WorldWideScience

Sample records for dual-color two-photon microscopy

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.

    Science.gov (United States)

    Brodehl, Andreas; Hedde, Per Niklas; Dieding, Mareike; Fatima, Azra; Walhorn, Volker; Gayda, Susan; Šarić, Tomo; Klauke, Bärbel; Gummert, Jan; Anselmetti, Dario; Heilemann, Mike; Nienhaus, Gerd Ulrich; Milting, Hendrik

    2012-05-01

    Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.

  6. Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants*

    Science.gov (United States)

    Brodehl, Andreas; Hedde, Per Niklas; Dieding, Mareike; Fatima, Azra; Walhorn, Volker; Gayda, Susan; Šarić, Tomo; Klauke, Bärbel; Gummert, Jan; Anselmetti, Dario; Heilemann, Mike; Nienhaus, Gerd Ulrich; Milting, Hendrik

    2012-01-01

    Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies. PMID:22403400

  7. 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.

  8. 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.

  9. 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

  10. Axial range of conjugate adaptive optics in two-photon microscopy

    CERN Document Server

    Paudel, Hari P; Mertz, Jerome; Bifano, Thomas

    2015-01-01

    We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.

  11. 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.

  12. 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

  13. Polarization-resolved two-photon luminescence microscopy of V-groove arrays

    DEFF Research Database (Denmark)

    Beermann, J.; Novikov, S. M.; Holmgaard, T.

    2012-01-01

    Using two-photon luminescence (TPL) microscopy and local reflection spectroscopy we investigate electromagnetic field enhancement effects from a mu m-sized composition of 450-nm-deep V-grooves milled by focused ion beam in a thick gold film and assembled to feature, within the same structure...

  14. 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

  15. 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.

  16. Polarization-resolved two-photon luminescence microscopy of V-groove arrays

    DEFF Research Database (Denmark)

    Beermann, J.; Novikov, S. M.; Holmgaard, T.;

    2012-01-01

    Using two-photon luminescence (TPL) microscopy and local reflection spectroscopy we investigate electromagnetic field enhancement effects from a mu m-sized composition of 450-nm-deep V-grooves milled by focused ion beam in a thick gold film and assembled to feature, within the same structure...... obtained to evaluation of local field enhancements using TPL microscopy, especially when investigating extended structures exhibiting different radiation channels, are discussed. (C)2011 Optical Society of America...

  17. 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.

  18. Imaging zebrafish embryos by two-photon excitation time-lapse microscopy.

    Science.gov (United States)

    Carvalho, Lara; Heisenberg, Carl-Philipp

    2009-01-01

    The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. In particular, 2PE microscopy has been shown to be useful for imaging deep cell layers within the embryo and following tissue morphogenesis over long periods. This chapter describes how to use 2PE microscopy to study morphogenetic movements during early zebrafish embryonic development, providing a general blueprint for its use in zebrafish.

  19. Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

    Science.gov (United States)

    Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

    2012-08-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

  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. Tailored probes for atomic force microscopy fabricated by two-photon polymerization

    Science.gov (United States)

    Göring, Gerald; Dietrich, Philipp-Immanuel; Blaicher, Matthias; Sharma, Swati; Korvink, Jan G.; Schimmel, Thomas; Koos, Christian; Hölscher, Hendrik

    2016-08-01

    3D direct laser writing based on two-photon polymerization is considered as a tool to fabricate tailored probes for atomic force microscopy. Tips with radii of 25 nm and arbitrary shape are attached to conventionally shaped micro-machined cantilevers. Long-term scanning measurements reveal low wear rates and demonstrate the reliability of such tips. Furthermore, we show that the resonance spectrum of the probe can be tuned for multi-frequency applications by adding rebar structures to the cantilever.

  2. 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.

  3. 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.

  4. 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.

  5. Two-photon microscopy for imaging germinal centers and T follicular helper cells.

    Science.gov (United States)

    Clatworthy, Menna R

    2015-01-01

    One of the principle features of immune cells is their dynamic nature. Lymphocytes circulate in the blood between secondary lymphoid organs and tissues in an effort to maximize the likelihood of a rapid and appropriate immune response to invading pathogens and tissue damage. Conventional experimental techniques such as histology and flow cytometry have greatly increased our understanding of immune cells, but in the last decade, two-photon microscopy has revolutionized our ability to interrogate the dynamic behavior of immune cells, a facet so critical to their function. Two-photon microscopy relies on the excitation of fluorophores by simultaneous application of two photons of longer wavelength light. This allows a greater depth of imaging with minimal photodamage. Thus, living tissues can be imaged, including immune cells in lymph nodes. This technique has been used to interrogate the events occurring in a germinal center response and the interactions between cells in the germinal center, including T follicular helper cells (Tfh), germinal center B cells, and follicular dendritic cells (FDC). Herein, a method is described by which the interactions between Tfh and B cells within a germinal center in a popliteal lymph node can be imaged in a live mouse.

  6. 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.

  7. In vivo reactive neural plasticity investigation by means of correlative two photon: electron microscopy

    Science.gov (United States)

    Allegra Mascaro, A. L.; Cesare, P.; Sacconi, L.; Grasselli, G.; Mandolesi, G.; Maco, B.; Knott, G.; Huang, L.; De Paola, V.; Strata, P.; Pavone, F. S.

    2013-02-01

    In the adult nervous system, different populations of neurons correspond to different regenerative behavior. Although previous works showed that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury1, we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo. Time lapse two-photon imaging has been combined to laser nanosurgery2, 3 to define a temporal pattern of the degenerative event and to follow the structural rearrangement after injury. To characterize the damage and to elucidate the possible formation of new synaptic contacts on the sprouted branches of the lesioned CF, we combined two-photon in vivo imaging with block face scanning electron microscopy (FIB-SEM). Here we describe the approach followed to characterize the reactive plasticity after injury.

  8. 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.

  9. 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

  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. 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

  12. 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.

  13. 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.

  14. 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.

  15. Automated filtering of intrinsic movement artifacts during two-photon intravital microscopy.

    Directory of Open Access Journals (Sweden)

    Denis Soulet

    Full Text Available In vivo imaging using two-photon microscopy is an essential tool to explore the dynamic of physiological events deep within biological tissues for short or extended periods of time. The new capabilities offered by this technology (e.g. high tissue penetrance, low toxicity have opened a whole new era of investigations in modern biomedical research. However, the potential of using this promising technique in tissues of living animals is greatly limited by the intrinsic irregular movements that are caused by cardiac and respiratory cycles and muscular and vascular tone. Here, we show real-time imaging of the brain, spinal cord, sciatic nerve and myenteric plexus of living mice using a new automated program, named Intravital_Microscopy_Toolbox, that removes frames corrupted with motion artifacts from time-lapse videos. Our approach involves generating a dissimilarity score against precalculated reference frames in a specific reference channel, thus allowing the gating of distorted, out-of-focus or translated frames. Since the algorithm detects the uneven peaks of image distortion caused by irregular animal movements, the macro allows a fast and efficient filtering of the image sequence. In addition, extra features have been implemented in the macro, such as XY registration, channel subtraction, extended field of view with maximum intensity projection, noise reduction with average intensity projections, and automated timestamp and scale bar overlay. Thus, the Intravital_Microscopy_Toolbox macro for ImageJ provides convenient tools for biologists who are performing in vivo two-photon imaging in tissues prone to motion artifacts.

  16. Evaluation of cryo-preserved skin tissues using two-photon microscopy

    Science.gov (United States)

    Riemann, Iris; Beier, Axel; Schwarz, Martin; Dörr, Daniel; Stracke, Frank; Zimmermann, Heiko

    2010-02-01

    If no fresh skin samples can be obtained or used, it is important for research and industries to have models and stored tissue samples as close to the native state as possible at disposal. One way to preserve tissues for a longer timeframe is to use deep freezing cryo-techniques. Unfortunately much damage can be induced during the cooling and the thawing processes like disruption of cells and extra-cellular matrices due to the formation of ice crystals. This could lead to a disturbance of the united cell structure up to the point of a loss of cell viability. Two-photon microscopy is able to gather information about cells and tissue components via excitation of the autofluorescence deep inside the sample with a high resolution in both, frozen and thawed states. It is possible to monitor the samples before and after and, important, observe events during the freezing process like the formation of ice crystals. To determine the state of skin tissues after slow rate freezing and the quick process of vitrification, the samples were examined with two-photon microscopy. To establish an optimized freezing-protocol for skin tissues, morphological changes, changes in autofluorescence of endogenous fluorophores (NADH, keratin, flavins, elastin) or changes in second harmonic generation of collagen fibres could provide information about the quality of the used freezing parameters and protective additives and lead to an optimized freezing-protocol with a new set of parameters to obtain mostly intact tissue samples. Multiphoton microscopy has been established as a useful tool for optical in situ quality control of frozen tissues.

  17. 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.

  18. 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

  19. 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.

  20. Nanoshells for in vivo imaging using two-photon excitation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gao Liang; Nammalvar, Vengadesan [Department of Bioengineering, Rice University, Houston, TX 77005 (United States); Vadakkan, Tegy J, E-mail: lg3@rice.edu, E-mail: venkyn@rice.edu [Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 (United States)

    2011-09-07

    Gold nanoshells have been intensively investigated and applied to various biomedical fields because of their flexible optical tunability and biological compatibility. They hold great potential to serve as luminescent contrast agents excitable with near-infrared (NIR) lasers. In this paper, we describe the development of nanoshells with a peak of plasmon resonance at 800 nm and their subsequent use for in vivo blood vessel imaging using two-photon excitation microscopy at an excitation wavelength of 750 nm. We were able to image single nanoshell particles in blood vessels and generate optical contrast for blood vessel structure using luminescent signals. These results confirm the feasibility of engineering nanoshells with controlled optical properties for single-particle-based in vivo imaging.

  1. Nanoshells for in vivo imaging using two-photon excitation microscopy.

    Science.gov (United States)

    Gao, Liang; Vadakkan, Tegy J; Nammalvar, Vengadesan

    2011-09-07

    Gold nanoshells have been intensively investigated and applied to various biomedical fields because of their flexible optical tunability and biological compatibility. They hold great potential to serve as luminescent contrast agents excitable with near-infrared (NIR) lasers. In this paper, we describe the development of nanoshells with a peak of plasmon resonance at 800 nm and their subsequent use for in vivo blood vessel imaging using two-photon excitation microscopy at an excitation wavelength of 750 nm. We were able to image single nanoshell particles in blood vessels and generate optical contrast for blood vessel structure using luminescent signals. These results confirm the feasibility of engineering nanoshells with controlled optical properties for single-particle-based in vivo imaging.

  2. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.

    Science.gov (United States)

    Cahalan, Michael D; Parker, Ian

    2008-01-01

    The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the field of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.

  3. Two-photon microscopy with diffractive optical elements and spatial light modulators

    Directory of Open Access Journals (Sweden)

    Brendon O Watson

    2010-09-01

    Full Text Available Two-photon microscopy is often performed at slow frame rates, due to the need to serially scan all points in a field of view with a single laser beam. To overcome this problem, we have developed two optical methods that split and multiplex a laser beam across the sample. In the first method a diffractive optical element (DOE generates a fixed number of beamlets that are scanned in parallel, resulting in a corresponding increase in speed, or in signal-to-noise ratio, in time-lapse measurements. The second method uses a computer-controlled spatial light modulator (SLM, to generate any arbitrary spatio-temporal light pattern. With an SLM one can image or photostimulate any predefined region of the image, such as neurons or dendritic spines. In addition, SLMs can be used to mimic a large number of optical transfer functions, including light path corrections or as adaptive optical devices.

  4. 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.

  5. 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

  6. 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.

  7. Visualization of laser tattoo removal treatment effects in a mouse model by two-photon microscopy

    Science.gov (United States)

    Jang, Won Hyuk; Yoon, Yeoreum; Kim, Wonjoong; Kwon, Soonjae; Lee, Seunghun; Song, Duke; Choi, Jong Woon; Kim, Ki Hean

    2017-01-01

    Laser tattoo removal is an effective method of eliminating tattoo particles in the skin. However, laser treatment cannot always remove the unwanted tattoo completely, and there are risks of either temporary or permanent side effects. Studies using preclinical animal models could provide detailed information on the effects of laser treatment in the skin, and might help to minimize side effects in clinical practices. In this study, two-photon microscopy (TPM) was used to visualize the laser treatment effects on tattoo particles in both phantom specimens and in vivo mouse models. Fluorescent tattoo ink was used for particle visualization by TPM, and nanosecond (ns) and picosecond (ps) lasers at 532 nm were used for treatment. In phantom specimens, TPM characterized the fragmentation of individual tattoo particles by tracking them before and after the laser treatment. These changes were confirmed by field emission scanning electron microscopy (FE-SEM). TPM was used to measure the treatment efficiency of the two lasers at different laser fluences. In the mouse model, TPM visualized clusters of tattoo particles in the skin and detected their fragmentation after the laser treatment. Longitudinal TPM imaging observed the migration of cells containing tattoo particles after the laser treatment. These results show that TPM may be useful for the assessment of laser tattoo removal treatment in preclinical studies. PMID:28856046

  8. 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...

  9. 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.

  10. Two-photon microscopy measurement of CMRO2 using periarteriolar PO2 gradients(Conference Presentation)

    Science.gov (United States)

    Sakadžić, Sava; Yaseen, Mohammad A.; Jaswal, Rajeshwer S.; Roussakis, Emmanuel; Dale, Anders M.; Buxton, Richard B.; Vinogradov, Sergei A.; Boas, David A.; Devor, Anna

    2017-02-01

    The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. Measurements of CMRO2 with high spatio-temporal resolution are critically important for understanding how the brain copes with metabolic and blood perfusion changes associated with various clinical conditions, such as stroke, periinfarct depolarizations, and various microvasculopathies (e.g., Alzheimer's disease, chronic hypertension). CMRO2 measurements are also important for understanding the physiological underpinnings of functional Magnetic Resonance Imaging signals. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a novel method that allows estimation of CMRO2 based on a single measurement modality - two-photon phosphorescence lifetime microscopy (2PLM) imaging of the partial pressure of oxygen (PO2) in cortical tissue. CMRO2 is estimated by fitting the changes of tissue PO2 around cortical penetrating arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats, and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method has a spatial resolution of approximately 200 μm and it may provide CMRO2 measurements in individual cortical layers or within confined cortical regions such as in ischemic penumbra and the foci of functional activation.

  11. Changes in cortical microvasculature during misery perfusion measured by two-photon laser scanning microscopy.

    Science.gov (United States)

    Tajima, Yosuke; Takuwa, Hiroyuki; Kokuryo, Daisuke; Kawaguchi, Hiroshi; Seki, Chie; Masamoto, Kazuto; Ikoma, Yoko; Taniguchi, Junko; Aoki, Ichio; Tomita, Yutaka; Suzuki, Norihiro; Kanno, Iwao; Saeki, Naokatsu; Ito, Hiroshi

    2014-08-01

    This study aimed to examine the cortical microvessel diameter response to hypercapnia in misery perfusion using two-photon laser scanning microscopy (TPLSM). We evaluated whether the vascular response to hypercapnia could represent the cerebrovascular reserve. Cerebral blood flow (CBF) during normocapnia and hypercapnia was measured by laser-Doppler flowmetry through cranial windows in awake C57/BL6 mice before and at 1, 7, 14, and 28 days after unilateral common carotid artery occlusion (UCCAO). Diameters of the cortical microvessels during normocapnia and hypercapnia were also measured by TPLSM. Cerebral blood flow and the vascular response to hypercapnia were decreased after UCCAO. Before UCCAO, vasodilation during hypercapnia was found primarily in arterioles (22.9%±3.5%). At 14 days after UCCAO, arterioles, capillaries, and venules were autoregulatorily dilated by 79.5%±19.7%, 57.2%±32.3%, and 32.0%±10.8%, respectively. At the same time, the diameter response to hypercapnia in arterioles was significantly decreased to 1.9%±1.5%. A significant negative correlation was observed between autoregulatory vasodilation and the diameter response to hypercapnia in arterioles. Our findings indicate that arterioles play main roles in both autoregulatory vasodilation and hypercapnic vasodilation, and that the vascular response to hypercapnia can be used to estimate the cerebrovascular reserve.

  12. Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors.

    Science.gov (United States)

    Salomé, R; Kremer, Y; Dieudonné, S; Léger, J-F; Krichevsky, O; Wyart, C; Chatenay, D; Bourdieu, L

    2006-06-30

    Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.

  13. 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.

  14. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Bickford, Lissett; Sun Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah [Department of Bioengineering, Rice University, Houston, TX 77005 (United States)], E-mail: drezek@rice.edu

    2008-08-06

    We demonstrate the capability of using immunotargeted gold nanoshells as contrast agents for in vitro two-photon microscopy. The two-photon luminescence properties of different-sized gold nanoshells are first validated using near-infrared excitation at 780 nm. The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breast cancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imaging results are compared to normal breast cells. Five different imaging channels are simultaneously examined within the emission wavelength range of 451-644 nm. Our results indicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cells labeled with immunotargeted nanoshells occurs at an emission wavelength ranging from 590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescence from unlabeled cancer and normal cells remain imperceptible under the same conditions.

  15. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    Science.gov (United States)

    Bickford, Lissett; Sun, Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah

    2008-08-01

    We demonstrate the capability of using immunotargeted gold nanoshells as contrast agents for in vitro two-photon microscopy. The two-photon luminescence properties of different-sized gold nanoshells are first validated using near-infrared excitation at 780 nm. The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breast cancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imaging results are compared to normal breast cells. Five different imaging channels are simultaneously examined within the emission wavelength range of 451-644 nm. Our results indicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cells labeled with immunotargeted nanoshells occurs at an emission wavelength ranging from 590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescence from unlabeled cancer and normal cells remain imperceptible under the same conditions.

  16. 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.

  17. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  18. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    William L Rice

    Full Text Available BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF and second harmonic generation (SHG as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(PH, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(PH and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool

  19. 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.

  20. Dendritic spine classification using shape and appearance features based on two-photon microscopy.

    Science.gov (United States)

    Ghani, Muhammad Usman; Mesadi, Fitsum; Kanık, Sümeyra Demir; Argunşah, Ali Özgür; Hobbiss, Anna Felicity; Israely, Inbal; Ünay, Devrim; Taşdizen, Tolga; Çetin, Müjdat

    2017-03-01

    Neuronal morphology and function are highly coupled. In particular, dendritic spine morphology is strongly governed by the incoming neuronal activity. The first step towards understanding the structure-function relationships is to classify spine shapes into the main spine types suggested in the literature. Due to the lack of reliable automated analysis tools, classification is mostly performed manually, which is a time-intensive task and prone to subjectivity. We propose an automated method to classify dendritic spines using shape and appearance features based on challenging two-photon laser scanning microscopy (2PLSM) data. Disjunctive Normal Shape Models (DNSM) is a recently proposed parametric shape representation. We perform segmentation of spine images by applying DNSM and use the resulting representation as shape features. Furthermore, we use Histogram of oriented gradients (HOG) to extract appearance features. In this context, we propose a kernel density estimation (KDE) based framework for dendritic spine classification, which uses these shape and appearance features. Our shape and appearance features based approach combined with Neural Network (NN) correctly classifies 87.06% of spines on a dataset of 456 spines. Our proposed method outperforms standard morphological feature based approaches. Our KDE based framework also enables neuroscientists to analyze the separability of spine shape classes in the likelihood ratio space, which leads to further insights about nature of the spine shape analysis problem. Results validate that performance of our proposed approach is comparable to a human expert. It also enable neuroscientists to study shape statistics in the likelihood ratio space. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. 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.

  2. 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

  3. 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.

  4. Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

    Science.gov (United States)

    Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

    2016-03-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

  5. 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.

  6. 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...

  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. 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. 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.

  10. 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.

  11. NIR-to-NIR Two-Photon Scanning Laser Microscopy Imaging of Single Nanoparticles Doped by Yb(III) Complexes.

    Science.gov (United States)

    Bourdolle, Adrien; D'Aléo, Anthony; Philippot, Cécile; Baldeck, Patrice L; Guyot, Yannick; Dubois, Fabien; Ibanez, Alain; Andraud, Chantal; Brasselet, Sophie; Maury, Olivier

    2016-01-04

    The photophysical and nonlinear optical properties of water-soluble chromophore-functionalised tris-dipicolinate complexes [LnL3](3-) (Ln=Yb and Nd) are thoroughly studied, revealing that only the Yb(III) luminescence can be sensitized by a two-photon excitation process. The stability of the complex in water is strongly enhanced by embedding in dispersible organosilicate nanoparticles (NPs). Finally, the spectroscopic properties of [NBu4]3 [YbL3] are studied in solution and in the solid state. The high brightness of the NPs allows imaging them as single objects using a modified two-photon microscopy setup in a NIR-to-NIR configuration.

  12. 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.

  13. 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...

  14. 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

  15. Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

    Directory of Open Access Journals (Sweden)

    Chunqiang Li

    2016-01-01

    Full Text Available Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs, mast cells, vascular smooth muscle cells (SMCs. Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs which participate in the degradation of collagen.

  16. Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy.

    Science.gov (United States)

    Bharali, Dhruba J; Lucey, Derrick W; Jayakumar, Harishankar; Pudavar, Haridas E; Prasad, Paras N

    2005-08-17

    A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Automated image analysis for diameters and branching points of cerebral penetrating arteries and veins captured with two-photon microscopy.

    Science.gov (United States)

    Sugashi, Takuma; Yoshihara, Kouichi; Kawaguchi, Hiroshi; Takuwa, Hiroyuki; Ito, Hiroshi; Kanno, Iwao; Yamada, Yukio; Masamoto, Kazuto

    2014-01-01

    The present study was aimed to characterize 3-dimensional (3D) morphology of the cortical microvasculature (e.g., penetrating artery and emerging vein), using two-photon microscopy and automated analysis for their cross-sectional diameters and branching positions in the mouse cortex. We observed that both artery and vein had variable cross-sectional diameters across cortical depths. The mean diameter was similar for both artery (17 ± 5 μm) and vein (15 ± 5 μm), and there were no detectable differences over depths of 50-400 μm. On the other hand, the number of branches was slightly increased up to 400-μm depth for both the artery and vein. The mean number of branches per 0.1 mm vessel length was 1.7 ± 1.2 and 3.8 ± 1.6 for the artery and vein, respectively. This method allows for quantification of the large volume data of microvascular images captured with two-photon microscopy. This will contribute to the morphometric analysis of the cortical microvasculature in functioning brains.

  2. Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging

    Science.gov (United States)

    Thériault, Gabrielle; Cottet, Martin; Castonguay, Annie; McCarthy, Nathalie; De Koninck, Yves

    2014-01-01

    Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general. PMID:24904284

  3. Cell and brain tissue imaging of the flavonoid fisetin using label-free two-photon microscopy.

    Science.gov (United States)

    Krasieva, Tatiana B; Ehren, Jennifer; O'Sullivan, Thomas; Tromberg, Bruce J; Maher, Pamela

    2015-10-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Characterizing the intracellular distribution of metabolites in intact Chlamydia-infected cells by Raman and two-photon microscopy.

    Science.gov (United States)

    Szaszák, Márta; Chang, Jiun Chiun; Leng, Weinan; Rupp, Jan; Ojcius, David M; Kelley, Anne Myers

    2013-06-01

    Chlamydia species are obligate intracellular pathogens that proliferate only within infected cells. Currently, there are no known techniques or systems that can probe the spatial distribution of metabolites of interest within intact Chlamydia-infected cells. Here we investigate the ability of Raman microscopy to probe the chemical composition of different compartments (nucleus, inclusion, and cytoplasm) of Chlamydia trachomatis-infected epithelial cells. The overall intensity of the Raman spectrum is greatest in the inclusions and lowest in the cytoplasm in fixed cells. Difference spectra generated by normalizing to the intensity of the strong 1004 cm(-1) phenylalanine line show distinct differences among the three compartments. Most notably, the concentrations of adenine are greater in both the inclusions and the nucleus than in the cytoplasm, as seen by Raman microscopy. The source of the adenine was explored through a complementary approach, using two-photon microscopy imaging. Autofluorescence measurements of living, infected cells show that the adenine-containing molecules, NAD(P)H and FAD, are present mainly in the cytoplasm, suggesting that these molecules are not the source of the additional adenine signal in the nucleus and inclusions. Experiments of infected cells stained with a DNA-binding dye, Hoechst 33258, reveal that most of the DNA is present in the nucleus and the inclusions, suggesting that DNA/RNA is the main source of the additional Raman adenine signal in the nucleus and inclusions. Thus, Raman and two-photon microscopy are among the few non-invasive methods available to investigate cells infected with Chlamydia and, together, should also be useful for studying infection by other intracellular pathogens that survive within intracellular vacuoles.

  5. 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.

  6. Two-photon luminescence contrast by tip-sample coupling in femtosecond near-field optical microscopy

    Science.gov (United States)

    Horneber, Anke; Wackenhut, Frank; Braun, Kai; Wang, Xiao; Wang, Jiyong; Zhang, Dai; Meixner, Alfred J.

    2017-01-01

    We investigate the role of tip-sample interaction in nonlinear optical scanning near-field microscopy. The experiment was performed by tightly focusing femtosecond laser pulses onto a sharp gold tip that was positioned in close proximity to the surface of a sample with gold nanostructures on a Si-substrate by shear force feedback. The nonlinear optical signal consists of two-photon photoluminescence and second harmonic signal from the gold tip and the gold nanostructures. These signals can be used to characterize different coupling parameters such as geometry, material and width of the tip-sample gap and enable to reveal the mechanism responsible for the image contrast. Under the excitation with 776-nm and 110-fs laser pulses nonlinear imaging is almost background free and yields super resolution showing features with dimensions significantly below the diffraction limit with a signal intensity following quadratic excitation power law.

  7. Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser.

    Science.gov (United States)

    Kawakami, Ryosuke; Sawada, Kazuaki; Sato, Aya; Hibi, Terumasa; Kozawa, Yuichi; Sato, Shunichi; Yokoyama, Hiroyuki; Nemoto, Tomomi

    2013-01-01

    In vivo two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than several hundred micrometers from the brain surface. We developed a novel semiconductor-laser-based light source with a wavelength of 1030 nm that can generate pulses of 5-picosecond duration with 2-W output power, and a 20-MHz repetition rate. We also developed a system to secure the head of the mouse under an upright microscope stage that has a horizontal adjustment mechanism. We examined the penetration depth while imaging the H-Line mouse brain and demonstrated that our newly developed laser successfully images not only cortex pyramidal neurons spreading to all cortex layers at a superior signal-to-background ratio, but also images hippocampal CA1 neurons in a young adult mouse.

  8. Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium 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

    2014-07-01

    Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular 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. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium 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.

  9. 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

  10. The use of two-photon microscopy to study the biological effects of focused ultrasound on the brain

    Science.gov (United States)

    Burgess, Alison; Cho, Eunice E.; Shaffaf, Leila; Nhan, Tam; Poon, Charissa; Hynynen, Kullervo

    2012-03-01

    Focused ultrasound (FUS) has been used to successfully disrupt the blood-brain barrier (BBB), aiding in the delivery of therapeutic agents to the brain and leading to improvements in disease pathology. Although significant progress has been made in the development of FUS technology, there is still a lack of understanding of the biophysical mechanisms of the BBB disruption and the microscopic effects of this disruption on brain cells. In this study, we combine a custom built ultrasound transducer with two-photon microscopy to conduct real time monitoring of BBB disruption in vivo. We have manufactured and tested a single element piezoelectric transducer with frequencies ranging from 1.15 to 1.30 MHz. Sonications were performed using 0.07-0.25 MPa estimated in situ pressure, 10 ms pulses, 1 Hz pulse repetition frequency for a total duration of 120 s in the presence of microbubbles. BBB disruption was observed through a cranial window created in the rat skull after intravenous injection of dextran conjugated- Texas Red (MW: 10,000 - 70,000 Da). Using this experimental setup, we have observed and characterized 3 different leakage patterns following BBB disruption. Our results indicate that varying the acoustic power leading to in situ pressure changes, may allow us to control the mechanism of BBB disruption. Furthermore, we have labelled astrocytes in vivo in order to visualize the effects of FUS on this cell population. Combination of our custom transducers with two-photon microscopy will allow significant advancement in allow significant advancement in the understanding of the mechanisms and cellular effects of FUS-induced BBB disruption.

  11. In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy

    Science.gov (United States)

    Le, Viet-Hoan; Lee, Seunghun; Lee, Seungwon; Wang, Taejun; Hyuk Jang, Won; Yoon, Yeoreum; Kwon, Soonjae; Kim, Hyekang; Lee, Seung-Woo; Hean Kim, Ki

    2017-01-01

    Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies. PMID:28276477

  12. 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.

  13. Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation.

    Science.gov (United States)

    Liese, Jan; Rooijakkers, Suzan H M; van Strijp, Jos A G; Novick, Richard P; Dustin, Michael L

    2013-06-01

    Staphylococcus (S.) aureus is a frequent cause of severe skin infections. The ability to control the infection is largely dependent on the rapid recruitment of neutrophils (PMN). To gain more insight into the dynamics of PMN migration and host-pathogen interactions in vivo, we used intravital two-photon (2-P) microscopy to visualize S. aureus skin infections in the mouse. Reporter S. aureus strains expressing fluorescent proteins were developed, which allowed for detection of the bacteria in vivo. By employing LysM-EGFP mice to visualize PMN, we observed the rapid appearance of PMN in the extravascular space of the dermis and their directed movement towards the focus of infection, which led to the delineation of an abscess within 1 day. Moreover, tracking of transferred labelled bone-marrow neutrophils showed that PMN localization to the site of infection is dependent on the presence of G-protein-coupled receptors on the PMN, whereas Interleukin-1 receptor was required on host cells other than PMN. Furthermore, the S. aureus complement inhibitor Ecb could block PMN accumulation at thesite of infection. Our results establish that 2-P microscopy is a powerful tool to investigate the orchestration of the immune cells, S. aureus location and gene expression in vivo on a single cell level.

  14. 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.

  15. Upconverting nanoparticles: a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging.

    Science.gov (United States)

    Park, Yong Il; Lee, Kang Taek; Suh, Yung Doug; Hyeon, Taeghwan

    2015-03-21

    Lanthanide-doped upconverting nanoparticles (UCNPs) have recently attracted enormous attention in the field of biological imaging owing to their unique optical properties: (1) efficient upconversion photoluminescence, which is intense enough to be detected at the single-particle level with a (nonscanning) wide-field microscope setup equipped with a continuous wave (CW) near-infrared (NIR) laser (980 nm), and (2) resistance to photoblinking and photobleaching. Moreover, the use of NIR excitation minimizes adverse photoinduced effects such as cellular photodamage and the autofluorescence background. Finally, the cytotoxicity of UCNPs is much lower than that of other nanoparticle systems. All these advantages can be exploited simultaneously without any conflicts, which enables the establishment of a novel UCNP-based platform for wide-field two-photon microscopy. UCNPs are also useful for multimodal in vivo imaging because simple variations in the composition of the lattice atoms and dopant ions integrated into the particles can be easily implemented, yielding various distinct biomedical activities relevant to magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). These multiple functions embedded in a single type of UCNPs play a crucial role in precise disease diagnosis. The application of UCNPs is extended to therapeutic fields such as photodynamic and photothermal cancer therapies through advanced surface conjugation schemes.

  16. Effects of cholesterol on plasma membrane lipid order in MCF-7 cells by two-photon microscopy

    Science.gov (United States)

    Zeng, Yixiu; Chen, Jianling; Yang, Hongqin; Wang, Yuhua; Li, Hui; Xie, Shusen

    2014-09-01

    Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.

  17. In vivo monitoring of intracellular chloroplast movements in intact leaves of C4 plants using two-photon microscopy.

    Science.gov (United States)

    Ryu, Jeongeun; Nam, Hyoseok; Kim, Hae Koo; Joo, Yongjoon; Lee, Sang Joon; Kim, Ki Hean

    2014-10-01

    Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light-induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two-photon microscopy (TPM) system was adapted to monitor the intracellular 3-dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto-second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub-epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions.

  18. NEAR-IR TWO PHOTON MICROSCOPY IMAGING OF SILICA NANOPARTICLES FUNCTIONALIZED WITH ISOLATED SENSITIZED Yb(III) CENTERS

    Energy Technology Data Exchange (ETDEWEB)

    Lapadula, Giuseppe; Bourdolle, Adrien; Allouche, Florian; Conley, Matthew P.; Maron, Laurent; Lukens, Wayne W.; Guyot, Yannick; Andraud, Chantal; Brasselet, Sophie; Copé; ret, Christophe; Maury, Olivier; Andersen, Richard A.

    2013-01-12

    Bright nano objects emitting in the near infrared with a maximal cross section of 41.4 x 103 GM (Goppert Mayer), were prepared by implanting ca. 180 4,4 diethylaminostyryl 2,2 bipyridine (DEAS) Yb(III) complexes on the surface of 12 nm silica nanoparticles. The surface complexes ([DEAS Ln SiO2], Ln =Y,Yb) were characterized using IR, solid state NMR, UV Vis, EXAFS spectroscopies in combination with the preparation and characterization of similar molecular analogues by analytical techniques (IR, solution NMR, UV Vis, X ray crystallography) as well as DFT calculations. Starting from the partial dehydroxylation of the silica at 700 C on high vacuum having 0.8 OH.nm 2, the grafting of Ln(N(SiMe3)2)3 generate ≤SiO Ln(N(SiMe3)2)2, which upon thermal step and coordination of the DEAS chromophore yields (≤SiO)3Ln(DEAS). Surface and molecular analogues display similar properties, in terms of DEAS binding constants absorption maxima and luminescence properties (intense emission band assigned to a ligand centered CT fluorescence and life time) in the solid state, consistent with the molecular nature of the surface species. The densely functionalized nanoparticles can be dispersed via ultra-sonication in small ca. 15-20 nm aggregates (1 to 6 elementary particles) that were detected using two photon microscopy imaging at 720 nm excitation, making them promising nano objects for bio imaging.

  19. 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.

  20. 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.

  1. Brain refractive index measured in vivo with high-NA defocus-corrected full-field OCT and consequences for two-photon microscopy.

    Science.gov (United States)

    Binding, Jonas; Ben Arous, Juliette; Léger, Jean-François; Gigan, Sylvain; Boccara, Claude; Bourdieu, Laurent

    2011-03-14

    Two-photon laser scanning microscopy (2PLSM) is an important tool for in vivo tissue imaging with sub-cellular resolution, but the penetration depth of current systems is potentially limited by sample-induced optical aberrations. To quantify these, we measured the refractive index n' in the somatosensory cortex of 7 rats in vivo using defocus optimization in full-field optical coherence tomography (ff-OCT). We found n' to be independent of imaging depth or rat age. From these measurements, we calculated that two-photon imaging beyond 200 µm into the cortex is limited by spherical aberration, indicating that adaptive optics will improve imaging depth.

  2. 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

  3. 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.

  4. 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

  5. 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 .

  6. Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confo-cal laser scanning microscopy

    Institute of Scientific and Technical Information of China (English)

    张春阳; 李艳平; 马辉; 李素文; 薛绍白; 陈瓞延

    2001-01-01

    Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Lund, F. W.; Lomholt, M. A.; Solanko, L. M.

    2012-01-01

    Background: Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute...... to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter...... probe has utility for prolonged live-cell imaging of sterol transport. Results: We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements...

  12. Photon Emission and Reabsorption Processes in CH3NH3PbBr3 Single Crystals Revealed by Time-Resolved Two-Photon-Excitation Photoluminescence Microscopy

    Science.gov (United States)

    Yamada, Takumi; Yamada, Yasuhiro; Nakaike, Yumi; Wakamiya, Atsushi; Kanemitsu, Yoshihiko

    2017-01-01

    The dynamical processes of radiative recombination of photocarriers and reabsorption of emitted photons in CH3NH3PbBr3 single crystals are studied using time-resolved two-photon-excitation photoluminescence (PL) microscopy. We find that the PL spectrum and its decay dynamics depend on the excitation-depth profile. As the excitation depth increases, the PL spectrum becomes asymmetric, the peak energy redshifts, and the PL decay time becomes longer. These observations can be well explained by a simple model including photon recycling (photon emission and reabsorption) in thick samples with strong band-to-band transitions and high radiative recombination efficiencies.

  13. Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Lund Frederik W

    2012-10-01

    Full Text Available Abstract Background Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE, an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results We found that BChol is very photostable under two-photon (2P-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t > ~5 s, a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle

  14. 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.

  15. 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.

  16. 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.

  17. Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording and pharmacology

    Directory of Open Access Journals (Sweden)

    Manuel eLevy

    2012-12-01

    Full Text Available Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1 hyperpolarization by intracellular current injection, (2 pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3 GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5 to 10 nA, which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be

  18. Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

    Directory of Open Access Journals (Sweden)

    Jong Kang Park

    2017-03-01

    Full Text Available Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD, can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.

  19. 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.

  20. 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.

  1. 4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM).

    Science.gov (United States)

    Lavagnino, Zeno; Sancataldo, Giuseppe; d'Amora, Marta; Follert, Philipp; De Pietri Tonelli, Davide; Diaspro, Alberto; Cella Zanacchi, Francesca

    2016-04-01

    In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces.

  2. Analysis of Recombination in CdTe Heterostructures With Time-Resolved Two-Photon Excitation Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kuciauskas, Darius; Wernsing, Keith; Jensen, Soren Alkaersig; Barnes, Teresa M.; Myers, Thomas H.; Bartels, Randy A.

    2016-11-01

    We used time-resolved photoluminescence micro-scopy to analyze charge carrier transport and recombination in CdTe double heterostructures fabricated by molecular beam epitaxy (MBE). This allowed us to determine the charge carrier mobility in this system, which was found to be 500-625 cm2/(V s). Charge carrier lifetimes in the 15-100 ns range are limited by the interface recombination, and the data indicate higher interface recombination velocity near extended defects. This study describes a new method to analyze the spatial distribution of the interface recombination velocity and the interface defects in semiconductor heterostructures.

  3. 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...

  4. 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.

  5. 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

  6. Routine dual-color immunostaining with a 3-antibody cocktail improves the detection of small cancers in prostate needle biopsies.

    Science.gov (United States)

    Tolonen, Teemu T; Kujala, Paula M; Laurila, Marita; Tirkkonen, Mika; Ilvesaro, Joanna; Tuominen, Vilppu J; Tammela, Teuvo L J; Isola, Jorma

    2011-11-01

    We performed dual-color immunostaining with a 3-antibody cocktail (α-methylacyl coenzyme-A racemase, CK34betaE12, and p63) on prostate biopsies from 200 patients. Current practice (hematoxylin and eosin staining followed by dual-color immunostaining on selected cases) was compared with a protocol in which routine dual-color immunostaining was provided in all cases. In the original pathology reports, adenocarcinoma was diagnosed in 87/200 (43%) patients. Small foci interpreted as putative cancers were detected with dual-color immunostaining in 14/113 patients who were originally diagnosed with a nonmalignant lesion. All of the suggested cancerous foci were independently reevaluated by 5 pathologists. A diagnosis of adenocarcinoma was assessed by consensus in 8 cases, and atypical small acinar proliferation was diagnosed in 1 case. Consensus was not reached in 5 cases. Six of the foci reclassified as cancer were of Gleason score 3 + 3 = 6, while 2 were graded as Gleason score 4 + 4 = 8. The feasibility of routine dual-color immunostaining was also tested by analyzing the time spent on microscopic assessment. Because small, atypical lesions expressing α-methylacyl coenzyme-A racemase (blue chromogen) were easy to detect using dual-color immunostaining, the microscopic analysis of dual-color immunostaining and hematoxylin-eosin staining was faster than that of hematoxylin-eosin staining alone that was later followed by dual-color immunostaining in selected cases (median 251 seconds versus 299 seconds, P < .0001). We concluded that routine dual-color immunostaining of all prostate biopsies would produce better diagnostic sensitivity with a smaller microscopy workload for the pathologist. However, minute foci interpreted as cancer with dual-color immunostaining need to be confirmed with hematoxylin-eosin staining, and minimal criteria for a definitive diagnosis of cancer are still lacking. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view.

    Science.gov (United States)

    Kremer, Y; Léger, J-F; Lapole, R; Honnorat, N; Candela, Y; Dieudonné, S; Bourdieu, L

    2008-07-07

    Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their use has been limited until now by their small scanning range and by the spatial and temporal dispersions of the laser beam going through the deflectors. We show that the use of AOD of large aperture (13mm) compared to standard deflectors allows accessing much larger field of view while minimizing spatio-temporal distortions. An acousto-optic modulator (AOM) placed at distance of the AOD is used to compensate spatial and temporal dispersions. Fine tuning of the AOM-AOD setup using a frequency-resolved optical gating (GRENOUILLE) allows elimination of pulse front tilt whereas spatial chirp is minimized thanks to the large aperture AOD.

  8. Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

    Science.gov (United States)

    Lees, Robert M; Peddie, Christopher J; Collinson, Lucy M; Ashby, Michael C; Verkade, Paul

    2017-01-01

    Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. A novel technique for the in vivo imaging of autoimmune diabetes development in the pancreas by two-photon microscopy.

    Directory of Open Access Journals (Sweden)

    Ken Coppieters

    Full Text Available Type 1 diabetes (T1D is characterized by the immune-mediated destruction of beta cells in the pancreas. Little is known about the in vivo dynamic interactions between T cells and beta cells or the kinetic behavior of other immune cell subsets in the pancreatic islets. Utilizing multiphoton microscopy we have designed a technique that allows for the real-time visualization of diabetogenic T cells and dendritic cells in pancreatic islets in a live animal, including their interplay with beta cells and the vasculature. Using a custom designed stage, the pancreas was surgically exposed under live conditions so that imaging of islets under intact blood pressure and oxygen supply became possible. We demonstrate here that this approach allows for the tracking of diabetogenic leukocytes as well as vascularization phenotype of islets and accumulation of dendritic cells in islets during diabetes pathogenesis. This technique should be useful in mapping crucial kinetic events in T1D pathogenesis and in testing the impact of immune based interventions on T cell migration, extravasation and islet destruction.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo.

    Directory of Open Access Journals (Sweden)

    Claudia Laperchia

    Full Text Available Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype.With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity of dendritic cells (DCs, and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.

  15. In Vivo Time-Course Imaging of Tumor Angiogenesis in Colorectal Liver Metastases in the Same Living Mice Using Two-Photon Laser Scanning Microscopy

    Directory of Open Access Journals (Sweden)

    Koji Tanaka

    2012-01-01

    Full Text Available In vivo real-time visualization of the process of angiogenesis in secondary tumors in the same living animals presents a major challenge in metastasis research. We developed a technique for intravital imaging of colorectal liver metastasis development in live mice using two-photon laser scanning microscopy (TPLSM. We also developed time-series TPLSM in which intravital TPLSM procedures were performed several times over periods of days to months. Red fluorescent protein-expressing colorectal cancer cells were inoculated into the spleens of green fluorescent protein-expressing mice. First- and second-round intravital TPLSM allowed visualization of viable cancer cells (red in hepatic sinusoids or the space of Disse. Third-round intravital TPLSM demonstrated liver metastatic colonies consisting of viable cancer cells and surrounding stroma with tumor vessels (green. In vivo time-course imaging of tumor angiogenesis in the same living mice using time-series TPLSM could be an ideal tool for antiangiogenic drug evaluation, reducing the effects of interindividual variation.

  16. The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.

    Science.gov (United States)

    Niesner, Raluca; Andresen, Volker; Neumann, Jens; Spiecker, Heinrich; Gunzer, Matthias

    2007-10-01

    Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.

  17. 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.

  18. A method to investigate radial glia cell behavior using two-photon time-lapse microscopy in an ex vivo model of spinal cord development.

    Directory of Open Access Journals (Sweden)

    Janelle M.P. Pakan

    2014-04-01

    Full Text Available The mammalian central nervous system (CNS develops from multipotent progenitor cells, which proliferate and differentiate into the various cell types of the brain and spinal cord. Despite the wealth of knowledge from progenitor cell culture studies, there is a significant lack of understanding regarding dynamic progenitor cell behavior over the course of development. This is in part due to shortcomings in the techniques available to study these processes in living tissues as they are occurring. In order to investigate cell behavior under physiologically relevant conditions we established an ex vivo model of the developing rat spinal cord. This method allows us to directly observe specific populations of cells ex vivo in real time and over extended developmental periods as they undergo proliferation, migration and differentiation in the CNS. Previous investigations of progenitor cell behavior have been limited in either spatial or temporal resolution (or both due to the necessity of preserving tissue viability and avoiding phototoxic effects of fluorescent imaging. The method described here overcomes these obstacles. Using two-photon and confocal microscopy and transfected organotypic spinal cord slice cultures we have undertaken detailed imaging of a unique population of neural progenitors, radial glial cells. This method uniquely enables analysis of large populations as well as individual cells; ultimately resulting in a 4D dataset of progenitor cell behavior for up to seven days during embryonic development. This approach can be adapted to study a variety of cell populations at different stages of development using appropriate promoter driven fluorescent protein expression. The ability to control the tissue micro-environment makes this ex vivo method a powerful tool to elucidate the underlying molecular mechanisms regulating cell behavior during embryonic development.

  19. 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.

  20. Dual-Colored DNA Comb Polymers for Single Molecule Rheology

    Science.gov (United States)

    Mai, Danielle; Marciel, Amanda; Schroeder, Charles

    2014-03-01

    We report the synthesis and characterization of branched biopolymers for single molecule rheology. In our work, we utilize a hybrid enzymatic-synthetic approach to graft ``short'' DNA branches to ``long'' DNA backbones, thereby producing macromolecular DNA comb polymers. The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs): ``short'' branches consist of Cy5-labeled dNTPs and a terminal azide group, and ``long'' backbones contain dibenzylcyclooctyne-modified (DBCO) dNTPs. In this way, we utilize strain-promoted, copper-free cycloaddition ``click'' reactions for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, comb polymers can be labeled with an intercalating dye (e.g., YOYO) for dual-color fluorescence imaging. We characterized these materials using gel electrophoresis, HPLC, and optical microscopy, with atomic force microscopy in progress. Overall, DNA combs are suitable for single molecule dynamics, and in this way, our work holds the potential to improve our understanding of topologically complex polymer melts and solutions.

  1. 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.

  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. 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.

  4. Non-linear imaging and characterization of atherosclerotic arterial tissue using combined two photon fluorescence, second-harmonic generation and CARS microscopy

    Science.gov (United States)

    Cicchi, Riccardo; Matthäus, Christian; Meyer, Tobias; Lattermann, Annika; Dietzek, Benjamin; Brehm, Bernhard R.; Popp, Jürgen; Pavone, Francesco S.

    2014-02-01

    Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires a morpho-functional approach. Multimodal non-linear microscopy has the potential to bridge this gap by providing morpho-functional information on the examined tissues in a label-free way. Here we employed multiple non-linear microscopy techniques, including CARS, TPF, and SHG to provide intrinsic optical contrast from various tissue components in both arterial wall and atherosclerotic plaques. CARS and TPF microscopy were used to respectively image lipid depositions within plaques and elastin in the arterial wall. Cholesterol deposition in the lumen and collagen in the arterial wall were selectively imaged by SHG microscopy and distinguished by forward-backward SHG ratio. Image pattern analysis allowed characterizing collagen organization in different tissue regions. Different values of fiber mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

  5. 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.

  6. Dual-Color Click Beetle Luciferase Heteroprotein Fragment Complementation Assays

    Science.gov (United States)

    Villalobos, Victor; Naik, Snehal; Bruinsma, Monique; Dothager, Robin S.; Pan, Mei-Hsiu; Samrakandi, Mustapha; Moss, Britney; Elhammali, Adnan; Piwnica-Worms, David

    2010-01-01

    Summary Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically-relevant time scales. Herein we describe a novel set of reversible, multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discreet pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a novel candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells. PMID:20851351

  7. 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.

  8. Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

    Science.gov (United States)

    Chizhik, Anna M; Stein, Simon; Dekaliuk, Mariia O; Battle, Christopher; Li, Weixing; Huss, Anja; Platen, Mitja; Schaap, Iwan A T; Gregor, Ingo; Demchenko, Alexander P; Schmidt, Christoph F; Enderlein, Jörg; Chizhik, Alexey I

    2016-01-13

    Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

  9. 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.

  10. 2000-fold parallelized dual-color STED fluorescence nanoscopy.

    Science.gov (United States)

    Bergermann, Fabian; Alber, Lucas; Sahl, Steffen J; Engelhardt, Johann; Hell, Stefan W

    2015-01-12

    Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 "intensity minima"). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes.

    Science.gov (United States)

    Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

    2016-03-02

    Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application.

  16. 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.

  17. 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.

  18. Three-Dimensional Reconstruction of Nuclear Envelope Architecture Using Dual-Color Metal-Induced Energy Transfer Imaging.

    Science.gov (United States)

    Chizhik, Anna M; Ruhlandt, Daja; Pfaff, Janine; Karedla, Narain; Chizhik, Alexey I; Gregor, Ingo; Kehlenbach, Ralph H; Enderlein, Jörg

    2017-09-20

    The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color metal-induced energy transfer (MIET), we determine the axial distance between Lap2β and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively. Using MIET imaging, we reconstruct the 3D profile of the nuclear envelope over the whole basal area, with an axial resolution of a few nanometers. This result demonstrates that optical microscopy can achieve nanometer axial resolution in biological samples and without recourse to complex interferometric approaches.

  19. 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)

  20. 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.

  1. 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.

  2. 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....

  3. 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.

  4. 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

  5. Dual-color decompositions at one-loop level in Yang-Mills theory

    CERN Document Server

    Du, Yi-Jian; Fu, Chih-Hao

    2014-01-01

    In this work, we extend the construction of dual color decomposition in Yang-Mills theory to one-loop level, i.e., we show how to write one-loop integrands in Yang-Mills theory to the dual DDM-form and the dual trace-form. In dual forms, integrands are decomposed in terms of color-ordered one-loop integrands for color scalar theory with proper dual color coefficients.In dual DDM decomposition, The dual color coefficients can be obtained directly from BCJ-form by applying Jacobi-like identities for kinematic factors. In dual trace decomposition, the dual trace factors can be obtained by imposing one-loop KK relations, reflection relation and their relation with the kinematic factors in dual DDM-form.

  6. 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.

  7. 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.

  8. 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.

  9. Two-photon-excited autofluorescence and second-harmonic generation microscopy for the visualization of penetration of TiO2 and ZnO nanoparticles into human tooth tissue ex vivo

    Science.gov (United States)

    Trunina, Natalia A.; Popov, Alexey P.; Lademann, Jürgen; Tuchin, Valery V.; Myllylä, Risto; Darvin, Maxim E.

    2012-06-01

    Penetration of nanoparticles into tooth tissues is of significant interest in solving problems related to reduction of tooth sensitivity, enamel strengthening and restoration and cosmetic bleaching. In this work we demonstrate two-photonexcited autofluorescence and second-harmonic generation microscopy for visualization of penetration of TiO2 and ZnO nanoparticles into tooth tissues.

  10. 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.

  11. 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.

  12. 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)

  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. 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....

  15. 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.

  16. 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.

  17. 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...

  18. 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.

  19. Enhancement of two-photon photoluminescence and SERS for low-coverage gold films

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Frydendahl, Christian

    2016-01-01

    Electromagnetic field enhancement (FE) effects occurring in thin gold films 3-12-nm are investigated with two-photon photoluminescence (TPL) and Raman scanning optical microscopies. The samples are characterized using scanning electron microscopy images and linear optical spectroscopy. TPL images...

  20. 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.

  1. 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

  2. Eu/Tb codoped spindle-shaped fluorinated hydroxyapatite nanoparticles for dual-color cell imaging.

    Science.gov (United States)

    Ma, Baojin; Zhang, Shan; Qiu, Jichuan; Li, Jianhua; Sang, Yuanhua; Xia, Haibing; Jiang, Huaidong; Claverie, Jerome; Liu, Hong

    2016-06-02

    Lanthanide doped fluorinated hydroxyapatite (FAp) nanoparticles are promising cell imaging nanomaterials but they are excited at wavelengths which do not match the light sources usually found in a commercial confocal laser scanning microscope (CLSM). In this work, we have successfully prepared spindle-shaped Eu/Tb codoped FAp nanoparticles by a hydrothermal method. Compared with single Eu doped FAp, Eu/Tb codoped FAp can be excited by a 488 nm laser, and exhibit both green and red light emission. By changing the amounts of Eu and Tb peaks, the emission in the green region (500-580 nm) can be decreased to the benefit of the emission in the red region (580-720 nm), thus reaching a balanced dual color emission. Using MC3T3-E1 cells co-cultured with Eu/Tb codoped FAp nanoparticles, it is observed that the nanoparticles are cytocompatible even at a concentration as high as 800 μg ml(-1). The Eu/Tb codoped FAp nanoparticles are located in the cytoplasm and can be monitored by dual color-green and red imaging with a single excitation light at 488 nm. At a concentration of 200 μg ml(-1), the cytoplasm is saturated in 8 hours, and Eu/Tb codoped FAp nanoparticles retain their fluorescence for at least 3 days. The cytocompatible Eu/Tb codoped FAp nanoparticles with unique dual color emission will be of great use for cell and tissue imaging.

  3. 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.

  4. 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).

  5. 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.

  6. 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/.

  7. 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.

  8. 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.

  9. 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.

  10. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    Science.gov (United States)

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.

  11. 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.

  12. 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

  13. CaRuby-Nano: a novel high affinity calcium probe for dual color imaging.

    Science.gov (United States)

    Collot, Mayeul; Wilms, Christian D; Bentkhayet, Asma; Marcaggi, Païkan; Couchman, Kiri; Charpak, Serge; Dieudonné, Stéphane; Häusser, Michael; Feltz, Anne; Mallet, Jean-Maurice

    2015-03-31

    The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

  14. Detection of Embryo Sex Chromosome by Dual Color Fluorescent In-Situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    刘群; 朱桂金

    2003-01-01

    Summary: In order to evaluate the effects of sex chromosomal mosaicism on the accuracy of single-cell gender diagnosis, sex chromosomes of 21 normal fertilized embryos were detected by dual colorfluorescent in-situ hybridization (FISH). The results showed that 4 embryos had sex chromosomalmosaicism (19%) and the remaining 17 showed uniformly XX or XY signals in all blastomeres. Inconclusion, identification of sex by dual color FISH analysis of a single cell was accurate and efficient,and sex chromosomal mosaicism would not affect preimplantation gender diagnosis.

  15. 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.

  16. 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.

  17. 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.

  18. In vivo cell kinetics of the bone marrow transplantation using dual colored transgenic rat system

    Science.gov (United States)

    Kai, Kotaro; Teraoka, Satoshi; Adachi, Yasushi; Ikehara, Susumu; Murakami, Takashi; Kobayashi, Eiji

    2008-02-01

    Because bone marrow is an adequate site for bone marrow stem cells, intra-bone marrow - bone marrow transplantation (IBM-BMT) is an efficient strategy for bone marrow transplantation (BMT). However, the fate of the transplanted cells remains unclear. Herein, we established a dual-colored transgenic rat system utilizing green fluorescent protein (GFP) and a luciferase (luc) marker. We then utilized this system to investigate the in vivo kinetics of transplanted bone marrow cells (BMCs) after authentic intravenous (IV)-BMT or IBM-BMT. The in vivo fate of the transplanted cells was tracked using an in vivo luminescent imaging technique; alterations in peripheral blood chimerism were also followed using flow cytometry. IBM-BMT and IV-BMT were performed using syngeneic and allogeneic rat combinations. While no difference in the proliferation pattern was observed between the two treatment groups at 7 days after BMT, different distribution patterns were clearly observed during the early phase. In the IBM-BMT-treated rats, the transplanted BMCs were engrafted immediately at the site of the injected bone marrow and expanded more rapidly than in the IV-BMT-treated rats during this phase. Graft-versus-host disease was also visualized. Our bio-imaging system using dual-colored transgenic rats is a powerful tool for performing quantitative and morphological assessments in vivo.

  19. Dual-color encoded DNAzyme nanostructures for multiplexed detection of intracellular metal ions in living cells.

    Science.gov (United States)

    Zhou, Wenjiao; Liang, Wenbing; Li, Daxiu; Yuan, Ruo; Xiang, Yun

    2016-11-15

    The detection of intracellular metal ions is of great importance in understanding metal homeostasis in cells and related diseases, and yet it remains a significant challenge to achieve this goal. Based on a new self-assembled and dual-color encoded DNAzyme nanostructure, we describe here an approach for multiplexed sensing of UO2(2+) and Pb(2+) in living cells. The fluorescently quenched nanoprobes can be prepared by simple thermal annealing of four ssDNAs containing the metal ion-dependent enzymatic and substrate sequences. The self-assembly formation of the nanostructures are verified by native polyacrylamide gel electrophoresis. The target metal ions can cleave the substrate sequences in the DNAzyme nanostructures to recover fluorescent emissions at different wavelengths for sensitive and selective in vitro multiplexed detection of UO2(2+) and Pb(2+) with the detection limits of 0.6nM and 3.9nM, respectively. Importantly, we demonstrate that these nanoprobes are stable in cell lysates and can enter cells without the aid of any transfection agents for simultaneous imaging intracellular UO2(2+) and Pb(2+). Moreover, the nanoprobes offer excellent biocompatibility and non-cytotoxicity. With these unique features, the dual-color encoded nanostructures presented here can thus offer new opportunities for multiplexed detection of specific intracellular species. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. 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.

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

    Science.gov (United States)

    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.

  2. 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.

  3. Cellular resolution multiplexed FLIM tomography with dual-color Bessel beam.

    Science.gov (United States)

    Xu, Dongli; Zhou, Weibin; Peng, Leilei

    2017-02-01

    Fourier multiplexed FLIM (FmFLIM) tomography enables multiplexed 3D lifetime imaging of whole embryos. In our previous FmFLIM system, the spatial resolution was limited to 25 μm because of the trade-off between the spatial resolution and the imaging depth. In order to achieve cellular resolution imaging of thick specimens, we built a tomography system with dual-color Bessel beam. In combination with FmFLIM, the Bessel FmFLIM tomography system can perform parallel 3D lifetime imaging on multiple excitation-emission channels at a cellular resolution of 2.8 μm. The image capability of the Bessel FmFLIM tomography system was demonstrated by 3D lifetime imaging of dual-labeled transgenic zebrafish embryos.

  4. Dual-color ultrasensitive bright-field RNA in situ hybridization with RNAscope.

    Science.gov (United States)

    Wang, Hongwei; Su, Nan; Wang, Li-Chong; Wu, Xingyong; Bui, Son; Nielsen, Allissa; Vo, Hong-Thuy; Luo, Yuling; Ma, Xiao-Jun

    2014-01-01

    In situ hybridization (ISH) techniques have been important to the study of gene expression signatures in cells and tissues. The ability to detect multiple targets simultaneously is especially valuable, since it allows dissecting gene expression of distinct cell types with precise cellular and subcellular resolution within morphological context. Recently, we have reported using a novel dual-color ultrasensitive bright-field RNA in situ hybridization for detection of clonally restricted immunoglobulin light chain mRNA expression in B cell lymphomas. Here, we present detailed protocols of RNAscope 2-Plex assays for FFPE tissue sections. The protocols describe the tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis, as well as emphasize the critical steps for ensuring successful staining.

  5. Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles

    Science.gov (United States)

    Guo, Longhua; Xu, Shaohua; Ma, Xiaoming; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2016-09-01

    Colorimetric enzyme-linked immunosorbent assay utilizing 3‧-3-5‧-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3‧-3-5‧-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.

  6. 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.

  7. 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}}.

  8. 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...

  9. 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.

  10. 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

  11. Adaptive optics for in vivo two-photon calcium imaging of neuronal networks

    Science.gov (United States)

    Meimon, Serge; Conan, Jean-Marc; Mugnier, Laurent M.; Michau, Vincent; Cossart, Rosa; Malvache, Arnaud

    2014-03-01

    The landscape of biomedical research in neuroscience has changed dramatically in recent years as a result of spectacular progress in dynamic microscopy. However, the optical accessibility of deep brain structures or deeper regions of the surgically exposed hippocampus (a few 100 microns typically) remains limited, due to volumic aberrations created by the sample inhomogeneities. Adaptive optics can correct for these aberrations. Our goal is to realize a novel adaptive optics module dedicated to in vivo two-photon calcium imaging of the hippocampus. The key issue in adaptive optics is the ability to perform an accurate and reliable wavefront sensing. In two- photon microscopy indirect methods are required. Two families of approaches have been proposed so far, the modal sensorless technique and a method based on pupil segmentation. We present here a formal comparison of these approaches, in particular as a function of the amount of aberrations.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. A Dual Color Immunohistochemistry Assay for Measurement of Cereblon in Multiple Myeloma Patient Samples.

    Science.gov (United States)

    Ren, Yan; Wang, Maria; Couto, Suzana; Hansel, Donna E; Miller, Karen; Lopez-Girona, Antonia; Bjorklund, Chad C; Gandhi, Anita K; Thakurta, Anjan; Chopra, Rajesh; Breider, Michael

    Clinical interest in the measurement of Cereblon (CRBN), the primary target of the IMiDs immunomodulatory drugs lenalidomide and pomalidomide, has been fueled by its essential requirement for antitumor or immunomodulatory activity of both drugs in multiple myeloma (MM). However, limited analyses of clinical samples for CRBN gene expression or protein levels have utilized unvalidated reagents and assays, raising uncertainty about the interpretation of these results. We previously described a highly specific rabbit monoclonal antibody CRBN65 against 65-76 AA of human Cereblon. Here we describe a validated dual color bright-field Cereblon/CD138 immunohistochemical (IHC) assay utilizing CRBN65 and a commercial mouse monoclonal CD138 antibody. Sensitivity and specificity of the assay was determined and assay precision was shown for both cytoplasmic and nuclear Cereblon in MM bone marrow samples with coefficient of variation values of 5% and 2%, respectively. The dual IHC assay was effective for detecting a continuous range of Cereblon levels in 22 MM patient bone marrow core biopsies and aspirate clots, as shown by average cytoplasmic H-scores ranging from 63 to 267 and nuclear H-scores ranging from 17 to 250. Interpathologist comparison of MM sample H-scores by 3 pathologists demonstrated good concordance (R=0.73). This dual assay demonstrated superior Cereblon IHC measurement in MM samples compared with the single IHC assay using a published commercial rabbit polyclonal Cereblon antibody and could be used to explore the potential utility of Cereblon as a biomarker in the clinic.

  6. Single-gene dual-color reporter cell line to analyze RNA synthesis in vivo.

    Science.gov (United States)

    Palangat, Murali; Larson, Daniel R

    2016-07-01

    RNA synthesis occurs through the multi-step process of transcription which consists of initiation, elongation, termination, and cleavage of the nascent RNA. In recent years, post-initiation events have attracted considerable attention as regulatory steps in gene expression. In particular, changes in elongation rate have been proposed to alter RNA fate either through changes in RNA secondary structure or recruitment of trans-acting factors, but systematic approaches for perturbing and measuring elongation rate are currently lacking. Here, we describe a system for precisely measuring elongation dynamics for single nascent transcripts at a single gene locus in human cell lines. The system is based on observing the production of fluorescently labeled RNA stem loops which flank a region of interest. The region of interest can be altered using flp recombinases, thus allowing one to study the effects of cis-acting sequences on transcription rate. The dual-color RNAs which are made during this process are exported and translated, thus enabling visualization of each step in gene expression.

  7. Two-photon photodynamic properties of TBO-AuNR-in-shell nanoparticles (Conference Presentation)

    Science.gov (United States)

    Wu, Cheng-Han; Yeh, Chen-Sheng; Cheng, Fong-Yu; Tsai, Zen-Uong; Liu, Tzu-Ming

    2016-03-01

    Photodynamic therapy (PDT) is a light-activated chemotherapeutic treatment that utilizes singlet oxygen and reactive oxygen species induced oxidative reactions to react with surrounding biological substrates, which either kills or irreversibly damages malignant cells. We used multiphoton nonlinear optical microscopy to observe the photo-dynamic effects of TBO-AuNR-in-shell NPs. Excited by femtosecond Cr:forsterite laser operating at 1230nm, singlet oxygen were generated through a plasmon-enhanced two-photon nonlinear optical process. For cells took up NPs, this photodynamic effect can kill the cell. From nonlinear optical microscopy images, we found they shrunk after 3 minutes of illumination.

  8. A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay.

    Science.gov (United States)

    Bartlett, J M S; Campbell, Fiona M; Ibrahim, Merdol; O'Grady, Anthony; Kay, Elaine; Faulkes, Catherine; Collins, Nadine; Starczynski, Jane; Morgan, John M; Jasani, Bharat; Miller, Keith

    2011-01-01

    We performed a multicenter assessment of a new HER2 dual-color chromogenic in situ hybridization (CISH) test and herein report on concordance of CISH data with fluorescence in situ hybridization (FISH) data and intraobserver and interlaboratory scoring consistency. HER2 results were evaluated using duplicate cores from 30 breast cancers in 5 laboratories using the Ventana HER2 dual-color ISH assay (Ventana Medical Systems, Cambridgeshire, England) and in 1 central laboratory using a standard FISH assay. Overall 93.3% of cases were successfully analyzed by CISH across the 5 participating laboratories. There was excellent concordance (98.0% overall) for diagnosis of HER2 amplification by CISH compared with FISH. Intraobserver variability (7.7%) and intersite variability (9.1%) of absolute HER2/chromosome enumeration probe 17 ratios were tightly controlled across all participating laboratories. The Ventana HER2 dual-color ISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national and international guidelines for performance of ISH-based diagnostic tests.

  9. A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay.

    LENUS (Irish Health Repository)

    Bartlett, J M S

    2011-01-01

    We performed a multicenter assessment of a new HER2 dual-color chromogenic in situ hybridization (CISH) test and herein report on concordance of CISH data with fluorescence in situ hybridization (FISH) data and intraobserver and interlaboratory scoring consistency. HER2 results were evaluated using duplicate cores from 30 breast cancers in 5 laboratories using the Ventana HER2 dual-color ISH assay (Ventana Medical Systems, Cambridgeshire, England) and in 1 central laboratory using a standard FISH assay. Overall 93.3% of cases were successfully analyzed by CISH across the 5 participating laboratories. There was excellent concordance (98.0% overall) for diagnosis of HER2 amplification by CISH compared with FISH. Intraobserver variability (7.7%) and intersite variability (9.1%) of absolute HER2\\/chromosome enumeration probe 17 ratios were tightly controlled across all participating laboratories. The Ventana HER2 dual-color ISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national and international guidelines for performance of ISH-based diagnostic tests.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

    Science.gov (United States)

    Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

    2013-10-01

    The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. [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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Multi-immunoreaction-based dual-color capillary electrophoresis for enhanced diagnostic reliability of thyroid gland disease.

    Science.gov (United States)

    Woo, Nain; Kim, Su-Kang; Kang, Seong Ho

    2017-08-04

    Thyroid-stimulating hormone (TSH) secretion plays a critical role in regulating thyroid gland function and circulating thyroid hormones (i.e., thyroxine (T4) and triiodothyronine (T3)). A novel multi-immunoreaction-based dual-color capillary electrophoresis (CE) technique was investigated in this study to assess its reliability in diagnosing thyroid gland disease via simultaneous detection of TSH, T3, and T4 in a single run of CE. Compared to the conventional immunoreaction technique, multi-immunoreaction of biotinylated streptavidin antibodies increased the selectivity and sensitivity for individual hormones in human blood samples. Dual-color laser-induced fluorescence (LIF) detection-based CE performed in a running buffer of 25mM Na2B4O7-NaOH (pH 9.3) allowed for fast, simultaneous quantitative analysis of three target thyroid hormones using different excited wavelengths within 3.2min. This process had excellent sensitivity and detection limits of 0.05-5.32 fM. The results showed 1000-100,000 times higher detection sensitivity than previous methods. Method validation with enzyme linked immunosorbent assay for application with human blood samples showed that the CE method was not significantly different at the 98% confidence level. Therefore, the developed CE-LIF method has the advantages of high detection sensitivity, faster analysis time, and smaller sample amount compared to the conventional methods The combined multi-immunoreaction and dual-color CE-LIF method should have increased diagnostic reliability for thyroid gland disease compared to conventional methods based on its highly sensitive detection of thyroid hormones using a single injection and high-throughput screening. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. 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.

  13. 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.

  14. Characterization of cytoplasmic Gag-gag interactions by dual-color z-scan fluorescence fluctuation spectroscopy.

    Science.gov (United States)

    Fogarty, Keir H; Chen, Yan; Grigsby, Iwen F; Macdonald, Patrick J; Smith, Elizabeth M; Johnson, Jolene L; Rawson, Jonathan M; Mansky, Louis M; Mueller, Joachim D

    2011-03-16

    Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs.

    Science.gov (United States)

    Cevenini, Luca; Michelini, Elisa; D'Elia, Marcello; Guardigli, Massimo; Roda, Aldo

    2013-01-01

    Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ(9)-tetrahydrocannabinol as major constituent.

  16. 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).

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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).

  4. Thermodynamic free-energy minimization for unsupervised fusion of dual-color infrared breast images

    Science.gov (United States)

    Szu, Harold; Miao, Lidan; Qi, Hairong

    2006-04-01

    function [A] may vary from the point tumor to its neighborhood, we could not rely on neighborhood statistics as did in a popular unsupervised independent component analysis (ICA) mathematical statistical method, we instead impose the physics equilibrium condition of the minimum of Helmholtz free-energy, H = E - T °S. In case of the point breast cancer, we can assume the constant ground state energy E ° to be normalized by those benign neighborhood tissue, and then the excited state can be computed by means of Taylor series expansion in terms of the pixel I/O data. We can augment the X-ray mammogram technique with passive IR imaging to reduce the unwanted X-rays during the chemotherapy recovery. When the sequence is animated into a movie, and the recovery dynamics is played backward in time, the movie simulates the cameras' potential for early detection without suffering the PD=0.1 search uncertainty. In summary, we applied two satellite-grade dual-color IR imaging cameras and advanced military (automatic target recognition) ATR spectrum fusion algorithm at the middle wavelength IR (3 - 5μm) and long wavelength IR (8 - 12μm), which are capable to screen malignant tumors proved by the time-reverse fashion of the animated movie experiments. On the contrary, the traditional thermal breast scanning/imaging, known as thermograms over decades, was IR spectrum-blind, and limited to a single night-vision camera and the necessary waiting for the cool down period for taking a second look for change detection suffers too many environmental and personnel variabilities.

  5. 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.

  6. 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.

  7. 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 ...

  8. 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.

  9. 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-.

  10. 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.

  11. 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.

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

    Science.gov (United States)

    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.

  13. (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.

  14. 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...

  15. 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.

  16. 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.

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Two-photon absorption-induced photoacoustic imaging of Rhodamine B dyed polyethylene spheres using a femtosecond laser.

    Science.gov (United States)

    Langer, Gregor; Bouchal, Klaus-Dieter; Grün, Hubert; Burgholzer, Peter; Berer, Thomas

    2013-09-23

    In the present paper we demonstrate the possibility to image dyed solids, i.e. Rhodamine B dyed polyethylene spheres, by means of two-photon absorption-induced photoacoustic scanning microscopy. A two-photon luminescence image is recorded simultaneously with the photoacoustic image and we show that location and size of the photoacoustic and luminescence image match. In the experiments photoacoustic signals and luminescence signals are generated by pulses from a femtosecond laser. Photoacoustic signals are acquired with a hydrophone; luminescence signals with a spectrometer or an avalanche photo diode. In addition we derive the expected dependencies between excitation intensity and photoacoustic signal for single-photon absorption, two-photon absorption and for the combination of both. In order to verify our setup and evaluation method the theoretical predictions are compared with experimental results for liquid and solid specimens, i.e. a carbon fiber, Rhodamine B solution, silicon, and Rhodamine B dyed microspheres. The results suggest that the photoacoustic signals from the Rhodamine B dyed microspheres do indeed stem from two-photon absorption.

  8. A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging.

    Science.gov (United States)

    Jathoul, Amit P; Grounds, Helen; Anderson, James C; Pule, Martin A

    2014-11-24

    Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λ(max)=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

  9. Solution-Grown ZnO Films toward Transparent and Smart Dual-Color Light-Emitting Diode.

    Science.gov (United States)

    Huang, Xiaohu; Zhang, Li; Wang, Shijie; Chi, Dongzhi; Chua, Soo Jin

    2016-06-22

    An individual light-emitting diode (LED) capable of emitting different colors of light under different bias conditions not only allows for compact device integration but also extends the functionality of the LED beyond traditional illumination and display. Herein, we report a color-switchable LED based on solution-grown n-type ZnO on p-GaN/n-GaN heterojunction. The LED emits red light with a peak centered at ∼692 nm and a full width at half-maximum of ∼90 nm under forward bias, while it emits green light under reverse bias. These two lighting colors can be switched repeatedly by reversing the bias polarity. The bias-polarity-switched dual-color LED enables independent control over the lighting color and brightness of each emission with two-terminal operation. The results offer a promising strategy toward transparent, miniaturized, and smart LEDs, which hold great potential in optoelectronics and optical communication.

  10. 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

  11. 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...

  12. 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.

  13. 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)

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Two-photon absorption and transient photothermal imaging of pigments in tissues

    Science.gov (United States)

    Ye, Tong; Fu, Dan; Matthews, Thomas E.; Hong, Lian; Simon, John D.; Warren, Warren S.

    2008-02-01

    As a main pigment in skin tissues, melanin plays an important role in photo-protecting skin from UV radiation. However, melanogenesis may be altered due to disease or environmental factors; for example, sun exposure may cause damage and mutation of melanocytes and induce melanoma. Imaging pigmentation changes may provide invaluable information to catch the malignant transformation in its early stage and in turn improve the prognosis of patients. We have demonstrated previously that transmission mode, two-photon, one- or two-color absorption microscopy could provide remarkable contrast in imaging melanin in skin. In this report we demonstrate significantly improved sensitivity, so that we are now able to image in epi-mode (or back reflection) in two-photon absorption. This improvement makes possible for us to characterize the different types of pigmentation on the skin in vivo at virtually any location. Another finding is that we can also image transient photothermal dynamics due to the light absorption of melanin. By carefully choosing excitation and probe wavelengths, we might be able to image melanin in different structures under different micro-environments in skin, which could provide useful photochemical and photophysical insights in understanding how pigments are involved in photoprotection and photodamage of cells.

  20. In situ electrical and thermal monitoring of printed electronics by two-photon mapping.

    Science.gov (United States)

    Pastorelli, Francesco; Accanto, Nicolò; Jørgensen, Mikkel; van Hulst, Niek F; Krebs, Frederik C

    2017-06-19

    Printed electronics is emerging as a new, large scale and cost effective technology that will be disruptive in fields such as energy harvesting, consumer electronics and medical sensors. The performance of printed electronic devices relies principally on the carrier mobility and molecular packing of the polymer semiconductor material. Unfortunately, the analysis of such materials is generally performed with destructive techniques, which are hard to make compatible with in situ measurements, and pose a great obstacle for the mass production of printed electronics devices. A rapid, in situ, non-destructive and low-cost testing method is needed. In this study, we demonstrate that nonlinear optical microscopy is a promising technique to achieve this goal. Using ultrashort laser pulses we stimulate two-photon absorption in a roll coated polymer semiconductor and map the resulting two-photon induced photoluminescence and second harmonic response. We show that, in our experimental conditions, it is possible to relate the total amount of photoluminescence detected to important material properties such as the charge carrier density and the molecular packing of the printed polymer material, all with a spatial resolution of 400 nm. Importantly, this technique can be extended to the real time mapping of the polymer semiconductor film, even during the printing process, in which the high printing speed poses the need for equally high acquisition rates.

  1. 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.

  2. 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.

  3. Multi-beam two-photon imaging of fast Ca2+ signals in the Langendorff mouse heart.

    Science.gov (United States)

    Hammer, Karin; Lipp, Peter; Kaestner, Lars

    2014-11-03

    Although the role of calcium (Ca(2+)) in excitation-contraction coupling in the heart can be comprehensively studied at the cellular level, propagation of Ca(2+) signals intercellularly requires tissue-based investigations. To access cells below the epicardium, an optical-sectioning technique is necessary. Multi-photon microscopy allows reliable imaging for penetration to depths of up to 0.5 mm. Here, we provide a protocol that uses multibeam two-photon microscopy for measuring Ca(2+) signals in a Langendorff-perfused mouse heart.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Autocorrelation measurement of femtosecond laser pulses based on two-photon absorption in GaP photodiode

    Energy Technology Data Exchange (ETDEWEB)

    Chong, E. Z.; Watson, T. F.; Festy, F., E-mail: frederic.festy@kcl.ac.uk [Biomaterials, Biomimetics and Biophotonics Division, King' s College London—Dental Institute, SE1 9RT London (United Kingdom)

    2014-08-11

    Semiconductor materials which exhibit two-photon absorption characteristic within a spectral region of interest can be useful in building an ultra-compact interferometric autocorrelator. In this paper, we report on the evidence of a nonlinear absorption process in GaP photodiodes which was exploited to measure the temporal profile of femtosecond Ti:sapphire laser pulses with a tunable peak wavelength above 680 nm. The two-photon mediated conductivity measurements were performed at an average laser power of less than a few tenths of milliwatts. Its suitability as a single detector in a broadband autocorrelator setup was assessed by investigating the nonlinear spectral sensitivity bandwidth of a GaP photodiode. The highly favourable nonlinear response was found to cover the entire tuning range of our Ti:sapphire laser and can potentially be extended to wavelengths below 680 nm. We also demonstrated the flexibility of GaP in determining the optimum compensation value of the group delay dispersion required to restore the positively chirped pulses inherent in our experimental optical system to the shortest pulse width possible. With the rise in the popularity of nonlinear microscopy, the broad two-photon response of GaP and the simplicity of this technique can provide an alternative way of measuring the excitation laser pulse duration at the focal point of any microscopy systems.

  15. 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

  16. 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.

  17. Two-photon imaging of lymphoma cells targeted by gold nanoparticles

    Institute of Scientific and Technical Information of China (English)

    Xiaochao Qu; Jing Wang; Cuiping Yao; Zhenxi Zhang

    2008-01-01

    Gold nanoparticles (NPs) have highly efficient multi-photon-induced luminescence. In this paper, we record the two-photon images of gold NPs, lymphoma cell line Karpas 299, and Karpas 299 incubated with 30-nm-diameter gold NPs and ACT-1 antibody conjugates (Au30-ACT-1 conjugates) by using a multi-photon microscopy system. Due to the specific conjugation of ACT-1 antibody and celt membrane receptor CD25, gold NPs are only bound to the surface of cell membrane of Karpas 299. The luminescence intensity of gold NPs is higher than that of cells at 750-nm laser excitation. By comparing the images of Karpas 299 cells incubated with and without gold NPs, it is found that by means of gold NPs, we can get clear cell images with lower excitation power. Their excellent optical and chemical properties make gold NPs an attractive contrast agent for cellular imaging.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual-color bioluminescence reporter.

    Science.gov (United States)

    Kwon, Hyuck Joon; Ohmiya, Yoshihiro; Yasuda, Kazunori

    2014-12-01

    A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red-emitting Phrixothrix hirtus luciferase (PxRe) and a blue-emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non-mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual-color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes.

  5. Transformation of odor selectivity from projection neurons to single mushroom body neurons mapped with dual-color calcium imaging.

    Science.gov (United States)

    Li, Hao; Li, Yiming; Lei, Zhengchang; Wang, Kaiyu; Guo, Aike

    2013-07-16

    Although the response properties of most neurons are, to a large extent, determined by the presynaptic inputs that they receive, comprehensive functional characterization of the presynaptic inputs of a single neuron remains elusive. Toward this goal, we introduce a dual-color calcium imaging approach that simultaneously monitors the responses of a single postsynaptic neuron together with its presynaptic axon terminal inputs in vivo. As a model system, we applied the strategy to the feed-forward connections from the projection neurons (PNs) to the Kenyon cells (KCs) in the mushroom body of Drosophila and functionally mapped essentially all PN inputs for some of the KCs. We found that the output of single KCs could be well predicted by a linear summation of the PN input signals, indicating that excitatory PN inputs play the major role in generating odor-selective responses in KCs. When odors failed to activate KC output, local calcium transients restricted to individual postsynaptic sites could be observed in the KC dendrites. The response amplitudes of the local transients often correlated linearly with the presynaptic response amplitudes, allowing direct assay of the strength of single synaptic sites. Furthermore, we found a scaling relationship between the total number of PN terminals that a single KC received and the average synaptic strength of these PN-KC synapses. Our strategy provides a unique perspective on the process of information transmission and integration in a model neural circuit and may be broadly applicable for the study of the origin of neuronal response properties.

  6. Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution.

    Science.gov (United States)

    Yasunaga, Mayu; Nakajima, Yoshihiro; Ohmiya, Yoshihiro

    2014-09-01

    Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution.

  7. 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.

  8. 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.

  9. 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.

  10. 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.)

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. 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.

  16. 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...

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Development and application of biological techniques to two-photon photodynamic therapy

    Science.gov (United States)

    Khurana, Mamta; Karotki, Aliaksandr; Moriyama, Eduardo H.; Akens, Margarete K.; Wilson, Brian C.

    2007-06-01

    Two-photon (2-γ) photodynamic therapy (PDT) as opposed to "standard" one-photon (1-γ) PDT with Visudyne has recently been suggested as a targeted treatment alternative for wet-form age-related macular degeneration (AMD) and other neovascular diseases. AMD is a major cause of severe vision loss in the older population. It occurs due to growth of new leaky blood vessels (neovasculature) from the choriocapillaris, which results in destruction of photoreceptors in the fovea and loss of central vision. Damage outside the diseased region is always a concern, due to photosensitizer accumulation and its 1-γ excitation. Highly targeted 2-γ excitation, due to its non-linear intensity dependence, intrinsically avoids out-of-focus damage to healthy tissues and so could be valuable for wet-AMD. We have previously developed a quantitative approach for comparing the 2-γ efficacy of photosensitizers in vitro. In this study, we report further the development of ex vivo and in vivo techniques. A mouse mesenteric vessel has been investigated as the ex vivo model of neovasculature. For the in vivo studies, we have explored a mouse dorsal skin-fold window chamber model. Two-photon PDT is delivered using tightly focused ~300 fs laser pulses from a Ti:sapphire laser operating at 850 nm with 90 MHz pulse repetition rate. Confocal microscopy coupled to the laser was used to visualize the vessel's/cell's response before, during and after the treatment. We are able to demonstrate quantitative biological techniques to evaluate efficacy of 2-γ PDT photosensitizers in vivo.

  3. Clinical, cytogenetic and dual-color FISH studies on five cases of myelodysplastic syndrome or acute myeloid leukemia patients with 1;7 translocation

    Institute of Scientific and Technical Information of China (English)

    申咏梅; 薛永权; 李建勇; 潘金兰; 吴亚芳

    2003-01-01

    Objective To study the clinical and cytogenetic characteristics of four patients with myel odysplastic syndrome (MDS) and one with acute myeloid leukemia experiencing t(1; 7).Methods Five patients seen in our hospital from 1992 to 2001 were diagnosed as MDS and acute myelocytic leukemia (AML) according to the French-American-British (FAB) criteria. Chromosomes were prepared using the direct method as well as 24-hou r unstimulated cultures of fresh heparinized bone marrow for each subject, while R-banding was used to analyze karyotypes. Dual-color fluorescence in situ hy bridization (FISH) using SpectrumRed and SpectrumGreen directly labeled chromoso me 1-specific α-satellite DNA probe (red) and chromosome 7- specific α-sat ellite DNA probe (green) was performed for three cases. Results Of the five patients, three had 1;7 translocation due to along history of expos ure to benzene. In three cases, dual-color FISH resulted in three red signals and two green ones, in which one red signal adjoining one green signal in 27.6% , 84% and 18.5% metaphases, respectively. Conclusions Exposure to benzene may be the cause for Chinese MDS and AML patients with t(1;7 ) translocation. The result of dual-color FISH convincingly confirmed that the centromere of the derivative chromosome 7p/1q resulting from 1;7 translocation was made up of centromeres from both chromosomes 1 and 7.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.)

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  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. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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].

  4. Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.

    Directory of Open Access Journals (Sweden)

    Haruki Odaka

    Full Text Available Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

  5. Genetically-Encoded Yellow Fluorescent cAMP Indicator with an Expanded Dynamic Range for Dual-Color Imaging

    Science.gov (United States)

    Odaka, Haruki; Arai, Satoshi; Inoue, Takafumi; Kitaguchi, Tetsuya

    2014-01-01

    Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics. PMID:24959857

  6. Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study

    DEFF Research Database (Denmark)

    Carrer, Dolores C.; Vermehren, Charlotte; Bagatolli, Luis

    2008-01-01

    that are in turn separated by particular structures we named "canyons". These canyons start in the surface as a wrinkle, eventually closing and going all the way inside the epidermis as a distinct structure that reaches the stratum basale. This structure, described previously in the epidermis of mouse skin...

  7. Enhancement of Squeezing in Two-Photon Jaynes-Cummings Model with Atomic Measurement

    Institute of Scientific and Technical Information of China (English)

    YE Sai-Yun

    2006-01-01

    We investigate the squeezing properties of the cavity field in the degenerate two-photon Jaynes-Cummings model. Compared with the one-photon Jaynes-Cummings model, the squeezing is more pronounced in the case of two-photon Jaynes-Cummings model under certain conditions.

  8. Event-by-event simulation of nonclassical effects in two-photon interference experiments

    NARCIS (Netherlands)

    Michielsen, K.; Jin, F.; Delina, M.; Raedt, H. De

    2012-01-01

    A corpuscular simulation model for second-order intensity interference phenomena is discussed. It is shown that both the visibility V = 1/2 predicted for two-photon interference experiments with two independent sources and the visibility V = 1 predicted for two-photon interference experiments with a

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

    Science.gov (United States)

    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.

  10. Effect of morphology and solvent on two-photon absorption of nano zinc oxide

    Energy Technology Data Exchange (ETDEWEB)

    Kavitha, M.K. [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695547, Kerala (India); Haripadmam, P.C.; Gopinath, Pramod; Krishnan, Bindu [Department of Physics, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695547, Kerala (India); John, Honey, E-mail: honey@iist.ac.in [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695547, Kerala (India)

    2013-05-15

    Highlights: ► ZnO nanospheres and triangular structures synthesis by novel precipitation technique. ► The effect of precursor concentration on the size and shape of nano ZnO. ► Open aperture Z-scan measurements of the ZnO nanoparticle dispersions. ► Nanospheres exhibit higher two photon absorption coefficient than triangular nanostructures. ► Nanospheres dispersed in water exhibit higher two photon absorption coefficient than its dispersion in 2-propanol. - Abstract: In this paper, we report the effect of morphology and solvent on the two-photon absorption of nano zinc oxide. Zinc oxide nanoparticles in two different morphologies like nanospheres and triangular nanostructures are synthesized by novel precipitation technique and their two-photon absorption coefficient is measured using open aperture Z-scan technique. Experimental results show that the zinc oxide nanospheres exhibit higher two-photon absorption coefficient than the zinc oxide triangular nanostructures. The zinc oxide nanospheres dispersed in water exhibit higher two-photon absorption coefficient than that of its dispersion in 2-propanol. The zinc oxide nanospheres dispersed in water shows a decrease in two-photon absorption coefficient with an increase in on-axis irradiance. The result confirms the dependence of shape and solvent on the two-photon absorption of nano zinc oxide.

  11. Production of e, $\\mu$ and $\\tau$ Pairs in Untagged Two-Photon Collisions at LEP

    CERN Document Server

    Acciarri, M; Aguilar-Benítez, M; Ahlen, S P; Alcaraz, J; Alemanni, G; Allaby, James V; Aloisio, A; Alverson, G; Alviggi, M G; Ambrosi, G; Anderhub, H; Andreev, V P; Angelescu, T; Anselmo, F; Arefev, A; Azemoon, T; Aziz, T; Bagnaia, P; Baksay, L; Ball, R C; Banerjee, S; Banerjee, Sw; Banicz, K; Barczyk, A; Barillère, R; Barone, L; Bartalini, P; Baschirotto, A; Basile, M; Battiston, R; Bay, A; Becattini, F; Becker, U; Behner, F; Berdugo, J; Berges, P; Bertucci, B; Betev, B L; Bhattacharya, S; Biasini, M; Biland, A; Bilei, G M; Blaising, J J; Blyth, S C; Bobbink, Gerjan J; Böck, R K; Böhm, A; Boldizsar, L; Borgia, B; Boucham, A; Bourilkov, D; Bourquin, Maurice; Boutigny, D; Braccini, S; Branson, J G; Brigljevic, V; Brock, I C; Buffini, A; Buijs, A; Burger, J D; Burger, W J; Busenitz, J K; Cai, X D; Campanelli, M; Capell, M; Cara Romeo, G; Carlino, G; Cartacci, A M; Casaus, J; Castellini, G; Cavallari, F; Cavallo, N; Cecchi, C; Cerrada-Canales, M; Cesaroni, F; Chamizo-Llatas, M; Chang, Y H; Chaturvedi, U K; Chekanov, S V; Chemarin, M; Chen, A; Chen, G; Chen, G M; Chen, H F; Chen, H S; Chen, M; Chiefari, G; Chien, C Y; Cifarelli, Luisa; Cindolo, F; Civinini, C; Clare, I; Clare, R; Cohn, H O; Coignet, G; Colijn, A P; Colino, N; Commichau, V; Costantini, S; Cotorobai, F; de la Cruz, B; Csilling, Akos; Dai, T S; D'Alessandro, R; De Asmundis, R; Degré, A; Deiters, K; Denes, P; De Notaristefani, F; DiBitonto, Daryl; Diemoz, M; Van Dierendonck, D N; Di Lodovico, F; Dionisi, C; Dittmar, Michael; Dominguez, A; Doria, A; Dorne, I; Dova, M T; Drago, E; Duchesneau, D; Duinker, P; Durán, I; Dutta, S; Easo, S; Efremenko, Yu V; El-Mamouni, H; Engler, A; Eppling, F J; Erné, F C; Ernenwein, J P; Extermann, Pierre; Fabre, M; Faccini, R; Falciano, S; Favara, A; Fay, J; Fedin, O; Felcini, Marta; Fenyi, B; Ferguson, T; Ferroni, F; Fesefeldt, H S; Fiandrini, E; Field, J H; Filthaut, Frank; Fisher, P H; Fisk, I; Forconi, G; Fredj, L; Freudenreich, Klaus; Furetta, C; Galaktionov, Yu; Ganguli, S N; García-Abia, P; Gau, S S; Gentile, S; Gerald, J; Gheordanescu, N; Giagu, S; Goldfarb, S; Goldstein, J; Gong, Z F; Gougas, Andreas; Gratta, Giorgio; Grünewald, M W; Gupta, V K; Gurtu, A; Gutay, L J; Hartmann, B; Hasan, A; Hatzifotiadou, D; Hebbeker, T; Hervé, A; Van Hoek, W C; Hofer, H; Hong, S J; Hoorani, H; Hou, S R; Hu, G; Innocente, Vincenzo; Janssen, H; Jenkes, K; Jin, B N; Jones, L W; de Jong, P; Josa-Mutuberria, I; Kasser, A; Khan, R A; Kamrad, D; Kamyshkov, Yu A; Kapustinsky, J S; Karyotakis, Yu; Kaur, M; Kienzle-Focacci, M N; Kim, D; Kim, D H; Kim, J K; Kim, S C; Kim, Y G; Kinnison, W W; Kirkby, A; Kirkby, D; Kirkby, Jasper; Kiss, D; Kittel, E W; Klimentov, A; König, A C; Kopp, A; Korolko, I; Koutsenko, V F; Krämer, R W; Krenz, W; Kunin, A; Ladrón de Guevara, P; Landi, G; Lapoint, C; Lassila-Perini, K M; Laurikainen, P; Lebeau, M; Lebedev, A; Lebrun, P; Lecomte, P; Lecoq, P; Le Coultre, P; Leggett, C; Le Goff, J M; Leiste, R; Leonardi, E; Levchenko, P M; Li Chuan; Lin, C H; Lin, W T; Linde, Frank L; Lista, L; Liu, Z A; Lohmann, W; Longo, E; Lu, W; Lü, Y S; Lübelsmeyer, K; Luci, C; Luckey, D; Luminari, L; Lustermann, W; Ma Wen Gan; Maity, M; Majumder, G; Malgeri, L; Malinin, A; Maña, C; Mangeol, D J J; Mangla, S; Marchesini, P A; Marin, A; Martin, J P; Marzano, F; Massaro, G G G; McNally, D; Mele, S; Merola, L; Meschini, M; Metzger, W J; Von der Mey, M; Mi, Y; Mihul, A; Van Mil, A J W; Mirabelli, G; Mnich, J; Molnár, P; Monteleoni, B; Moore, R; Morganti, S; Moulik, T; Mount, R; Müller, S; Muheim, F; Muijs, A J M; Nahn, S; Napolitano, M; Nessi-Tedaldi, F; Newman, H; Niessen, T; Nippe, A; Nisati, A; Nowak, H; Oh, Yu D; Opitz, H; Organtini, G; Ostonen, R; Palomares, C; Pandoulas, D; Paoletti, S; Paolucci, P; Park, H K; Park, I H; Pascale, G; Passaleva, G; Patricelli, S; Paul, T; Pauluzzi, M; Paus, C; Pauss, Felicitas; Peach, D; Pei, Y J; Pensotti, S; Perret-Gallix, D; Petersen, B; Petrak, S; Pevsner, A; Piccolo, D; Pieri, M; Pinto, J C; Piroué, P A; Pistolesi, E; Plyaskin, V; Pohl, M; Pozhidaev, V; Postema, H; Produit, N; Prokofev, D; Prokofiev, D O; Rahal-Callot, G; Raja, N; Rancoita, P G; Rattaggi, M; Raven, G; Razis, P A; Read, K; Ren, D; Rescigno, M; Reucroft, S; Van Rhee, T; Riemann, S; Riles, K; Rind, O; Robohm, A; Rodin, J; Roe, B P; Romero, L; Rosier-Lees, S; Rosselet, P; Van Rossum, W; Roth, S; Rubio, Juan Antonio; Ruschmeier, D; Rykaczewski, H; Salicio, J; Sánchez, E; Sanders, M P; Sarakinos, M E; Sarkar, S; Sassowsky, M; Sauvage, G; Schäfer, C; Shchegelskii, V; Schmidt-Kärst, S; Schmitz, D; Schmitz, P; Schneegans, M; Scholz, N; Schopper, Herwig Franz; Schotanus, D J; Schwenke, J; Schwering, G; Sciacca, C; Sciarrino, D; Servoli, L; Shevchenko, S; Shivarov, N; Shoutko, V; Shukla, J; Shumilov, E; Shvorob, A V; Siedenburg, T; Son, D; Sopczak, André; Soulimov, V; Smith, B; Spillantini, P; Steuer, M; Stickland, D P; Stone, H; Stoyanov, B; Strässner, A; Strauch, K; Sudhakar, K; Sultanov, G G; Sun, L Z; Susinno, G F; Suter, H; Swain, J D; Tang, X W; Tauscher, Ludwig; Taylor, L; Ting, Samuel C C; Ting, S M; Tonutti, M; Tonwar, S C; Tóth, J; Tully, C; Tuchscherer, H; Tung, K L; Uchida, Y; Ulbricht, J; Uwer, U; Valente, E; Van de Walle, R T; Vesztergombi, G; Vetlitskii, I; Viertel, Gert M; Vivargent, M; Völkert, R; Vogel, H; Vogt, H; Vorobev, I; Vorobyov, A A; Vorvolakos, A; Wadhwa, M; Wallraff, W; Wang, J C; Wang, X L; Wang, Z M; Weber, A; Wittgenstein, F; Wu, S X; Wynhoff, S; Xu, J; Xu, Z Z; Yang, B Z; Yang, C G; Yao, X Y; Ye, J B; Yeh, S C; You, J M; Zalite, A; Zalite, Yu; Zemp, P; Zeng, Y; Zhang, Z; Zhang, Z P; Zhou, B; Zhou, Y; Zhu, G Y; Zhu, R Y; Zichichi, Antonino; Ziegler, F

    1997-01-01

    The two-photon collision reaction e+e- --> e+e-l+l- has been studied at root(s) ~ 91 GeV using the L3 detector at LEP for l = e, muon , tau. We have analysed untagged configurations where the two photons are quasi-real. Good agreement is found between our measurements and the order alpha**4 QED expectation.

  12. 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

  13. Two-Photon Interference with the Type Ⅱ Spontaneous Parametric Down-Conversion

    Institute of Scientific and Technical Information of China (English)

    江云坤; 史保森; 李剑; 段开敏; 范晓锋; 郭光灿

    2001-01-01

    The two-photon polarized entangled state is generated from the type Ⅱ spontaneous parametric down-conversion pumped by a femtosecond pulse. The two-photon interference is observed in the Hong-Ou-Mandel interferometer. The high visibility of the interference is restored with narrow band interference filters placed in front of the detectors.

  14. Description of states of two-photon interference in optical gating Michelson interferometer

    Science.gov (United States)

    Pongophas, Ekkarat; Sinsarp, Asawin; Suwanna, Sujin; Chunwachirasiri, Withoon; Singhsomroje, Wisit

    2015-07-01

    The interference of two photons in the optical gating Michelson interferometer is investigated. 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 an equivalent description of the two-photon states which is the interference of four cases of two-photon traveling states, as implied by the evolution analysis. Additionally, the space-time domain analysis reveals that the classical interference of high-intensity light source is identical to the two-photon interference in the quantum regime except for a multiplicative factor of (n 2), where n is the number of photons.

  15. 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.

  16. Band Structure of Photonic Crystals Fabricated by Two-Photon Polymerization

    Directory of Open Access Journals (Sweden)

    Mikhail V. Rybin

    2015-01-01

    Full Text Available We study theoretically the band-gap structures of several types of three-dimensional photonic crystals with the fcc lattice symmetry: synthetic opals, inverted yablonovite and woodpile. The samples of inverted yablonovite, inverted yablonovite with a glassy superstructure and woodpile are fabricated by two-photon polymerization through a direct laser writing technique, which allows the creation of complex three-dimensional photonic crystals with a resolution better than 100 nm. A material is polymerized along the trace of a moving laser focus, thus enabling the fabrication of any desirable three-dimensional structure by direct “recording” into the volume of a photosensitive material. The correspondence of the structures of the fabricated samples to the expected fcc lattices is confirmed by scanning electron microscopy. We discuss theoretically how the complete photonic band-gap is modified by structural and dielectric parameters. We demonstrate that the photonic properties of opal and yablonovite are opposite: the complete photonic band gap appears in the inverted opal, and direct yablonovite is absent in direct opal and inverted yablonovite.

  17. Nanomechanical probing of soft matter through hydrophobic AFM tips fabricated by two-photon polymerization

    Science.gov (United States)

    Suriano, Raffaella; Zandrini, Tommaso; De Marco, Carmela; Osellame, Roberto; Turri, Stefano; Bragheri, Francesca

    2016-04-01

    Atomic force microscopy (AFM) nanoindentation of soft materials is a powerful tool for probing mechanical properties of biomaterials. Though many results have been reported in this field over the last decade, adhesion forces between the tip and the sample hinder the elastic modulus measurement when hydrophilic soft samples are investigated. Here, two-photon polymerization (2PP) technology was used to fabricate hydrophobic perfluoropolyether-based AFM tips. The hydrophobic 2PP tips allowed us to overcome the limitations of commercial and functionalized tips as well as to successfully measure the elastic modulus of medically relevant soft materials in air. Our results obtained in the characterization of poly(dimethyl siloxane) and polyethylene glycol hydrogels showed lower adhesion forces over a larger measurement range when compared to measurements performed with commercial tips. The elastic moduli measured by means of hydrophobic 2PP AFM tips were also found to be comparable to those obtained using conventional techniques for macroscopic samples. We successfully showed that the hydrophobic AFM tips developed by this highly versatile technology enable the study of mechanical properties of soft matter, benefiting from reduced sample-tip interactions, and a custom-made shape and dimension of the tips.

  18. Multispot two-photon imaging of mice heart tissue detecting calcium waves

    Science.gov (United States)

    de Mauro, C.; Cecchetti, C. A.; Alfieri, D.; Borile, G.; Mongillo, M.; Pavone, F. S.

    2012-06-01

    High rate, full field image acquisition in multiphoton imaging is achievable by parallelization of the excitation and of the detection paths. Via a Diffractive Optical Elements (DOEs) which splits a pulsed laser, and a spatial resolved descanned detection path, a new approach to microscopy has been developed. By exploiting the three operating mode, single beam, 16 beamlets or 64 beamlets, the best experimental conditions can be found by adapting the power per beamlet. This Multiphoton Multispot system (MCube) has been characterized in thick tissue samples, and subsequently used for the first time for Ca2+ imaging of acute heart slices. A test sample with fixed mice heart slices with embedded sub-resolution fluorescent beads has been used to test the capability of optical axial resolution up to ~200 microns in depth. Radial and axial resolutions of 0.6 microns and 3 microns have been respectively obtained with a 40X water immersion objective, getting close to the theoretical limit. Then images of heart slices cardiomyocites, loaded with Fluo4-AM have been acquired. The formation of Ca2+ waves during electrostimulated beating has been observed, and the possibility of easily acquire full frame images at 15 Hz (16 beamlets) has been demonstrated, towards the in vivo study of time resolved cellular dynamics and arrhythmia trigger mechanisms in particular. A very high speed two-photon Random Access system for in vivo electrophysiological studies, towards the correlation of voltage and calcium signals in arrhythmia phenomena, is now under developing at Light4tech.

  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. 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)].

  1. Nanomechanical probing of soft matter through hydrophobic AFM tips fabricated by two-photon polymerization.

    Science.gov (United States)

    Suriano, Raffaella; Zandrini, Tommaso; De Marco, Carmela; Osellame, Roberto; Turri, Stefano; Bragheri, Francesca

    2016-04-15

    Atomic force microscopy (AFM) nanoindentation of soft materials is a powerful tool for probing mechanical properties of biomaterials. Though many results have been reported in this field over the last decade, adhesion forces between the tip and the sample hinder the elastic modulus measurement when hydrophilic soft samples are investigated. Here, two-photon polymerization (2PP) technology was used to fabricate hydrophobic perfluoropolyether-based AFM tips. The hydrophobic 2PP tips allowed us to overcome the limitations of commercial and functionalized tips as well as to successfully measure the elastic modulus of medically relevant soft materials in air. Our results obtained in the characterization of poly(dimethyl siloxane) and polyethylene glycol hydrogels showed lower adhesion forces over a larger measurement range when compared to measurements performed with commercial tips. The elastic moduli measured by means of hydrophobic 2PP AFM tips were also found to be comparable to those obtained using conventional techniques for macroscopic samples. We successfully showed that the hydrophobic AFM tips developed by this highly versatile technology enable the study of mechanical properties of soft matter, benefiting from reduced sample-tip interactions, and a custom-made shape and dimension of the tips.

  2. Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye.

    Science.gov (United States)

    Imanishi, Yoshikazu; Batten, Matthew L; Piston, David W; Baehr, Wolfgang; Palczewski, Krzysztof

    2004-02-01

    Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 +/- 1.1 microm in length and 0.8 +/- 0.2 microm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65-/- mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat-/- mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.

  3. 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.

  4. 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.

  5. 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

  6. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer

    DEFF Research Database (Denmark)

    Pedersen, Marianne; Rasmussen, Birgitte Bruun

    2009-01-01

    obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative...... invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios...

  7. 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.

  8. Two-photon absorption and two-photon circular dichroism of L-tryptophan in the near to far UV region

    Science.gov (United States)

    Vesga, Yuly; Hernandez, Florencio E.

    2017-09-01

    Herein we report on the first measurements of the two-photon absorption (TPA) spectrum of L-tryptophan in DMSO solution in the near to far UV region and the two-photon circular dichroism (TPCD) signal corresponding to a transition at 200 nm. We demonstrate the application of the Double L-scan technique in the near to far UV region to perform polarization dependent TPA measurements of chiral molecules. TPCD measurements below 400 nm reveal that chiral molecules in solution, such as tryptophan/DMSO, can undergo photochemical reactions in front of prolonged exposure to UV radiation.

  9. Induced structural defects in Ti-doped ZnO and its two-photon-excitation

    Science.gov (United States)

    Martínez Julca, Milton A.; Rivera, Ivonnemary; Santillan Mercado, Jaime; Sierra, Heidy; Perales-Pérez, Oscar

    2016-03-01

    ZnO is a well-known luminescent material that reacts with light to generate free radicals enabling its use in cancer treatment by Photodynamic Therapy (PDT). Unfortunately, up to know, the photo-excitation of ZnO-based materials' requires excitation with ultraviolet light, which limits their biomedical applications. In this regard, this work investigates the effect of Ti species incorporation into the lattice of ZnO nanoparticles (NPs) with the aim of improving the corresponding optical properties and enabling the two-photoexcitation with 690nm-light (near infrared light). A modified polyol-based route was used to synthesize pure and Ti-doped (9% at.) ZnO NPs. X-ray diffraction confirmed the formation of ZnO-wurtzite whereas Scanning Electron Microscopy confirmed the formation of monodispersed 100-nm NPs. Raman Spectroscopy measurements evidenced the presence of zinc interstitials (Zni) and oxygen vacancies (VO) in the host oxide strcuture. Asynthesized NPs were excited using the technique of two-photon fluorescence microscopy (TPFM). The photoluminescence (PL) spectra generated from the analysis of TPFM images revealed a high emission peak presence in the green region (555 nm) that was assigned to VO. Also, a weak but noticeable band at 420 nm was detected, which is attributed to electron transition from the shallow donor level of Zni to the valence band. These PL transitions will favor triplet states formation necessary to yield cytotoxic reactive oxygen species. Furthermore, the presence of the PL peaks confirmed the Ti-ZnO NPs capacity to be excited by 690-nm light, thus, opening new possibilities for this NPs to be used in lightinduced bio-medical applications.

  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. Dispersion spreading of biphotons in optical fibres and two-photon interference

    CERN Document Server

    Brida, G; Genovèse, M; Gramegna, M; Krivitsky, L A

    2006-01-01

    We present the first observation of two-photon polarization interference structure in the second-order Glauber's correlation function of two-photon light generated via type-II spontaneous parametric down-conversion. In order to obtain this result, two-photon light is transmitted through an optical fibre and the coincidence distribution is analyzed by means of the START-STOP method. Beyond the experimental demonstration of an interesting effect in quantum optics, these results also have considerable relevance for quantum communications.

  12. Coalescence and Anti-Coalescence Interference of Two-Photon Wavepacket in a Beam Splitter

    Institute of Scientific and Technical Information of China (English)

    WANG Kai-Ge; YANG Guo-Jian

    2004-01-01

    @@ We study theoretically the interference of a two-photon wavepacket in a beam splitter. We find that the spectrum symmetry for the two-photon wavepacket dominates the perfect coalescence and anti-coalescence interference.The coalescence interference is unrelated to photon entanglement. Only the anti-coalescence interference has evidence of photon entanglement. We prove that the two-photon wavepacket with an anti-symmetric spectrum is transparent to pass the 50/50 beam splitter, showing perfect anti-coalescence interference.

  13. The two-photon exchange contribution to elastic electron-nucleon scattering at large momentum transfer

    Energy Technology Data Exchange (ETDEWEB)

    Andrei V. Afanasev; Stanley J. Brodsky; Carl E. Carlson; Yu-Chun Chen; Marc Vanderhaeghen

    2005-01-01

    We estimate the two-photon exchange contribution to elastic electron-proton scattering at large momentum transfer by using a quark-parton representation of virtual Compton scattering. We thus can relate the two-photon exchange amplitude to the generalized parton distributions which also enter in other wide angle scattering processes. We find that the interference of one- and two-photon exchange contribution is able to substantially resolve the difference between electric form factor measurements from Rosenbluth and polarization transfer experiments.

  14. Dispersion spreading of biphotons in optical fibers and two-photon interference.

    Science.gov (United States)

    Brida, G; Chekhova, M V; Genovese, M; Gramegna, M; Krivitsky, L A

    2006-04-14

    We present the first observation of two-photon polarization interference structure in the second-order Glauber correlation function of two-photon light generated via type-II spontaneous parametric down-conversion. In order to obtain this result, two-photon light is transmitted through an optical fiber and the coincidence distribution is analyzed by means of the start-stop method. Beyond the experimental demonstration of an interesting effect in quantum optics, these results also have considerable relevance for quantum communications.

  15. Search for a Higgs boson decaying into two photons in the CMS detector

    Indian Academy of Sciences (India)

    Roberta Volpe; on behalf of the CMS Collaboration

    2012-11-01

    A search for a Higgs boson decaying into two photons in collisions at the LHC at a centre-of-mass energy of 7 TeV is presented. The analysis is performed on a dataset corresponding to 1.66 fb-1 of data recorded in 2011 by the CMS experiment. Limits are set on the cross-section of a Standard Model Higgs boson decaying into two photons, and on the cross-section of a fermiophobic Higgs boson decaying into two photons.

  16. Two-photon absorption of [2.2]paracyclophane derivatives in solution: A theoretical investigation

    Science.gov (United States)

    Ferrighi, Lara; Frediani, Luca; Fossgaard, Eirik; Ruud, Kenneth

    2007-12-01

    The two-photon absorption of a class of [2.2]paracyclophane derivatives has been studied using quadratic response and density functional theories. For the molecules investigated, several effects influencing the two-photon absorption spectra have been investigated, such as side-chain elongation, hydrogen bonding, the use of ionic species, and solvent effects, the latter described by the polarizable continuum model. The calculations have been carried out using a recent parallel implementation of the polarizable continuum model in the DALTON code. Special attention is given to those aspects that could explain the large solvent effect on the two-photon absorption cross sections observed experimentally for this class of compounds.

  17. 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.

  18. Four-State Model for Three-Branch Molecule's Two-Photon Absorption Properties

    Institute of Scientific and Technical Information of China (English)

    SU Yan; WANG Pei-Ji; ZHAO Peng; RONG Zhen-Yu

    2006-01-01

    @@ We present a four-state model for calculating the two-photon absorption of multi-branched molecules by using the time-depended function method. The numerical results indicate that the two-photon absorption cross section has a strong enhancement for three-branch molecules compared to two-branch structures. The maximal two-photon-absorption cross section is 2.358 × 10-47 cm 4 s/photon. At the same time, the charge-transfer process for the charge-transfer states is visualized in order to explain mechanism about the maximal TPA cross section.

  19. Dicke Coherent Narrowing in Two-Photon and Raman Spectroscopy of Thin Vapour Cells

    CERN Document Server

    Dutier, G; Hamdi, I; Maurin, I; Saltiel, S; Bloch, D; Ducloy, M; Dutier, Gabriel; Todorov, Petko; Hamdi, Ismah\\`{e}ne; Maurin, Isabelle; Saltiel, Solomon; Bloch, Daniel; Ducloy, Martial

    2005-01-01

    The principle of coherent Dicke narrowing in a thin vapour cell, in which sub-Doppler spectral lineshapes are observed under a normal irradiation for a l/2 thickness, is generalized to two-photon spectroscopy. Only the sum of the two wave vectors must be normal to the cell, making the two-photon scheme highly versatile. A comparison is provided between the Dicke narrowing with copropagating fields, and the residual Doppler-broadening occurring with counterpropagating geometries. The experimental feasibility is discussed on the basis of a first observation of a two-photon resonance in a 300 nm-thick Cs cell. Extension to the Raman situation is finally considered.

  20. T20-insensitive HIV-1 from naive patients exhibits high viral fitness in a novel dual-color competition assay on primary cells.

    Science.gov (United States)

    Neumann, Thomas; Hagmann, Isabel; Lohrengel, Sabine; Heil, Marintha L; Derdeyn, Cynthia A; Kräusslich, Hans-Georg; Dittmar, Matthias T

    2005-03-15

    The relationship between sensitivity to antiviral drugs and viral fitness is of paramount importance in understanding the long-term implications of clinical resistance. Here we report the development of a novel recombinant virus assay to study entry inhibitor-resistant HIV variants using a biologically relevant cell type, primary CD4 T-cells. We have modified the replication-competent molecular clone HIV(NL4-3) to express a reporter protein (Renilla luciferase), Green Fluorescent Protein (EGFP), or Red Fluorescent Protein (DsRed2) upon infection, thus allowing quantification of replication. Luciferase-expressing virus was used to evaluate drug sensitivity, while co-infection with viruses carrying the green and red fluorescent proteins was employed in the competitive fitness assay. Using envelope proteins from three T20 insensitive variants, lower levels of resistance were observed in primary CD4 T-cells than had been previously reported for cell lines. Importantly, dual-color competition assays demonstrated comparable or higher fitness for these variants despite their reduced T20 sensitivity. We conclude that reduced sensitivity to T20 is compatible with high viral fitness in the absence of selection pressure. Thus, simultaneously measuring both resistance and viral fitness using this newly described dual-color competition assay will likely provide important information about resistant viral variants that emerge during therapy with entry inhibitors.

  1. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.

    Science.gov (United States)

    Shao, Shipeng; Zhang, Weiwei; Hu, Huan; Xue, Boxin; Qin, Jinshan; Sun, Chaoying; Sun, Yuao; Wei, Wensheng; Sun, Yujie

    2016-05-19

    Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

  2. Two-photon vibrational excitation of air by long-wave infrared laser pulses

    CERN Document Server

    Palastro, J P; Johnson, L A; Hafizi, B; Wahlstrand, J K; Milchberg, H M

    2016-01-01

    Ultrashort long-wave infrared (LWIR) laser pulses can resonantly excite vibrations in N2 and O2 through a two-photon transition. The absorptive, vibrational component of the ultrafast optical nonlinearity grows in time, starting smaller than, but quickly surpassing, the electronic, rotational, and vibrational refractive components. The growth of the vibrational component results in a novel mechanism of 3rd harmonic generation, providing an additional two-photon excitation channel, fundamental + 3rd harmonic. The original and emergent two-photon excitations drive the resonance exactly out of phase, causing spatial decay of the absorptive, vibrational nonlinearity. This nearly eliminates two-photon vibrational absorption. Here we present simulations and analytical calculations demonstrating how these processes modify the ultrafast optical nonlinearity in air. The results reveal nonlinear optical phenomena unique to the LWIR regime of ultrashort pulse propagation in atmosphere.

  3. 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.

  4. LANTHANIDE ENHANCE LUMINESCENCE (LEL) WITH ONE AND TWO PHOTON EXCITATION OF QUANTUM DYES LANTHANIDE (III) - MACROCYCLES

    Science.gov (United States)

    Title: Lanthanide Enhance Luminescence (LEL) with one and two photon excitation of Quantum Dyes? Lanthanide(III)-Macrocycles Principal Author:Robert C. Leif, Newport InstrumentsSecondary Authors:Margie C. Becker, Phoenix Flow Systems Al Bromm, Virginia Commonw...

  5. Solving Two Kinds of JC Models Relating to Two-Photon Process by Supersymmetric Transformation

    Institute of Scientific and Technical Information of China (English)

    FAN Hong-Yi; Wei-Jun

    2004-01-01

    We propose two kinds of new Jaynes Cummings models relating to two-photon process by using the supersymmetric unitary transformation. The corresponding energy eigenvalues and eigenvectors are obtained.

  6. Two-photon absorption properties of a new series of 2CTσ chromophores

    Science.gov (United States)

    Zhou, Yu-fang; Meng, Fan-qing; Zhao, Xian; Xu, Dong; Jiang, Min-hu

    2000-10-01

    We have designed and synthesized a new series of two-photon ASPT-like charge transfer moieties linked by σ-bond spacers to N-position of pyridine cycle. Both theoretical and experimental results show there is no linear absorption in 600-1300 nm, so two-photon properties can be expected in this range. Two-photon absorption (TPA) cross-sections were calculated by using INDO/CI and SOS methods. The results show that those compounds possess large cross-sections as well as appropriate absorption wavelengths. Also the magnitude of the cross-section changes regularly with the number of the σ-bond spacers. These imply that they are good candidates for two-photon devices.

  7. LANTHANIDE ENHANCE LUMINESCENCE (LEL) WITH ONE AND TWO PHOTON EXCITATION OF QUANTUM DYES LANTHANIDE (III) - MACROCYCLES

    Science.gov (United States)

    Title: Lanthanide Enhance Luminescence (LEL) with one and two photon excitation of Quantum Dyes? Lanthanide(III)-Macrocycles Principal Author:Robert C. Leif, Newport InstrumentsSecondary Authors:Margie C. Becker, Phoenix Flow Systems Al Bromm, Virginia Commonw...

  8. Observation of Nondegenerate Two-Photon Gain in GaAs

    CERN Document Server

    Reichert, Matthew; Salamo, Greg; Hagan, David J; Van Stryland, Eric W

    2016-01-01

    Two-photon lasers require materials with large two-photon gain (2PG) coefficients and low linear and nonlinear losses. Our previous demonstration of large enhancement of two-photon absorption in semiconductors for very different photon energies translates directly into enhancement of 2PG. We experimentally demonstrate nondegenerate 2PG in optically excited bulk GaAs via femtosecond pump-probe measurements. 2PG is isolated from other pump induced effects through the difference between measurements performed with parallel and perpendicular polarizations of pump and probe. An enhancement in the 2PG coefficient of nearly two orders-of-magnitude is reported. The results point a possible way toward two-photon semiconductor lasers.

  9. Absolute Frequency Measurement of Rubidium 5S-7S Two-Photon Transitions

    CERN Document Server

    Morzynski, Piotr; Ablewski, Piotr; Gartman, Rafal; Gawlik, Wojciech; Maslowski, Piotr; Nagorny, Bartlomiej; Ozimek, Filip; Radzewicz, Czeslaw; Witkowski, Marcin; Ciurylo, Roman; Zawada, Michal

    2013-01-01

    We report the absolute frequency measurements of rubidium 5S-7S two-photon transitions with a cw laser digitally locked to an atomic transition and referenced to an optical frequency comb. The narrow, two-photon transition, 5S-7S (760 nm) insensitive to first order in a magnetic field, is a promising candidate for frequency reference. The performed tests yield the transition frequency with accuracy better than reported previously.

  10. 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.

  11. Influence of Two Photon Absorption on Soliton Self-Frequency Shift

    DEFF Research Database (Denmark)

    Steffensen, Henrik; Rottwitt, Karsten; Jepsen, Peter Uhd;

    2011-01-01

    The creation of mid-infrared supercontinua necessitates the use of soft-glass fibers. However, some materials, like chalcogenide, have a substantial two photon absorption. We introduce a model for soliton self-frequency shift that successfully includes this effect.......The creation of mid-infrared supercontinua necessitates the use of soft-glass fibers. However, some materials, like chalcogenide, have a substantial two photon absorption. We introduce a model for soliton self-frequency shift that successfully includes this effect....

  12. Two-photon ionization of atomic hydrogen with elliptically polarized light

    Science.gov (United States)

    Kassaee, A.; Rustgi, M. L.; Long, S. A. T.

    1988-01-01

    The theory of two-photon ionization of a hydrogenic state in the nonrelativistic dipole approximation is generalized for elliptically polarized light. An application to the metastable 2S state of atomic hydrogen is made. Significant differences in the angular distribution of the outgoing electrons are found depending upon the polarization of the photons. It is claimed that two-photon ionization employing elliptically polarized photons from lasers may provide an additional test for the theories of multiphoton ionization.

  13. Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches.

    Science.gov (United States)

    Izquierdo-Serra, Mercè; Gascón-Moya, Marta; Hirtz, Jan J; Pittolo, Silvia; Poskanzer, Kira E; Ferrer, Èric; Alibés, Ramon; Busqué, Félix; Yuste, Rafael; Hernando, Jordi; Gorostiza, Pau

    2014-06-18

    Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.

  14. 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.

  15. Two-photon cooperative emission in the presence of athermal electromagnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Enaki, N.A.; Mihalache, D

    1997-05-15

    The possibility of cooperative spontaneous two-photon emission of an extended radiators system and the influence of the external thermal electromagnetic field on the spontaneous emission rate, in such a system, are investigated. It is concluded that, in an external electromagnetic field, the two-photon cooperative emission rate increases significantly. The importance of this effect on the emission of gamma rays from inverted long-lived isomers triggered by X-ray thermal fields, is emphasized.

  16. Engineering Two-Atom Thermal Entanglement via Two-Photon Process

    Institute of Scientific and Technical Information of China (English)

    GUO Yan-Qing; ZHOU Ling; SONG He-Shan; YI Xue-Xi

    2004-01-01

    We study that two atoms simultaneously interact with a single mode thermal field via different couplings and different spontaneous emission rates when two-photon process is involved. It is found that we indeed can employ the different couplings to produce the two-atom thermal entanglement in two-photon process. The different atomic spontaneous emission rates are also utilizable in generating thermal entanglement. We also investigate the effect of the can obtain a strong and steady entanglement.

  17. Two-Photon Exchange Corrections to Single Spin Asymmetry of Neutron and 3He

    Institute of Scientific and Technical Information of China (English)

    CHEN Dian-Yong; DONG Yu-Bing

    2011-01-01

    In a simple hadronic model, the two-photon exchange contributions to the single spin asymmetries for the nucleon and the 3He are estimated. The results show that the elastic contributions of two-photon exchange to the single spin asymmetries for the nucleon are rather small while those for the 3He are relatively large. Besides the strong angular dependence, the twophoton contributions to the single spin asymmetry for the 3He are very sensitive to the momentum transfer.

  18. Synthesis, crystals of centrosymmetric triphenylamine chromophores bearing prodigious two-photon absorption cross-section and biological imaging

    Science.gov (United States)

    Wang, Shichao; Xu, Shasha; Wang, Yiming; Tian, Xiaohe; Zhang, Yujin; Wang, Chuankui; Wu, Jieying; Yang, Jiaxiang; Tian, Yupeng

    2017-02-01

    Two centrosymmetric D-π-D type triphenylamine chromophores with long π-conjugated bridge and strong electron-donating moiety were designed, synthesized and fully characterized. The crystal analysis revealed that multiple Csbnd H ⋯ π interactions existed in two chromophores, which played a crucial role in generating molecular 1D chains and 2D layers structures. Linear and nonlinear optical properties of the chromophores were systematically investigated with the aid of theoretical calculations. Two chromophores both exhibited intense and wide-dispersed one-photon/two-photon excited fluorescence, bear prodigious 2PA cross section (δ). Especially for Dye2, with ethyoxyl groups, displayed the strong 2PA activity, large cross-sections (δmax > 16,000 GM) and high NLO efficiency (δmax/MW > 16 GM/(g·mol)) in the range of 680-830 nm in DMF. In addition, one- and two-photon fluorescence microscopy images of HepG2 cells incubated with Dye2 were obtained and found that Dye2 could effectively uptake toward living cells and display a uniformly localized in cytosolic space.

  19. Simultaneous Two-photon in Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex.

    Science.gov (United States)

    Łukasiewicz, Kacper; Robacha, Magdalena; Bożycki, Łukasz; Radwanska, Kasia; Czajkowski, Rafał

    2016-01-01

    This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo. The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging. The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAV(mCherry)) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

  20. Lighting the Way to See Inside Two-Photon Absorption Materials: Structure-Property Relationship and Biological Imaging.

    Science.gov (United States)

    Zhang, Qiong; Tian, Xiaohe; Zhou, Hongping; Wu, Jieying; Tian, Yupeng

    2017-02-23

    ions, including transition metals and lanthanides, can serve as an important part of the structure to control the intramolecular charge-transfer process that drives the 2PA process. As templates, transition metal ions can assemble simple to more sophisticated ligands in a variety of multipolar arrangements resulting in interesting and tailorable electronic and optical properties, depending on the nature of the metal center and the energetics of the metal-ligand interactions, such as intraligand charge-transfer (ILCT) and metal-ligand charge-transfer (MLCT) processes. Lanthanide complexes are attractive for a number of reasons: (i) their visible emissions are quite long-lived; (ii) their absorption and emission can be tuned with the aid of appropriate photoactive ligands; (iii) the accessible energy-transfer path between the photo-active ligands and the lanthanide ion can facilitate efficient lanthanide-based 2PA properties. Thus, the above materials with excellent 2PA properties should be applied in two-photon applications, especially two-photon fluorescence microscopy (TPFM) and related emission-based applications. Furthermore, the progress of research into the use of those new 2PA materials with moderate 2PA cross section in the near-infrared region, good Materials 2017, 10, 223 2 of 37 biocompatibility, and enhanced two-photon excited fluorescence for two-photon bio-imaging is summarized. In addition, several possible future directions in this field are also discussed (146 references).

  1. Lighting the Way to See Inside Two-Photon Absorption Materials: Structure–Property Relationship and Biological Imaging

    Science.gov (United States)

    Zhang, Qiong; Tian, Xiaohe; Zhou, Hongping; Wu, Jieying; Tian, Yupeng

    2017-01-01

    ions, including transition metals and lanthanides, can serve as an important part of the structure to control the intramolecular charge-transfer process that drives the 2PA process. As templates, transition metal ions can assemble simple to more sophisticated ligands in a variety of multipolar arrangements resulting in interesting and tailorable electronic and optical properties, depending on the nature of the metal center and the energetics of the metal-ligand interactions, such as intraligand charge-transfer (ILCT) and metal-ligand charge-transfer (MLCT) processes. Lanthanide complexes are attractive for a number of reasons: (i) their visible emissions are quite long-lived; (ii) their absorption and emission can be tuned with the aid of appropriate photoactive ligands; (iii) the accessible energy-transfer path between the photo-active ligands and the lanthanide ion can facilitate efficient lanthanide-based 2PA properties. Thus, the above materials with excellent 2PA properties should be applied in two-photon applications, especially two-photon fluorescence microscopy (TPFM) and related emission-based applications. Furthermore, the progress of research into the use of those new 2PA materials with moderate 2PA cross section in the near-infrared region, good biocompatibility, and enhanced two-photon excited fluorescence for two-photon bio-imaging is summarized. In addition, several possible future directions in this field are also discussed (146 references). PMID:28772584

  2. Lighting the Way to See Inside Two-Photon Absorption Materials: Structure–Property Relationship and Biological Imaging

    Directory of Open Access Journals (Sweden)

    Qiong Zhang

    2017-02-01

    properties. The metal ions, including transition metals and lanthanides, can serve as an important part of the structure to control the intramolecular charge-transfer process that drives the 2PA process. As templates, transition metal ions can assemble simple to more sophisticated ligands in a variety of multipolar arrangements resulting in interesting and tailorable electronic and optical properties, depending on the nature of the metal center and the energetics of the metal-ligand interactions, such as intraligand charge-transfer (ILCT and metal-ligand charge-transfer (MLCT processes. Lanthanide complexes are attractive for a number of reasons: (i their visible emissions are quite long-lived; (ii their absorption and emission can be tuned with the aid of appropriate photoactive ligands; (iii the accessible energy-transfer path between the photo-active ligands and the lanthanide ion can facilitate efficient lanthanide-based 2PA properties. Thus, the above materials with excellent 2PA properties should be applied in two-photon applications, especially two-photon fluorescence microscopy (TPFM and related emission-based applications. Furthermore, the progress of research into the use of those new 2PA materials with moderate 2PA cross section in the near-infrared region, good Materials 2017, 10, 223 2 of 37 biocompatibility, and enhanced two-photon excited fluorescence for two-photon bio-imaging is summarized. In addition, several possible future directions in this field are also discussed (146 references.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. In vivo spectral imaging of different cell types in the small intestine by two-photon excited autofluorescence

    Science.gov (United States)

    Orzekowsky-Schroeder, Regina; Klinger, Antje; Martensen, Björn; Blessenohl, Maike; Gebert, Andreas; Vogel, Alfred; Hüttmann, Gereon

    2011-11-01

    Spectrally resolved two-photon excited autofluorescence imaging is used to distinguish different cell types and functional areas during dynamic processes in the living gut. Excitation and emission spectra of mucosal tissue and tissue components are correlated to spectra of endogenous chromophores. We show that selective excitation with only two different wavelengths within the tuning range of a Ti:sapphire femtosecond laser system yields excellent discrimination between enterocytes, antigen presenting cells and lysosomes based on the excitation and emission properties of their autofluorescence. The method is employed for time-lapse microscopy over up to 8 h. Changes of the spectral signature with the onset of photodamage are demonstrated, and their origin is discussed.

  8. 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

  9. MRT letter: Two-photon excitation-based 2pi light-sheet system for nano-lithography.

    Science.gov (United States)

    Mohan, Kavya; Mondal, Partha Pratim

    2015-01-01

    We propose two-photon excitation-based light-sheet technique for nano-lithography. The system consists of 2π-configured cylindrical lens system with a common geometrical focus. Upon superposition, the phase-matched counter-propagating light-sheets result in the generation of identical and equi spaced nano-bump pattern. Study shows a feature size of as small as few tens of nanometers with a inter-bump distance of few hundred nanometers. This technique overcomes some of the limitations of existing nano-lithography techniques, thereby, may pave the way for mass-production of nano-structures. Potential applications can also be found in optical microscopy, plasmonics, and nano-electronics. © 2014 Wiley Periodicals, Inc.

  10. 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.

  11. Note: Derivation of two-photon circular dichroism - Addendum to "two-photon circular dichroism" [J. Chem. Phys. 62, 1006 (1975)

    OpenAIRE

    Friese, Daniel Henrik

    2015-01-01

    Published version, also available at http://dx.doi.org/10.1063/1.4930017 This addendum shows the detailed derivation of the fundamental equations for two-photon circular dichroism which are given in a very condensed form in the original publication [I. Tinoco, J. Chem. Phys. 62, 1006 (1975)]. In addition, some minor errors are corrected and some of the derivations in the original publication are commented.

  12. Measurement of degenerate two-photon absorption spectra of a series of developed two-photon initiators using a dispersive white light continuum Z-scan

    Science.gov (United States)

    Ajami, Aliasghar; Husinsky, Wolfgang; Tromayer, Maximilian; Gruber, Peter; Liska, Robert; Ovsianikov, Aleksandr

    2017-08-01

    To achieve efficient micro- and nanostructuring based on two-photon polymerization (2PP), the development and evaluation of specialized two-photon initiators (2PIs) are essential. Hence, a reliable method to determine the two-photon absorption (2PA) spectra of the synthesized 2PIs used for 2PP structuring is crucial. A technique by which absolute visible-to-near-infrared 2PA spectra of degenerate nature can be determined via performing a single dispersive white-light continuum (WLC) Z-scan has been realized. Using a dispersed white light beam containing 8 fs pulses at wavelengths ranging from 650 nm to 950 nm, the nonlinear transmittance as a function of the sample position can be measured for all spectral components by performing a single scan along the laser beam propagation direction. In this work, the 2PA spectrum of three different 2PIs was determined using this technique. 2PP structuring was also accomplished using the developed 2PIs at different wavelengths. Tuning the wavelength of the laser to match the peak of the 2PA spectra of the developed 2PIs resulted in lower intensity thresholds and facilitated higher structuring speeds. As an example, using M2CMK 2PI for 2PP, the scanning speed can be increased up to 5 folds when the laser wavelength is tuned to 760 nm (i.e., 2PA maximum) instead of the conventionally used 800 nm.

  13. Pseudopotential calculations and photothermal lensing measurements of two-photon absorption in solids

    Energy Technology Data Exchange (ETDEWEB)

    White, W.T. III

    1985-11-04

    We have studied two-photon absorption in solids theoretically and experimentally. We have shown that it is possible to use accurate band structure techniques to compute two-photon absorption spectra within 15% of measured values in a wide band-gap material, ZnS. The empirical pseudopotential technique that we used is significantly more accurate than previous models of two-photon absorption in zinc blende materials, including present tunneling theories (which are essentially parabolic-band results in disguise) and the nonparabolic-band formalism of Pidgeon et al. and Weiler. The agreement between our predictions and previous measurements allowed us to use ZnS as a reference material in order to validate a technique for measuring two-photon absorption that was previously untried in solids, pulsed dual-beam thermal lensing. With the validated technique, we examined nonlinear absorption in one other crystal (rutile) and in several glasses, including silicates, borosilicates, and one phosphate glass. Initially, we believed that the absorption edges of all the materials were comparable; however, subsequent evidence suggested that the effective band-gap energies of the glasses were above the energy of two photons in our measurement. Therefore, we attribute the nonlinear absorption that we observed in glasses to impurities or defects. The measured nonlinear absorption coefficients were of the order of a few cm/TW in the glasses and of the order of 10 cm/GW in the crystals, four orders of magnitude higher than in glasses. 292 refs.

  14. Localized Polymerization Using Single Photon Photoinitiators in Two-photon process for Fabricating Subwavelength Structures

    CERN Document Server

    Ummethala, Govind; Chaudhary, Raghvendra P; Hawal, Suyog; Saxena, Sumit; Shukla, Shobha

    2016-01-01

    Localized polymerization in subwavelength volumes using two photon dyes has now become a well-established method for fabrication of subwavelength structures. Unfortunately, the two photon absorption dyes used in such process are not only expensive but also proprietary. LTPO-L is an inexpensive, easily available single photon photoinitiator and has been used extensively for single photon absorption of UV light for polymerization. These polymerization volumes however are not localized and extend to micron size resolution having limited applications. We have exploited high quantum yield of radicals of LTPO-Lfor absorption of two photons to achieve localized polymerization in subwavelength volumes, much below the diffraction limit. Critical concentration (10wt%) of LTPO-Lin acrylate (Sartomer) was found optimal to achieve subwavelength localized polymerization and has been demonstrated by fabricating 2D/3D complex nanostructures and functional devices such as variable polymeric gratings with nanoscaled subwavelen...

  15. Coherent control of non-resonant two-photon transition in molecular system

    Institute of Scientific and Technical Information of China (English)

    Zhang Hui; Zhang Shi-An; Wang Zu-Geng; Sun Zhen-Rong

    2010-01-01

    In this paper,we study theoretically and experimentally the coherent control of non-resonant two-photon transition in a molecular system (Perylene dissolved in chloroform solution) by shaping the femtosecond pulses with simple phase patterns (cosinusoidal and π phase step-function shape).The control efficiency of the two-photon transition probability is correlated with both the laser field and the molecular absorption bandwidth.Our results demonstrate that,the two-photon transition probability in a molecular system can be reduced but not completely eliminated by manipulating the laser field,and the control efficiency is minimal when the molecular absorption bandwidth is larger than twice the laser spectral bandwidth.

  16. In vivo two-photon calcium imaging in the visual system.

    Science.gov (United States)

    Ohki, Kenichi; Reid, R Clay

    2014-04-01

    Two-photon imaging of calcium-sensitive dyes in vivo has become a common tool used by neuroscientists, largely because of the development of bolus loading techniques, which can label every neuron in a local circuit with calcium-sensitive dye. Like multielectrode recordings, two-photon imaging paired with bolus loading provides a method for monitoring many neurons at once, but, in addition, it provides a means for determining the precise location of every neuron. Thus, it is an ideal method for studying the fine-scale functional architecture of the cortex and guiding the experimenter to individual neurons that can be targeted for further anatomical study. Two-photon calcium imaging enables study of the fine structure of functional maps in the visual cortex in cats and rodents. In mice, it can allow the characterization of specific cell types when paired with transgenic or retrograde labeling.

  17. Investigation of two-photon absorption induced excited state absorption in a fluorenyl-based chromophore.

    Science.gov (United States)

    Li, Changwei; Yang, Kun; Feng, Yan; Su, Xinyan; Yang, Junyi; Jin, Xiao; Shui, Min; Wang, Yuxiao; Zhang, Xueru; Song, Yinglin; Xu, Hongyao

    2009-12-03

    Two-photon absorption induced excited state absorption in the solution of a new fluorenyl-based chromophore is investigated by a time-resolved pump-probe technique using femtosecond pulses. With the help of an additional femtosecond open-aperture Z-scan technique, numerical simulations based on a three-energy level model are used to interpret the experimental results, and we determine the nonlinear optical parameters of this new chromophore uniquely. Large two-photon absorption cross section and excited state absorption cross section for singlet excited state are obtained, indicating a good candidate for optical limiting devices. Moreover, the influence of two-beam coupling induced energy transfer in neat N,N'-dimethylformamide solvent is also considered, although this effect is strongly restrained by the instantaneous two-photon absorption.

  18. Visualization of two-photon Rabi oscillations in evanescently coupled optical waveguides

    Energy Technology Data Exchange (ETDEWEB)

    Ornigotti, M; Valle, G Della; Fernandez, T Toney; Laporta, P; Longhi, S [Dipartimento di Fisica and Istituto di Fotonica e Nanotecnologie del CNR, Politecnico di Milano, Piazza L. da Vinci 32, I-20133 Milano (Italy); Coppa, A; Foglietti, V [Istituto di Fotonica e Nanotecnologie del CNR, sezione di Roma, Via Cineto Romano 42, 00156 Roma (Italy)], E-mail: longhi@fisi.polimi.it

    2008-04-28

    An optical analogue of two-photon Rabi oscillations, occurring in a three-level atomic or molecular system coherently driven by two detuned laser fields, is theoretically proposed and experimentally demonstrated using three evanescently coupled optical waveguides realized on an active glass substrate. The optical analogue stems from the formal analogy between spatial propagation of light waves in the three-waveguide structure and the coherent temporal evolution of populations in a three-level atomic medium driven by two laser fields under two-photon resonance. In our optical experiment, two-photon Rabi oscillations are thus visualized as a slow spatial oscillatory exchange of light power between the two outer waveguides of the structure with a small excitation of the central waveguide.

  19. Selective two-photon excitation of a vibronic state by correlated photons.

    Science.gov (United States)

    Oka, Hisaki

    2011-03-28

    We theoretically investigate the two-photon excitation of a molecular vibronic state by correlated photons with energy anticorrelation. A Morse oscillator having three sets of vibronic states is used, as an example, to evaluate the selectivity and efficiency of two-photon excitation. We show that a vibrational mode can be selectively excited with high efficiency by the correlated photons, without phase manipulation or pulse-shaping techniques. This can be achieved by controlling the quantum correlation so that the photon pair concurrently has two pulse widths, namely, a temporally narrow width and a spectrally narrow width. Though this concurrence is seemingly contradictory, we can create such a photon pair by tailoring the quantum correlation between two photons.

  20. Observation of single- and two-photon beating between independent Raman scattering

    CERN Document Server

    Chen, Li-Qing; Zhang, Guo-Wan; Ou, Z Y; Zhang, Weiping

    2010-01-01

    By using spontaneous Raman processes in the high gain regime, we produce two independent Raman Stokes fields from an atomic ensemble. Temporal beating is observed between the two directly generated Stokes fields in a single realization. The beat frequency is found to be a result of an AC Stark frequency shift effect. However, due to the spontaneous nature of the process, the phases of the two Stokes fields change from one realization to another so that the beat signal disappears after average over many realizations. On the other hand, the beat signal is recovered in a two-photon correlation measurement, showing a two-photon interference effect. The two-photon beat signal enables us to obtain dephasing information in the Raman process. The dephasing effect is found to depend on the temperature of the atomic medium.

  1. 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.

  2. 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.

  3. 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.

  4. Three-dimensional protein networks assembled by two-photon activation.

    Science.gov (United States)

    Gatterdam, Volker; Ramadass, Radhan; Stoess, Tatjana; Fichte, Manuela A H; Wachtveitl, Josef; Heckel, Alexander; Tampé, Robert

    2014-05-26

    Spatial and temporal control over chemical and biological processes plays a key role in life and material sciences. Here we synthesized a two-photon-activatable glutathione (GSH) to trigger the interaction with glutathione S-transferase (GST) by light at superior spatiotemporal resolution. The compound shows fast and well-confined photoconversion into the bioactive GSH, which is free to interact with GST-tagged proteins. The GSH/GST interaction can be phototriggered, changing its affinity over several orders of magnitude into the nanomolar range. Multiplexed three-dimensional (3D) protein networks are simultaneously generated in situ through two-photon fs-pulsed laser-scanning excitation. The two-photon activation facilitates the three-dimensional assembly of protein structures in real time at hitherto unseen resolution in time and space, thus opening up new applications far beyond the presented examples.

  5. Polarization properties of optical phase conjugation by two-photon resonant degenerate four-wave mixing

    Science.gov (United States)

    Kauranen, Martti; Gauthier, Daniel J.; Malcuit, Michelle S.; Boyd, Robert W.

    1989-08-01

    We develop a semiclassical theory of the polarization properties of phase conjugation by two-photon resonant degenerate four-wave mixing. The theory includes the effects of saturation by the pump waves. We solve the density-matrix equations of motion in steady state for a nonlinear medium consisting of stationary atoms with a ground and excited state connected by two-photon transitions. As an illustration of the general results, we consider an S0-->S0 two-photon transition, which is known to lead to perfect polarization conjugation in the limit of third-order theory. We show that the fidelity of the polarization-conjugation process is degraded for excessively large pump intensities. The degradation can occur both due to transfer of population to the excited state and due to nonresonant Stark shifts. Theoretical results are compared to those of a recent experiment [Malcuit, Gauthier, and Boyd, Opt. Lett. 13, 663 (1988)].

  6. Highly selective population of two excited states in nonresonant two-photon absorption

    Institute of Scientific and Technical Information of China (English)

    Zhang Hui; Zhang Shi-An; Sun Zhen-Rong

    2011-01-01

    A nonresonant two-photon absorption process can be manipulated by tailoring the ultra-short laser pulse.In this paper,we theoretically demonstrate a highly selective population of two excited states in the nonresonant two-photon absorption process by rationally designing a spectral phase distribution.Our results show that one excited state is maximally populated while the other state population is widely tunable from zero to the maximum value.We believe that the theoretical results may play an important role in the selective population of a more complex nonlinear process comprising nonresonant two-photon absorption,such as resonance-mediated(2+1)-three-photon absorption and (2+1)-resonant multiphoton ionization.

  7. 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.

  8. 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.

  9. Robust spatial-polarization hyperentanglement distribution of two-photon systems against collective noise

    Science.gov (United States)

    Gao, Cheng-Yan; Wang, Guan-Yu; Alzahrani, Faris; Hobiny, Aatef; Deng, Fu-Guo

    2017-03-01

    Hyperentanglement is a significant resource for high-capacity quantum communication. Here we present a robust spatial-polarization hyperentanglement distribution scheme for two-photon systems. The error on the polarization states of two-photon systems transmitted from two paths can be corrected resorting to the robust time-bin entanglement which suffers little from the channel noise. The spatial bit-flip error takes place with a very small probability and the spatial phase-flip error can be precluded by adjusting the path-length of spatial modes. Using this scheme, the two parties in quantum communication can share a maximally hyperentangled state of two-photon systems in a deterministic way, which will improve the efficiency of quantum communication largely.

  10. One- and two-photon scattering from generalized V-type atoms

    OpenAIRE

    Sánchez-Burillo, Eduardo; Martín-Moreno, Luis; Zueco, David; García-Ripoll, Juan José

    2016-01-01

    The one- and two-photon scattering matrix S is obtained analytically for a one-dimensional waveguide and a point-like scatterer with N excited levels (generalized V -type atom). We argue that the two-photon scattering matrix contains sufficient information to distinguish between different level structures which are equivalent for single-photon scattering, such as a V -atom with N = 2 excited levels and two two-level systems. In particular, we show that the scattering with the V -type atom exh...

  11. A direct frequency comb for two-photon transition spectroscopy in a cesium vapor

    Institute of Scientific and Technical Information of China (English)

    Zhang Yi-Chi; Wu Ji-Zhou; Li Yu-Qing; Jin Li; Ma Jie; Wang Li-Rong; Zhao Yan-Ting; Xiao Lian-Tuan; Jia Suo-Tang

    2012-01-01

    A phase-stabilized femtosecond frequency comb is used to measure high-resolution spectra of two-photon transition 62S1/2-62P1/2,3/2-82S1/2 in a cesium vapor.The broadband laser output from a femtosecond frequency comb is split into counter-propagating parts,shaped in an original way,and focused into a room-temperature cesium vapor.We obtain high-resolution two-photon spectroscopy by scanning the repetition rate of femtosecond frequency comb,and through absolute frequency measurements.

  12. Manipulation of multiple electromagnetically induced two-photon transparency in a six-level atomic system

    Institute of Scientific and Technical Information of China (English)

    Jia Wen-Zhi; Wang Shun-Jin

    2009-01-01

    In the five-level K-type atomic system, by using another control field to couple the excited level of the coupling transition to the sixth higher excited level, a six-level atomic system is constructed. In this system, the multiple electromagnetically induced two-photon transparency has been investigated. What is more, if choosing the parameters of the control fields properly the triple transparency window will reduce to a double one which means that the multiple electromagnetically induced two-photon transparency can be manipulated in this system. The physical interpretation of these phenomena is given in terms of the dressed states and the dark states.

  13. Two-photon laser fabrication of three-dimensional silver microstructures with submicron scale linewidth

    Energy Technology Data Exchange (ETDEWEB)

    Tsutsumi, Naoto; Nagata, Kazuya; Sakai, Wataru [Kyoto Institute of Technology, Department of Macromolecular Science and Engineering, Graduate School of Science and Technology, Kyoto (Japan)

    2011-05-15

    We show three-dimensional silver microstructures with a submicron scale linewidth fabricated via two-photon photoreduction of silver ions in a poly(N-vinylpyrrolidone) (PVP) matrix. Femtosecond laser at 508 nm directly excites the carbonyl group of PVP via two-photon excitation to reduce silver ions. Lone pair electrons in PVP stabilized silver ions and lower molecular weight of PVP prevented silver clusters growing larger. The effect of molecular weight of PVP on linewidth of silver nanowire is investigated. (orig.)

  14. 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.

  15. Two-photon photoemission from metals induced by picosecond laser pulses

    Science.gov (United States)

    Bechtel, J. H.; Smith, W. L.; Bloembergen, N.

    1977-01-01

    We have measured the two-photon photoemission current density from tungsten, tantalum, and molybdenum when irradiated by 532-nm wavelength radiation. This wavelength was produced by the second-harmonic radiation of single picosecond laser pulses from a mode-locked neodymium-doped yttrium-aluminum-garnet laser. The results are interpreted in terms of both a simple temperature-independent two-photon photoemission effect and a generalization of the Fowler-DuBridge theory of photoemission. The laser polarization dependence of the emitted current is also reported.

  16. Two-photon quantum walks in an elliptical direct-write waveguide array

    CERN Document Server

    Owens, J O; Biggerstaff, D N; Goggin, M E; Fedrizzi, A; Linjordet, T; Ams, M; Marshall, G D; Twamley, J; Withford, M J; White, A G

    2011-01-01

    Integrated optics provides an ideal test bed for the emulation of quantum systems via continuous-time quantum walks. Here we study the evolution of two-photon states in an elliptic array of waveguides. We characterise the photonic chip via coherent-light tomography and use the results to predict distinct differences between temporally indistinguishable and distinguishable two-photon inputs which we then compare with experimental observations. Our work highlights the feasibility for emulation of coherent quantum phenomena in three-dimensional waveguide structures.

  17. THE TWO-PHOTON DEGENERATE JAYNES-CUMMINGS MODEL WITH AND WITHOUT ROTATING-WAVE APPROXIMATION

    Institute of Scientific and Technical Information of China (English)

    ZHOU LING; SONG HE-SHAN; YAO LI

    2001-01-01

    We take into account the two-photon process and generalize the Jaynes-Cummings (JC) model to the case of atomic level degenerate in the projections of the angular momenta, and we establish two-photon degenerate JC models with and without the rotating-wave approximation (RWA) quantum theory. Comparing the atom population inversion of the generalized JC model with that of the original JC model, we found that the revival period of the degenerate JC model becomes longer and the maximum amplitude of atomic inversion decreases with RWA. Without RWA, the quantum chaos of the generalized JC model is much weaker than that of the original JC model

  18. Dynamics of Two-Photon Lasers with Λ Atomic Level Configuration

    Institute of Scientific and Technical Information of China (English)

    YANG Peng; QIAN Feng; HUANG Hong-Bin; XIE Xia; ZHANG Ya-Jun

    2006-01-01

    We derive the dimensionless dynamic equations of two-photon lasers with A atomic level configuration by using the quantum Langevin equation method with the considerations of atomic coherence and injected classical fields.Then we analyze the stability and the chaotic dynamics of the two-photon laser by calculating the bifurcation diagram and the maximum Lyapunov exponent (MLE). Our results show that the Lorenz strange attractors and one-focus strange attractors can exist in this system, and the chaos can be induced or inhibited by the injected classical fields via Hopfbifurcations or crises, while the atomic coherence induces chaos via crises, and inhibit chaos via Hopf bifurcation or crises.

  19. Two-photon exchange correction to $2S$-$2P$ splitting in muonic helium

    CERN Document Server

    Carlson, Carl E; Vanderhaeghen, Marc

    2016-01-01

    We calculate the two-photon exchange correction to the Lamb shift in muonic helium atoms within the dispersion relations framework. Part of the effort entailed making analytic fits to the electron-$^3$He quasielastic scattering data set, for purposes of doing the dispersion integrals. Our result is that the energy of the 2$S$ state is shifted downwards by two-photon exchange effects by 15.14(49) meV, in good accord with the result obtained from a potential model and effective field theory calculation.

  20. Nonsequential Two-Photon Double Ionization of Atoms: Identifying the Mechanism

    CERN Document Server

    F\\orre, Morten; Nepstad, Raymond

    2010-01-01

    We develop an approximate model for the process of direct (nonsequential) two-photon double ionization of atoms. Employing the model, we calculate (generalized) total cross sections as well as energy-resolved differential cross sections of helium for photon energies ranging from 39 to 54 eV. A comparison with results of \\textit{ab initio} calculations reveals that the agreement is at a quantitative level. We thus demonstrate that this complex ionization process is fully described by the simple model, providing insight into the underlying physical mechanism. Finally, we use the model to calculate generalized cross sections for the two-photon double ionization of neon in the nonsequential regime.

  1. Near IR two photon absorption of cyanines dyes: application to optical power limiting at telecommunication wavelengths

    Science.gov (United States)

    Bouit, Pierre-Antoine; Wetzel, Guillaume; Feneyrou, Patrick; Bretonnière, Yann; Kamada, Kenji; Maury, Olivier; Andraud, Chantal

    2008-02-01

    The design and synthesis of symmetrical and unsymmetrical heptamethine cyanines is reported. These chromophores present significant two-photon cross section in the 1400-1600 nm spectral range. In addition, they display optical power limiting (OPL) properties. OPL curves were interpreted on the basis of two-photon absorption (2PA) followed by excited state absorption (ESA). Finally, these molecules present several relevant properties (nonlinear absorption properties, two-step gram scale synthesis, high solubility, good thermal stability), which could lead to numerous practical applications in material science (solid state optical limiting, signal processing) or in biology (imaging).

  2. New insight in boron chemistry: Application in two-photon absorption

    Science.gov (United States)

    Bolze, F.; Hayek, A.; Sun, X. H.; Baldeck, P. L.; Bourgogne, C.; Nicoud, J.-F.

    2011-07-01

    Two groups of one-dimensional (1D) boron containing two-photon absorbing fluorophores have been prepared and characterized. One group includes boron atoms incorporated in the conjugated or pseudo conjugated central core and the other contain a boron cluster as an acceptor group at one end of the fluorophores. Two boron containing central cores (with two boron atoms) have been explored: the cyclodiborazane and the pyrazabole moieties. The chosen boron cluster, p-carborane, contains 10 boron atoms. All the prepared fluorophores present high two-photon absorption cross-sections. Some water-soluble as well as lipophylic dyes have been prepared and used in bio-imaging.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Free electron laser induced two-photon photoconductivity in Hg1-xCdxTe

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The Beijing free electron laser (BFEL) has been employed for the first time to study the nonlinear photoconductivity characteristics of the typical infrared photoelectronic material Hg1-xCdxTe. Taking advantage of the high photon flux density of BFEL, we have investigated the photoconductivity characteristics in Hg1-xCdxTe induced by two-photon absorption by means of the photoconductivity technique, observed the photoconductivity signals saturation, and studied the two-photon photoconductivity characteristics on different bias voltages across the sample.

  8. Search for a Higgs Boson Decaying into Two Photons 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 de Walle, R T; 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

    A Higgs particle produced in association with a Z boson and decaying into two photons is searched for in the data collected by the L3 experiment at LEP. All possible decay modes of the Z boson are investigated. No signal is observed in 447.5 pb^-1 of data recorded at centre-of-mass energies up to 209 GeV. Limits on the branching fraction of the Higgs boson decay into two photons as a function of the Higgs mass are derived. A lower limit on the mass of a fermiophobic Higgs boson is set at 105.4 GeV at 95% confidence level.

  9. Temporal behavior of low-amplitude two-photon screening-photovoltaic grey spatial solitons

    Institute of Scientific and Technical Information of China (English)

    JI Xuan-mang; JIANG Qi-chang; WANG Jin-lai; LIU Jin-song

    2011-01-01

    The time-dependent formation of one-dimensional two-photon screening-photovoltaic (PV) grey spatial solitons under low-amplitude conditions is presented theoretically. The time-dependent propagation equation of two-photon screening- photovoltaic solitons is obtained by the numerical method. The results indicate that as the time evolves, the intensity width of grey screening-photovoltaic spatial solitons decreases monotonously to a minimum value towards the steady state. The higher the ratio of soliton peak intensity to the dark irradiation intensity, the narrower the width of grey solitons within the propagation time.

  10. Gold Core Mesoporous Organosilica Shell Degradable Nanoparticles for Two-Photon Imaging and Gemcitabine Monophosphate Delivery

    KAUST Repository

    Rhamani, Saher

    2017-09-12

    The synthesis of gold core degradable mesoporous organosilica shell nanoparticles is described. The nanopaticles were very efficient for two-photon luminescence imaging of cancer cells and for in vitro gemcitabine monophosphate delivery, allowing promising theranostic applications in the nanomedicine field.

  11. Two-photon upconversion affected by intermolecule correlations near metallic nanostructures

    Science.gov (United States)

    Osaka, Yoshiki; Yokoshi, Nobuhiko; Ishihara, Hajime

    2016-04-01

    We investigate an efficient two-photon upconversion process in more than one molecule coupled to an optical antenna. In the previous paper [Y. Osaka et al., Phys. Rev. Lett. 112, 133601 (2014), 10.1103/PhysRevLett.112.133601], we considered the two-photon upconversion process in a single molecule within one-dimensional input-output theory and revealed that controlling the antenna-molecule coupling enables the efficient upconversion with radiative loss in the antenna suppressed. In this paper, aiming to propose a way to enhance the total probability of antenna-photon scattering, we extend the model to the case of multiple molecules. In general, the presence of more than one molecule decreases the upconversion probability because they equally share the energy of the two photons. However, it is shown that we can overcome the difficulty by controlling the intermolecule coupling. Our result implies that, without increasing the incident photon number (light power), we can enlarge the net probability of the two-photon upconversion.

  12. Experimental method for the determination of two-photon cross sections using four-wave mixing

    Science.gov (United States)

    Burris, J.; Mcilrath, T. J.

    1985-01-01

    The two-photon absorption cross section for the R22 + S12(J double prime = 9 1/2) transition in nitric oxide's gamma band has been determined. The value is in good agreement with previous measurements on several other NO transitions. The technique described here can be used to obtain accurate cross sections for other diatomic molecules.

  13. Higgs decay into two photons from a 3HDM with flavor symmetry

    Energy Technology Data Exchange (ETDEWEB)

    Aranda, Alfredo, E-mail: fefo@ucol.mx [Facultad de Ciencias, CUICBAS, Universidad de Colima, Colima (Mexico); Dual C-P Institute of High Energy Physics (Mexico); Bonilla, Cesar, E-mail: rasec.cmbd@gmail.com [Facultad de Ciencias Físico–Matemáticas, Benemérita Universidad Autónoma de Puebla (Mexico); Anda, Francisco de, E-mail: franciscojosedea@gmail.com [Departamento de Fisica, CUCEI, Universidad de Guadalajara (Mexico); Delgado, Antonio, E-mail: antonio.delgado@nd.edu [Department of Physics, University of Notre Dame, Notre Dame, IN 46556 (United States); Hernández-Sánchez, Jaime, E-mail: jaimeh@ece.buap.mx [Dual C-P Institute of High Energy Physics (Mexico); Facultad de Ciencias de la Electrónica, Benemérita Universidad Autónoma de Puebla, Apdo. Postal 542, 72570 Puebla, Puebla (Mexico)

    2013-08-09

    In this short Letter we show that the excess of events in the decay of Higgs to two photons reported by ATLAS and CMS can be easily accommodated in a flavor renormalizable three Higgs doublet model (3HDM). The model is consistent with all fermion masses, mixing angles, and flavor changing neutral current constraints.

  14. One-bit photon polarization in two-photon experiments. An information mechanics perspective

    Science.gov (United States)

    Kantor, Frederick W.

    1991-03-01

    Two-photon experiments of Aspect, Grangier, and Roger, directed toward testing Einstein, Podolsky, and Rosen's thought experiment, are seen in the context of Kantor's information mechanics as illustrating some consequences of the fact that the amount of information represented by the photon's polarization is one bit.

  15. Higgs boson decay into two photons in an electromagnetic background field

    DEFF Research Database (Denmark)

    Nielsen, N. K.

    2014-01-01

    The amplitude for Higgs boson decay into two photons in a homogeneous and time-independent magnetic field is investigated by proper-time regularization in a gauge-invariant manner and is found to be singular at large field values. The singularity is caused by the component of the charged vector...

  16. Fabrication of 3D nano/microelectrodes via two-photon-polymerization

    DEFF Research Database (Denmark)

    Abaddi, Mohammed Al-; Sasso, Luigi; Dimaki, Maria

    2012-01-01

    The integration of two-photon polymerization technology with standard microfabrication techniques is imperative for the use of this tool in micro- and nanotechnology and especially for the future commercialization of the technology. In this work, we report a novel method for the fabrication of 3D...

  17. Solvent effects on optical properties of a newly synthesized two-photon polymerization initiator: BPYPA

    Institute of Scientific and Technical Information of China (English)

    Guo Ya-Hui; Sun Yuan-Hong; Tao Li-Min; Zhao Ke; Wang Chuan-Kui

    2005-01-01

    Time-dependent hybrid density functional theory in combination with polarized continuum model is applied to study the solvent effects on the geometrical and electronic structures as well as one- and two-photon absorption processes,of a newly synthesized asymmetrical charge-transfer organic molecule bis-(4-bromo-phenyl)-[4-(2-pyridin-4-yl-vinyl)phenyl]-amine (BPYPA). There exist two charge-transfer states for the compound in visible region. The two-photon absorption cross section calculated by a three-state model and solvatochromic shift of the charge-transfer states are found to be solvent-dependent, where a nonmonotonic behaviour with respect to the polarity of the solvents is observed. The numerical results show that the organic molecule exhibits a rather large two-photon absorption cross section as compared with the compound 4-trans-[p-(N, N-Di-n-butylamino)-p-stilbenyl vinyl] pyridine (DBASVP) reported previously, and is predicted to be a good two-photon polymerization initiator. The hydrogen-bond effect is analysed. The computational results are in good agreement with the measurements.

  18. Decay and coherence of two-photon excited yellow orthoexcitons in Cu2O

    NARCIS (Netherlands)

    Karpinska, Katarzyna; Mostovoy, M; van der Vegte, MA; Revcolevschi, A; van Loosdrecht, PHM

    2005-01-01

    Photoluminescence excitation spectroscopy has revealed a highly efficient two-photon excitation method to produce a cold, uniformly distributed high density excitonic gas in bulk cuprous oxide. A study of the time evolution of the density, temperature, and chemical potential of the exciton gas shows

  19. Experimental method for the determination of two-photon cross sections using four-wave mixing

    Science.gov (United States)

    Burris, J.; Mcilrath, T. J.

    1985-01-01

    The two-photon absorption cross section for the R22 + S12(J double prime = 9 1/2) transition in nitric oxide's gamma band has been determined. The value is in good agreement with previous measurements on several other NO transitions. The technique described here can be used to obtain accurate cross sections for other diatomic molecules.

  20. Dependence of the two-photon photoluminescence yield of gold nanostructures on the laser pulse duration

    DEFF Research Database (Denmark)

    Biagioni, P.; Celebrano, M.; Savoini, M.

    2009-01-01

    Two-photon photoluminescence (TPPL) from gold nanostructures is becoming one of the most relevant tools for plasmon-assisted biological imaging and photothermal therapy as well as for the investigation of plasmonic devices. Here we study the yield of TPPL as a function of the temporal width δ of ...

  1. 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.

  2. Long vs. short distance dispersive two-photon $K_{L} \\to \\mu^{+} \\mu^{-}$ amplitude

    CERN Document Server

    Eeg, Jan O; Picek, I

    1999-01-01

    We report on the calculation of the two-loop electroweak, two-photon mediated short-distance dispersive K_L \\to \\mu^+\\mu^- decay amplitude. QCD corrections change the sign of this contribution and reduce it by an order of magnitude. The resulting amplitude enables us to provide a constraint on the otherwise uncertain long-distance dispersive amplitude.

  3. Observation of high-$p_{T}$ jets in two-photon interactions

    CERN Document Server

    Bartel, Wulfrin; Dittmann, P; Eichler, R; Felst, R; Haidt, Dieter; Krehbiel, H; Meier, K; Naroska, Beate; O'Neill, L H; Steffen, P; Wenninger, Horst; Zhang, Y; Elsen, E E; Helm, M; Petersen, A; Warming, P; Weber, G; Bethke, Siegfried; Drumm, H; Heintze, J; Heinzelmann, G; Hellenbrand, K H; Heuer, R D; Von Krogh, J; Lennert, P; Kawabata, S; Matsumura, H; Nozaki, T; Olsson, J; Rieseberg, H; Wagner, A; Bell, A; Foster, F; Hughes, G; Wriedt, H; Allison, J; Ball, A H; Bamford, G; Barlow, R; Bowdery, C K; Duerdoth, I P; Hassard, J F; King, B T; Loebinger, F K; MacBeth, A A; McCann, H; Mills, H E; Murphy, P G; Stephens, K; Clarke, D; Goddard, M C; Marshall, R; Pearce, G F; Kobayashi, T; Komamiya, S; Koshiba, M; Minowa, M; Nosaki, M; Orito, S; Sato, A; Suda, T; Takeda, H; Totsuka, Y; Watanabe, Y; Yamada, S; Yanagisawa, C

    1981-01-01

    Events with a characteristic two-jet topology have been observed in two-photon interactions. The production cross section is found to be higher than the point-like gamma gamma -qq cross section, which is approached only at transverse momenta larger than 3 GeV/c. (11 refs).

  4. Measurement Induced Enhancement of Squeezing in Nondegenerate Two-Photon Jaynes-Cummings Model

    Institute of Scientific and Technical Information of China (English)

    YE Sai-Yun

    2006-01-01

    Squeezing properties in the nondegenerate two-photon Jaynes-Cummings model are investigated. The effects of direct selective atomic measurement and the application of the classical field followed by atomic measurement are analyzed. Different values of the parameters of the classical field are taken into account. It is found that the field squeezing can be enhanced by measurement.

  5. Two-photon excited highly polarized and directional upconversion emission from slab organic crystals

    NARCIS (Netherlands)

    Fang, Hong-Hua; Chen, Qi-Dai; Yang, Jie; Xia, Hong; Ma, Yu-Guang; Wang, Hai-Yu; Sun, Hong-Bo; Fang, Honghua

    2010-01-01

    Effective upconversion emission from an organic crystal of cyano-substituted oligo (p-phenylenevinylene) (CNDPASDB) based on two-photon absorption is presented. Frequency upconverted cavityless lasing, or amplified spontaneous emission, from the crystal pumped by a femtosecond laser of 800 nm was ob

  6. Carbon quantum dot-NO photoreleaser nanohybrids for two-photon phototherapy of hypoxic tumors.

    Science.gov (United States)

    Fowley, Colin; McHale, Anthony P; McCaughan, Bridgeen; Fraix, Aurore; Sortino, Salvatore; Callan, John F

    2015-01-04

    We report a conjugate between carbon quantum dots and a NO photoreleaser able to photogenerate the anticancer NO radical via an energy transfer mechanism. This nanohybrid proved toxic to cancer cells in vitro and significantly reduced tumor volume in mice bearing human xenograft BxPC-3 pancreatic tumors upon two-photon excitation with the highly biocompatible 800 nm light.

  7. Probing Electron-Phonon Interaction through Two-Photon Interference in Resonantly Driven Semiconductor Quantum Dots

    DEFF Research Database (Denmark)

    Reigue, Antoine; Iles-Smith, Jake; Lux, Fabian

    2017-01-01

    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...

  8. Sub-diffraction positioning of a two-photon excited and optically trapped quantum dot

    DEFF Research Database (Denmark)

    Jauffred, L.; Kyrsting, A.; Christensen, Eva Arnspang;

    2014-01-01

    Colloidal quantum dots are luminescent long-lived probes that can be two-photon excited and manipulated by a single laser beam. Therefore, quantum dots can be used for simultaneous single molecule visualization and force manipulation using an infra-red laser. Here, we show that even a single opti...

  9. Two-photon up-conversion affected by inter-molecule correlations near metallic nanostructure

    CERN Document Server

    Osaka, Yoshiki; Ishihara, Hajime

    2016-01-01

    We investigate an efficient two-photon up-conversion process in more than one molecule coupled to an optical antenna. In the previous work [Y. Osaka et al., PRL 112, 133601 (2014)], we considered the two-photon up-conversion process in a single molecule within one-dimensional input-output theory, and revealed that controlling the antenna-molecule coupling enables the efficient up-conversion with radiative loss in the antenna suppressed. In this work, aiming to propose a way to enhance the total probability of antenna-photon scattering, we extend the model to the case of multiple molecules. In general, the presence of more than one molecule decreases the up-conversion probability because they equally share the energy of the two photons. However, it is shown that we can overcome the difficulty by controlling the inter-molecule coupling. Our result implies that, without increasing the incident photon number (light power), we can enlarge the net probability of the two-photon up-conversion.

  10. Atomic Dipole Squeezing in the Correlated Two-Mode Two-Photon Jaynes-Cummings Model

    Science.gov (United States)

    Dong, Zhengchao; Zhao, Yonglin

    1996-01-01

    In this paper, we study the atomic dipole squeezing in the correlated two-mode two-photon JC model with the field initially in the correlated two-mode SU(1,1) coherent state. The effects of detuning, field intensity and number difference between the two field modes are investigated through numerical calculation.

  11. Engineering two-photon high-dimensional states through quantum interference

    CSIR Research Space (South Africa)

    Zhang, YI

    2016-02-01

    Full Text Available the storage and processing potential of quantum information systems. We demonstrate the controlled engineering of two-photon high-dimensional states entangled in their orbital angular momentum through Hong-Ou-Mandel interference. We prepare a large range...

  12. New cubic perovskites for one- and two-photon water splitting using the computational materials repository

    DEFF Research Database (Denmark)

    Castelli, Ivano Eligio; Landis, David; Thygesen, Kristian Sommer

    2012-01-01

    screening of around 19 000 oxides, oxynitrides, oxysulfides, oxyfluorides, and oxyfluoronitrides in the cubic perovskite structure with PEC applications in mind. We address three main applications: light absorbers for one- and two-photon water splitting and high-stability transparent shields to protect...

  13. Two-photon excitation spectra of Cr3 :K2NaScF6

    Science.gov (United States)

    Bartram, R. H.; Wein, G. R.; Hamilton, D. S.; Sliwczuk, U.; Rinzler, A. G.

    Two-photon excitation (TPE) spectra of Cr3+:K2NaScF6 exhibit unexpected features including a forbidden transition, extended progressions, a split zero-phonon line and anomalous polarization anisotropy. These features are explained by departures from standard approximations.

  14. A study of Two Photon Decays of Charmonium Resonances Formed in Proton Anti-Proton Annihilations

    Energy Technology Data Exchange (ETDEWEB)

    Pedlar, Todd Kristofer [Northwestern Univ., Evanston, IL (United States)

    1999-06-01

    In this dissertation we describe the results of an investigation of the production of charmonium states (ηc, η'c, χ0 and χ2) in Fermilab experiment E835 via antiproton-proton annihilation and their detection via their decay into two photons.

  15. Background-Free Optical Sampling System Using Si Avalanche Photodiode as Two-Photon Absorber

    Institute of Scientific and Technical Information of China (English)

    Kenji; Taira; Ryo; Ohta; Yasuyuki; Ozeki; Yutaka; Fukuchi; Kazuhiro; Katoh; Kazuro; Kikuchi

    2003-01-01

    The introduction of a double-chopping scheme eliminates the background level in the optical sampling system, where a Si avalanche photodiode acts as a two-photon absorber. We successfully demonstrate background-free optical sampling of 40-GHz and 160-GHz pulse trains.

  16. Simultaneous two-photon activation of type-I photodynamic therapy agents.

    Science.gov (United States)

    Fisher, W G; Partridge, W P; Dees, C; Wachter, E A

    1997-08-01

    The excitation and emission properties of several psoralen derivatives are compared using conventional single-photon excitation and simultaneous two-photon excitation (TPE). Two-photon excitation is effected using the output of a mode-locked titanium: sapphire laser, the near infrared output of which is used to promote nonresonant TPE directly. Specifically, the excitation spectra and excited-state properties of 8-methoxypsoralen and 4'-aminomethyl-4,5,8-trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two-photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two-photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamaging near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage.

  17. Rapid Prototyping of Chemical Microsensors Based on Molecularly Imprinted Polymers Synthesized by Two-Photon Stereolithography.

    Science.gov (United States)

    Gomez, Laura Piedad Chia; Spangenberg, Arnaud; Ton, Xuan-Anh; Fuchs, Yannick; Bokeloh, Frank; Malval, Jean-Pierre; Tse Sum Bui, Bernadette; Thuau, Damien; Ayela, Cédric; Haupt, Karsten; Soppera, Olivier

    2016-07-01

    Two-photon stereolithography is used for rapid prototyping of submicrometre molecularly imprinted polymer-based 3D structures. The structures are evaluated as chemical sensing elements and their specific recognition properties for target molecules are confirmed. The 3D design capability is exploited and highlighted through the fabrication of an all-organic molecularly imprinted polymeric microelectromechanical sensor.

  18. Superradiant dye solution laser with two-photon picosecond optical pumping

    Energy Technology Data Exchange (ETDEWEB)

    Prokhorenko, V.I.; Tikhonov, E.A.; Shpak, M.T.

    1981-01-01

    A superradiant (superfluorescent) dye solution laser with two-photon picosecond pumping was constructed for the first time. A preliminary study was made of the principal characteristics of the output radiation of this laser which performed up-conversion of the frequency of the pump radiation. The physical mechanisms governing the operation of lasers of this type were analyzed.

  19. Two-photon photoemission from a copper cathode in an X -band photoinjector

    Science.gov (United States)

    Li, H.; Limborg-Deprey, C.; Adolphsen, C.; McCormick, D.; Dunning, M.; Jobe, K.; Raubenheimer, T.; Vrielink, A.; Vecchione, T.; Wang, F.; Weathersby, S.

    2016-02-01

    This paper presents two-photon photoemission from a copper cathode in an X -band photoinjector. We experimentally verified that the electron bunch charge from photoemission out of a copper cathode scales with laser intensity (I) square for 400 nm wavelength photons. We compare this two-photon photoemission process with the single photon process at 266 nm. Despite the high reflectivity (R ) of the copper surface for 400 nm photons (R =0.48 ) and higher thermal energy of photoelectrons (two-photon at 200 nm) compared to 266 nm photoelectrons, the quantum efficiency of the two-photon photoemission process (400 nm) exceeds the single-photon process (266 nm) when the incident laser intensity is above 300 GW /cm2 . At the same laser pulse energy (E ) and other experimental conditions, emitted charge scales inversely with the laser pulse duration. A thermal emittance of 2.7 mm-mrad per mm root mean square (rms) was measured on our cathode which exceeds by sixty percent larger compared to the theoretical predictions, but this discrepancy is similar to previous experimental thermal emittance on copper cathodes with 266 nm photons. The damage of the cathode surface of our first-generation X -band gun from both rf breakdowns and laser impacts mostly explains this result. Using a 400 nm laser can substantially simplify the photoinjector system, and make it an alternative solution for compact pulsed electron sources.

  20. A scheme to realize time-bin entanglement between two photons that never interacted

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    We propose a scheme of entangling two photons from two separated sources.Our proposal which is inspired by the time-bin entanglement developed recently,provides a novel alternative for revealing contradiction between quantum nonlocality and local realism based on two independent single photon sources.