefit of MOFs and luminescent behaviour of organic dyes but also creates a new synergistic two-photon-pumped lasing functionality, opening a new avenue for the future creation of solid-state photonic materials and devices.

  8. Tuning Ag29 nanocluster light emission from red to blue with one and two-photon excitation

    Science.gov (United States)

    Russier-Antoine, Isabelle; Bertorelle, Franck; Hamouda, Ramzi; Rayane, Driss; Dugourd, Philippe; Sanader, Željka; Bonačić-Koutecký, Vlasta; Brevet, Pierre-François; Antoine, Rodolphe

    2016-01-01

    We demonstrate that the tuning of the light emission from red to blue in dihydrolipoic acid (DHLA) capped Ag29 nanoclusters can be trigged with one and two photon excitations. The cluster stoichiometry was determined with mass spectrometry and found to be Ag29(DHLA)12. In a detailed optical investigation, we show that these silver nanoclusters exhibit a strong red photoluminescence visible to the naked eye and characterized by a quantum yield of nearly ~2% upon one-photon excitation. In the nonlinear optical (NLO) study of the properties of the clusters, the two-photon excited fluorescence spectra were recorded and their first hyperpolarizability obtained. The two-photon absorption cross-section at ~800 nm for Ag29(DHLA)12 is higher than 104 GM and the hyperpolarizability is 106 × 10-30 esu at the same excitation wavelength. The two-photon excited fluorescence spectrum appears strongly blue-shifted as compared to the one-photon excited spectrum, displaying a broad band between 400 and 700 nm. The density functional theory (DFT) provides insight into the structural and electronic properties of Ag29(DHLA)12 as well as into interplay between metallic subunit or core and ligands which is responsible for unique optical properties.We demonstrate that the tuning of the light emission from red to blue in dihydrolipoic acid (DHLA) capped Ag29 nanoclusters can be trigged with one and two photon excitations. The cluster stoichiometry was determined with mass spectrometry and found to be Ag29(DHLA)12. In a detailed optical investigation, we show that these silver nanoclusters exhibit a strong red photoluminescence visible to the naked eye and characterized by a quantum yield of nearly ~2% upon one-photon excitation. In the nonlinear optical (NLO) study of the properties of the clusters, the two-photon excited fluorescence spectra were recorded and their first hyperpolarizability obtained. The two-photon absorption cross-section at ~800 nm for Ag29(DHLA)12 is higher than 104

  9. Two-photon quantum interference in plasmonics: theory and applications.

    Science.gov (United States)

    Gupta, S Dutta; Agarwal, G S

    2014-01-15

    We report perfect two-photon quantum interference with near-unity visibility in a resonant tunneling plasmonic structure in folded Kretschmann geometry. This is despite absorption-induced loss of unitarity in plasmonic systems. The effect is traced to perfect destructive interference between the squares of amplitude reflection and transmission coefficients. We further highlight yet another remarkable potential of coincidence measurements as a probe with better resolution as compared to standard spectroscopic techniques. The finer features show up in both angle resolved and frequency resolved studies.

  10. Chromophore design for large two-photon absorption

    Science.gov (United States)

    Dudley, Christopher

    2014-11-01

    Conjugated oligothiophene chromophores are compared and studied for designing large linear and nonlinear absorption cross-sections. Optical properties of chromophores synthesized by the Naval Research Laboratory are modeled to construct a design factor of merit to predict and understand two-photon absorption (TPA) designs. Computer modeling to optimize parameters to produce photo active chromophores is conducted. Geometry, π-center (electron relay) and the electron donor or acceptor groups attached to the π-centers are considered for importance in TPA. This work could serve equally well as guide for quick back of the envelop research or industrial design verifications as well as an outline for introducing computation methods to students.

  11. New two-photon based nanoscopic modalities and optogenetics

    DEFF Research Database (Denmark)

    Glückstad, Jesper

    -matter interaction on these scales involves the combination of optimal light-sculpting [4] with the use of optimized shapes in micro-robotics structures [5]. Microfabrication processes such as two-photon photo-polymerization offer three-dimensional resolutions for creating custom-designed monolithic microstructures...... that can be equipped with optical trapping handles for convenient mechanical control using only optical forces [6]. These microstructures illustrated above can be effectively handled with simultaneous top- and side-view on our BioPhotonics Workstation to undertake six-degree-of-freedom optical actuation...

  12. Two-photon polymerization of immune cell scaffolds

    DEFF Research Database (Denmark)

    Olsen, Mark Holm

    and easy to use chip integrated migration platform. Free-form constructs with three-dimensional (3D) microporosity were fabricated by two-photon polymerization inside the closed microchannel of an injection molded commercially available polymer chip for analysis of directed cell migration. Acrylate...... also present a poly (ethylene glycol) diacrylate (PEGDA) based strategy to fabricate soft 3D hydrogel scaffolds. Our experiments with the hydrogel confirm we can control the mechanical properties and introduce biochemical cues on the surface that are recognized by fibroblast cells. Finally we present...

  13. The Nelson Model with Less Than Two Photons

    CERN Document Server

    Galtbayar, A; Yajima, K

    2002-01-01

    We study the spectral and scattering theory of the Nelson model for an atom interacting with a photon field in the subspace with less than two photons. For the free electron-photon system, the spectral property of the reduced Hamiltonian in the center of mass coordinates and the large time dynamics are determined. If the electron is under the influence of the nucleus via spatially decaying potentials, we locate the essential spectrum, prove the absence of singular continuous spectrum and the existence of the ground state, and construct wave operators giving the asymptotic dynamics.

  14. Two-photon tomography using on-chip quantum walks

    CERN Document Server

    Titchener, James; Sukhorukov, Andrey

    2016-01-01

    We present a conceptual approach to quantum tomography based on first expanding a quantum state across extra degrees of freedom and then exploiting the introduced sparsity to perform reconstruction. We formulate its application to photonic circuits, and show that measured spatial photon correlations at the output of a specially tailored discrete-continuous quantum-walk can enable full reconstruction of any two-photon spatially entangled and mixed state at the input. This approach does not require any tunable elements, so is well suited for integration with on-chip superconducting photon detectors.

  15. Two Photon Decays of Charmonia from Lattice QCD

    Energy Technology Data Exchange (ETDEWEB)

    Jozef Dudek; Robert Edwards

    2006-07-12

    We make the first calculation in lattice QCD of two-photon decays of mesons. Working in the charmonium sector, using the LSZ reduction to relate a photon to a sum of hadronic vector eigenstates, we compute form-factors in both the space-like and time-like domains for the transitions {eta}{sub c} {yields} {gamma}*{gamma}* and {chi}{sub c0} {yields} {gamma}*{gamma}*. At the on-shell point we find approximate agreement with experimental world-average values.

  16. Quantum teleportation of one- and two-photon superposition states

    Institute of Scientific and Technical Information of China (English)

    李英; 张天才; 张俊香; 谢常德

    2003-01-01

    Quantum teleportation of one- and two-photon superposition states based on EPR entanglement of continuouswave two-mode squeezed state is discussed. The fidelities of teleportation are deduced for two different input quantum states. The dependence of the fidelity on the parameters of EPR entanglement and the gain of the classical channels are shown numerically. Comparing with the teleportation of Fock state and coherent state, it is pointed out that for given EPR entanglement and classical gain, the higher the nonclassicality of the input state, the lower the accessible fidelity of teleportation.

  17. Spectral Features of FM Spectroscopy of Two-Photon Interactions

    Institute of Scientific and Technical Information of China (English)

    夏慧荣; JohnL.Hall

    1994-01-01

    The spectral features of FM two-photon resonant interaction processes have been calculated for five different frequency modulation versions of counter-propagating incident fields. It is found that the proposed new modulation version (case b in the text) provides novel spectral features for a completely canceled absorption and a sharp dispersion shape at the fundamental beat note. Moreover, its absorption feature appears at the second harmonic of the RF modulation frequency generated by the joint modes via six interaction pathways without mutual phase shift. Such features persist even when the effects of the second-order sidebands of the incident fields are taken into account. Application potentials are emphasized.

  18. Inclusive $D*^{+-}$ Production in Two-Photon Collisions at LEP

    CERN Document Server

    Achard, P; Aguilar-Benítez, M; Alcaraz, J; Alemanni, G; Allaby, James V; Aloisio, A; Alviggi, M G; Anderhub, H; Andreev, V P; Anselmo, F; Arefev, A; Azemoon, T; Aziz, T; Bagnaia, P; Bajo, A; Baksay, G; Baksay, L; Baldew, S V; Banerjee, S; Banerjee, Sw; Barczyk, A; Barillère, R; Bartalini, P; Basile, M; Batalova, N; Battiston, R; Bay, A; Becattini, F; Becker, U; Behner, F; Bellucci, L; Berbeco, R; Berdugo, J; Berges, P; Bertucci, B; Betev, B L; Biasini, M; Biglietti, M; Biland, A; Blaising, J J; Blyth, S C; Bobbink, Gerjan J; Böhm, A; Boldizsar, L; Borgia, B; Bottai, S; Bourilkov, D; Bourquin, Maurice; Braccini, S; Branson, J G; Brochu, F; Burger, J D; Burger, W J; Cai, X D; Capell, M; Cara Romeo, G; Carlino, G; Cartacci, A M; Casaus, J; Cavallari, F; Cavallo, N; Cecchi, C; Cerrada, M; Chamizo-Llatas, M; Chang, Y H; Chemarin, M; Chen, A; Chen, G; Chen, G M; Chen, H F; Chen, H S; Chiefari, G; Cifarelli, Luisa; Cindolo, F; Clare, I; Clare, R; Coignet, G; Colino, N; Costantini, S; de la Cruz, B; Cucciarelli, S; van Dalen, J A; De Asmundis, R; Déglon, P L; Debreczeni, J; Degré, A; Deiters, K; Della Volpe, D; Delmeire, E; Denes, P; De Notaristefani, F; De Salvo, A; Diemoz, M; Dierckxsens, M; Dionisi, C; Dittmar, Michael; Doria, A; Dova, M T; Duchesneau, D; Echenard, B; Eline, A; El-Mamouni, H; Engler, A; Eppling, F J; Ewers, A; Extermann, Pierre; Falagán, M A; Falciano, S; Favara, A; Fay, J; Fedin, O; Felcini, Marta; Ferguson, T; Fesefeldt, H S; Fiandrini, E; Field, J H; Filthaut, Frank; Fisher, P H; Fisher, W; Fisk, I; Forconi, G; Freudenreich, Klaus; Furetta, C; Galaktionov, Yu; Ganguli, S N; García-Abia, P; Gataullin, M; Gentile, S; Giagu, S; Gong, Z F; Grenier, G; Grimm, O; Grünewald, M W; Guida, M; van Gulik, R; Gupta, V K; Gurtu, A; Gutay, L J; Haas, D; Hakobyan, R S; Hatzifotiadou, D; Hebbeker, T; Hervé, A; Hirschfelder, J; Hofer, H; Hohlmann, M; Holzner, G; Hou, S R; Hu, Y; Jin, B N; Jones, L W; de Jong, P; Josa-Mutuberria, I; Käfer, D; Kaur, M; Kienzle-Focacci, M N; Kim, J K; Kirkby, Jasper; Kittel, E W; Klimentov, A; König, A C; Kopal, M; Koutsenko, V F; Kräber, M H; Krämer, R W; Krenz, W; Krüger, A; Kunin, A; Ladrón de Guevara, P; Laktineh, I; Landi, G; Lebeau, M; Lebedev, A; Lebrun, P; Lecomte, P; Lecoq, P; Le Coultre, P; Le Goff, J M; Leiste, R; Levtchenko, M; Levchenko, P M; Li, C; Likhoded, S A; Lin, C H; Lin, W T; Linde, Frank L; Lista, L; Liu, Z A; Lohmann, W; Longo, E; Lü, Y S; Lübelsmeyer, K; Luci, C; Luminari, L; Lustermann, W; Ma Wen Gan; Malgeri, L; Malinin, A; Maña, C; Mangeol, D J J; Mans, J; Martin, J P; Marzano, F; Mazumdar, K; McNeil, R R; Mele, S; Merola, L; Meschini, M; Metzger, W J; Mihul, A; Milcent, H; Mirabelli, G; Mnich, J; Mohanty, G B; Muanza, G S; Muijs, A J M; Musicar, B; Musy, M; Nagy, S; Natale, S; Napolitano, M; Nessi-Tedaldi, F; Newman, H; Niessen, T; Nisati, A; Nowak, H; Ofierzynski, R A; Organtini, G; Palomares, C; Pandoulas, D; Paolucci, P; Paramatti, R; Passaleva, G; Patricelli, S; Paul, T; Pauluzzi, M; Paus, C; Pauss, Felicitas; Pedace, M; Pensotti, S; Perret-Gallix, D; Petersen, B; Piccolo, D; Pierella, F; Pioppi, M; Piroué, P A; Pistolesi, E; Plyaskin, V; Pohl, M; Pozhidaev, V; Pothier, J; Prokofiev, D O; Prokofev, D; Quartieri, J; Rahal-Callot, G; Rahaman, M A; Raics, P; Raja, N; Ramelli, R; Rancoita, P G; Ranieri, R; Raspereza, A V; Razis, P A; Ren, D; Rescigno, M; Reucroft, S; Riemann, S; Riles, K; Roe, B P; Romero, L; Rosca, A; Rosier-Lees, S; Roth, S; Rosenbleck, C; Roux, B; Rubio, Juan Antonio; Ruggiero, G; Rykaczewski, H; Sakharov, A; Saremi, S; Sarkar, S; Salicio, J; Sánchez, E; Sanders, M P; Schäfer, C; Shchegelskii, V; Schmidt-Kärst, S; Schmitz, D; Schopper, Herwig Franz; Schotanus, D J; Schwering, G; Sciacca, C; Servoli, L; Shevchenko, S; Shivarov, N; Shoutko, V; Shumilov, E; Shvorob, A V; Siedenburg, T; Son, D; Spillantini, P; Steuer, M; Stickland, D P; Stoyanov, B; Strässner, A; Sudhakar, K; Sultanov, G G; Sun, L Z; Sushkov, S V; Suter, H; Swain, J D; Szillási, Z; Tang, X W; Tarjan, P; Tauscher, Ludwig; Taylor, L; Tellili, B; Teyssier, D; Timmermans, C; Ting, Samuel C C; Ting, S M; Tonwar, S C; Tóth, J; Tully, C; Tung, K L; Ulbricht, J; Valente, E; Van, R T; De Walle, M; Veszpremi, V; Vesztergombi, G; Vetlitskii, I; Vicinanza, D; Viertel, Gert M; Villa, S; Vivargent, M; Vlachos, S; Vodopyanov, I; Vogel, H; Vogt, H; Vorobev, I; Vorobyov, A A; Wadhwa, M; Wallraff, W; Wang, X L; Wang, Z M; Weber, M; Wienemann, P; Wilkens, H; Wynhoff, S; Xia, L; Xu, Z Z; Yamamoto, J; Yang, B Z; Yang, C G; Yang, H J; Yang, M; Yeh, S C; Zalite, A; Zalite, Yu; Zhang, Z P; Zhao, J; Zhu, G Y; Zhu, R Y; Zhuang, H L; Zichichi, A; Zilizi, G; Zimmermann, B; Zöller, M

    2002-01-01

    Inclusive D^{*+-} production in two-photon collisions is studied with the L3 detector at LEP, using 683 pb^{-1} of data collected at centre-of-mass energies from 183 to 208 GeV. Differential cross sections are determined as functions of the transverse momentum and pseudorapidity of the D^{*+-} mesons in the kinematic region 1 GeV e^+e^-D^{*+-}X)$ in this kinematical region is measured and the sigma(e^+e^- ---> e^+e^- cc{bar}X) cross section is derived. The measurements are compared with next-to-leading order perturbative QCD calculations.

  19. Two-photon photoassociative spectroscopy of ultracold 88-Sr

    CERN Document Server

    de Escobar, Y N Martinez; Pellegrini, P; Nagel, S B; Traverso, A; Yan, M; Côté, R; Killian, T C

    2008-01-01

    We present results from two-photon photoassociative spectroscopy of the least-bound vibrational level of the X$^1\\Sigma_g^+$ state of the $^{88}$Sr$_2$ dimer. Measurement of the binding energy allows us to determine the s-wave scattering length, $a_{88}=-1.4(6) a_0$. For the intermediate state, we use a bound level on the metastable $^1S_0$-$^3P_1$ potential, which provides large Franck-Condon transition factors and narrow one-photon photoassociative lines that are advantageous for observing quantum-optical effects such as Autler-Townes resonance splittings.

  20. Two-photon photoassociative spectroscopy of ultracold Sr88

    Science.gov (United States)

    Martinez de Escobar, Y. N.; Mickelson, P. G.; Pellegrini, P.; Nagel, S. B.; Traverso, A.; Yan, M.; Côté, R.; Killian, T. C.

    2008-12-01

    We present results from two-photon photoassociative spectroscopy of the least-bound vibrational level of the XΣg+1 state of the Sr288 dimer. Measurement of the binding energy allows us to determine the s -wave scattering length a88=-1.4(6)a0 . For the intermediate state, we use a bound level on the metastable S01-P13 potential, which provides large Franck-Condon transition factors and narrow one-photon photoassociative lines that are advantageous for observing quantum-optical effects such as Autler-Townes resonance splittings.

  1. Correction-free remotely scanned two-photon in vivo mouse retinal imaging

    Institute of Scientific and Technical Information of China (English)

    Adi Schejter Bar-Noam; Nairouz Farah; Shy Shoham

    2016-01-01

    Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications.The in vivotwo-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue.However,to date,this has only been possible using relatively complex adaptive-optics systems.Here,the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality,optically sectioned fundus images.By remotely scanning the focus using an electronically tunable lens,high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired,thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging.Moreover,the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator,GCaMP6s.These results and the simplicity of the new add-on optics are an important step toward several structural,functional,and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.

  2. Optical limiting effect in a two-photon absorption dye doped solid matrix

    Science.gov (United States)

    He, Guang S.; Bhawalkar, Jayant D.; Zhao, Chan F.; Prasad, Paras N.

    1995-10-01

    We recently reported a new lasing dye, trans-4-[p-(N-ethyl-N-hydroxylethylamino)styryl]-N-methylpyridinium tetraphenylborate (ASPT), which has also been shown to possess a strong two-photon absorption (TPA) and subsequent frequency upconversion fluorescence behavior when excited with near infrared laser radiation. Based on the TPA mechanism, a highly efficient optical limiting performance has been demonstrated in a 2 cm long ASPT-doped epoxy rod pumped with 1.06 μm Q-switched laser pulses at 50-250 MW/cm2 intensity levels. The measured nonlinear absorption coefficient reached 6 cm/GW for the tested sample of dopant concentration d0=4×10-3 M/L. The molecular TPA cross section of ASPT in the epoxy matrix is estimated as σ2=2.5×10-18 cm4/GW or σ2'=4.7×10-46 cm4/photon/s, respectively. Two-photon pumped cavity lasing is also observed in an ASPT-doped polymer rod.

  3. Two-photon pumped cavity lasing in novel dye doped bulk matrix rods

    Science.gov (United States)

    He, Guang S.; Zhao, Chan F.; Bhawalkar, Jayant D.; Prasad, Paras N.

    1995-12-01

    Trans-4-[p-(N-ethyl-N-hydroxyethylamino)styryl]-N-methylpyridi that possesses a much greater two-photon absorption cross section and much stronger upconversion fluorescence emission than common organic dyes (such as rhodamine), when excited with near infrared laser radiation. Utilizing ASPT doped bulk polymer rods, two-photon pumped frequency upconverted cavity lasing has been accomplished using a Q-switched Nd:YAG laser as the pump source. The wavelength and pulse duration were ˜600 nm and 3-6 ns, respectively, for the cavity lasing; whereas the corresponding values for pump pulses were 1.06 μm and ˜10 ns, respectively. For a 7 mm long sample rod with a dopant concentration d0=8×10-3 M/L, the conversion efficiency from the absorbed pump energy to the cavity lasing output was ˜3.5% at a pump energy level of 1.3 mJ. The lasing lifetime, in terms of pulse numbers, was more than 4×104 pulses at 2 Hz repetition rate and room temperature.

  4. Properties of two-photon pumped cavity lasing in novel dye doped solid matrices

    Energy Technology Data Exchange (ETDEWEB)

    He, G.S.; Bhawalkar, J.D.; Zhao, C.; Prasad, P.N. [State Univ. of New York, Buffalo, NY (United States). Dept. of Chemistry

    1996-05-01

    Two-photon pumped frequency upconversion cavity lasing at {approximately}600 nm is accomplished in three types of dye-doped solid rods pumped with {approximately}10 ns and 1.06-{micro}m IR laser pulses. The dopant is a new dye, trans-4-[p-(N-ethyl-N-(hydroxyethyl)amino)styryl]-N-methylpyridinium tetraphenylborate, abbreviated as ASPT, which possesses a greater two-photon absorption cross section and stronger upconversion fluorescence emission than common commercial dyes (such as rhodamine). Three different materials were chosen as solid matrices: poly(2-hydroxyethyl methacrylate), VYCOR porous glass, and sol-gel glass. Using a Q-switched Nd:YAG pulse laser as the pump source, strong cavity lasing could be achieved in these three ASPT doped solid rods as well as in ASPT solution in a liquid cell. The spectral, temporal, and spatial characteristics of the cavity lasing output have been systematically investigated. The measured output-input characteristics, lasing lifetime, and damage threshold for the three different rods are presented.

  5. Polarization-Sensitive Two-Photon Microscopy Study of the Organization of Liquid-Crystalline DNA

    Science.gov (United States)

    Mojzisova, Halina; Olesiak, Joanna; Zielinski, Marcin; Matczyszyn, Katarzyna; Chauvat, Dominique; Zyss, Joseph

    2009-01-01

    Abstract Highly concentrated DNA solutions exhibit self-ordering properties such as the generation of liquid-crystalline phases. Such organized domains may play an important role in the global chromatin topology but can also be used as a simple model for the study of more complex 3D DNA structures. In this work, using polarized two-photon fluorescence microscopy, we report on the orientation of DNA molecules in liquid-crystalline phases. For this purpose, we analyze the signal emitted by fluorophores that are noncovalently bound to DNA strands. In nonlinear processes, excitation occurs exclusively in the focal volume, which offers advantages such as the reduction of photobleaching of out-of-focus molecules and intrinsic 3D sectioning capability. Propidium iodide and Hoechst, two fluorophores with different DNA binding modes, have been considered. Polarimetric measurements show that the dyes follow the alignment with respect to the DNA strands and allow the determination of the angles between the emission dipoles and the longitudinal axis of the DNA double strand. These results provide a useful starting point toward the application of two-photon polarimetry techniques to determine the local orientation of condensed DNA in physiological conditions. PMID:19843467

  6. Fully integrated reflection-mode photoacoustic/two-photon microscopy in vivo (Conference Presentation)

    Science.gov (United States)

    Song, Liang; Song, Wei; Zhang, Yang; Zheng, Wei

    2016-03-01

    Using a water-immersion optical objective in conjunction with a miniature 40-MHz ultrasonic transducer, we developed reflection-mode photoacoustic microscopy with a transverse resolution as high as 320 nm. Here, we further integrated two-photon microscopy capability into the system to enable multimodality in vivo biomedical imaging at submicron resolution. As a result, the system is capable of tri-modality label-free imaging of microvasculature, collagen, and cell morphology, based on the contrast of optical absorption, second-harmonic generation, and autofluorescence, respectively. In addition, we demonstrated simultaneous microscopic imaging of neuron and microvasculature in the brain cortex of a living mouse, which may offer new opportunities for studying the mechanisms of neurovascular coupling.

  7. Synthesis and Nonlinear Optical Properties of a New Two-photon Polymerization Initiator: DPAMOB with a Large TPA Cross-section

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xian; YU Xiao-Qiang; ZHANG Bao-Qin; FENG Yun-Guo; TAO Xu-Tang; JIANG Min-Hua

    2006-01-01

    E,E-1,4-Bis(4′-N,N-diphenylaminostyryl)-2,5-dimethoxybenzene (DPAMOB) has been synthesized by a simple and effective solid phase Wittig reaction and characterized by 1H NMR spectra and elemental analysis. Linear absorption, single-photon induced fluorescence and two-photon induced fluorescence spectra were experimentally studied. The new dye has a large two-photon absorption (TPA) cross-section of σr= 1007.2 GM [1 GM= 1 × 10-50results confirm that DPAMOB is a good TPA chromophore and can successfully initiate two-photon photopolymerization of ethoxylated trimethylolpropane triacrylate esters (SR454). Finally, a microstructure has been fabricated by use of DPAMOB as initiator.

  8. Two-Photon Holographic Stimulation of ReaChR

    Science.gov (United States)

    Chaigneau, Emmanuelle; Ronzitti, Emiliano; Gajowa, Marta A.; Soler-Llavina, Gilberto J.; Tanese, Dimitrii; Brureau, Anthony Y. B.; Papagiakoumou, Eirini; Zeng, Hongkui; Emiliani, Valentina

    2016-01-01

    Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue. PMID:27803649

  9. Inclusive D*(+/-) production in two photon collisions at LEP

    CERN Document Server

    Prokofiev, Denis Olegovich

    2001-01-01

    In this thesis I present my results on the measurement of the open charm production in two-photon collision events done with the L3 detector at Large Electron Positron machine (LEP). The data sample was collected from 1997 through 2000 at center-of-mass energies ranging from 183 GeV to 209 GeV, corresponding to a total integrated luminosity of 683.4pb −1. The open charm production in two-photon collision events extrapolated to the full phase space is estimated to be: s&parl0;e+e-&rarrr;e +e-cc&d1;X&parr0;=9 23±69±109±222pb. The differential cross sections d s /dpT(D*±) and d s /d:η(D*±): are also measured as functions of transverse momentum pT(D*±) and the absolute value of pseudorapidity :η(D*±):, respectively. A fit to the data estimating the relative contributions of Direct and Resolved open charm production mechanisms is performed, giving (28.7 ± 5.6)% and (71.3 ± 8.8)%, respectively. Using those relative fractions, the Direct and Resolved process cross sections yield: s&p...

  10. High-order dispersion effects in two-photon interference

    Science.gov (United States)

    Mazzotta, Zeudi; Cialdi, Simone; Cipriani, Daniele; Olivares, Stefano; Paris, Matteo G. A.

    2016-12-01

    Two-photon interference and Hong-Ou-Mandel (HOM) effect are relevant tools for quantum metrology and quantum information processing. In optical coherence tomography, the HOM effect is exploited to achieve high-resolution measurements with the width of the HOM dip being the main parameter. On the other hand, applications like dense coding require high-visibility performance. Here we address high-order dispersion effects in two-photon interference and study, theoretically and experimentally, the dependence of the visibility and the width of the HOM dip on both the pump spectrum and the downconverted photon spectrum. In particular, a spatial light modulator is exploited to experimentally introduce and manipulate a custom phase function to simulate the high-order dispersion effects. Overall, we show that it is possible to effectively introduce high-order dispersion effects on the propagation of photons and also to compensate for such effect. Our results clarify the role of the different dispersion phenomena and pave the way for optimization procedures in quantum technological applications involving PDC photons and optical fibers.

  11. Theory of Two-Photon Absorptions in Graphene Fragments

    Science.gov (United States)

    Aryanpour, K.; Shukla, A.; Mazumdar, S.; Sandhu, A.; Roberts, A.

    2012-02-01

    Electron-electron correlations in graphene is currently an active field of research [1-3]. The carbon atoms in graphene have the same sp^2 hybridization as in strongly correlated π-conjugated polymer systems. The low energy behavior in graphene however appears to be reasonably described within the one-electron Dirac massless fermions model. Historically, the occurrence of the lowest two-photon state below the optical one-photon state provided the strongest proof for strong electron correlations in linear polyenes [4]. We systematically study the Coulomb interaction effects on the ground state and nonlinear absorptions in graphene fragments as a function of system size, beginning from the smallest stable fragment coronene. We report high order calculations of one- vs two-photon spin singlet and triplet states, in coronene, hexabenzocoronene and other molecular fragments that clearly indicate the strong role of electron-electron interactions. We will discuss the implications of our work on molecular systems for the thermodynamic limit of graphene. [4pt] [1] Siegel David A.; et al., PNAS, v108, 28, 11365-11369 (2011)[0pt] [2] Gr"onqvist J. H.; et al., arXiv: 1107.5653v1[0pt] [3] Uchoa B.; et al., arXiv: 1109.1577v1[0pt] [4] Ramasesha S.; et al., J. Chem. Phys. 80, 3278 (1984)

  12. Nonresonant two-photon transitions in length and velocity gauges

    Science.gov (United States)

    Jentschura, U. D.

    2016-08-01

    We reexamine the invariance of two-photon transition matrix elements and corresponding two-photon Rabi frequencies under the "gauge" transformation from the length to the velocity gauge. It is shown that gauge invariance, in the most general sense, only holds at exact resonance, for both one-color as well as two-color absorption. The arguments leading to this conclusion are supported by analytic calculations which express the matrix elements in terms of hypergeometric functions, and ramified by a "master identity" which is fulfilled by off-diagonal matrix elements of the Schrödinger propagator under the transformation from the velocity to the length gauge. The study of the gauge dependence of atomic processes highlights subtle connections between the concept of asymptotic states, the gauge transformation of the wave function, and infinitesimal damping parameters for perturbations and interaction Hamiltonians that switch off the terms in the infinite past and future [of the form exp(-ɛ |t |)] . We include a pertinent discussion.

  13. Simultaneous two-photon excitation of photodynamic therapy agents

    Science.gov (United States)

    Wachter, Eric A.; Partridge, W. P., Jr.; Fisher, Walter G.; Dees, Craig; Petersen, Mark G.

    1998-07-01

    The spectroscopic and photochemical properties of several photosensitive compounds are compared using conventional single-photon excitation (SPE) and simultaneous two-photon excitation (TPE). TPE is achieved using a mode-locked titanium:sapphire laser, the near infrared output of which allows direct promotion of non-resonant TPE. Excitation spectra and excited state properties of both type I and type II photodynamic therapy (PDT) agents are examined. In general, while SPE and TPE selection rules may be somewhat different, the excited state photochemical properties are equivalent for both modes of excitation. In vitro promotion of a two-photon photodynamic effect is demonstrated using bacterial and human breast cancer models. These results suggest that use of TPE may be beneficial for PDT, since the technique allows replacement of visible or ultraviolet excitation with non- damaging near infrared light. Further, a comparison of possible excitation sources for TPE indicates that the titanium:sapphire laser is exceptionally well suited for non- linear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate; these features combine to effect efficient PDT activation with minimal potential for non-specific biological damage.

  14. A [111]-Cut Si Hemisphere Two-Photon Response Photodetector

    Institute of Scientific and Technical Information of China (English)

    LIU Xiu-Huan; CHEN Zhan-Guo; JIA Gang; WANG Hai-Yan; GAO Yan-Jun; LI Yi1

    2011-01-01

    Properties of two-photon response in a [lll]-cut nearly-intrinsic Si hemisphere photodetector are studied. The measured photocurrent of the photodetector responding to the 1.32μm continuous wave laser shows a quadratic dependence on the coupled optical power and is saturated with the bias voitage. Also, the photocurrent is independent of polarization. Such properties are in good agreement with the theory of two-photon absorption. The isotropic photocurrent generated from the [lll]-cut Si hemisphere is compared to the anisotropic one induced in the [110]-cut Si sample and the ratio of Xxxxx /Xxxyy for silicon performing at 1.32μm is calculated to be 2.4 via the fitted function of the anisotropic photocurrent from the [110]-cut sample.%Properties of two-photon response in a [111]-cut nearly-intrinsic Si hemisphere photodetector are studied.The measured photocurrent of the photodetector responding to the 1.32 μm continuous wave laser shows a quadratic dependence on the coupled optical power and is saturated with the bias voltage.Also,the photocurrent is independent of polarization.Such properties are in good agreement with the theory of two-photon absorption.The isotropic photocurrent generated from the [111]-cut Si hemisphere is compared to the anisotropic one induced in the [110]-cut Si sample and the ratio of Xxxxx /Xxxyy for silicon performing at 1.32μm is calculated to be 2.4via the fitted function of the anisotropic photocurrent from the [110]-cut sample.Silicon materials have a variety of applications in microelectronics and silicon optoelectronics and are still attractive to relevant researchers.Commercial Si photodetectors are largely designed based on singlephoton absorption (SPA).However,nonlinear characteristics have been exhibited in silicon devices.Specifically,two-photon absorption (TPA) has attracted much attention in such devices of Si p-n and p-i-n photodiodes,Si waveguides and Si avalanche diodes,etc.for the autocorrelation measurements of

  15. Measurement of Ultra-Short Single-Photon Pulse Duration with Two-Photon Interference

    Institute of Scientific and Technical Information of China (English)

    LV Fan; SUN Fang-Wen; ZOU Chang-Ling; HAN Zheng-Fu; GUO Guang-Can

    2011-01-01

    We proposed a protocol of measuring the duration of ultra-short single-photon pulse with two-photon interference.The pulse duration can be obtained from the width of the visibility of two-photon Hong-Ou-Mandel interference or the indistinguishability of the two photons. Moreover, the shape of a single-photon pulse can be measured with ultra-short single-photon pulses through the two-photon interference.%@@ We proposed a protocol of measuring the duration of ultra-short single-photon pulse with two-photon interference.The pulse duration can be obtained from the width of the visibility of two-photon Hong-Ou-Mandel interference or the indistinguishability of the two photons.Moreover, the shape of a single-photon pulse can be measured with ultra-short single-photon pulses through the two-photon interference.

  16. Flexible non-diffractive vortex microscope for three-dimensional depth-enhanced super-localization of dielectric, metal and fluorescent nanoparticles

    Science.gov (United States)

    Bouchal, Petr; Bouchal, Zdeněk

    2017-10-01

    In the past decade, probe-based super-resolution using temporally resolved localization of emitters became a groundbreaking imaging strategy in fluorescence microscopy. Here we demonstrate a non-diffractive vortex microscope (NVM), enabling three-dimensional super-resolution fluorescence imaging and localization and tracking of metal and dielectric nanoparticles. The NVM benefits from vortex non-diffractive beams (NBs) creating a double-helix point spread function that rotates under defocusing while maintaining its size and shape unchanged. Using intrinsic properties of the NBs, the dark-field localization of weakly scattering objects is achieved in a large axial range exceeding the depth of field of the microscope objective up to 23 times. The NVM was developed using an upright microscope Nikon Eclipse E600 operating with a spiral lithographic mask optimized using Fisher information and built into an add-on imaging module or microscope objective. In evaluation of the axial localization accuracy the root mean square error below 18 nm and 280 nm was verified over depth ranges of 3.5 μm and 13.6 μm, respectively. Subwavelength gold and polystyrene beads were localized with isotropic precision below 10 nm in the axial range of 3.5 μm and the axial precision reduced to 30 nm in the extended range of 13.6 μm. In the fluorescence imaging, the localization with isotropic precision below 15 nm was demonstrated in the range of 2.5 μm, whereas in the range of 8.3 μm, the precision of 15 nm laterally and 30–50 nm axially was achieved. The tracking of nanoparticles undergoing Brownian motion was demonstrated in the volume of 14 × 10 × 16 μm3. Applicability of the NVM was tested by fluorescence imaging of LW13K2 cells and localization of cellular proteins.

  17. Quantitative read-out of Al2O3:C,Mg-based fluorescent nuclear track detectors using a commercial confocal microscope

    CERN Document Server

    Greilich, Steffen; Niklas, Martin; Lauer, Florian; Bestvater, Felix; Jäkel, Oliver

    2014-01-01

    Fluorescent nuclear track detectors (FNTD) show great potential for applications in ion-beam therapy research, such as dosimetry, advanced beam characterization, in-vivo use or as radiobiological assay. A essential feature of FNTDs is their ability to assess the energy loss of single ions yielding for example LET estimations. This article describes the basic characterisations of FNTDs and our read-out system (a Zeiss LSM710 confocal laser scanning microscope) to enable quantative measurements of energy loss.

  18. Simple approach to three-color two-photon microscopy by a fiber-optic wavelength convertor.

    Science.gov (United States)

    Li, Kuen-Che; Huang, Lynn L H; Liang, Jhih-Hao; Chan, Ming-Che

    2016-11-01

    A simple approach to multi-color two-photon microscopy of the red, green, and blue fluorescent indicators was reported based on an ultra-compact 1.03-μm femtosecond laser and a nonlinear fiber. Inside the nonlinear fiber, the 1.03-μm laser pulses were simultaneously blue-shifted to 0.6~0.8 μm and red-shifted to 1.2~1.4 μm region by the Cherenkov radiation and fiber Raman gain effects. The wavelength-shifted 0.6~0.8 μm and 1.2~1.4 μm radiations were co-propagated with the residual non-converted 1.03-μm pulses inside the same nonlinear fiber to form a fiber-output three-color femtosecond source. The application of the multi-wavelength sources on multi-color two-photon fluorescence microscopy were also demonstrated. Overall, due to simple system configuration, convenient wavelength conversion, easy wavelength tunability within the entire 0.7~1.35 μm bio-penetration window and less requirement for high power and bulky light sources, the simple approach to multi-color two-photon microscopy could be widely applicable as an easily implemented and excellent research tool for future biomedical and possibly even clinical applications.

  19. Two-photon transitions driven by a combination of diode and femtosecond lasers

    CERN Document Server

    Moreno, Marco P; Felinto, Daniel; Vianna, Sandra S

    2012-01-01

    We report on the combined action of a cw diode laser and a train of ultrashort pulses when each of them drives one step of the 5S-5P-5D two-photon transition in rubidium vapor. The fluorescence from the 6P_{3/2} state is detected for a fixed repetition rate of the femtosecond laser while the cw-laser frequency is scanned over the rubidium D_{2} lines. This scheme allows for a velocity selective spectroscopy in a large spectral range including the 5D_{3/2} and 5D_{5/2} states. The results are well described in a simplified frequency domain picture, considering the interaction of each velocity group with the cw laser and a single mode of the frequency comb.

  20. In-vivo two-photon imaging of the honey bee antennal lobe

    CERN Document Server

    Haase, Albrecht; Trona, Federica; Anfora, Gianfranco; Vallortigara, Giorgio; Antolini, Renzo; Vinegoni, Claudio

    2010-01-01

    Due to the honey bee's importance as a simple neural model, there is a great need for new functional imaging modalities. Herein we report on the use of two-photon microscopy for in-vivo functional and morphological imaging of the honey bee's olfactory system focusing on its primary centers, the antennal lobes (ALs). Our imaging platform allows for simultaneously obtaining both morphological measurements of the AL and in-vivo calcium recording of neural activities. By applying external odor stimuli to the bee's antennas, we were able to record the characteristic odor response maps. Compared to previous works where conventional fluorescence microscopy is used, our approach offers all the typical advantages of multi-photon imaging, providing substantial enhancement in both spatial and temporal resolutions while minimizing photo-damages and autofluorescence contribution with a four-fold improvement in the functional signal. Moreover, the multi-photon associated extended penetration depth allows for functional ima...

  1. Fiber-optic raster scanning two-photon endomicroscope using a tubular piezoelectric actuator

    Science.gov (United States)

    Do, Dukho; Yoo, Hongki; Gweon, Dae-Gab

    2014-06-01

    A nonresonant, fiber-optic raster scanning endomicroscope was developed using a quarter-tubular piezoelectric (PZT) actuator. A fiber lever mechanism was utilized to enhance the small actuation range of the tubular PZT actuator and to increase its field-of-view. Finite element method simulation of the endoscopic probe was conducted for various conditions to maximize its scanning range. After fabricating the probe using a double clad fiber, we obtained two-photon fluorescence images using raster beam scanning of the fiber. The outer diameter of the probe was 3.5 mm and its rigid distal length was 30 mm including a high numerical aperture gradient index lens. These features are sufficient for input into the instrumental channel of a commercial colonoscope or gastroscope to obtain high resolution images in vivo.

  2. Electric field allowed molecular transitions for one and two photon excitation microscopy.

    Science.gov (United States)

    Mondal, Partha Pratim; Diaspro, Alberto

    2008-07-01

    We propose an excitation technique for observing single and two photon excitation in those molecules for which such transitions are forbidden by the selection rules. This is possible by the application of an external electric field that perturbs the molecular orbitals, thereby resulting in a significant shift of energy levels. Such a shift of energy levels may bring those levels in resonance with the radiation field which is normally forbidden by selection rules. Further, parity of the these states may significantly improve the emission process. The external electric field results in the mixing of excited (short lifetime) and metastable states (long lifetime), thus reducing the lifetime of metastable (or near metastable) states. This may provide an effective channel for allowing transition from the metastable states. An application of electric field may result in the excitation of poorly excitable biomolecules. This excitation technique may find applications in single- and multi-photon fluorescence microscopy, bioimaging and optical devices.

  3. Two-Photon-Absorption Induced Superradiance of a New Organic Dye PSPS

    Institute of Scientific and Technical Information of China (English)

    周广勇; 王东; 王筱梅; 杨胜军; 许心光; 赵显; 邵宗书; 蒋民华

    2002-01-01

    The linear and nonlinear optical properties of a new two-photon absorption (TPA) dye, trans-4-(4'-pyrrolidinyl styryl)-N-methyl pyridinium methyl sulfate (abbreviated as PSPS) is reported. Intense red superradiance with a peak located at 625nm can be observed from PSPS solution in benzyl alcohol when pumped by a focused picosecond laser beam operated at 1064nm. The lifetimes of one-photon absorption (OPA) and TPA fluorescence were measured to be 370 and 384ps, respectively. The pulse widths of OPA and TPA superradiance were 60 and 58 ps, respectively. The highest net upconversion efficiency from the absorbed pump laser to the upconverted superradiance is 8.3% at the pump energy of 0.6 mJ.

  4. Two-photon Absorption In Quantum Dots,quantum Dashes And Related Materials

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Ravinder

    2009-08-31

    We have proposed the use of USQDs for various deep-tissue biological imaging applications, notably wavelength-multiplexed multicolor imaging and intra-nuclear studies such as those involving cell apoptosis, and have studied the issue of maximizing two-photon absorption-induced fluorescence (TPAF) signals from CdSe/ZnS USQDs to be used for this application. In particular, using 2 nm USQDs, we have shown that the TPAF signal at 780 nm is ~ 8 times that at 850 nm and 68 times that at 900 nm, two wavelengths that have been used in previous studies using CdSe/ZnS SQDs for deep-tissue imaging of biological studies via TPAF .

  5. Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

    Science.gov (United States)

    Zhao, Ming; Zhang, Han; Li, Yu; Ashok, Amit; Liang, Rongguang; Zhou, Weibin; Peng, Leilei

    2014-01-01

    In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM. PMID:24876996

  6. Novel Bis-β-diketone-type Ligand and Its Copper and Zinc Complexes for Two-photon Biological Imaging

    Institute of Scientific and Technical Information of China (English)

    ZHOU Shuang-sheng; XUE Xuan; WEI Dong; JIANG Bo; WANG Jia-feng; LU Cheng-hua

    2012-01-01

    A curcumin derivative ligand,1,7-bis(3-methoxyl-4-oxyethylacetate)phenyl-1,6-heptadiene-3,5-diketone (diethyl acetatecurcumin,abbreviated as HL),and its Cu(Ⅱ) and Zn(Ⅱ) complexes have been synthesized and characterized by elemental analyses,infrared(IR),1H NMR and molar conductivity.The experimental results show that the resulting complexes bear strong two-photon excited fluorescence(TPEF) in N,N-dimethyformamide solvent,which has been proven to be potentially useful for two-photon microscopy imaging in living cells.In addition,cytotoxicity tests show that the low-micromolar concentrations of metal-ligand complex(ML2) did not cause significant reduction in cell viability over a pcriod of,at least,24 h and should be safe for further biological studies.

  7. Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy.

    Science.gov (United States)

    Zhao, Ming; Zhang, Han; Li, Yu; Ashok, Amit; Liang, Rongguang; Zhou, Weibin; Peng, Leilei

    2014-05-01

    In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.

  8. Effects of Ox-LDL on Macrophages NAD(P)H Autofluorescence Changes by Two-photon Microscopy

    CERN Document Server

    Lin, Ching-Ting; Lee, Szu-Yuan; Lu, Long-Sheng; Wu, Chau-Chung; Dong, Chen-Yuan; Lin, Chii-Wann

    2007-01-01

    Ox-LDL uptakes by macrophage play a critical role in the happening of atherosclerosis. Because of its low damage on observed cells and better signal-to- background ratio, two-photon excitation fluorescence microscopy is used to observe NAD(P)H autofluorescence of macrophage under difference cultured conditions- bare cover glass, coated with fibronectin or poly-D-lysine. The results show that the optimal condition is fibronectin coated surface, on which, macrophages profile can be clearly identified on NAD(P)H autofluorescence images collected by two-photon microscopy. Moreover, different morphology and intensities of autofluorescence under different conditions were observed as well. In the future, effects of ox-LDL on macrophages will be investigated by purposed system to research etiology of atherosclerosis.

  9. Electromagnetically induced absorption and transparency in an optical-rf two-photon coupling configuration

    Energy Technology Data Exchange (ETDEWEB)

    Fu Guangsheng [College of Physical Science and Technology, Hebei University, Baoding 071002 (China); Li Xiaoli [College of Physical Science and Technology, Hebei University, Baoding 071002 (China)], E-mail: xiaolixiaoli001@yahoo.com.cn; Zhuang Zhonghong; Zhang Lianshui; Yang Lijun; Li Xiaowei; Han Li [College of Physical Science and Technology, Hebei University, Baoding 071002 (China); Manson, Neil B.; Wei Changjiang [Laser Physics Center, Research School of Physical Sciences and Engineering, Australian Nation University, Canberra, ACT 0200 (Australia)

    2008-01-07

    We study electromagnetically induced absorption (EIA) and transparency (EIT) in an optical-rf two-photon coupling configuration. It is shown that the interference effect due to interacting dark resonances results in an EIA for a resonant two-photon coupling and this EIA is observed to evolve into an EIT when there is a detuning in the two-photon coupling.

  10. Two-photon absorption properties of cationic 1,4-bis(styryl)benzene derivative and its inclusion complexes with cyclodextrins.

    Science.gov (United States)

    Nag, Okhil Kumar; Nayak, Rati Ranjan; Lim, Chang Su; Kim, In Hong; Kyhm, Kwangseuk; Cho, Bong Rae; Woo, Han Young

    2010-07-29

    Two-photon absorption properties of 1,4-bis{4'-[N,N-bis(6''-trimethylammoniumhexyl)amino]styryl}benzene tetrabromide (C1) and its inclusion complexes (ICs) with cyclodextrins (CDs) have been studied. Upon complexation with CDs, the absorption spectra of C1 showed a slight red shift, whereas the emission spectra showed a blue shift with concomitant increase in the fluorescence quantum efficiency. A Stern-Volmer study using K(3)Fe(CN)(6) as a quencher revealed significant reduction in the photoinduced charge transfer quenching, in accord with the IC formation. Comparison of the spectroscopic results reveals that C1 forms increasingly more stable ICs in the order C1/beta-CD < C1/gamma-CD < C1/(3gamma:beta)-CD (gamma-CD/beta-CD 3:1, mole ratio). Moreover, the two-photon action cross section of C1 increased from 200 GM for C1 to 400 GM for C1/beta-CD, 460 GM for C1/gamma-CD, and 650 GM for C1/(3gamma:beta)-CD, respectively. Furthermore, the two-photon microscopy images of HeLa cells stained with C1 emitted strong two-photon excited fluorescence in the plasma membrane. These results provide a useful guideline for the development of efficient two-photon materials for bioimaging applications.

  11. Two-Photon Study on the Electronic Interactions between the First Excited Singlet States in Carotenoid-Tetrapyrrole Dyads

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Pen-Nan [Technische Universitat Braunschweig (Germany); Pillai, Smitha [Arizona State Univ., Tempe, AZ (United States); Gust, Devens [Arizona State Univ., Tempe, AZ (United States); Moore, Thomas A. [Arizona State Univ., Tempe, AZ (United States); Moore, Ana L. [Arizona State Univ., Tempe, AZ (United States); Walla, Peter J. [Technische Universitat Braunschweig (Germany)

    2011-03-22

    Electronic interactions between the first excited states (S1) of carotenoids (Car) of different conjugation lengths (8-11 double bonds) and phthalocyanines (Pc) in different Car-Pc dyad molecules were investigated by two-photon spectroscopy and compared with Car S1-chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective two-photon excitation of the Car S1 state allowed sensitive monitoring of the flow of energy between Car S1 and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S1 and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked Car-Pc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S1 → Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc → Car S1 energy transfer. In phenylamino-linked Car-Pc dyads in toluene extremely large electronic interactions between the Car S1 state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S1-Pc Qy interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.

  12. Carbon nanodots featuring efficient FRET for two-photon photodynamic cancer therapy with a low fs laser power density.

    Science.gov (United States)

    Wang, Jing; Zhang, Zehui; Zha, Shuai; Zhu, Yinyan; Wu, Peiyi; Ehrenberg, Benjamin; Chen, Ji-Yao

    2014-11-01

    The 5,10,15,20-tetrakis(1-methyl 4-pyridinio) porphyrins (TMPyP), a photosensitizer used for photodynamic therapy of cancers (PDT), were linked to carbon dots (CDots) to form the conjugates of CDot-TMPyP by the electrostatic force. The 415 nm emission band of CDots was well overlapped with the absorption band of TMPyP, so that the Cdots in conjugates can work as donor to transfer the energy to TMPyP moiety by fluorescence resonance energy transfer (FRET) with an FRET efficiency of 45%, determined by the fluorescence lifetime change between the free CDots and conjugated CDots. The two-photon absorption cross section (TPACS) of TMPyP is as low as 110 GM and the TMPyP thus be not suitable for two-photon PDT. Whereas the CDots have high TPACS, and their TPACS are excitation wavelength dependent with the maximum value of 15000 GM at 700 nm. Therefore, the conjugates of CDot-TMPyP were explored for two-photon excitation (TPE) PDT. The two-photon image of CDot-TMPyP in Hela cells was clearly seen under the excitation of a 700 nm femto-second (fs) laser. The singlet oxygen production of CDot-TMPyP was also much higher than that of TMPyP alone under TPE of a 700 nm fs laser. The in vitro PDT killing was further achieved with CDot-TMPyP by TPE of the 700 nm fs laser. Particularly herein the low power density of fs laser from unfocused laser beam was successfully used to carry out the TPE PDT, because of the high TPACS of CDots. These results demonstrate that the CDot-TMPyP conjugates are promising for TPE PDT and needed to investigate further. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Clinical multiphoton tomography and clinical two-photon microendoscopy

    Science.gov (United States)

    König, Karsten; Bückle, Rainer; Weinigel, Martin; Elsner, Peter; Kaatz, Martin

    2009-02-01

    We report on applications of high-resolution clinical multiphoton tomography based on the femtosecond laser system DermaInspectTM with its flexible mirror arm in Australia, Asia, and Europe. Applications include early detection of melanoma, in situ tracing of pharmacological and cosmetical compounds including ZnO nanoparticles in the epidermis and upper dermis, the determination of the skin aging index SAAID as well as the study of the effects of anti-aging products. In addition, first clinical studies with novel rigid high-NA two-photon 1.6 mm GRIN microendoscopes have been conducted to study the effect of wound healing in chronic wounds (ulcus ulcera) as well as to perform intrabody imaging with subcellular resolution in small animals.

  14. Two-Photon Micromaser with Initial Atomic Coherence

    Institute of Scientific and Technical Information of China (English)

    SUN Wei-Hui; DU Si-De; CHEN Xiao-Shuang

    2005-01-01

    @@ We investigate the quantum dynamics ora two-photon micromaser pumped by atoms injected in the superpositionstate of the upper and intermediate levels. We simulate a master equation governing the system by the MonteCarlo wavefunction approach and analyse the steady-state behaviour as a function of the atomic transit time.The atomic coherence can effectively enhance the intensity and sub-Poissonian of the cavity field as comparedwith the atomic mixture. It is also discovered that the phase of the cavity field can be shifted by adjusting thedetuning between the atom and field. This result shows that it is possible to manipulate the phase of the cavityfield by detuning, due to atomic coherence.

  15. Two-photon resonant, stimulated processes in krypton and xenon

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J.C.

    1988-11-01

    Both on-axis and conical emissions have been observed following two-photon pumping of the 5p states of krypton and the 6p', 7p, 8p, and 4f states of xenon. In the former case, coherent emissions from the 5p states to the 5s are observed, and in the latter case, many p..-->..s, d..-->..p, and f..-->..d cascade emissions are observed. By analogy to the well-studied alkali and alkaline earth examples, the emissions are discussed in terms of amplified spontaneous emission (ASE), stimulated hyper-Raman scattering, and parametric four-wave mixing. The physical processes responsible for the conical emission and for intensity anomalies in the xenon p..-->..s emissions are not understood at present. Interference effects due to coherent cancellation between competing excitation pathways may be occurring. 4 refs., 3 figs.

  16. Whole brain imaging with Serial Two-Photon Tomography

    Directory of Open Access Journals (Sweden)

    Stephen P Amato

    2016-03-01

    Full Text Available Imaging entire mouse brains at submicron resolution has historically been a challenging undertaking and largely confined to the province of dedicated atlasing initiatives. The has limited systematic investigations into important areas of neuroscience, such as neural circuits, brain mapping and neurodegeneration. In this paper, we describe in detail Serial Two-Photon (STP tomography, a robust, reliable method for imaging entire brains with histological detail. We provide examples of how the basic methodology can be extended to other imaging modalities, such as optical coherence tomography, in order to provide unique contrast mechanisms. Furthermore we provide a survey of the research that STP tomography has enabled in the field of neuroscience, provide examples of how this technology enables quantitative whole brain studies, and discuss the current limitations of STP tomography-based approaches

  17. Two-photon assisted clock comparison to picosecond precision

    CERN Document Server

    Zhang, Shi-Wei; Yao, Yin-Ping; Wan, Ren-Gang; Zhang, Tong-Yi

    2015-01-01

    We have experimentally demonstrated a clock comparison scheme utilizing time-correlated photon pairs generated from the spontaneous parametric down conversion process of a laser pumped beta-barium borate crystal. The coincidence of two-photon events are analyzed by the cross correlation of the two time stamp sequences. Combining the coarse and fine part of the time differences at different resolutions, a 64 ps precision for clock synchronization has been realized. We also investigate the effects of hardware devices used in the system on the precision of clock comparison. The results indicate that the detector's time jitter and the background noise will degrade the system performance. With this method, comparison and synchronization of two remote clocks could be implemented with a precision at the level of a few tens of picoseconds.

  18. Measurement of bottom quark production in two photon collisions

    CERN Document Server

    Saremi, Sepehr

    2001-01-01

    The cross section for bottom quark production in two-photon collisions, sigma( e+e- → e+e- bb¯X), is measured for the first time. The measurement is performed with the L3 detector at the Large Electron Positron (LEP) collider at the European Center for Nuclear and Particle Physics (CERN). The data corresponds to 410 pb-1 taken at center-of-mass energies from 189 GeV to 202 GeV. Hadrons containing a bottom quark are identified by detecting electrons or muons from their semi-leptonic decays. The measured cross section is in excess of the Next to Leading Order QCD prediction by a factor of three.

  19. Two-photon transition form factor of c ¯ quarkonia

    Science.gov (United States)

    Chen, Jing; Ding, Minghui; Chang, Lei; Liu, Yu-xin

    2017-01-01

    The two-photon transition of c ¯c quarkonia are studied within a covariant approach based on the consistent truncation scheme of the quantum chromodynamics Dyson-Schwinger equation for the quark propagator and the Bethe-Salpeter equation for the mesons. We find the decay widths of ηc→γ γ and χc 0 ,2→γ γ in good agreement with experimental data. The obtained transition form factor of ηc→γ γ* for a wide range of spacelike photon-momentum-transfer squared is also in agreement with the experimental findings of the BABAR experiment. As a by-product, the decay widths of ηb,χb 0 ,2→γ γ and the transition form factor of ηb,χc 0 ,b 0→γ γ* are predicted, which await experimental testing.

  20. Nuclear two-photon decay in 0 +→0 + transitions

    Science.gov (United States)

    Kramp, J.; Habs, D.; Kroth, R.; Music, M.; Schirmer, J.; Schwalm, D.; Broude, C.

    1987-11-01

    The two-photon decay of the first excited 0 + state of 16O has been measured using the Heidelberg-Darmstadt crystal ball. A branching ratio of {Γ γγ}/{Γ tot} = (6.6±0.5) · 10 -4 was obtained. As in the cases of 40Ca and 90Zr previously reported by us, the 2γ decay of 16O proceeds via double E1 and M1 transitions of similar strength; the evidence is the observed interference term in the 2γ angular correlation. The ratio of the matrix elements {α E1 }/{χ} for 16O was restricted to the two inverse values (-6.2±1.5) or (-0.16±0.04). An interpretation of 2γ matrix elements observed for 16O, 40Ca and 90Zr in terms of the electric polarizabilities and magnetic susceptibility is given leading to a qualitative understanding of this decay mode.

  1. Spectral and lifetime endomicroscopic measurements using one and two-photon excitation

    Science.gov (United States)

    Ibrahim, A.; Poulon, F.; Zanello, M.; Habert, R.; Varlet, P.; Devaux, B.; Kudlinski, A.; Abi Haidar, D.

    2017-02-01

    Current surgical biopsy needs several days for the analysis process to be finished. Anatomopathologists provide analysis reports to the surgeon a few days after the surgical intervention, which makes it a lengthy decision making practice. In addition, the lack of precise guidance often leads to inaccuracies in the selection of tissue regions for biopsy and so necessitates repeating the operation sometimes. Our project aims at reducing this time as well as patient discomfort. In this context, we propose to develop a multimodal nonlinear endomicroscope providing several means of contrast. Among these contrast that are useful in the detection of tumor regions, we note imaging by linear and non-linear fluorescence, by second and third harmonic generation and by reflectance. In addition, this technique allows fluorescence lifetime and spectral measurements. Our endomicroscopic system is based on a new homemade customized double-clad photonic crystal fiber (DC-PCF). Finally, this double-clad micro structured optical fiber insures visible and near infrared excitation. This system was tested by measuring fluorescence lifetime and the spectral shape of a fixed tumoral brain sample in one and two photon excitations.

  2. Integrating two-photon microscopy and cryo-electron microscopy for studying the interaction of Cafeteria roenbergensis and CroV

    Science.gov (United States)

    Aghvami, Seyedmohammadali

    Cafeteria roenbergensis (Cro) is a marine zooplankton; its voracious appetite plays a significant role in regulating bacteria populations. The giant virus that lives within Cro, known as Cafeteria roenbergensis virus (CroV), has an important effect on the mortality of Cro populations. Although viral infections are extremely abundant in oceans, the complete procedure of the infection is still unknown. We study the infection process of Cro by CroV to find out whether the initial contact is through phagocytosis or CroV penetrating the host cell membrane directly. Cro is a moving at speed in the range of 10-100 um/s, therefore, there are many difficulties and challenges for traditional imaging techniques to study this viral-host interaction. We apply two-photon fluorescence microscopy to image this infection process. The image is taken at video rate (30 frame/s), which makes us able to catch the moment of interaction. We are able to image host and virus simultaneously where CroV is stained by SYBR gold dye and Cro is excited through NADH autofluorescence. For further structural biology study, we will obtain atomic level resolution information of infection. After catching the initial moment of infection, we will freeze the sample instantly and image it with cryo-electron microscope .

  3. Theoretical analysis on two-photon absorption spectroscopy in a confined four-level atomic system

    Institute of Scientific and Technical Information of China (English)

    Yuanyuan Li; Jintao Bai; Li Li; Yanpeng Zhang; Xun Hou

    2009-01-01

    We investigate theoretically two-photon absorption spectroscopy modified by a control field in a confined Y-type four-level system. Dicke-narrowing effect occurs both in two-photon absorption lines and the dips of transparency against two-photon absorption due to enhanced contribution of slow atoms. We also find that the suppression and the enhancement of two-photon absorption can be modified by changing the strength of the control field and the detuning of three laser fields. This control of two-photon absorption may have some applications in information processing and optical devices.

  4. Two-photon excited surface plasmon enhanced energy transfer between DAPI and gold nanoparticles: Opportunities in intra-cellular imaging and sensing

    Science.gov (United States)

    Zhang, Yinan; Birch, David J. S.; Chen, Yu

    2011-09-01

    We have demonstrated energy transfer between 4'-6-Diamidino-2-phenylindole (DAPI), a commonly used DNA label, and gold nanoparticles under two-photon excitation in solution using fluorescence lifetime imaging microscopy (FLIM). With comparable size and concentration, gold nanorods (GNRs) are shown to provide more efficient energy transfer than gold nanospheres (GNSs). We attribute this transfer enhancement effect to the longitudinal surface plasmon mode of GNRs overlapping with the excitation wavelength. Energy transfer under two-photon excitation between GNRs and DAPI has also been observed in cell culture and found to be in accord with the solution phase results.

  5. Advances in two photon scanning and scanless microscopy technologies for functional neural circuit imaging.

    Science.gov (United States)

    Schultz, Simon R; Copeland, Caroline S; Foust, Amanda J; Quicke, Peter; Schuck, Renaud

    2017-01-01

    Recent years have seen substantial developments in technology for imaging neural circuits, raising the prospect of large scale imaging studies of neural populations involved in information processing, with the potential to lead to step changes in our understanding of brain function and dysfunction. In this article we will review some key recent advances: improved fluorophores for single cell resolution functional neuroimaging using a two photon microscope; improved approaches to the problem of scanning active circuits; and the prospect of scanless microscopes which overcome some of the bandwidth limitations of current imaging techniques. These advances in technology for experimental neuroscience have in themselves led to technical challenges, such as the need for the development of novel signal processing and data analysis tools in order to make the most of the new experimental tools. We review recent work in some active topics, such as region of interest segmentation algorithms capable of demixing overlapping signals, and new highly accurate algorithms for calcium transient detection. These advances motivate the development of new data analysis tools capable of dealing with spatial or spatiotemporal patterns of neural activity, that scale well with pattern size.

  6. Probing Electron-Phonon Interaction through Two-Photon Interference in Resonantly Driven Semiconductor Quantum Dots

    Science.gov (United States)

    Reigue, Antoine; Iles-Smith, Jake; Lux, Fabian; Monniello, Léonard; Bernard, Mathieu; Margaillan, Florent; Lemaitre, Aristide; Martinez, Anthony; McCutcheon, Dara P. S.; Mørk, Jesper; Hostein, Richard; Voliotis, Valia

    2017-06-01

    We investigate the temperature dependence of photon coherence properties through two-photon interference (TPI) measurements from a single quantum dot (QD) under resonant excitation. We show that the loss of indistinguishability is related only to the electron-phonon coupling and is not affected by spectral diffusion. Through these measurements and a complementary microscopic theory, we identify two independent separate decoherence processes, both of which are associated with phonons. Below 10 K, we find that the relaxation of the vibrational lattice is the dominant contribution to the loss of TPI visibility. This process is non-Markovian in nature and corresponds to real phonon transitions resulting in a broad phonon sideband in the QD emission spectra. Above 10 K, virtual phonon transitions to higher lying excited states in the QD become the dominant dephasing mechanism, this leads to a broadening of the zero phonon line, and a corresponding rapid decay in the visibility. The microscopic theory we develop provides analytic expressions for the dephasing rates for both virtual phonon scattering and non-Markovian lattice relaxation.

  7. Two-Photon Excitation STED Microscopy with Time-Gated Detection.

    Science.gov (United States)

    Coto Hernández, Iván; Castello, Marco; Lanzanò, Luca; d'Amora, Marta; Bianchini, Paolo; Diaspro, Alberto; Vicidomini, Giuseppe

    2016-01-13

    We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture's complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source.

  8. Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope

    Science.gov (United States)

    Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

    2015-01-01

    We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy. PMID:26819828

  9. Imaging living cells with a combined high-resolution multi-photon-acoustic microscope

    Science.gov (United States)

    Schenkl, Selma; Weiss, Eike; Stark, Martin; Stracke, Frank; Riemann, Iris; Lemor, Robert; König, Karsten

    2007-02-01

    With increasing demand for in-vivo observation of living cells, microscope techniques that do not need staining become more and more important. In this talk we present a combined multi-photon-acoustic microscope with the possibility to measure synchronously properties addressed by ultrasound and two-photon fluorescence. Ultrasound probes the local mechanical properties of a cell, while the high resolution image of the two-photon fluorescence delivers insight in cell morphology and activity. In the acoustic part of the microscope an ultrasound wave, with a frequency of GHz, is focused by an acoustic sapphire lens and detected by a piezo electric transducer assembled to the lens. The achieved lateral resolution is in the range of 1μm. Contrast in the images arises mainly from the local absorption of sound in the cells, related to properties, such as mass density, stiffness and viscose damping. Additionally acoustic microscopy can access the cell shape and the state of the cell membrane as it is a intrinsic volume scanning technique.The optical part bases on the emission of fluorescent biomolecules naturally present in cells (e.g. NAD(P)H, protophorphyrin IX, lipofuscin, melanin). The nonlinear effect of two-photon absorption provides a high lateral and axial resolution without the need of confocal detection. In addition, in the near-IR cell damages are drastically reduced in comparison to direct excitation in the visible or UV. Both methods can be considered as minimal invasive, as they relay on intrinsic contrast mechanisms and dispense with the need of staining. First results on living cells are presented and discussed.

  10. Two-Photon Ghost Image and Interference-Diffraction

    Science.gov (United States)

    Shih, Y. H.; Sergienko, A. V.; Pittman, T. B.; Strekalov, D. V.; Klyshko, D. N.

    1996-01-01

    One of the most surprising consequences of quantum mechanics is entanglement of two or more distance particles. The two-particle entangled state was mathematically formulated by Schrodinger. Based on this unusual quantum behavior, EPR defined their 'physical reality' and then asked the question: 'Can Quantum-Mechanical Description of Physical Reality Be Considered Complete?' One may not appreciate EPR's criterion of physical reality and insist that 'no elementary quantum phenomenon is a phenomenon until it is a recorded phenomenon'. Optical spontaneous parametric down conversion (SPDC) is the most effective mechanism to generate an EPR type entangled two-photon state. In SPDC, an optical beam, called the pump, is incident on a birefringent crystal. The pump is intense enough so that nonlinear effects lead to the conversion of pump photons into pairs of photons, historically called signal and idler. Technically, the SPDC is said to be type-1 or type-2, depending on whether the signal and idler beams have parallel or orthogonal polarization. The SPDC conversion efficiency is typically on the order of 10(exp -9) to 10(exp -11), depending on the SPDC nonlinear material. The signal and idler intensities are extremely low, only single photon detection devices can register them. The quantum entanglement nature of SPDC has been demonstrated in EPR-Bohm experiments and Bell's inequality measurements. The following two experiments were recently performed in our laboratory, which are more closely related to the original 1935 EPR gedankenezperiment. The first experiment is a two-photon optical imaging type experiment, which has been named 'ghost image' by the physics community. The signal and idler beams of SPDC are sent in different directions, so that the detection of the signal and idler photons can be performed by two distant photon counting detectors. An aperture object (mask) is placed in front of the signal photon detector and illuminated by the signal beam through a

  11. Vasodilation by in vivo activation of astrocyte endfeet via two-photon calcium uncaging as a strategy to prevent brain ischemia

    Science.gov (United States)

    Chen, Yuanxin; Mancuso, James; Zhao, Zhen; Li, Xuping; Cheng, Jie; Roman, Gustavo; Wong, Stephen T. C.

    2013-12-01

    Decreased cerebral blood flow causes brain ischemia and plays an important role in the pathophysiology of many neurodegenerative diseases, including Alzheimer's disease and vascular dementia. In this study, we photomodulated astrocytes in the live animal by a combination of two-photon calcium uncaging in the astrocyte endfoot and in vivo imaging of neurovasculature and astrocytes by intravital two-photon microscopy after labeling with cell type specific fluorescent dyes. Our study demonstrates that photomodulation at the endfoot of a single astrocyte led to a 25% increase in the diameter of a neighboring arteriole, which is a crucial factor regulating cerebral microcirculation in downstream capillaries. Two-photon uncaging in the astrocyte soma or endfoot near veins does not show the same effect on microcirculation. These experimental results suggest that infrared photomodulation on astrocyte endfeet may be a strategy to increase cerebral local microcirculation and thus prevent brain ischemia.

  12. Multicolor excitation two-photon microscopy: in vivo imaging of cells and tissues

    Science.gov (United States)

    Li, Dong; Zheng, Wei; Qu, Jianan Y.

    2010-02-01

    Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system. Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular, with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early development of cancer in epithelial tissue.

  13. Two-photon polymerization for fabrication of biomedical devices

    Science.gov (United States)

    Ovsianikov, Aleksandr; Doraiswamy, Anand; Narayan, R.; Chichkov, B. N.

    2007-01-01

    Two-photon polymerization (2PP) is a novel technology which allows the fabrication of complex three-dimensional (3D) microstructures and nanostructures. The number of applications of this technology is rapidly increasing; it includes the fabrication of 3D photonic crystals [1-4], medical devices, and tissue scaffolds [5-6]. In this contribution, we discuss current applications of 2PP for microstructuring of biomedical devices used in drug delivery. While in general this sector is still dominated by oral administration of drugs, precise dosing, safety, and convenience are being addressed by transdermal drug delivery systems. Currently, main limitations arise from low permeability of the skin. As a result, only few types of pharmacological substances can be delivered in this manner [7]. Application of microneedle arrays, whose function is to help overcome the barrier presented by the epidermis layer of the skin, provides a very promising solution. Using 2PP we have fabricated arrays of hollow microneedles with different geometries. The effect of microneedle geometry on skin penetration is examined. Our results indicate that microneedles created using 2PP technique are suitable for in vivo use, and for integration with the next generation of MEMS- and NEMS-based drug delivery devices.

  14. Review of two-photon exchange in electron scattering

    Energy Technology Data Exchange (ETDEWEB)

    J. Arrington, P. G. Blunden, W. Melnitchouk

    2011-10-01

    We review the role of two-photon exchange (TPE) in electron-hadron scattering, focusing in particular on hadronic frameworks suitable for describing the low and moderate Q^2 region relevant to most experimental studies. We discuss the effects of TPE on the extraction of nucleon form factors and their role in the resolution of the proton electric to magnetic form factor ratio puzzle. The implications of TPE on various other observables, including neutron form factors, electroproduction of resonances and pions, and nuclear form factors, are summarized. Measurements seeking to directly identify TPE effects, such as through the angular dependence of polarization measurements, nonlinear epsilon contributions to the cross sections, and via e+p to e-p cross section ratios, are also outlined. In the weak sector, we describe the role of TPE and gamma-Z interference in parity-violating electron scattering, and assess their impact on the extraction of the strange form factors of the nucleon and the weak charge of the proton.

  15. Higgs decay into two photons in a warped extra dimension

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, Juliane; Hoerner, Clara; Malm, Raoul; Novotny, Kristiane; Schmell, Christoph [Johannes Gutenberg University, PRISMA Cluster of Excellence and Mainz Institute for Theoretical Physics, Mainz (Germany); Neubert, Matthias [Johannes Gutenberg University, PRISMA Cluster of Excellence and Mainz Institute for Theoretical Physics, Mainz (Germany); Cornell University, Department of Physics, LEPP, Ithaca, NY (United States)

    2014-05-15

    A detailed five-dimensional calculation of the Higgs-boson decay into two photons is performed in both the minimal and the custodially protected Randall-Sundrum (RS) model, where the Standard Model (SM) fields propagate in the bulk and the scalar sector lives on or near the IR brane. It is explicitly shown that the R{sub ξ} gauge invariance of the sum of diagrams involving bosonic fields in the SM also applies to the case of these RS scenarios. An exact expression for the h → γγ amplitude in terms of the five-dimensional (5D) gauge-boson and fermion propagators is presented, which includes the full dependence on the Higgs-boson mass. Closed expressions for the 5D W-boson propagators in theminimal and the custodial RS model are derived, which are valid to all orders in v{sup 2}/M{sup 2}{sub KK}. In contrast to the fermion case, the result for the bosonic contributions to the h → γγ amplitude is insensitive to the details of the localization of the Higgs profile on or near the IR brane. The various RS predictions for the rate of the pp → h → γγ process are compared with the latest LHC data, and exclusion regions for the RS model parameters are derived. (orig.)

  16. Two-Photon-Absorption Scheme for Optical Beam Tracking

    Science.gov (United States)

    Ortiz, Gerardo G.; Farr, William H.

    2011-01-01

    A new optical beam tracking approach for free-space optical communication links using two-photon absorption (TPA) in a high-bandgap detector material was demonstrated. This tracking scheme is part of the canonical architecture described in the preceding article. TPA is used to track a long-wavelength transmit laser while direct absorption on the same sensor simultaneously tracks a shorter-wavelength beacon. The TPA responsivity was measured for silicon using a PIN photodiode at a laser beacon wavelength of 1,550 nm. As expected, the responsivity shows a linear dependence with incident power level. The responsivity slope is 4.5 x 10(exp -7) A/W2. Also, optical beam spots from the 1,550-nm laser beacon were characterized on commercial charge coupled device (CCD) and complementary metal-oxide semiconductor (CMOS) imagers with as little as 13.7 microWatts of optical power (see figure). This new tracker technology offers an innovative solution to reduce system complexity, improve transmit/receive isolation, improve optical efficiency, improve signal-to-noise ratio (SNR), and reduce cost for free-space optical communications transceivers.

  17. Two-Photon Absorption in Conjugated Energetic Molecules.

    Science.gov (United States)

    Bjorgaard, Josiah A; Sifain, Andrew E; Nelson, Tammie; Myers, Thomas W; Veauthier, Jacqueline M; Chavez, David E; Scharff, R Jason; Tretiak, Sergei

    2016-07-07

    Time-dependent density functional theory (TD-DFT) was used to investigate the relationship between molecular structure and the one- and two-photon absorption (OPA and TPA, respectively) properties of novel and recently synthesized conjugated energetic molecules (CEMs). The molecular structures of CEMs can be strategically altered to influence the heat of formation and oxygen balance, two factors that can contribute to the sensitivity and strength of an explosive material. OPA and TPA are sensitive to changes in molecular structure as well, influencing the optical range of excitation. We found calculated vertical excitation energies to be in good agreement with experiment for most molecules. Peak TPA intensities were found to be significant and on the order of 10(2) GM. Natural transition orbitals for essential electronic states defining TPA peaks of relatively large intensity were used to examine the character of relevant transitions. Modification of molecular substituents, such as additional oxygen or other functional groups, produces significant changes in electronic structure, OPA, and TPA and improves oxygen balance. The results show that certain molecules are apt to undergo nonlinear absorption, opening the possibility for controlled, direct optical initiation of CEMs through photochemical pathways.

  18. Two-Photon-Exchange Effects and $\\Delta(1232)$ Deformation

    CERN Document Server

    Zhou, Hai-Qing

    2016-01-01

    The two-photon-exchange (TPE) contribution in $ep\\rightarrow ep\\pi ^0$ with $W=M_{\\Delta}$ and small $Q^2$ is calculated and its corrections to the ratios of electromagnetic transition form factors $R_{EM} = E_{1+}^{(3/2)}/M_{1+}^{(3/2)} $ and $R_{SM} = S_{1+}^{(3/2)}/M_{1+}^{(3/2)}$, are analysed. A simple hadronic model is used to estimate the TPE amplitude. Two phenomenological models, MAID2007 and SAID, are used to approximate the full $ep\\rightarrow ep\\pi ^0$ cross sections which contain both the TPE and the one-photon-exchange (OPE) contributions. The genuine the OPE amplitude is then extracted from an integral equation by iteration. We find that the TPE contribution is not sensitive to whether MAID or SAID is used as input in the region with $Q^2<2$ GeV$^2$. It gives small correction to $R_{EM}$ while for $R_{SM}$, the correction is about -10\\% at small $\\epsilon$ and about $1\\%$ at large $\\epsilon$ for $Q^2\\approx2.5$ GeV$^2$. The large correction from TPE at small $\\epsilon$ must be included in th...

  19. Time-resolved two-photon photoemission from metal surfaces

    CERN Document Server

    Weinelt, M

    2002-01-01

    The Rydberg-like series of image-potential states is a prototype system for loosely bound electrons at a metal surface. The electronic structure and the femtosecond dynamics of these states is studied by high-resolution energy-and time-resolved two-photon photoemission spectroscopy. The electron trapped in the image potential moves virtually freely laterally to the surface where it is subject to inelastic and quasielastic scattering processes which cause decay of population and phase relaxation. The influence of surface corrugation on these processes has been investigated for adsorbates on Cu(001) and stepped Cu(117) and Cu(119) surfaces which are vicinal to Cu(001). The dynamics depend on both the distance of the electron in front of the surface and the parallel momentum. For CO molecules on Cu(001) inelastic scattering into bulk states and adsorbate-induced resonances determine the decay rate. For small numbers of Cu adatoms on Cu(001) and the vicinal surfaces the decay rate of image-potential states is sig...

  20. Synergistic Two-Photon Absorption Enhancement in Photosynthetic Light Harvesting

    Science.gov (United States)

    Chen, Kuo-Mei; Chen, Yu-Wei; Gao, Ting-Fong

    2012-06-01

    The grand scale fixation of solar energies into chemical substances by photosynthetic reactions of light-harvesting organisms provides Earth's other life forms a thriving environment. Scientific explorations in the past decades have unraveled the fundamental photophysical and photochemical processes in photosynthesis. Higher plants, green algae, and light-harvesting bacteria utilize organized pigment-protein complexes to harvest solar power efficiently and the resultant electronic excitations are funneled into a reaction center, where the first charge separation process takes place. Here we show experimental evidences that green algae (Chlorella vulgaris) in vivo display a synergistic two-photon absorption enhancement in their photosynthetic light harvesting. Their absorption coefficients at various wavelengths display dramatic dependence on the photon flux. This newly found phenomenon is attributed to a coherence-electronic-energy-transfer-mediated (CEETRAM) photon absorption process of light-harvesting pigment-protein complexes of green algae. Under the ambient light level, algae and higher plants can utilize this quantum mechanical mechanism to create two entangled electronic excitations adjacently in their light-harvesting networks. Concerted multiple electron transfer reactions in the reaction centers and oxygen evolving complexes can be implemented efficiently by the coherent motion of two entangled excitons from antennae to the charge separation reaction sites. To fabricate nanostructured, synthetic light-harvesting apparatus, the paramount role of the CEETRAM photon absorption mechanism should be seriously considered in the strategic guidelines.

  1. Two-photon holographic optogenetics of neural circuits (Conference Presentation)

    Science.gov (United States)

    Yang, Weijian; Carrillo-Reid, Luis; Peterka, Darcy S.; Yuste, Rafael

    2016-03-01

    Optical manipulation of in vivo neural circuits with cellular resolution could be important for understanding cortical function. Despite recent progress, simultaneous optogenetic activation with cellular precision has either been limited to 2D planes, or a very small numbers of neurons over a limited volume. Here we demonstrate a novel paradigm for simultaneous 3D activation using a low repetition rate pulse-amplified fiber laser system and a spatial light modulator (SLM) to project 3D holographic excitation patterns on the cortex of mice in vivo for targeted volumetric 3D photoactivation. This method is compatible with two-photon imaging, and enables the simultaneous activation of multiple cells in 3D, using red-shifted opsins, such as C1V1 or ReaChR, while simultaneously imaging GFP-based sensors such as GCaMP6. This all-optical imaging and 3D manipulation approach achieves simultaneous reading and writing of cortical activity, and should be a powerful tool for the study of neuronal circuits.

  2. Plasma magnetic field diagnostic using two-photon Doppler-free LIF

    Science.gov (United States)

    Yoon, Young Dae; Bellan, Paul

    2015-11-01

    A detailed description of a new plasma B field diagnostic using Doppler-free two-photon laser-induced fluorescence is presented. The diagnostic is based on a method previously developed in the context of rubidium vapor experiments. Two counter-propagating 393nm diode laser beams are directed into an argon plasma to excite Ar-II ions from 3s2 3p4 4 s4P1 / 2 ⟶ 3s2 3p4 4 p4S3 / 2 ⟶ 3s2 3p4 4 d4P3 / 2 . These levels involve two similar (392.86 and 393.25nm) transition wavelengths, so the two counter-propagating beams effectively cancel out the Doppler effect. The excited ions then decay to the 3s2 3p4 4 p4P1 / 2 level, emitting a 324.98nm line which is to be detected by a photomultiplier tube. The Zeeman splitting -- normally unobservable because of the large Doppler broadening -- of the resultant fluorescence is then to be analyzed, yielding the magnetic field of the particular location. This method is expected to provide a 3-D localized, non-perturbing measurement of magnetic fields. An experimental implementation is currently in progress.

  3. Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues.

    Science.gov (United States)

    Durr, Nicholas J; Weisspfennig, Christian T; Holfeld, Benjamin A; Ben-Yakar, Adela

    2011-02-01

    Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼ 20) and large scattering coefficient (∼ 10 mm(-1)) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue.

  4. Effect of the coherent cancellation of the two-photon resonance on the generation of vacuum ultraviolet light by two-photon reasonantly enhanced four-wave mixing

    Energy Technology Data Exchange (ETDEWEB)

    Payne, M.G.; Garrett, W.R.; Judish, J.P.; Wunderlich, R.

    1988-11-01

    Many of the most impressive demonstrations of the efficient generation of vacuum ultraviolet (VUV) light have made use of two- photon resonantly enhanced four-wave mixing to generate light at ..omega../sub VUV/ = 2..omega../sub L1/ +- ..omega../sub L2/. The two-photon resonance state is coupled to the ground state both by two photons from the first laser, or by a photon from the second laser and one from the generated VUV beam. We show here that these two coherent pathways destructively interfere once the second laser is made sufficiently intense, thereby leading to an important limiting effect on the achievable conversion efficiency. 4 refs.

  5. Distribution of quantum information between an atom and two photons

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Bernhard

    2008-11-03

    The construction of networks consisting of optically interconnected processing units is a promising way to scale up quantum information processing systems. To store quantum information, single trapped atoms are among the most proven candidates. By placing them in high finesse optical resonators, a bidirectional information exchange between the atoms and photons becomes possible with, in principle, unit efficiency. Such an interface between stationary and ying qubits constitutes a possible node of a future quantum network. The results presented in this thesis demonstrate the prospects of a quantum interface consisting of a single atom trapped within the mode of a high-finesse optical cavity. In a two-step process, we distribute entanglement between the stored atom and two subsequently emitted single photons. The long atom trapping times achieved in the system together with the high photon collection efficiency of the cavity make the applied protocol in principle deterministic, allowing for the creation of an entangled state at the push of a button. Running the protocol on this quasi-stationary quantum interface, the internal state of the atom is entangled with the polarization state of a single emitted photon. The entanglement is generated by driving a vacuum-stimulated Raman adiabatic passage between states of the coupled atom-cavity system. In a second process, the atomic part of the entangled state is mapped onto a second emitted photon using a similar technique and resulting in a polarization-entangled two-photon state. To verify and characterize the photon-photon entanglement, we measured a violation of a Bell inequality and performed a full quantum state tomography. The results prove the prior atom-photon entanglement and demonstrate a quantum information transfer between the atom and the two emitted photons. This reflects the advantages of a high-finesse cavity as a quantum interface in future quantum networks. (orig.)

  6. Dynamical modeling of pulsed two-photon interference

    Science.gov (United States)

    Fischer, Kevin A.; Müller, Kai; Lagoudakis, Konstantinos G.; Vučković, Jelena

    2016-11-01

    Single-photon sources are at the heart of quantum-optical networks, with their uniquely quantum emission and phenomenon of two-photon interference allowing for the generation and transfer of nonclassical states. Although a few analytical methods have been briefly investigated for describing pulsed single-photon sources, these methods apply only to either perfectly ideal or at least extremely idealized sources. Here, we present the first complete picture of pulsed single-photon sources by elaborating how to numerically and fully characterize non-ideal single-photon sources operating in a pulsed regime. In order to achieve this result, we make the connection between quantum Monte-Carlo simulations, experimental characterizations, and an extended form of the quantum regression theorem. We elaborate on how an ideal pulsed single-photon source is connected to its photocount distribution and its measured degree of second- and first-order optical coherence. By doing so, we provide a description of the relationship between instantaneous source correlations and the typical experimental interferometers (Hanbury-Brown and Twiss, Hong-Ou-Mandel, and Mach-Zehnder) used to characterize such sources. Then, we use these techniques to explore several prototypical quantum systems and their non-ideal behaviors. As an example numerical result, we show that for the most popular single-photon source—a resonantly excited two-level system—its error probability is directly related to its excitation pulse length. We believe that the intuition gained from these representative systems and characters can be used to interpret future results with more complicated source Hamiltonians and behaviors. Finally, we have thoroughly documented our simulation methods with contributions to the Quantum Optics Toolbox in Python in order to make our work easily accessible to other scientists and engineers.

  7. Voltage-sensitive rhodol with enhanced two-photon brightness.

    Science.gov (United States)

    Kulkarni, Rishikesh U; Kramer, Daniel J; Pourmandi, Narges; Karbasi, Kaveh; Bateup, Helen S; Miller, Evan W

    2017-03-14

    We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue.

  8. Determining the Quark Charges by One and Two Photon Processes.

    Science.gov (United States)

    Janah, Arjun

    1982-05-01

    Testable predictions are presented, which may be used to decide between the gauge theories of integer and fractionally charged quarks (icq and fcq). Two distinctive features of icq are exploited, namely (a) presence of color non-singlet components in weak and electromagnetic currents and (b) possible liberation of color non-singlet states above a threshold energy. Consequences are sought in lepton-hadron interaction processes, taking into account the known "color-suppression" effect. Single photon/weak-boson processes such as (nu)N (--->) (nu)X distinguish between icq and fcq only above color-threshold. Experimental consequences of color-liberation in the above process are obtained. It is found that the gluon-parton contribution survives color-suppression to produce a significant rise in the structure functions when color-threshold is exceeded. Two-photon processes such as e('+)e('-) (--->) e('+)e('-) + 2 jets distinguish between the two theories even below color threshold. To obtain the icq predictions for this process, one must take into account (a) the (momentum -dependent) color suppression and (b) the added contribution from pair production of charged gluons. This is done, and it is observed that: (i) in icq, the ratio R('(gamma)(gamma)(2 jet)) is not simply a number given by the quark charges; it depends on the gluon mass, on kinematics and on the particular differential cross-section considered; (ii) the deviation of icq cross-sections from the fcq values depends crucially on whether one includes "untagged" events; if this is done, the deviation is large; the charged gluon contribution is mainly responsible for this deviation; the quark contribution is smaller than naively expected. Finally, comparison is made with experimental data on e('+)e('-) (--->) e('+)e('-) + 2 jets. Here, icq is found to be in better agreement than fcq, for a broad range of gluon masses. A suitably modified equivalent photon approximation is employed.

  9. Two-photon excitation photodynamic therapy with Photofrin

    Science.gov (United States)

    Karotki, Aliaksandr; Khurana, Mamta; Lepock, James R.; Wilson, Brian C.

    2005-09-01

    Photodynamic therapy (PDT) based on simultaneous two-photon (2-γ) excitation has a potential advantage of highly targeted treatment by means of nonlinear localized photosensitizer excitation. One of the possible applications of 2-γ PDT is a treatment of exodus age-related macular degeneration where highly targeted excitation of photosensitizer in neovasculature is vital for reducing collateral damage to healthy surrounding tissue. To investigate effect of 2-γ PDT Photofrin was used as an archetypal photosensitizer. First, 2-γ absorption properties of Photofrin in the 750 - 900 nm excitation wavelength range were investigated. It was shown that above 800 nm 2-γ interaction was dominant mode of excitation. The 2-γ cross section of Photofrin was rather small and varied between 5 and 10 GM (1 GM = 10-50 cm4s/photon) in this wavelength range. Next, endothelial cells treated with Photofrin were used to model initial effect of 2-γ PDT on neovasculature. Ultrashort laser pulses provided by mode-locked Ti:sapphire laser (pulse duration at the sample 300 fs, repetition rate 90 MHz, mean laser power 10 mW, excitation wavelength 850 nm) were used for the excitation of the photosensitizer. Before 2-γ excitation of the Photofrin cells formed a single continuous sheet at the bottom of the well. The tightly focused laser light was scanned repeatedly over the cell layer. After irradiation the cell layer of the control cells stayed intact while cells treated with photofrin became clearly disrupted. The light doses required were high (6300 Jcm(-2) for ~ 50% killing), but 2-γ cytotoxicity was unequivocally demonstrated.

  10. A Two- Photon Femtosecond Laser System for Three-Dimensional Microfabrication and Data Storage

    Institute of Scientific and Technical Information of China (English)

    蒋中伟; 周拥军; 袁大军; 黄文浩; 夏安东

    2003-01-01

    Utilizing the well-focused femtosecond laser with extreme high pulse intensity, we built a two-photon microfabrication and data storage system, which was introduced through several functional parts. Based on this homemade system, several three-dimensional microstructures were fabricated by two-photon polymerization, and three-dimensional data storage of six-layers was achieved by two-photon excitation with a photochromic material.

  11. Two-photon path-entangled states in multi-mode waveguides

    CERN Document Server

    Poem, Eilon; Silberberg, Yaron

    2012-01-01

    We experimentally show that two-photon path-entangled states can be coherently manipulated by multi-mode interference in multi-mode waveguides. By measuring the output two-photon spatial correlation function versus the phase of the input state, we show that multi-mode waveguides perform as nearly-ideal multi-port beam splitters at the quantum level, creating a large variety of entangled and separable multi-path two-photon states.

  12. Two-photon approximation in the theory of the electron recombination in hydrogen

    OpenAIRE

    Solovyev, D.; Labzowsky, L.

    2010-01-01

    A rigorous QED theory of the multiphoton decay of excited states in hydrogen atom is presented. The "two-photon" approximation is formulated which is limited by the one-photon and two-photon transitions including cascades transitions with two-photon links. This may be helpful for the strict description of the recombination process in hydrogen atom and, in principle, for the history of the hydrogen recombination in the early Universe.

  13. Description of the states of two-photon interference in an optical gating Michelson interferometer

    Science.gov (United States)

    Pongophas, Ekkarat; Sriklin, Watthana; Sinsarp, Asawin; Suwanna, Sujin; Chunwachirasiri, Withoon; Singhsomroje, Wisit

    2016-01-01

    We investigate the interference of two photons in an optical gating Michelson interferometer. The phenomenon is studied using two different representations of photons: the space-time domain and a step-by-step two-photon state evolution. Both representations lead to identical results. The evolution analysis describes the result by the interference of four two-photon traveling states, whereas the space-time domain analysis reveals that the classical interference of the high-intensity light source is identical to two-photon interference in the quantum regime, except for a multiplicative factor of (n2), where n is the number of photons.

  14. Two-photon induced photoluminescence and singlet oxygen generation from aggregated gold nanoparticles.

    Science.gov (United States)

    Jiang, Cuifeng; Zhao, Tingting; Yuan, Peiyan; Gao, Nengyue; Pan, Yanlin; Guan, Zhenping; Zhou, Na; Xu, Qing-Hua

    2013-06-12

    Metal nanoparticles have potential applications as bioimaging and photosensitizing agents. Aggregation effects are generally believed to be adverse to their biomedical applications. Here we have studied the aggregation effects on two-photon induced photoluminescence and singlet oxygen generation of Au nanospheres and Au nanorods of two different aspect ratios. Aggregated Au nanospheres and short Au nanorods were found to display enhanced two-photon induced photoluminescence and singlet oxygen generation capabilities compared to the unaggregated ones. The two-photon photoluminescence of Au nanospheres and short Au nanorods were enhanced by up to 15.0- and 2.0-fold upon aggregation, and the corresponding two-photon induced singlet oxygen generation capabilities were enhanced by 8.3 and 1.8-fold, respectively. The two-photon induced photoluminescence and singlet oxygen generation of the aggregated long Au nanorods were found to be lower than the unaggregated ones. These results support that the change in their two-photon induced photoluminescence and singlet oxygen generation originate from aggregation modulated two-photon excitation efficiency. This finding is expected to foster more biomedical applications of metal nanoparticles as Au nanoparticles normally exist in an aggregated form in the biological environments. Considering their excellent biocompatibility, high inertness, ready conjugation, and easy preparation, Au nanoparticles are expected to find more applications in two-photon imaging and two-photon photodynamic therapy.

  15. Time-reversed two-photon interferometry for phase super-resolution

    CERN Document Server

    Ogawa, Kazuhisa; Kobayashi, Hirokazu; Nakanishi, Toshihiro; Kitano, Masao

    2013-01-01

    We observed two-photon phase super-resolution in an unbalanced Michelson interferometer with classical Gaussian laser pulses. Our work is a time-reversed version of a two-photon interference experiment using an unbalanced Michelson interferometer. A measured interferogram exhibits two-photon phase super-resolution with a high visibility of 97.9% \\pm 0.4%. Its coherence length is about 22 times longer than that of the input laser pulses. It is a classical analogue to the large difference between the one- and two-photon coherence lengths of entangled photon pairs.

  16. Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.

    Science.gov (United States)

    Cai, Huawei; Singh, Ajay N; Sun, Xiankai; Peng, Fangyu

    2015-01-01

    To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron

  17. Two-photon absorption and spectroscopy of the lowest two-photon transition in small donor-acceptor-substituted organic molecules

    Science.gov (United States)

    Beels, Marten T.; Biaggio, Ivan; Reekie, Tristan; Chiu, Melanie; Diederich, François

    2015-04-01

    We determine the dispersion of the third-order polarizability of small donor-acceptor substituted organic molecules using wavelength-dependent degenerate four-wave mixing experiments in solutions with varying concentrations. We find that donor-acceptor-substituted molecules that are characterized by extremely efficient off-resonant nonlinearities also have a correspondingly high two-photon absorption cross section. The width and shape of the first two-photon resonance for these noncentrosymmetric molecules follows what is expected from their longest wavelength absorption peak, and the observed two-photon absorption cross sections are record high when compared to the available literature data, the size of the molecule, and the fundamental limit for two-photon absorption to the lowest excited state, which is essentially determined by the number of conjugated electrons and the excited-state energies. The two-photon absorption of the smallest molecule, which only has 16 electrons in its conjugated system, is one order of magnitude larger than for the molecule called AF-50, a reference molecule for two-photon absorption [O.-K. Kim et al., Chem. Mater. 12, 284 (2000), 10.1021/cm990662r].

  18. Application of magnetic and core-shell nanoparticles to determine enrofloxacin and its metabolite using laser induced fluorescence microscope.

    Science.gov (United States)

    Kim, Suji; Ko, Junga; Lim, H B

    2013-04-10

    A unique analytical method using nanoparticles and laser-induced fluorescence microscopy (LIFM) was developed to determine enrofloxacin in this work. For sample pretreatment, two different kinds of particles, i.e., synthesized dye-doped core-shell silica nanoparticles and magnetic micro-particles (MPs), were used for fluorescent tagging and concentrating the enrofloxacin, respectively. The antibody of enrofloxacin was immobilized on the synthesized FITC-doped core-shell nanoparticles, and the enrofloxacin target was extracted by the MPs. At this moment, the average number of antibodies on each core-shell silica nanoparticle was ~0.9, which was determined by the fluorescence ratiometric method. The described method was demonstrated for a meat sample to determine enrofloxacin using LIFM, and the result was compared with enzyme-linked immunosorbent assay (ELISA). The developed technique allowed the simplified analytical procedure, improved the detection limit about 54-fold compared to ELISA.

  19. Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures

    Science.gov (United States)

    Inagawa, H.; Toratani, Y.; Motohashi, K.; Nakamura, I.; Matsushita, M.; Fujiyoshi, S.

    2015-01-01

    We have developed a cryogenic fluorescence microscope system, the core of which is a reflecting objective that consists of spherical and aspherical mirrors. The use of an aspherical mirror allows the reflecting objective to have a numerical aperture (NA) of up to 0.99, which is close to the maximum possible NA of 1.03 in superfluid helium. The performance of the system at a temperature of 1.7 K was tested by recording a three-dimensional fluorescence image of individual quantum dots using excitation wavelengths (λex) of 532 nm and 635 nm. At 1.7 K, the microscope worked with achromatic and nearly diffraction-limited performance. The 1/e2 radius (Γ) of the point spread function of the reflecting objective in the lateral (xy) direction was 0.212 ± 0.008 μm at λex = 532 nm and was less than 1.2 times the simulated value for a perfectly polished objective. The radius Γ in the axial (z) direction was 0.91 ± 0.04 μm at λex = 532 nm and was less than 1.4 times the simulated value of Γ. The chromatic aberrations between the two wavelengths were one order of magnitude smaller than Γ in each direction. PMID:26239746

  20. A Novel Imaging Biomarker Extracted from Fluorescence Microscopic Imaging of TRA-8/DR5 Oligomers Predicts TRA-8 Therapeutic Efficacy in Breast and Pancreatic Cancer Mouse Models.

    Science.gov (United States)

    Kim, Harrison; Buchsbaum, Donald J; Zinn, Kurt R

    2016-06-01

    The aim of the study was to develop a reliable quantitative imaging biomarker from fluorescence microscopic imaging of TRA-8/death receptor 5 (DR5) oligomer to predict TRA-8 therapeutic efficacy in human breast and pancreatic cancer mouse models. Two breast (2LMP, SUM159) and two pancreatic (MIA PaCa-2, PANC1) cancer cell lines were used. 10(5) cells per cell line were placed in a culture dish and treated with Cy5.5-labeled TRA-8 overnight in vitro. Three fluorescence microphotographs (×20) were acquired from randomly selected areas, and about 300 cells were analyzed per cell line. Two-dimensional (2D) fluorescence signal distribution of Cy5.5-TRA-8 on each cell was measured. Gaussian curve fitting to the distribution was determined by the least square regression method, and the coefficient of determination (R (2)) of the fitting was found. In addition, two features of the best fitting Gaussian curve such as peak amplitude and the volume under the curve (VUC) were retrieved. A novel image biomarker was extracted by correlating the combination of R (2) value, peak amplitude, and the VUC with the logarithmic values of the half maximal inhibitory concentrations (IC50) of TRA-8 for the four cell lines or the percentage of tumor growth inhibition (%TGI) at a week of TRA-8 treatment in animal models. Cy5.5-TRA-8 binding to DR5 receptors resulted in an oligomer on each cell membrane, and its fluorescence signal distribution followed Gaussian curve. Peak amplitude of fluorescence signal in the oligomeric region, R (2) value of the Gaussian fitting, and the VUC in TRA-8-sensitive cells were significantly higher than those in resistant cells (p TRA-8 may serve as a predictive biomarker of TRA-8 therapy for cancer patients.