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  1. Multiphoton microscopy in life sciences.

    Science.gov (United States)

    König, K

    2000-11-01

    Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm(-2) to GW cm(-2), which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via

  2. Multiphoton Microscopy for Ophthalmic Imaging

    Directory of Open Access Journals (Sweden)

    Emily A. Gibson

    2011-01-01

    Full Text Available We review multiphoton microscopy (MPM including two-photon autofluorescence (2PAF, second harmonic generation (SHG, third harmonic generation (THG, fluorescence lifetime (FLIM, and coherent anti-Stokes Raman Scattering (CARS with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

  3. Multiphoton microscopy imaging of developing tooth germs

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    Pei-Yu Pan

    2014-01-01

    Conclusion: In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

  4. Multiphoton microscopy: An introduction to gastroenterologists

    Institute of Scientific and Technical Information of China (English)

    Hye Jin Cho; Hoon Jai Chun; Eun Sun Kim; Bong Rae Cho

    2011-01-01

    Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.

  5. Multiphoton microscopy in defining liver function

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    Thorling, Camilla A.; Crawford, Darrell; Burczynski, Frank J.; Liu, Xin; Liau, Ian; Roberts, Michael S.

    2014-09-01

    Multiphoton microscopy is the preferred method when in vivo deep-tissue imaging is required. This review presents the application of multiphoton microscopy in defining liver function. In particular, multiphoton microscopy is useful in imaging intracellular events, such as mitochondrial depolarization and cellular metabolism in terms of NAD(P)H changes with fluorescence lifetime imaging microscopy. The morphology of hepatocytes can be visualized without exogenously administered fluorescent dyes by utilizing their autofluorescence and second harmonic generation signal of collagen, which is useful in diagnosing liver disease. More specific imaging, such as studying drug transport in normal and diseased livers are achievable, but require exogenously administered fluorescent dyes. If these techniques can be translated into clinical use to assess liver function, it would greatly improve early diagnosis of organ viability, fibrosis, and cancer.

  6. Application of multiphoton microscopy in dermatological studies: A mini-review

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    Elijah Yew

    2014-09-01

    Full Text Available This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

  7. A review of biomedical multiphoton microscopy and its laser sources

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    Lefort, Claire

    2017-10-01

    Multiphoton microscopy (MPM) has been the subject of major development efforts for about 25 years for imaging biological specimens at micron scale and presented as an elegant alternative to classical fluorescence methods such as confocal microscopy. In this topical review, the main interests and technical requirements of MPM are addressed with a focus on the crucial role of excitation source for optimization of multiphoton processes. Then, an overview of the different sources successfully demonstrated in literature for MPM is presented, and their physical parameters are inventoried. A classification of these sources in function with their ability to optimize multiphoton processes is proposed, following a protocol found in literature. Starting from these considerations, a suggestion of a possible identikit of the ideal laser source for MPM concludes this topical review. Dedicated to Martin.

  8. Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy

    NARCIS (Netherlands)

    Antonello, J.; Werkhoven, T. van; Verhaegen, M.; Truong, H.H.; Keller, C.U.; Gerritsen, H.C.

    2014-01-01

    Optical aberrations have detrimental effects in multiphoton microscopy. These effects can be curtailed by implementing model-based wavefront sensorless adaptive optics, which only requires the addition of a wavefront shaping device, such as a deformable mirror (DM) to an existing microscope. The abe

  9. Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging.

    NARCIS (Netherlands)

    Andresen, V.; Alexander, S.; Heupel, W.M.; Hirschberg, M.; Hoffman, R.M.; Friedl, P.H.A.

    2009-01-01

    Multiphoton microscopy (MPM) is the method of choice for investigating cells and cellular functions in deep tissue sections and organs. Here we present the setup and applications of infrared-(IR-)MPM using excitation wavelengths above 1080 nm. IR-MPM enables the use of red fluorophores and

  10. Advances in renal (patho)physiology using multiphoton microscopy.

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    Sipos, A; Toma, I; Kang, J J; Rosivall, L; Peti-Peterdi, J

    2007-11-01

    Multiphoton excitation fluorescence microscopy is a state-of-the-art confocal imaging technique ideal for deep optical sectioning of living tissues. It is capable of performing ultrasensitive, quantitative imaging of organ functions in health and disease with high spatial and temporal resolution which other imaging modalities cannot achieve. For more than a decade, multiphoton microscopy has been successfully used with various in vitro and in vivo experimental approaches to study many functions of different organs, including the kidney. This study focuses on recent advances in our knowledge of renal (patho)physiological processes made possible by the use of this imaging technology. Visualization of cellular variables like cytosolic calcium, pH, cell-to-cell communication and signal propagation, interstitial fluid flow in the juxtaglomerular apparatus (JGA), real-time imaging of tubuloglomerular feedback (TGF), and renin release mechanisms are reviewed. A brief summary is provided of kidney functions that can be measured by in vivo quantitative multiphoton imaging including glomerular filtration and permeability, concentration, dilution, and activity of the intrarenal renin-angiotensin system using this minimally invasive approach. New visual data challenge a number of existing paradigms in renal (patho)physiology. Also, quantitative imaging of kidney function with multiphoton microscopy has tremendous potential to eventually provide novel non-invasive diagnostic and therapeutic tools for future applications in clinical nephrology.

  11. Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

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    Sun, Yen; Lo, Wen; Lin, Sung-Jan; Lin, Wei-Chou; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover, increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area. In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.

  12. Multimodal optoacoustic and multiphoton microscopy of human carotid atheroma

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    Markus Seeger

    2016-09-01

    Full Text Available Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

  13. Multiphoton microscopy as a diagnostic imaging modality for lung cancer

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    Pavlova, Ina; Hume, Kelly R.; Yazinski, Stephanie A.; Peters, Rachel M.; Weiss, Robert S.; Webb, Watt W.

    2010-02-01

    Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

  14. Reassignment of scattered emission photons in multifocal multiphoton microscopy.

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    Cha, Jae Won; Singh, Vijay Raj; Kim, Ki Hean; Subramanian, Jaichandar; Peng, Qiwen; Yu, Hanry; Nedivi, Elly; So, Peter T C

    2014-06-05

    Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.

  15. Spectroscopic analysis of skin intrinsic signals for multiphoton microscopy

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    Pena, Ana-Maria; Strupler, Mathias; Boulesteix, Thierry; Senni, Karim; Godeau, Gaston; Beaurepaire, Emmanuel; Schanne-Klein, Marie-Claire

    2006-02-01

    We recorded multiphoton images of human skin biopsies using endogenous sources of nonlinear optical signals. We detected simultaneously two-photon excited fluorescence (2PEF) from intrinsic fluorophores and second harmonic generation (SHG) from collagen. We observed SHG from fibrillar collagens in the dermis, whereas no SHG was detectable from the non fibrillar type IV collagen in the basal laminae. We compared these distinct behaviours of collagens I and IV in SHG microscopy to polarization-resolved surface SHG experiments on thin films of collagens I and IV molecules. We observed similar signals for both types of molecular films, except for the chiroptical contributions which are present only for collagen I and enhance the signal typically by a factor of 2. We concluded that SHG microscopy is a sensitive probe of the micrometer-scale structural organization of collagen in biological tissues. In order to elucidate the origin of the endogenous fluorescence signals, we recorded 2PEF spectra at various positions in the skin biopsies, and compared these data to in vitro spectroscopic analysis. In particular, we studied the keratin fluorescence and determined its 2PEF action cross section. We observed a good agreement between 2PEF spectra recorded in the keratinized upper layers of the epidermis and in a solution of purified keratin. Finally, to illustrate the capabilities of this technique, we recorded 2PEF/SHG images of skin biopsies obtained from patients of various ages.

  16. Improving Signal Levels in Intravital Multiphoton Microscopy using an Objective Correction Collar.

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    Muriello, Pamela A; Dunn, Kenneth W

    2008-04-01

    Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreased the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.

  17. The analysis of aging skin based on multiphoton microscopy

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    Wu, Shulian; Li, Hui; Zhang, Xiaoman; Li, Zhifang; Xu, Shufei

    2010-11-01

    Aging is a very important issue not only in dermatology, but also in cosmetic science. Cutaneous aging involves both chronological and photoaging aging process. The chronological aging is induced with the passage of time. And the photoaging skin is the extrinsic aging caused by sun exposure. The aim of this study is to use multiphoton microscopy (MPM) in vivo to assess intrinsic-age-related and photo-age-related difference. The changes of dermal collagen are measured in quantitively. The algorithm that we used automatically produced the transversal dermal map from MPM. Others, the texture of dermis are analyzed by Fourier transform and Gray Level Co-occurrence Matrix. And the object extraction in textured images is proposed based on the method in object edge extraction, and the aim of it is to detect the object hidden in the skin texture in difference aging skin. The result demonstrates that the approach is effective in detecting the object in epidermis and dermis textured image in different aging skin. It could help to further understand the aging mechanism.

  18. Label-free detection of breast masses using multiphoton microscopy.

    Directory of Open Access Journals (Sweden)

    Xiufeng Wu

    Full Text Available Histopathology forms the gold standard for the diagnosis of breast cancer. Multiphoton microscopy (MPM has been proposed to be a potentially powerful adjunct to current histopathological techniques. A label-free imaging based on two- photon excited fluorescence and second-harmonic generation is developed for differentiating normal breast tissues, benign, as well as breast cancer tissues. Human breast biopsies (including human normal breast tissues, benign as well as breast cancer tissues that are first imaged (fresh, unfixed, and unstained with MPM and are then processed for routine H-E histopathology. Our results suggest that the MPM images, obtained from these unprocessed biopsies, can readily distinguish between benign lesions and breast cancers. In the tissues of breast cancers, MPM showed that the tumor cells displayed marked cellular and nuclear pleomorphism. The tumor cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, infiltrated into disrupted connective tissue, leading to the loss of second-harmonic generation signals. For breast cancer, MPM diagnosis was 100% correct because the tissues of breast cancers did not have second-harmonic generation signals in MPM imaging. On the contrary, in benign breast masses, second-harmonic generation signals could be seen easily in MPM imaging. These observations indicate that MPM could be an important potential tool to provide label-free noninvasive diagnostic impressions that can guide surgeon in biopsy and patient management.

  19. Identification of intramural metastasis in esophageal cancer using multiphoton microscopy

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    Xu, Jian; Kang, Deyong; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, jiangbo; Chen, Jianxin

    2017-02-01

    Intramural metastasis (IM) of esophageal cancer is defined as metastasis from a primary lesion to the esophageal wall without intraepithelial cancer extension. Esophageal cancer with IM is more common and such cases indicate a poor prognosis. In esophageal surgery, if curative resection is possible, the complete removal of both primary tumor and associated IMs is required. Therefore, accurate diagnosis of IMs in esophageal cancer prior to surgery is of particular importance. Multiphoton microscopy (MPM) with subcellular resolution is well-suited for deep tissue imaging since many endogenous fluorophores of fresh biological tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Here, a study to identify IM in fresh tissue section using MPM is reported. In this study, the morphological and spectral differences between IM and surrounding tissue are described. These results show that MPM has the ability to accurately identify IM in esophageal tissues. With improvement of the penetration depth of MPM and the development of multiphton microendoscope, MPM may be a promising imaging technique for preoperative diagnosis of IMs in esophageal cancer in the future.

  20. Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mulholland, William J [PowderJect Centre for Gene and Drug delivery Research, Department of Engineering Science, University of Oxford OX2 6PE (United Kingdom); Kendall, Mark A F [PowderJect Centre for Gene and Drug delivery Research, Department of Engineering Science, University of Oxford OX2 6PE (United Kingdom); White, Nick [Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE (United Kingdom); Bellhouse, Brian J [PowderJect Centre for Gene and Drug delivery Research, Department of Engineering Science, University of Oxford OX2 6PE (United Kingdom)

    2004-11-21

    Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.

  1. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy

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    Segall Jeffrey E

    2005-05-01

    Full Text Available Abstract Background The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. Results Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1 the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2 direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. Conclusion Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

  2. Multiphoton excitation fluorescence microscopy in planar membrane systems.

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    Brewer, Jonathan; Bernardino de la Serna, Jorge; Wagner, Kerstin; Bagatolli, Luis A

    2010-07-01

    The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

  3. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

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    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  4. Acousto-optic multiphoton laser scanning microscopy and multiphoton photon counting spectroscopy: Applications and implications for optical neurobiology

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    Iyer, Vijay

    Multiphoton excitation of molecular probes has become an important tool in experimental neurobiology owing to the intrinsic optical sectioning and low light scattering it affords. Using molecular functional indicators, multiphoton excitation allows physiological signals within single neurons to be observed from within living brain tissue. Ideally, it would be possible to record from multiple sites located throughout the elaborately branching dendritic arbors, in order to study the correlations of structure and function both within and across experiments. However, existing multiphoton microscope systems based on scanning mirrors do not allow optical recordings to be obtained from more than a handful of sites simultaneously at the high rates required to capture the fast physiological signals of interest (>100Hz for Ca2+ signals, >1kHz for membrane potential transients). In order to overcome this limitation, two-dimensional acousto-optic deflection was employed, to allow an ultrafast laser beam suited for multiphoton excitation to be rapidly repositioned with low latency (˜15mus). This supports a random-access scanning mode in which the beam can repeatedly visit a succession of user-selected sites of interest within the microscope's field-of-view at high rates, with minimal sacrifice of pixel dwell time. This technique of acousto-optic multiphoton laser scanning microscope (AO-MPLSM) was demonstrated to allow the spatial profile of signals arising in response to physiological stimulation to be rapidly mapped. Means to compensate or avoid problems of dispersion which have hampered AO-MPLSM in the past are presented, with the latter being implemented. Separately, the combination of photon counting detection with multiphoton excitation, termed generally multiphoton photon counting spectroscopy (MP-PCS), was also considered, with particular emphasis on the technique of fluorescence correlation spectroscopy (FCS). MP-PCS was shown to allow information about molecular

  5. Ex vivo applications of multiphoton microscopy in urology

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    Jain, Manu; Mukherjee, Sushmita

    2016-03-01

    Background: Routine urological surgery frequently requires rapid on-site histopathological tissue evaluation either during biopsy or intra-operative procedure. However, resected tissue needs to undergo processing, which is not only time consuming but may also create artifacts hindering real-time tissue assessment. Likewise, pathologist often relies on several ancillary methods, in addition to H&E to arrive at a definitive diagnosis. Although, helpful these techniques are tedious and time consuming and often show overlapping results. Therefore, there is a need for an imaging tool that can rapidly assess tissue in real-time at cellular level. Multiphoton microscopy (MPM) is one such technique that can generate histology-quality images from fresh and fixed tissue solely based on their intrinsic autofluorescence emission, without the need for tissue processing or staining. Design: Fresh tissue sections (neoplastic and non-neoplastic) from biopsy and surgical specimens of bladder and kidney were obtained. Unstained deparaffinized slides from biopsy of medical kidney disease and oncocytic renal neoplasms were also obtained. MPM images were acquired using with an Olympus FluoView FV1000MPE system. After imaging, fresh tissues were submitted for routine histopathology. Results: Based on the architectural and cellular details of the tissue, MPM could characterize normal components of bladder and kidney. Neoplastic tissue could be differentiated from non-neoplastic tissue and could be further classified as per histopathological convention. Some of the tumors had unique MPM signatures not otherwise seen on H&E sections. Various subtypes of glomerular lesions were identified as well as renal oncocytic neoplasms were differentiated on unstained deparaffinized slides. Conclusions: We envision MPM to become an integral part of regular diagnostic workflow for rapid assessment of tissue. MPM can be used to evaluate the adequacy of biopsies and triage tissues for ancillary studies

  6. Optical tweezers and multiphoton microscopies integrated photonic tool for mechanical and biochemical cell processes studies

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    de Thomaz, A. A.; Faustino, W. M.; Fontes, A.; Fernandes, H. P.; Barjas-Castro, M. d. L.; Metze, K.; Giorgio, S.; Barbosa, L. C.; Cesar, C. L.

    2007-09-01

    The research in biomedical photonics is clearly evolving in the direction of the understanding of biological processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools. However, an integration of different photonic tools and a multimodal and functional approach will be necessary to access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for integration. Therefore, we developed such integrated tool by incorporating single and double Optical Tweezers with Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second Harmonic Generation microscopies together with optical manipulations. It also can acquire Fluorescence and SHG spectra of specific spots. Force, elasticity and viscosity measurements of stretched membranes can be followed by real time confocal microscopies. Also opticaly trapped living protozoas, such as leishmania amazonensis. Integration with CARS microscopy is under way. We will show several examples of the use of such integrated instrument and its potential to observe mechanical and biochemical processes at cell level.

  7. Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

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    Tung, Oi-Hong; Lee, Shyh-Yuan; Lai, Yu-Lin; Chen, How-Foo

    2011-06-01

    Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

  8. Towards in vivo breast skin characterization using multiphoton microscopy

    Science.gov (United States)

    Batista, Ana; Uchugonova, Aisada; Breunig, Hans Georg; König, Karsten

    2017-02-01

    Breast cancer, the most common type of cancer in women worldwide, as well as its treatment (e.g. radiation therapy) can affect the human skin. Multiphoton imaging could provide new insights into these skin alterations non-invasively and with high-resolution. As a preparation for a later investigation involving patients, areas of the breast and forearm skin of healthy volunteers were imaged using the clinically certified multiphoton imaging tomograph MPTflex based on endogenous skin autofluorescence and second-harmonic signals. Depth-resolved image stacks were acquired in consecutive weeks to explore the influence of hormonal variations on the skin properties. Both breasts were considered and up to three different areas were imaged per session. Acquisition parameters were optimized to minimize artifacts caused by breathing-motion. As a first result, skin properties, such as the epidermal thickness, appear to be influenced by hormonal variations.

  9. Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

    Science.gov (United States)

    Zhuo, S. M.; Chen, J. X.; Jiang, X. S.; Lu, K. C.; Xie, S. S.

    2008-08-01

    We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered-resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer.

  10. Multiphoton microscopy and image guided light activated therapy using nanomaterials (Conference Presentation)

    Science.gov (United States)

    Prasad, Paras N.

    2017-02-01

    This talk will focus on design and applications of nanomaterials exhibiting strong multiphoton upconversion for multiphoton microscopy as well as for image-guided and light activated therapy .1-3 Such processes can occur by truly nonlinear optical interactions proceeding through virtual intermediate states or by stepwise coupled linear excitations through real intermediate states. Multiphoton processes in biocompatible multifunctional nanoparticles allow for 3D deep tissue imaging. In addition, they can produce in-situ photon conversion of deep tissue penetrating near IR light into a needed shorter wavelength light for photo-activated therapy at a targeted site, thus overcoming the limited penetration of UV or visible light into biological media. We are using near IR emitters such as silicon quantum dots which also exhibit strong multiphoton excitation for multiphoton microscopy. Another approach involves nonlinear nanocrystals such as ZnO which can produce four wave mixing, sum frequency generation as well as second harmonic generation to convert a deep tissue penetrating Near IR light at the targeted biological site to a desired shorter wavelength light suitable for bio imaging or activation of a therapy. We have utilized this approach to activate a photosensitizer for photodynamic therapy. Yet another type of upconversion materials is rare-earth ion doped optical nanotransformers which transform a Near IR (NIR) light from an external source by sequential single photon absorption, in situ and on demand, to a needed wavelength. Applications of these nanotransformers in multiphoton photoacoustic imaging will also be presented. An exciting direction pursued by us using these multiphoton nanoparticles, is functional imaging of brain. Simultaneously, they can effect optogenetics for regioselective stimulation of neurons for providing an effective intervention/augmentation strategy to enhance the cognitive state and lead to a foundation for futuristic vision of super

  11. Distinguishing human normal or cancerous esophagus tissue ex vivo using multiphoton microscopy

    Science.gov (United States)

    Liu, N. R.; Chen, G. N.; Wu, S. S.; Chen, R.

    2014-02-01

    Application of multiphoton microscopy (MPM) to clinical cancer research has greatly developed over the last few years. In this paper, we mainly focus on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) for investigating esophageal cancer. We chiefly discuss the SHG/TPEF image and spectral characteristics of normal and cancerous esophagus submucosa with the combined multi-channel imaging mode and Lambda mode of a multiphoton microscope (LSM 510 META). Great differences can be detected, such as collagen content and morphology, glandular-shaped cancer cells, TPEF/SHG intensity ratio, and so on, which demonstrate that the multiphoton imaging technique has the potential ability for minimally-invasive early cancer diagnosis.

  12. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

    OpenAIRE

    Supatto, Willy; McMahon, Amy; Fraser, Scott E.; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and progr...

  13. Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Lin, Po-Yen; Lyu, Hong-Chou; Hsu, Chin-Ying Stephen; Chang, Chia-Seng; Kao, Fu-Jen

    2011-01-01

    In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and, despite their very similar spectral profiles, can be differentiated by lifetime analysis. Specifically, the carious dental tissue exhibits a greatly reduced autofluorescence lifetime, which result is consistent with the degree of demineralization, determined by micro-computed tomography. Our findings suggest that two-photon excited fluorescence lifetime imaging may be a promising tool for diagnosing and monitoring dental caries. PMID:21326645

  14. Differentiating the two main histologic categories of fibroadenoma tissue from normal breast tissue by using multiphoton microscopy.

    Science.gov (United States)

    Nie, Y T; Wu, Y; Fu, F M; Lian, Y E; Zhuo, S M; Wang, C; Chen, J X

    2015-04-01

    Multiphoton microscopy has become a novel biological imaging technique that allows cellular and subcellular microstructure imaging based on two-photon excited fluorescence and second harmonic generation. In this work, we used multiphoton microscopy to obtain the high-contrast images of human normal breast tissue and two main histologic types of fibroadenoma (intracanalicular, pericanalicular). Moreover, quantitative image analysis was performed to characterize the changes of collagen morphology (collagen content, collagen orientation). The results show that multiphoton microscopy combined with quantitative method has the ability to identify the characteristics of fibroadenoma including changes of the duct architecture and collagen morphology in stroma. With the advancement of multiphoton microscopy, we believe that the technique has great potential to be a real-time histopathological diagnostic tool for intraoperative detection of fibroadenoma in the future.

  15. Detection of the multiphoton signals in stained tissue using nonlinear optical microscopy

    Science.gov (United States)

    Zeng, Yaping; Xu, Jian; Kang, Deyong; Lin, Jiangbo; Chen, Jianxin

    2016-10-01

    Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging, has become a powerful, important tool for tissue imaging at the molecular level. Recently, MPM is also used to image hematoxylin and eosin (H and E)-stained sections in cancer diagnostics. However, several studies have showed that the MPM images of tissue stained with H and E are significantly different from unstained tissue sections. Our aim was to detect of the multiphoton signals in stained tissue by using MPM. In this paper, MPM was used to image histological sections of esophageal invasive carcinoma tissues stained with H, E, H and E and fresh tissue. To detect of the multiphoton signals in stained tissue, the emission spectroscopic of tissue stained with H, E, H and E were obtained. For comparison, the fresh tissues were also investigated. Our results showed that the tissue stained with H, E, H and E could be detected by their TPEF signals. While the tissue stained with H and fresh tissue could be detected by their TPEF and SHG signals. In this work, we detect of the multiphoton signals in stained tissue. These findings will be useful for choosing suitable staining method so to improve the quality of MPM imaging in the future.

  16. NI-78LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

    Science.gov (United States)

    Uckermann, Ortrud; Galli, Roberta; Geiger, Kathrin; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a

  17. Quantitative analysis of monocyte subpopulations in murine atherosclerotic plaques by multiphoton microscopy.

    Directory of Open Access Journals (Sweden)

    Abigail S Haka

    Full Text Available The progressive accumulation of monocyte-derived cells in the atherosclerotic plaque is a hallmark of atherosclerosis. However, it is now appreciated that monocytes represent a heterogeneous circulating population of cells that differ in functionality. New approaches are needed to investigate the role of monocyte subpopulations in atherosclerosis since a detailed understanding of their differential mobilization, recruitment, survival and emigration during atherogenesis is of particular importance for development of successful therapeutic strategies. We present a novel methodology for the in vivo examination of monocyte subpopulations in mouse models of atherosclerosis. This approach combines cellular labeling by fluorescent beads with multiphoton microscopy to visualize and monitor monocyte subpopulations in living animals. First, we show that multiphoton microscopy is an accurate and timesaving technique to analyze monocyte subpopulation trafficking and localization in plaques in excised tissues. Next, we demonstrate that multiphoton microscopy can be used to monitor monocyte subpopulation trafficking in atherosclerotic plaques in living animals. This novel methodology should have broad applications and facilitate new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.

  18. Real-time digital signal processing in multiphoton and time-resolved microscopy

    Science.gov (United States)

    Wilson, Jesse W.; Warren, Warren S.; Fischer, Martin C.

    2016-03-01

    The use of multiphoton interactions in biological tissue for imaging contrast requires highly sensitive optical measurements. These often involve signal processing and filtering steps between the photodetector and the data acquisition device, such as photon counting and lock-in amplification. These steps can be implemented as real-time digital signal processing (DSP) elements on field-programmable gate array (FPGA) devices, an approach that affords much greater flexibility than commercial photon counting or lock-in devices. We will present progress toward developing two new FPGA-based DSP devices for multiphoton and time-resolved microscopy applications. The first is a high-speed multiharmonic lock-in amplifier for transient absorption microscopy, which is being developed for real-time analysis of the intensity-dependence of melanin, with applications in vivo and ex vivo (noninvasive histopathology of melanoma and pigmented lesions). The second device is a kHz lock-in amplifier running on a low cost (50-200) development platform. It is our hope that these FPGA-based DSP devices will enable new, high-speed, low-cost applications in multiphoton and time-resolved microscopy.

  19. Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues.

    Directory of Open Access Journals (Sweden)

    Roberta Galli

    Full Text Available Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

  20. Potential of ultraviolet widefield imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Brewer, Jonathan R.; Bagatolli, Luis

    2011-01-01

    Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP...... found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could...

  1. Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy

    CERN Document Server

    Vinegoni, C; Luo, W; Marks, D L; Ralston, T; Tan, W

    2005-01-01

    An integrated microscope that combines different optical techniques for simultaneous imaging is demonstrated. The microscope enables spectral-domain optical coherence microscopy based on optical backscatter, and multi-photon microscopy for the detection of two-photon fluorescence and second harmonic generation signals. The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. In addition, the contemporary analysis of the spectroscopic features can enhance contrast by differentiating among different tissue components.

  2. Label-free discrimination of normal and pulmonary cancer tissues using multiphoton fluorescence ratiometric microscopy

    Science.gov (United States)

    Wang, Chun-Chin; Wu, Ruei-Jr; Lin, Sung-Jan; Chen, Yang-Fang; Dong, Chen-Yuan

    2010-07-01

    We performed multiphoton excited autofluorescence and second harmonic generation microscopy for the distinction of normal, lung adenocarcinoma (LAC), and squamous cell carcinoma (SCC) specimens. In addition to morphological distinction, we derived quantitative metrics of cellular redox ratios for cancer discrimination. Specifically, the redox ratios of paired normal/SCC and normal/LAC specimens were found to be 0.53±0.05/0.41±0.06 and 0.56±0.02/0.35±0.06, respectively. The lower redox ratios in cancer specimens, indicating an increase in metabolic activity. These results show that the combination of morphological multiphoton imaging along with redox ratio indices can be used for the discrimination of normal and pulmonary cancer tissues.

  3. In vivo multiphoton fluorescence microscopy of epithelial precancer

    Science.gov (United States)

    Zheng, Wei; Li, Dong; Zeng, Yan; Qu, Jianan Y.

    2011-03-01

    Most human cancers arise from epithelium, the superficial layer covering the exterior of body or lining the internal body cavities. Endogenous fluorophores such as aromatic amino acids, reduced nicotinamide adenine dinucleotide (NADH), flavoprotein (FAD), keratin, collagen, and elastin can provide abundant information to reveal the changes in biochemistry, metabolism, and morphology of living tissues. Thus, autofluorescence spectroscopy and microscopy have been recognized as potential tools for discrimination of cancer from normal tissues. However, current fluorescence diagnostic studies mostly rely on spectral analysis or morphological differentiation. It is challenged since the emission spectra of endogenous fluorophores are broad and usually overlapping with each other and the fluorescence intensity could be affected by many factors. In this study, we instrumented a nonlinear optical microscopy system to characterize the morphologic and biochemical features in the epithelial precancer in vivo. The 7,12-dimethylbenz(a)anthracenetreated hamster cheek pouch were used as a living animal carcinogenesis model. And the autofluorescence signals of NADH, collagen and elastin were recorded by a time- and spectral- resolved detection system. The results show that there are obvious differences in the morphology of three-dimensional autofluorescence images between normal and precancerous epithelial tissues. The fluorescence lifetime of NADH and the SHG signal from collagen could provide additional approaches to identify cancer from normal tissue.

  4. Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy.

    Science.gov (United States)

    Turcotte, Raphaël; Rutledge, Danette J; Bélanger, Erik; Dill, Dorothy; Macklin, Wendy B; Côté, Daniel C

    2016-08-19

    Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo.

  5. Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy

    Science.gov (United States)

    Turcotte, Raphaël; Rutledge, Danette J.; Bélanger, Erik; Dill, Dorothy; Macklin, Wendy B.; Côté, Daniel C.

    2016-08-01

    Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo.

  6. Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

    Science.gov (United States)

    Yu, Jiun-Yann; Kuo, Chun-Hung; Holland, Daniel B.; Chen, Yenyu; Ouyang, Mingxing; Blake, Geoffrey A.; Zadoyan, Ruben; Guo, Chin-Lin

    2011-11-01

    Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

  7. 3D super-resolved in vitro multiphoton microscopy by saturation of excitation

    CERN Document Server

    Nguyen, Anh Dung; Bouwens, Arno; Vanholsbeeck, Frédérique; Egrise, Dominique; Van Simayes, Gaetan; Emplit, Philippe; Goldman, Serge; Gorza, Simon-Pierre

    2015-01-01

    We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples.

  8. Characterization of corneal damage from Pseudomonas aeruginosa infection by the use of multiphoton microscopy

    Science.gov (United States)

    Chang, Yu-Lin; Chen, Wei-Liang; Lo, Wen; Chen, Shean-Jen; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2010-11-01

    Using multiphoton autofluorescence (MAF) and second harmonic generation (SHG) microscopy, we investigate the morphology and the structure of the corneal epithelium and stroma collagen of bovine cornea following injection of Pseudomonas aeruginosa. We found that corneal epithelial cells are damaged and stromal collagen becoming increasingly autofluorescent with time. We also characterized infected cornea cultured for 0, 6, 12, and 24 h by quantitative ratiometric MAF to SHG index (MAFSI) analysis. MAFSI results show that the destruction of the stromal collagen corresponds to a decrease in SHG intensity and increase of MAF signal with time.

  9. Quantitative analysis on collagen morphology in aging skin based on multiphoton microscopy

    Science.gov (United States)

    Wu, Shulian; Li, Hui; Yang, Hongqin; Zhang, Xiaoman; Li, Zhifang; Xu, Shufei

    2011-04-01

    Multiphoton microscopy was employed for monitoring the structure changes of mouse dermis collagen in the intrinsic- or the extrinsic-age-related processes in vivo. The characteristics of textures in different aging skins were uncovered by fast Fourier transform in which the orientation index and bundle packing of collagen were quantitatively analyzed. Some significant differences in collagen-related changes are found in different aging skins, which can be good indicators for the statuses of aging skins. The results are valuable to the study of aging skin and also of interest to biomedical photonics.

  10. Real-time in vivo imaging collagen in lymphedematous skin using multiphoton microscopy.

    Science.gov (United States)

    Wu, Xiufeng; Zhuo, Shuangmu; Chen, Jianxin; Liu, Ningfei

    2011-01-01

    Changes of dermal collagen are characteristic for chronic lymphedema. To evaluate these changes, a real-time imaging based on two-photon excited fluorescence and second-harmonic generation was developed for investigating collagen of lymphedematous mouse and rat tail skin in vivo. Our findings showed that the technique could image the morphological changes and distribution of collagen in lymphedematous mouse and rat tail skin in vivo. More importantly, it may allow visualization of dynamic collagen alteration during the progression of lymphedema. Our findings demonstrated that multiphoton microscopy may have potential in a clinical setting as an in vivo diagnostic and monitoring system for therapy in lymphology.

  11. Quantitative characterization of articular cartilage using Mueller matrix imaging and multiphoton microscopy

    Science.gov (United States)

    Ellingsen, Pa˚L. Gunnar; Lilledahl, Magnus Borstad; Aas, Lars Martin Sandvik; Davies, Catharina De Lange; Kildemo, Morten

    2011-11-01

    The collagen meshwork in articular cartilage of chicken knee is characterized using Mueller matrix imaging and multiphoton microscopy. Direction and degree of dispersion of the collagen fibers in the superficial layer are found using a Fourier transform image-analysis technique of the second-harmonic generated image. Mueller matrix images are used to acquire structural data from the intermediate layer of articular cartilage where the collagen fibers are too small to be resolved by optical microscopy, providing a powerful multimodal measurement technique. Furthermore, we show that Mueller matrix imaging provides more information about the tissue compared to standard polarization microscopy. The combination of these techniques can find use in improved diagnosis of diseases in articular cartilage, improved histopathology, and additional information for accurate biomechanical modeling of cartilage.

  12. 多焦点多光子显微技术及其研究进展%Multifocal Multiphoton Microscopy and Its Research Progress

    Institute of Scientific and Technical Information of China (English)

    刘立新

    2011-01-01

    Multifocal multiphoton microscopy ( MMM) can greatly improve the utilization of the excitation light and imaging speed, and it can also acquire three-dimensional fluorescence microscopic images by multiphoton excitation with the advantages of high speed, reduced photobleaching and photodamage, enhanced penetration depth and high signal to noise ratio. The realization and research progresses of multifocal multiphoton microscopy are discussed in detail, which include simultaneous time- and spectrum-resolved multifocal multiphoton microscopy(STSR-MMM) , multifocal multiphoton microscopy based on time-correlated single photon counting( TCSPC-MMM ) , multifocal multiphoton microscopy using a field of view zoom scanning protocol and stochastic scanning multifocal multiphoton microscopy (SS-MMM) , etc.%多焦点多光子显微技术(multifocal multiphoton microscopy,MMM)提高了激发光能的利用率和成像速度,可以实现样品的三维快速多光子激发荧光显微成像,并具有对活体样品损伤小,成像深度大,图像信噪比高等优点.详细阐述了MMM的实现方法及其研究进展,包括同时时间和光谱分辨的MMM(simultaneous time- and spectrum-resolved multifocal multiphoton microscopy,STSR-MMM)、基于时间相关单光子计数技术的MMM( multifocal multiphoton microscopy based on time-correlated single photon counting,TCSPC-MMM)、基于随机扫描的MMM(stochastic scanning multifocal multiphoton microscopy,SS-MMM)、基于固定光路系统的变视场扫描的MMM等技术.

  13. Multiphoton microscopy as a diagnostic tool for pathological analysis of sentinel lymph nodes

    Science.gov (United States)

    Lemiere, J.; Douady, J.; Estève, F.; Salameire, D.; Lantuejoul, S.; Lorimier, P.; Ricard, C.; van der Sanden, B.; Vial, J.-C.

    2009-02-01

    Multiphoton microscopy has shown a powerful potential for biomedical in vivo and ex vivo analysis of tissue sections and explants. Studies were carried out on several animal organs such as brain, arteries, lungs, and kidneys. One of the current challenges is to transfer to the clinic the knowledge and the methods previously developed in the labs at the preclinical level. For tumour staging, physicians often remove the lymph nodes that are localized at the proximity of the lesion. In case of breast cancer or melanoma, sentinel lymph node protocol is performed: pathologists randomly realize an extensive sampling of formol fixed nodes. However, the duration of this protocol is important and its reliability is not always satisfactory. The aim of our study was to determine if multiphoton microscopy would enable the fast imaging of lymph nodes on important depths, with or without exogenous staining. Experiments were first conducted on pig lymph nodes in order to test various dyes and to determine an appropriate protocol. The same experiments were then performed on thin slices of human lymph nodes bearing metastatic melanoma cells. We obtained relevant images with both endofluorescence plus second-harmonic generation and xanthene dyes. They show a good contrast between tumour and healthy cells. Furthermore, images of pig lymph nodes were recorded up to 120μm below the surface. This new method could then enable a faster diagnosis with higher efficiency for the patient. Experiments on thicker human lymph nodes are currently underway in order to validate these preliminary results.

  14. Rapid visualization of grain boundaries in monolayer MoS2 by multiphoton microscopy

    Science.gov (United States)

    Karvonen, Lasse; Säynätjoki, Antti; Huttunen, Mikko J.; Autere, Anton; Amirsolaimani, Babak; Li, Shisheng; Norwood, Robert A.; Peyghambarian, Nasser; Lipsanen, Harri; Eda, Goki; Kieu, Khanh; Sun, Zhipei

    2017-06-01

    Grain boundaries have a major effect on the physical properties of two-dimensional layered materials. Therefore, it is important to develop simple, fast and sensitive characterization methods to visualize grain boundaries. Conventional Raman and photoluminescence methods have been used for detecting grain boundaries; however, these techniques are better suited for detection of grain boundaries with a large crystal axis rotation between neighbouring grains. Here we show rapid visualization of grain boundaries in chemical vapour deposited monolayer MoS2 samples with multiphoton microscopy. In contrast to Raman and photoluminescence imaging, third-harmonic generation microscopy provides excellent sensitivity and high speed for grain boundary visualization regardless of the degree of crystal axis rotation. We find that the contrast associated with grain boundaries in the third-harmonic imaging is considerably enhanced by the solvents commonly used in the transfer process of two-dimensional materials. Our results demonstrate that multiphoton imaging can be used for fast and sensitive characterization of two-dimensional materials.

  15. PScan 1.0: flexible software framework for polygon based multiphoton microscopy

    Science.gov (United States)

    Li, Yongxiao; Lee, Woei Ming

    2016-12-01

    Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo imaging because of precise control over the scanning and detection system. To date, there have been several flexible software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed imaging systems.

  16. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis.

    Science.gov (United States)

    Supatto, Willy; McMahon, Amy; Fraser, Scott E; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

  17. Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

    Science.gov (United States)

    Dumas, D; Gaborit, N; Grossin, L; Riquelme, B; Gigant-Huselstein, C; De Isla, N; Gillet, P; Netter, P; Stoltz, J F

    2004-01-01

    Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

  18. Characteristics of scar margin dynamic with time based on multiphoton microscopy.

    Science.gov (United States)

    Zhu, Xiaoqin; Zhuo, Shuangmu; Zheng, Liqin; Jiang, Xingshan; Chen, Jianxin; Lin, Bifang

    2011-03-01

    Scar margins dynamic with time were quantitatively characterized using multiphoton microscopy (MPM). 2D large-area and 3D focused images of elastin and collagen at scar margins were obtained to extract quantitative parameters. An obvious boundary was observed at the scar margin, showing altered morphological patterns of elastin and collagen on both sides. Content alteration of elastin and collagen between the two sides of boundary were defined to characterize scar margins from different individuals. The statistical results from 15 normal scar samples strongly demonstrated that content alteration degree of elastin and collagen had decreasing tendency with the increase of patient age or scar duration, consistent with the fact of normal scars regressing spontaneously over time. It indicated that alteration degree can potentially serve as quantitative indicators to examine wound healing and scar progression over time. With the advent of clinical portable multiphoton endoscopes, the MPM technique can be applied in tracking scar formation and progression in vivo by examination of scar margin.

  19. Optimal spectral filtering in soliton self-frequency shift for deep-tissue multiphoton microscopy

    Science.gov (United States)

    Wang, Ke; Qiu, Ping

    2015-05-01

    Tunable optical solitons generated by soliton self-frequency shift (SSFS) have become valuable tools for multiphoton microscopy (MPM). Recent progress in MPM using 1700 nm excitation enabled visualizing subcortical structures in mouse brain in vivo for the first time. Such an excitation source can be readily obtained by SSFS in a large effective-mode-area photonic crystal rod with a 1550-nm fiber femtosecond laser. A longpass filter was typically used to isolate the soliton from the residual in order to avoid excessive energy deposit on the sample, which ultimately leads to optical damage. However, since the soliton was not cleanly separated from the residual, the criterion for choosing the optimal filtering wavelength is lacking. Here, we propose maximizing the ratio between the multiphoton signal and the n'th power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS when the soliton shows dramatic overlapping with the residual. This optimization is based on the most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally. Its application to MPM may reduce tissue damage, while maintaining high signal levels for efficient deep penetration.

  20. In vivo imaging of spinal cord in contusion injury model mice by multi-photon microscopy

    Science.gov (United States)

    Oshima, Y.; Horiuchi, H.; Ogata, T.; Hikita, A.; Miura, H.; Imamura, T.

    2014-03-01

    Fluorescent imaging technique is a promising method and has been developed for in vivo applications in cellular biology. In particular, nonlinear optical imaging technique, multi-photon microscopy has make it possible to analyze deep portion of tissues in living animals such as axons of spinal code. Traumatic spinal cord injuries (SCIs) are usually caused by contusion damages. Therefore, observation of spinal cord tissue after the contusion injury is necessary for understanding cellular dynamics in response to traumatic SCI and development of the treatment for traumatic SCI. Our goal is elucidation of mechanism for degeneration of axons after contusion injuries by establishing SCI model and chronic observation of injured axons in the living animals. Firstly we generated and observed acute SCI model by contusion injury. By using a multi-photon microscope, axons in dorsal cord were visualized approximately 140 micron in depth from the surface. Immediately after injury, minimal morphological change of spinal cord was observed. At 3 days after injury, spinal cord was swelling and the axons seem to be fragmented. At 7 days after injury, increased degradation of axons could be observed, although the image was blurred due to accumulation of the connective tissue. In the present study, we successfully observed axon degeneration after the contusion SCI in a living animal in vivo. Our final goal is to understand molecular mechanisms and cellular dynamics in response to traumatic SCIs in acute and chronic stage.

  1. Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung

    Science.gov (United States)

    Pavlova, Ina; Hume, Kelly R.; Yazinski, Stephanie A.; Flanders, James; Southard, Teresa L.; Weiss, Robert S.; Webb, Watt W.

    2012-03-01

    Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.

  2. Label-free imaging of rat spinal cords based on multiphoton microscopy

    Science.gov (United States)

    Liao, Chenxi; Wang, Zhenyu; Zhou, Linquan; Zhu, Xiaoqin; Liu, Wenge; Chen, Jianxin

    2016-10-01

    As an integral part of the central nervous system, the spinal cord is a communication cable between the body and the brain. It mainly contains neurons, glial cells, nerve fibers and fiber tracts. The recent development of the optical imaging technique allows high-resolution imaging of biological tissues with the great potential for non-invasively looking inside the body. In this work, we evaluate the imaging capacity of multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) for the cells and extracellular matrix in the spinal cord at molecular level. Rat spinal cord tissues were sectioned and imaged by MPM to demonstrate that MPM is able to show the microstructure including white matter, gray matter, ventral horns, dorsal horns, and axons based on the distinct intrinsic sources in each region of spinal cord. In the high-resolution and high-contrast MPM images, the cell profile can be clearly identified as dark shadows caused by nuclei and encircled by cytoplasm. The nerve fibers in white matter region emitted both SHG and TPEF signals. The multiphoton microscopic imaging technique proves to be a fast and effective tool for label-free imaging spinal cord tissues, based on endogenous signals in biological tissue. It has the potential to extend this optical technique to clinical study, where the rapid and damage-free imaging is needed.

  3. Insights on proximity effect and multiphoton induced luminescence from gold nanospheres in far field optical microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Borglin, Johan [Biomedical Photonics Group, Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 412 96 Gothenburg (Sweden); Department of Physics, University of Gothenburg, Kemivägen 10, 412 96 Gothenburg (Sweden); Guldbrand, Stina [Department of Physics, University of Gothenburg, Kemivägen 10, 412 96 Gothenburg (Sweden); Evenbratt, Hanne [Pharmaceutical Technology, Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemigården 4, 412 96 Gothenburg (Sweden); Kirejev, Vladimir; Ericson, Marica B., E-mail: marica.ericson@chem.gu.se [Biomedical Photonics Group, Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 412 96 Gothenburg (Sweden); Grönbeck, Henrik [Department of Applied Physics, Chalmers University of Technology, Kemivägen 9, 412 96 Gothenburg (Sweden)

    2015-12-07

    Gold nanoparticles can be visualized in far-field multiphoton laser-scanning microscopy (MPM) based on the phenomena of multiphoton induced luminescence (MIL). This is of interest for biomedical applications, e.g., for cancer diagnostics, as MPM allows for working in the near-infrared (NIR) optical window of tissue. It is well known that the aggregation of particles causes a redshift of the plasmon resonance, but its implications for MIL applying far-field MPM should be further exploited. Here, we explore MIL from 10 nm gold nanospheres that are chemically deposited on glass substrates in controlled coverage gradients using MPM operating in NIR range. The substrates enable studies of MIL as a function of inter-particle distance and clustering. It was shown that MIL was only detected from areas on the substrates where the particle spacing was less than one particle diameter, or where the particles have aggregated. The results are interpreted in the context that the underlying physical phenomenon of MIL is a sequential two-photon absorption process, where the first event is driven by the plasmon resonance. It is evident that gold nanospheres in this size range have to be closely spaced or clustered to exhibit detectable MIL using far-field MPM operating in the NIR region.

  4. Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging

    Science.gov (United States)

    Chen, Wei-Liang; Chou, Chen-Kuan; Lin, Ming-Gu; Chen, Yang-Fang; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Tsai, Tsung-Hua; Kim, Ki-Hean; Kim, Daekeun; So, Peter T. C.; Lin, Sung-Jan; Dong, Chen-Yuan

    2009-09-01

    Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.

  5. USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EGGS

    Science.gov (United States)

    Multiphoton laser scanning microscopy (MPLSM) is a promising tool to study the tissue distribution of environmental chemical contaminants during fish early life stages. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon that a...

  6. Monitoring changes of tumor microenvironment in colorectal submucosa using multiphoton microscopy.

    Science.gov (United States)

    Qiu, Jingting; Jiang, Weizhong; Yang, Yinghong; Feng, Changyin; Chen, Zhifen; Guan, Guoxian; Zhuo, Shuangmu; Chen, Jianxin

    2015-01-01

    Recently, targeting tumor microenvironment has become a novel approach for cancer therapy. Collagen is one of important components of tissue microenvironment, and has been considered as a new visible target for cancer therapy. In this work, multiphoton microscopy (MPM) was used to monitor the changes of collagen in tumor microenvironment during tumor progression. It was found that MPM facilitates imaging of tumor cells and collagen. MPM images in different tumor microenvironment during tumor progression shows obvious increase in cell number and collagen degration. In addition, the quantitative analysis of collagen content and orientation index in tumor microenvironment shows significant alteration during tumor progression. These results suggest that MPM has the ability to monitor the changes of collagen morphology in tumor microenvironment and quantify content and orientation index of collagen during tumor progression. Therefore this technique is a powerful imaging tool for the investigation of targeting tumor microenvironment for cancer therapy. © Wiley Periodicals, Inc.

  7. Pathological process investigation of Jadassohn-Pellizzari anetoderma based on multiphoton microscopy

    Science.gov (United States)

    Chen, Jianxin; Zhao, Jingjun; Yang, Yinghong; Zhuo, Shuangmu; Jiang, Xingshan; Tian, Wei; Ye, Xiaoyin; Lin, Lihang

    2009-08-01

    Multiphoton microscopy based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was firstly employed to investigate pathological process from the unaffected skin to the erythematous phase and to finally affected skin of Jadassohn-Pellizzari anetoderma. Our study showed that the normal elastic fibers in unaffected skin were almost completely absent in erthematous skin tissue, then replaced by a lot of elastic fibers with granular morphology in affected skin. The obvious change of collagen fibers and occurrence of inflammatory cell infiltration in erthematous tissue suggested that variation of these two components were also main pathogenesis of anetoderma except for deficiency of elastic fibers. Our results give a sight to noninvasively study the pathological process of skin disease and the other disease with tight relation to variation of collagen and elastic fibers in skin dermis and cell in skin epidermis.

  8. The first decade of using multiphoton microscopy for high-power kidney imaging.

    Science.gov (United States)

    Peti-Peterdi, János; Burford, James L; Hackl, Matthias J

    2012-01-15

    In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans.

  9. Differentiating fibroadenoma and ductal carcinoma in situ from normal breast tissue by multiphoton microscopy

    Science.gov (United States)

    Nie, Yuting; Wu, Yan; Lian, Yuane; Fu, Fangmeng; Wang, Chuan; Chen, Jianxin

    2014-09-01

    Fibroadenoma (FA) is the most common benign tumor of the female breast and several studies have reported that women with it have increased risk of breast cancer. While the ductal carcinoma in situ (DCIS) is a very early form of breast cancer. Thus, early detections of FA and DCIS are critical for improving breast tumor outcome and survival. In this paper, we use multiphoton microscopy (MPM) to obtain the high-contrast images of fresh, unfixed, unstained human breast specimens (normal breast tissue, FA and DCIS). Our results show that MPM has the ability to identify the characteristics of FA and DCIS including changes of duct architecture and collagen morphology. These results are consistent with the histological results. With the advancement of MPM, the technique has potential ability to serve as a real-time noninvasive imaging tool for early detection of breast tumor.

  10. Spectral behavior of second harmonic signals from organic and non-organic materials in multiphoton microscopy

    Directory of Open Access Journals (Sweden)

    Tobias Ehmke

    2015-08-01

    Full Text Available Multimodal nonlinear microscopy allows imaging of highly ordered biological tissue due to spectral separation of nonlinear signals. This requires certain knowledge about the spectral distribution of the different nonlinear signals. In contrast to several publications we demonstrate a factor of 1 2 2 relating the full width at half maximum of a gaussian laser pulse spectrum to the corresponding second harmonic pulse spectrum in the spatial domain by using a simple theoretical model. Experiments on monopotassium phosphate crystals (KDP-crystals and on porcine corneal tissue support our theoretical predictions. Furthermore, no differences in spectral width were found for epi- and trans-detection of the second harmonic signal. Overall, these results may help to build an optimized multiphoton setup for spectral separation of nonlinear signals.

  11. Structural and dynamical aspects of skin studied by multiphoton excitation fluorescence microscopy-based methods

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Brewer, Jonathan R.; Bagatolli, Luis

    2013-01-01

    This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes......' parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe--2,7-bis-(2...... excised skin, including applications of fluctuation correlation spectroscopy on transdermal penetration of liposomes are presented and discussed. The data from the different studies reported reveal the intrinsic heterogeneity of skin and also prove these strategies to be powerful noninvasive tools...

  12. Monitoring the progression from intraductal carcinoma to invasive ductal carcinoma based on multiphoton microscopy

    Science.gov (United States)

    Wu, Yan; Fu, Fangmeng; Lian, Yuane; Nie, Yuting; Zhuo, Shuangmu; Wang, Chuan; Chen, Jianxin

    2015-09-01

    Intraductal carcinoma is a precancerous lesion of the breast and the immediate precursor of invasive ductal carcinoma. Multiphoton microscopy (MPM) was used to monitor the progression from intraductal carcinoma to invasive ductal carcinoma, which can improve early detection of precursor lesions and halt progression to invasive neoplastic disease. It was found that MPM has the capability to reveal the qualitative changes in features of cells, structure of basement membranes, and architecture of collagens during the development from intraductal carcinoma to invasive ductal carcinoma, as well as the quantitative alterations in nuclear area, circle length of basement membrane, and collagen density. Combined with intra-fiberoptic ductoscopy or transdermal biopsy needle, MPM has the potential to provide immediate histological diagnosis of tumor progression in the field of breast carcinoma.

  13. Coherent beam control through inhomogeneous media in multi-photon microscopy

    Science.gov (United States)

    Paudel, Hari Prasad

    Multi-photon fluorescence microscopy has become a primary tool for high-resolution deep tissue imaging because of its sensitivity to ballistic excitation photons in comparison to scattered excitation photons. The imaging depth of multi-photon microscopes in tissue imaging is limited primarily by background fluorescence that is generated by scattered light due to the random fluctuations in refractive index inside the media, and by reduced intensity in the ballistic focal volume due to aberrations within the tissue and at its interface. We built two multi-photon adaptive optics (AO) correction systems, one for combating scattering and aberration problems, and another for compensating interface aberrations. For scattering correction a MEMS segmented deformable mirror (SDM) was inserted at a plane conjugate to the objective back-pupil plane. The SDM can pre-compensate for light scattering by coherent combination of the scattered light to make an apparent focus even at a depths where negligible ballistic light remains (i.e. ballistic limit). This problem was approached by investigating the spatial and temporal focusing characteristics of a broad-band light source through strongly scattering media. A new model was developed for coherent focus enhancement through or inside the strongly media based on the initial speckle contrast. A layer of fluorescent beads under a mouse skull was imaged using an iterative coherent beam control method in the prototype two-photon microscope to demonstrate the technique. We also adapted an AO correction system to an existing in three-photon microscope in a collaborator lab at Cornell University. In the second AO correction approach a continuous deformable mirror (CDM) is placed at a plane conjugate to the plane of an interface aberration. We demonstrated that this "Conjugate AO" technique yields a large field-of-view (FOV) advantage in comparison to Pupil AO. Further, we showed that the extended FOV in conjugate AO is maintained over a

  14. 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.

  15. Tunable fibre-coupled multiphoton microscopy with a negative curvature fibre.

    Science.gov (United States)

    Sherlock, Ben; Yu, Fei; Stone, Jim; Warren, Sean; Paterson, Carl; Neil, Mark A A; French, Paul M W; Knight, Jonathan; Dunsby, Chris

    2016-07-01

    Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core and cladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort optical pulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Its attenuation was measured to be <0.3 dB m(-1) over the range 600-850 nm and the GVD was -180 ± 70 fs(2)  m(-1) at 800 nm. Using an average fibre output power of ∼20 mW and pulse repetition rate of 80 MHz, the NCF enabled pulses with a duration of <200 fs to be transmitted through a length of 1.5 m of fibre over a tuning range of 180 nm without the need for dispersion compensation. In a 4 m fibre, temporal and spectral pulse widths were maintained to within 10% of low power values up to the maximum fibre output power achievable with the laser system used of 278 mW at 700 nm, 808 mW at 800 nm and 420 mW at 860 nm. When coupled to a multiphoton microscope, it enabled imaging of ex vivo tissue using excitation wavelengths from 740 nm to 860 nm without any need for adjustments to the set-up.

  16. Multimodal microscopy and the stepwise multi-photon activation fluorescence of melanin

    Science.gov (United States)

    Lai, Zhenhua

    The author's work is divided into three aspects: multimodal microscopy, stepwise multi-photon activation fluorescence (SMPAF) of melanin, and customized-profile lenses (CPL) for on-axis laser scanners, which will be introduced respectively. A multimodal microscope provides the ability to image samples with multiple modalities on the same stage, which incorporates the benefits of all modalities. The multimodal microscopes developed in this dissertation are the Keck 3D fusion multimodal microscope 2.0 (3DFM 2.0), upgraded from the old 3DFM with improved performance and flexibility, and the multimodal microscope for targeting small particles (the "Target" system). The control systems developed for both microscopes are low-cost and easy-to-build, with all components off-the-shelf. The control system have not only significantly decreased the complexity and size of the microscope, but also increased the pixel resolution and flexibility. The SMPAF of melanin, activated by a continuous-wave (CW) mode near-infrared (NIR) laser, has potential applications for a low-cost and reliable method of detecting melanin. The photophysics of melanin SMPAF has been studied by theoretical analysis of the excitation process and investigation of the spectra, activation threshold, and photon number absorption of melanin SMPAF. SMPAF images of melanin in mouse hair and skin, mouse melanoma, and human black and white hairs are compared with images taken by conventional multi-photon fluorescence microscopy (MPFM) and confocal reflectance microscopy (CRM). SMPAF images significantly increase specificity and demonstrate the potential to increase sensitivity for melanin detection compared to MPFM images and CRM images. Employing melanin SMPAF imaging to detect melanin inside human skin in vivo has been demonstrated, which proves the effectiveness of melanin detection using SMPAF for medical purposes. Selective melanin ablation with micrometer resolution has been presented using the Target system

  17. Super-resolved multimodal multiphoton microscopy with spatial frequency-modulated imaging

    CERN Document Server

    Field, Jeffrey J; Domingue, Scott R; Motz, Alyssa M Allende; DeLuca, Keith F; DeLuca, Jennifer G; Kuciauskas, Darius; Levi, Dean H; Squier, Jeff A; Bartels, Randy A

    2015-01-01

    Super-resolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all super-resolution imaging techniques reported to date rely on real energy states of probe molecules to circumvent the diffraction limit, preventing super-resolved imaging of contrast mechanisms that occur via virtual energy states such as harmonic generation (HG). Here we report a super-resolution technique based on SPatIal Frequency modulated Imaging (SPIFI) that permits super-resolved nonlinear microscopy with any contrast mechanism, and with single-pixel detection. We show multimodal super-resolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2$\\eta$ below the diffraction limit, where $\\eta$ is the highest power of the nonlinear intensity response. MP-SPIFI has the potential to not only pro...

  18. Multiphoton microscopy for skin wound healing study in terms of cellular metabolism and collagen regeneration

    Science.gov (United States)

    Deka, Gitanjal; Okano, Kazunori; Wu, Wei-Wen; Kao, Fu-Jen

    2014-02-01

    Multiphoton microscopy was employed to study normal skin wound healing in live rats noninvasively. Wound healing is a process involving series of biochemical events. This study evaluates the regeneration of collagen and change in cellular metabolic activity during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), respectively. In eukaryotic cells ATP is the molecule that holds the energy for cellular functioning. Whereas NADH is an electron donor in the metabolic pathways, required to generate ATP. Fluorescence lifetime of NADH free to protein bound ratio was evaluated to determine the relative metabolic activity. The FLIM data were acquired by a TCSPC system using SPCM software and analyzed by SPCImage software. Additionally, polarization resolved SHG signals were also collected to observe the changes in optical birefringence and hence the anisotropy of regenerated collagens from rat wound biopsy samples. Mat lab programming was used to process the data to construct the anisotropy images. Results indicated that, cells involved in healing had higher metabolic activity during the first week of healing, which decreases gradually and become equivalent to normal skin upon healing completes. A net degradation of collagen during the inflammatory phase and net regeneration starting from day 5 were observed in terms of SHG signal intensity change. Polarization resolved SHG imaging of the wound biopsy sample indicates higher value of anisotropy in proliferative phase, from day 4th to 8th, of wound formation; however the anisotropy decreases upon healing.

  19. Comparing in vivo pump-probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions

    Science.gov (United States)

    Wilson, Jesse W.; Degan, Simone; Gainey, Christina S.; Mitropoulos, Tanya; Simpson, Mary Jane; Zhang, Jennifer Y.; Warren, Warren S.

    2015-05-01

    We demonstrate a multimodal approach that combines a pump-probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump-probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump-probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100 μm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents.

  20. Multiphoton microscopy for imaging infectious keratitis: demonstration of the pattern of microbial spread in an experimental model

    Science.gov (United States)

    Sun, Yen; Lo, Wen; Wu, Ruei-Jhih; Lin, Sung-Jan; Lin, Wei-Chou; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this study is to assess the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging and monitoring the disease progress of infectious keratitis in an experimental model, and to investigate the possible correlation of tissue architecture with spreading patterns of pathogens in an experimental model. Porcine eyes are to be obtained from slaughter house and processed and placed in organ culture system. Fungal infections by common pathogens of infectious keratitis are to be induced in porcine cornea buttons. Multiphoton fluorescence and SHG microscopy will be used for imaging and for monitoring the progression and extension of tissue destruction and possibly the pattern of pathogen spreading. We found that SHG imaging is useful in identifying alterations to collagen architecture while autofluorescence microscopy can be used to visualize the fungi and cells within the stroma. In summary, multiphoton fluorescence and second harmonic generation microscopy can non-invasively demonstrate and monitor tissue destruction associated with infectious keratitis. The pattern of pathogen spreading and its correlation with the tissue architecture can also be shown, which can be useful for future studies of the tissue-microbial interactions for infectious keratitis.

  1. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    Science.gov (United States)

    Skala, Melissa Caroline

    2007-12-01

    Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in

  2. Preclinical study of using multiphoton microscopy to diagnose liver cancer and differentiate benign and malignant liver lesions

    Science.gov (United States)

    Yan, Jun; Zhuo, Shuangmu; Chen, Gang; Wu, Xiufeng; Zhou, Dong; Xie, Shusen; Jiang, Jiahao; Ying, Mingang; Jia, Fan; Chen, Jianxin; Zhou, Jian

    2012-02-01

    Recently, the miniaturized multiphoton microscopy (MPM) and multiphoton probe allow the clinical use of multiphoton endoscopy for diagnosing cancer via ``optical biopsy''. The purpose of this study was to establish MPM optical diagnostic features for liver cancer and evaluate the sensitivity, specificity, and accuracy of MPM optical diagnosis. Firstly, we performed a pilot study to establish the MPM diagnostic features by investigating 60 surgical specimens, and found that high-resolution MPM images clearly demonstrated apparent differences between benign and malignant liver lesions in terms of their tissue architecture and cell morphology. Cancer cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, were identified by MPM images, which were comparable to hematoxylin-eosin staining images. Secondly, we performed a blinded study to evaluate the sensitivity, specificity, and accuracy of MPM optical diagnosis by investigating another 164 specimens, and found that the sensitivity, specificity, and accuracy of MPM diagnosis was 96.32%, 96.43%, and 96.34%, respectively. In conclusion, it is feasible to use MPM to diagnose liver cancer and differentiate benign and malignant liver lesions. This preclinical study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive ``optical biopsy'' for liver lesions in the near future.

  3. In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

    Science.gov (United States)

    Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-05-01

    Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

  4. Non-invasive short-term assessment of retinoids effects on human skin in vivo using multiphoton microscopy.

    Science.gov (United States)

    Tancrède-Bohin, E; Baldeweck, T; Decencière, E; Brizion, S; Victorin, S; Parent, N; Faugere, J; Souverain, L; Bagot, M; Pena, A-M

    2015-04-01

    The occlusive patch test developed for assessing topical retinoids activity in human skin has been extended as a short-term screening protocol for anti-ageing agents. In this model, biopsies are performed at the end of the occlusion period for morphological and immuno-histochemistry analysis. Multiphoton microscopy is a recent non-invasive imaging technique that combined with image processing tools allows the in vivo quantification of human skin modifications. To validate with gold standards of anti-ageing that are retinoids, the relevance of multiphoton microscopy for kinetic and quantitative assessment in this model. Twenty women, aged 50-65 years, were enrolled. Retinol 0.3% (RO) and Retinoic acid 0.025% (RA) were applied to the dorsal photo-damaged side of their forearm under occlusive patches for 12 days. A patch alone was applied to a third area as control. Evaluation was performed at day D0, D12 (end of treatment), D18 and D32 using multiphoton microscopy. Epidermal thickness, normalized area of the dermal-epidermal junction (DEJ) and melanin density were estimated using 3D image processing tools. Main significant results are: Epidermal thickening at D12, D18 and D32 with RO and at D12, D18 with RA vs. baseline and vs. Increased DEJ undulation at D32 with RO and at D12 with RA vs. baseline and vs. Decreased melanin content with RO (at D12 and D18 vs. baseline and at D32 vs. baseline and vs. control) and with RA (at D12 vs. baseline). This study shows that multiphoton microscopy associated to specific 3D image processing tools allows cutaneous effects induced by topical retinoids in this in vivo model to be non-invasively detected, quantified and followed over time. This innovative approach could be applied to the evaluation of other active compounds. © 2014 European Academy of Dermatology and Venereology.

  5. Identification of tumor cells infiltrating into connective tissue in esophageal cancer by multiphoton microscopy

    Science.gov (United States)

    Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

    2016-10-01

    Esophageal cancer is one of the most common malignancies of the gastrointestinal cancers and carries poorer prognosis than other gastrointestinal cancers. In general practice, the depth of tumor infiltration in esophageal wall is crucial to establishing appropriate treatment plan which is established by detecting the tumor infiltration depth. Connective tissue is one of the main structures that form the esophageal wall. So, identification of tumor cells infiltrating into connective tissue is helping for detecting the tumor infiltration depth. Our aim is to evaluate whether multiphoton microscopy (MPM) can be used to detect tumor cells infiltrating into connective tissue in the esophageal cancer. MPM is well-suited for real-time detecting morphologic and cellular changes in fresh tissues since many endogenous fluorophores of fresh tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). In this work, microstructure of tumor cells and connective tissue are first studied. Then, morphological changes of collagen fibers after the infiltration of tumor cells are shown. These results show that MPM has the ability to detect tumor cells infiltrating into connective tissue in the esophageal cancer. In the future, MPM may be a promising imaging technique for detecting tumor cells in esophageal cancer.

  6. Imaging Mitochondrial Organization in Living Primate Oocytes and Embryos using Multiphoton Microscopy

    Science.gov (United States)

    Squirrell, J.M.; Schramm, R.D.; Paprocki, A.M.; Wokosin, D.L.; Bavister, B.D.

    2016-01-01

    We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction. PMID:12807671

  7. Multimodal imaging of vocal fold scarring in a rabbit model by multiphoton microscopy

    Science.gov (United States)

    Kazarine, Alexei; Bouhabel, Sarah; Douillette, Annie H.; Kost, Karen; Li-Jessen, Nicole Y. K.; Mongeau, Luc; Wiseman, Paul W.

    2017-02-01

    Vocal fold scarring as a result of injury or disease can lead to voice disorders which can significantly affect the quality of life. During the scarring process, the normally elastic tissue of the vocal fold lamina propria is replaced by a much stiffer collagen-based fibrotic tissue, which impacts the fold's ability to vibrate. Surgical removal of this tissue is often ineffective and can result in further scarring. Injectable biomaterials, a form of tissue engineering, have been proposed as a potential solution to reduce existing scars or prevent scarring altogether. In order to properly evaluate the effectiveness of these new materials, multiphoton microscopy emerges as an effective tool due to its intrinsic multiple label free contrast mechanisms that highlight extracellular matrix elements. In this study, we evaluate the spatial distribution of collagen and elastin fibers in a rabbit model using second harmonic generation (SHG), third harmonic generation (THG) and two photon autofluorescence (TPAF) applied to unlabeled tissue sections. In comparison to traditional methods that rely on histological staining or immunohistochemistry, SHG, THG and TPAF provide a more reliable detection of these native proteins. The evaluation of collagen levels allows us to follow the extent of scarring, while the presence of elastin fibers is thought to be indicative of the level of healing of the injured fold. Using these imaging modalities, we characterize the outcome of injectable biomaterial treatments in order to direct future treatments for tissue engineering.

  8. Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy.

    Science.gov (United States)

    Cui, Jason Z; Tehrani, Arash Y; Jett, Kimberly A; Bernatchez, Pascal; van Breemen, Cornelis; Esfandiarei, Mitra

    2014-09-01

    In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.

  9. Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

    Science.gov (United States)

    Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

    2014-09-01

    Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

  10. Identifying three different architectural subtypes of mammary ductal carcinoma in situ using multiphoton microscopy

    Science.gov (United States)

    Wu, Yan; Fu, Fangmeng; Lian, Yuane; Nie, Yuting; Zhuo, shuangmu; Wang, Chuan; Chen, Jianxin

    2015-10-01

    Ductal carcinoma in situ (DCIS) is often considered as the precursor of invasive breast cancer, and the risk of DCIS progression to IBC has been estimated based on the evaluation of pathological features, among which the architectural subtype is the most common one. In this study, multiphoton microscopy (MPM) is applied to identify three different architectural subtypes of DCIS (solid, cribriform and comedo). It is found that MPM has the capability to visualize the proliferating pattern of tumor cells, the presence of intraluminal necrosis and the morphology of basement membrane, which are all taken into account in subtyping DCIS. In addition, MPM also can be used to quantify the cellular metabolism, for quantitatively identifying tumor staging during tumor progression. This result highlights the potential of MPM as an advanced technique to assess the pathological characters of the breast tumor in real-time and reflect the degree of tumor progression in vivo, by integrating into the intra-fiberoptic ductoscopy or transdermal biopsy needle.

  11. Non-descanned multifocal multiphoton microscopy with a multianode photomultiplier tube

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Jae Won; Yew, Elijah Y. S. [Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA (United States); Kim, Daekeun [Department of Mechanical Engineering, Dankook University (Korea, Republic of); Subramanian, Jaichandar [Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA (United States); Nedivi, Elly [Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA (United States); Departments of Brain and Cognitive Sciences, and Biology, Massachusetts Institute of Technology, Cambridge, MA (United States); So, Peter T. C. [Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA (United States)

    2015-08-15

    Multifocal multiphoton microscopy (MMM) improves imaging speed over a point scanning approach by parallelizing the excitation process. Early versions of MMM relied on imaging detectors to record emission signals from multiple foci simultaneously. For many turbid biological specimens, the scattering of emission photons results in blurred images and degrades the signal-to-noise ratio (SNR). We have recently demonstrated that a multianode photomultiplier tube (MAPMT) placed in a descanned configuration can effectively collect scattered emission photons from each focus into their corresponding anodes significantly improving image SNR for highly scattering specimens. Unfortunately, a descanned MMM has a longer detection path resulting in substantial emission photon loss. Optical design constraints in a descanned geometry further results in significant optical aberrations especially for large field-of-view (FOV), high NA objectives. Here, we introduce a non-descanned MMM based on MAPMT that substantially overcomes most of these drawbacks. We show that we improve signal efficiency up to fourfold with limited image SNR degradation due to scattered emission photons. The excitation foci can also be spaced wider to cover the full FOV of the objective with minimal aberrations. The performance of this system is demonstrated by imaging interneuron morphological structures deep in the brains of living mice.

  12. Hindlimb heating increases vascular access of large molecules to murine tibial growth plates measured by in vivo multiphoton imaging.

    Science.gov (United States)

    Serrat, Maria A; Efaw, Morgan L; Williams, Rebecca M

    2014-02-15

    Advances in understanding the molecular regulation of longitudinal growth have led to development of novel drug therapies for growth plate disorders. Despite progress, a major unmet challenge is delivering therapeutic agents to avascular-cartilage plates. Dense extracellular matrix and lack of penetrating blood vessels create a semipermeable "barrier," which hinders molecular transport at the vascular-cartilage interface. To overcome this obstacle, we used a hindlimb heating model to manipulate bone circulation in 5-wk-old female mice (n = 22). Temperatures represented a physiological range of normal human knee joints. We used in vivo multiphoton microscopy to quantify temperature-enhanced delivery of large molecules into tibial growth plates. We tested the hypothesis that increasing hindlimb temperature from 22°C to 34°C increases vascular access of large systemic molecules, modeled using 10, 40, and 70 kDa dextrans that approximate sizes of physiological regulators. Vascular access was quantified by vessel diameter, velocity, and dextran leakage from subperichondrial plexus vessels and accumulation in growth plate cartilage. Growth plate entry of 10 kDa dextrans increased >150% at 34°C. Entry of 40 and 70 kDa dextrans increased plates, suggesting that temperature could be a noninvasive strategy for modulating delivery of therapeutics to impaired growth plates of children.

  13. Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy.

    Science.gov (United States)

    Raub, C B; Putnam, A J; Tromberg, B J; George, S C

    2010-12-01

    Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (∼200 mg ml⁻¹). Young's modulus (E) measurements from acellular collagen gels (range 0.5-12 kPa) exhibited a power-law concentration dependence (range 3-9 mg ml⁻¹) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5-27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ∼5-200 mg ml⁻¹). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R²=0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R²=0.83, exponents of -0.7 and -1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.

  14. Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

    Science.gov (United States)

    Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

    2017-02-24

    An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP](mi2004) zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

  15. Characterization of dermal structural assembly in normal and pathological connective tissues by intrinsic signal multiphoton optical microscopy

    Science.gov (United States)

    Lyubovitsky, Julia G.; Xu, Xiaoman; Sun, Chung-ho; Andersen, Bogi; Krasieva, Tatiana B.; Tromberg, Bruce J.

    2008-02-01

    Employing a reflectance multi-photon microscopy (MPM) technique, we developed novel method to quantitatively study the three-dimensional assembly of structural proteins within bulk of dermal ECMs. Using a structurally simplified model of skin with enzymatically dissected epidermis, we find that low resolution MPM clearly discriminates between normal and pathological dermis. High-resolution images revealed that the backscattered MPM signals are affected by the assembly of collagen fibrils and fibers within this system. Exposure of tissues to high concentrations of potentially denaturing chemicals also resulted in the reduction of SHG signals from structural proteins which coincided with the appearance of aggregated fluorescent structures.

  16. Potential of ultraviolet widefield imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Brewer, Jonathan R.; Bagatolli, Luis;

    2011-01-01

    Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) exc...

  17. The layered resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy

    Science.gov (United States)

    Zhuo, Shuangmu; Chen, Jianxin; Jiang, Xingshan; Xie, Shusen; Chen, Rong; Cao, Ning; Zou, Qilian; Xiong, Shuyuan

    2007-08-01

    The layered-resolved microstructure and spectroscopy of mouse oral mucosa are obtained using a combination of multiphoton imaging and spectral analysis with different excitation wavelengths. In the keratinizing layer, the keratinocytes microstructure can be characterized and the keratinizing thickness can be measured. The keratin fluorescence signal can be further characterized by emission maxima at 510 nm. In the epithelium, the cellular microstructure can be quantitatively visualized with depth and the epithelium thickness can be determined by multiphoton imaging excited at 730 nm. The study also shows that the epithelial spectra excited at 810 nm, showing a combination of NADH and FAD fluorescence, can be used for the estimation of the metabolic state in epithelium. Interestingly, a second-harmonic generation (SHG) signal from DNA was observed for the first time within the epithelial layer in backscattering geometry and provides the possibility of analyzing the chromatin structure. In the stroma, the combination of multiphoton imaging and spectral analysis excited at 850 nm in tandem can obtain quantitative information regarding the biomorphology and biochemistry of stroma. Specifically, the microstructure of collagen, minor salivary glands and elastic fibers, and the optical property of the stroma can be quantitatively displayed. Overall, these results suggest that the combination of multiphoton imaging and spectral analysis with different excitation wavelengths has the potential to provide important and comprehensive information for early diagnosis of oral cancer.

  18. The layered-resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhuo Shuangmu [Key Laboratory of Optoelectronic Science and Technology for Medicine, Fujian Normal University, Ministry of Education, Fuzhou 350007 (China); Chen Jianxin [Key Laboratory of Optoelectronic Science and Technology for Medicine, Fujian Normal University, Ministry of Education, Fuzhou 350007 (China); Jiang Xingshan [Key Laboratory of Optoelectronic Science and Technology for Medicine, Fujian Normal University, Ministry of Education, Fuzhou 350007 (China); Xie Shusen [Key Laboratory of Optoelectronic Science and Technology for Medicine, Fujian Normal University, Ministry of Education, Fuzhou 350007 (China); Chen Rong [Key Laboratory of Optoelectronic Science and Technology for Medicine, Fujian Normal University, Ministry of Education, Fuzhou 350007 (China); Cao Ning [Fujian Medical University, Fuzhou 350004 (China); Zou Qilian [Fujian Medical University, Fuzhou 350004 (China); Xiong Shuyuan [Fujian Medical University, Fuzhou 350004 (China)

    2007-08-21

    The layered-resolved microstructure and spectroscopy of mouse oral mucosa are obtained using a combination of multiphoton imaging and spectral analysis with different excitation wavelengths. In the keratinizing layer, the keratinocytes microstructure can be characterized and the keratinizing thickness can be measured. The keratin fluorescence signal can be further characterized by emission maxima at 510 nm. In the epithelium, the cellular microstructure can be quantitatively visualized with depth and the epithelium thickness can be determined by multiphoton imaging excited at 730 nm. The study also shows that the epithelial spectra excited at 810 nm, showing a combination of NADH and FAD fluorescence, can be used for the estimation of the metabolic state in epithelium. Interestingly, a second-harmonic generation (SHG) signal from DNA was observed for the first time within the epithelial layer in backscattering geometry and provides the possibility of analyzing the chromatin structure. In the stroma, the combination of multiphoton imaging and spectral analysis excited at 850 nm in tandem can obtain quantitative information regarding the biomorphology and biochemistry of stroma. Specifically, the microstructure of collagen, minor salivary glands and elastic fibers, and the optical property of the stroma can be quantitatively displayed. Overall, these results suggest that the combination of multiphoton imaging and spectral analysis with different excitation wavelengths has the potential to provide important and comprehensive information for early diagnosis of oral cancer.

  19. Snapshot coherence-gated direct wavefront sensing for multi-photon microscopy

    NARCIS (Netherlands)

    Van Werkhoven, T.I.M.; Antonello, J.; Truong, H.H.; Verhaegen, M.; Gerritsen, H.C.; Keller, C.U.

    2014-01-01

    Deep imaging in turbid media such as biological tissue is challenging due to scattering and optical aberrations. Adaptive optics has the potential to compensate the tissue aberrations. We present a wavefront sensing scheme for multi-photon scanning microscopes using the pulsed, near-infrared light r

  20. Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

    Science.gov (United States)

    Pena, Ana-Maria; Fagot, Dominique; Olive, Christian; Michelet, Jean-François; Galey, Jean-Baptiste; Leroy, Frédéric; Beaurepaire, Emmanuel; Martin, Jean-Louis; Colonna, Anne; Schanne-Klein, Marie-Claire

    2010-09-01

    Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

  1. Usefulness of Intravital Multiphoton Microscopy in Visualizing Study of Mouse Cochlea and Volume Changes in the Scala Media

    Directory of Open Access Journals (Sweden)

    Hyun Mi Ju

    2017-07-01

    Full Text Available Conventional microscopy has limitations in viewing the cochlear microstructures due to three-dimensional spiral structure and the overlying bone. But these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM. By using near-infrared lasers for multiphoton excitation, intravital MPM can detect endogenous fluorescence and second harmonic generation of tissues. In this study, we used intravital MPM to visualize various cochlear microstructures without any staining and non-invasively analyze the volume changes of the scala media (SM without removing the overlying cochlear bone. The intravital MPM images revealed various tissue types, ranging from thin membranes to dense bone, as well as the spiral ganglion beneath the cochlear bone. The two-dimensional, cross-sectional, and serial z-stack intravital MPM images also revealed the spatial dilation of the SM in the temporal bone of pendrin-deficient mice. These findings suggest that intravital MPM might serve as a new method for obtaining microanatomical information regarding the cochlea, similar to standard histopathological analyses in the animal study for the cochlea. Given the capability of intravital MPM for detecting an increase in the volume of the SM in pendrin-deficient mice, it might be a promising new tool for assessing the pathophysiology of hearing loss in the future.

  2. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    Science.gov (United States)

    Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

    2014-07-01

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

  3. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    Energy Technology Data Exchange (ETDEWEB)

    Darvin, M E; Richter, H; Zhu, Y J; Meinke, M C; Knorr, F; Lademann, J [Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Charité - Universitätsmedizin Berlin (Germany); Gonchukov, S A [National Research Nuclear University ' ' MEPhI' ' (Russian Federation); Koenig, K [JenLab GmbH, Schillerstr. 1, 07745 Jena (Germany)

    2014-07-31

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)

  4. Non-invasive discrimination between pancreatic islets and exocrine cells using multiphoton microscopy

    Science.gov (United States)

    Wu, Binlin; Li, Ge; Hao, Mingming; Mukherjee, Sushmita

    2015-03-01

    In this study, we propose a non-invasive method to distinguish pancreatic islet cells from exocrine cell clusters using multiphoton (MP) imaging. We demonstrate the principle of distinguishing them based on autofluorescence. The results show that MP imaging has a potential to distinguish pancreatic islets from exocrine cells. This ability to distinguish the two cell types could have many applications, such as the examination of fresh pancreatic biopsies when staining is not possible or desirable.

  5. Selection of proper objective lens for the higher-order multiphoton microscopy at the 1700-nm window

    Science.gov (United States)

    Wen, Wenhui; Qiu, Ping

    2017-02-01

    The 1700-nm window has emerged as a promising excitation window for multiphoton microscopy (MPM). On one hand, the combined low tissue absorption and scattering make this window well suited for deep-tissue MPM; on the other hand, the long excitation wavelength makes higher-order MPM in biological tissues feasible, e.g., recently 4-photon fluorescence MPM in mouse brain has been demonstrated. Objective lens is a key optical component in the entire MPM setup. Multiphoton signal levels are largely dependent on the transmittance of objective lens. Here we demonstrate experimental results of transmittance measurement of two water immersion objective lenses commonly used for MPM at the 1700-nm window, covering both the excitation and the signal window. Our target application is MPM of even higher order excited at this window, i.e., 4th harmonic generation (FHG) imaging and 5-photon fluorescence generation. Our results show that, although the customized objective lens offers higher transmittance at the excitation window, it suffers from dramatically degraded transmittance at the signal window, compared with the non-customized objective lens. These results will offer guidelines for selection of proper objective lens for higher-order MPM at the 1700-nm window.

  6. Optical characters and texture maps of skin and the aging mechanism by use of multiphoton microscopy and optical coherence tomography

    Science.gov (United States)

    Wu, Shulian; Li, Hui; Zhang, Xiaoman; Huang, Yudian; Xu, Xiaohui

    2012-03-01

    Cutaneous aging is a complicated biological process affecting different constituents of skin, which can be divided into two types: the chronological aging and the photo-aging. The two cutaneous aging processes often co-exist accompanying with each other. The effects are often overlapped including changes in epithelium and dermis. The degeneration of collagen is a major factor in dermal alteration with aging. In this study, multiphoton microscopy (MPM) with its high resolution imaging and optical coherence tomography (OCT) with its depth resolved imaging were used to study the anti-aging dermatology in vivo. It was attempted to make the optical parameter and texture feature to evaluate the process of aging skin using mathematical image processing. The links among optical parameter, spectrum and texture feature in collagen with aging process were established to uncover mechanism of aging skin.

  7. In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhuo, Shuangmu, E-mail: shuangmuzhuo@gmail.com, E-mail: hanry-yu@nuhs.edu.sg [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Laser and Optoelectronics Technology, Fujian Normal University, Fuzhou 350007 (China); Yan, Jie [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, MD 11 #04-01A, 117599 Singapore (Singapore); Kang, Yuzhan [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Xu, Shuoyu [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Peng, Qiwen [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Mechanobiology Institute, 5A Engineering Drive 1, T-Lab #05-01, 117411 Singapore (Singapore); and others

    2014-07-14

    Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

  8. Non-invasive in vivo characterization of skin wound healing using label-free multiphoton microscopy (Conference Presentation)

    Science.gov (United States)

    Jones, Jake D.; Majid, Fariah; Ramser, Hallie; Quinn, Kyle P.

    2017-02-01

    Non-healing ulcerative wounds, such as diabetic foot ulcers, are challenging to diagnose and treat due to their numerous possible etiologies and the variable efficacy of advanced wound care products. Thus, there is a critical need to develop new quantitative biomarkers and diagnostic technologies that are sensitive to wound status in order to guide care. The objective of this study was to evaluate the utility of label-free multiphoton microscopy for characterizing wound healing dynamics in vivo and identifying potential differences in diabetic wounds. We isolated and measured an optical redox ratio of FAD/(NADH+FAD) autofluorescence to provide three-dimensional maps of local cellular metabolism. Using a mouse model of wound healing, in vivo imaging at the wound edge identified a significant decrease in the optical redox ratio of the epidermis (p≤0.0103) between Days 3 through 14 compared to Day 1. This decrease in redox ratio coincided with a decrease in NADH fluorescence lifetime and thickening of the epithelium, collectively suggesting a sensitivity to keratinocyte hyperproliferation. In contrast to normal wounds, we have found that keratinocytes from diabetic wounds remain in a proliferative state at later time points with a lower redox ratio at the wound edge. Microstructural organization and composition was also measured from second harmonic generation imaging of collagen and revealed differences between diabetic and non-diabetic wounds. Our work demonstrates label-free multiphoton microscopy offers potential to provide non-invasive structural and functional biomarkers associated with different stages of skin wound healing, which may be used to detect delayed healing and guide treatment.

  9. Smart microscope: an adaptive optics learning system for aberration correction in multiphoton confocal microscopy.

    Science.gov (United States)

    Albert, O; Sherman, L; Mourou, G; Norris, T B; Vdovin, G

    2000-01-01

    Off-axis aberrations in a beam-scanning multiphoton confocal microscope are corrected with a deformable mirror. The optimal mirror shape for each pixel is determined by a genetic learning algorithm, in which the second-harmonic or two-photon fluorescence signal from a reference sample is maximized. The speed of the convergence is improved by use of a Zernike polynomial basis for the deformable mirror shape. This adaptive optical correction scheme is implemented in an all-reflective system by use of extremely short (10-fs) optical pulses, and it is shown that the scanning area of an f:1 off-axis parabola can be increased by nine times with this technique.

  10. In vivo measurements of cutaneous melanin across spatial scales: using multiphoton microscopy and spatial frequency domain spectroscopy.

    Science.gov (United States)

    Saager, Rolf B; Balu, Mihaela; Crosignani, Viera; Sharif, Ata; Durkin, Anthony J; Kelly, Kristen M; Tromberg, Bruce J

    2015-06-01

    The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ~30-65 μm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R² = 0.8895). SFDS melanin distribution thickness is correlated to MPM values (R² = 0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types.

  11. Correlating intravital multi-photon microscopy to 3D electron microscopy of invading tumor cells using anatomical reference points.

    Directory of Open Access Journals (Sweden)

    Matthia A Karreman

    Full Text Available Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.

  12. Modulation of the pupil function of microscope objective lens for multifocal multi-photon microscopy using a spatial light modulator

    Science.gov (United States)

    Matsumoto, Naoya; Okazaki, Shigetoshi; Takamoto, Hisayoshi; Inoue, Takashi; Terakawa, Susumu

    2014-02-01

    We propose a method for high precision modulation of the pupil function of a microscope objective lens to improve the performance of multifocal multi-photon microscopy (MMM). To modulate the pupil function, we adopt a spatial light modulator (SLM) and place it at the conjugate position of the objective lens. The SLM can generate an arbitrary number of spots to excite the multiple fluorescence spots (MFS) at the desired positions and intensities by applying an appropriate computer-generated hologram (CGH). This flexibility allows us to control the MFS according to the photobleaching level of a fluorescent protein and phototoxicity of a specimen. However, when a large number of excitation spots are generated, the intensity distribution of the MFS is significantly different from the one originally designed due to misalignment of the optical setup and characteristics of the SLM. As a result, the image of a specimen obtained using laser scanning for the MFS has block noise segments because the SLM could not generate a uniform MFS. To improve the intensity distribution of the MFS, we adaptively redesigned the CGH based on the observed MFS. We experimentally demonstrate an improvement in the uniformity of a 10 × 10 MFS grid using a dye solution. The simplicity of the proposed method will allow it to be applied for calibration of MMM before observing living tissue. After the MMM calibration, we performed laser scanning with two-photon excitation to observe a real specimen without detecting block noise segments.

  13. Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease

    Science.gov (United States)

    Chen, Jianxin; Zhuo, Shuangmu; Jiang, Xingshan; Zhu, Xiaoqin; Zheng, Liqin; Xie, Shusen; Lin, Bifang; Zeng, Haishan

    2011-05-01

    Multiphoton microscopy was used to study the extracellular matrix of keloid at the molecular level without tissue fixation and staining. Direct imaging of collagen and elastin was achieved by second harmonic generation and two-photon excited fluorescence, respectively. The morphology and quantity of collagen and elastin in keloid were characterized and quantitatively analyzed in comparison to normal skin. The study demonstrated that in keloid, collagen content increased in both the upper dermis and the deep dermis, while elastin mostly showed up in the deep dermis and its quantity is higher compared to normal skin. This suggests the possibility that abnormal fibroblasts synthesized an excessive amount of collagen and elastin at the beginning of keloid formation, corresponding to the observed deep dermis, while after a certain time point, the abnormal fibroblast produced mostly collagen, corresponding to the observed upper dermis. The morphology of collagen and elastin in keloid was disrupted and presented different variations. In the deep dermis, elastic fibers showed node structure, while collagen showed obviously regular gaps between adjacent bundles. In the upper dermis, collagen bundles aligned in a preferred direction, while elastin showed as sparse irregular granules. This new molecular information provided fresh insight about the development process of keloid.

  14. Identification of the boundary between normal breast tissue and invasive ductal carcinoma during breast-conserving surgery using multiphoton microscopy

    Science.gov (United States)

    Deng, Tongxin; Nie, Yuting; Lian, Yuane; Wu, Yan; Fu, Fangmeng; Wang, Chuan; Zhuo, Shuangmu; Chen, Jianxin

    2014-11-01

    Breast-conserving surgery has become an important way of surgical treatment for breast cancer worldwide nowadays. Multiphoton microscopy (MPM) has the ability to noninvasively visualize tissue architectures at the cellular level using intrinsic fluorescent molecules in biological tissues without the need for fluorescent dye. In this study, MPM is used to image the microstructures of terminal duct lobular unit (TDLU), invasive ductal carcinoma and the boundary region between normal and cancerous breast tissues. Our study demonstrates that MPM has the ability to not only reveal the morphological changes of the cuboidal epithelium, basement membrane and interlobular stroma but also identify the boundary between normal breast tissue and invasive ductal carcinoma, which correspond well to the Hematoxylin and Eosin (H and E) images. Predictably, MPM can monitor surgical margins in real time and provide considerable accuracy for resection of breast cancerous tissues intraoperatively. With the development of miniature, real-time MPM imaging technology, MPM should have great application prospects during breast-conserving surgery.

  15. Multi-photon microscopy with a low-cost and highly efficient Cr:LiCAF laser

    Science.gov (United States)

    Sakadić, Sava; Demirbas, Umit; Mempel, Thorsten R.; Moore, Anna; Ruvinskaya, Svetlana; Boas, David A.; Sennaroglu, Alphan; Kartner, Franz X.; Fujimoto, James G.

    2009-01-01

    Multi-photon microscopy (MPM) is a powerful tool for biomedical imaging, enabling molecular contrast and integrated structural and functional imaging on the cellular and subcellular level. However, the cost and complexity of femtosecond laser sources that are required in MPM are significant hurdles to widespread adoption of this important imaging modality. In this work, we describe femtosecond diode pumped Cr:LiCAF laser technology as a low cost alternative to femtosecond Ti:Sapphire lasers for MPM. Using single mode pump diodes which cost only $150 each, a diode pumped Cr:LiCAF laser generates ~70-fs duration, 1.8-nJ pulses at ~800 nm wavelengths, with a repetition rate of 100 MHz and average output power of 180 mW. Representative examples of MPM imaging in neuroscience, immunology, endocrinology and cancer research using Cr:LiCAF laser technology are presented. These studies demonstrate the potential of this laser source for use in a broad range of MPM applications. PMID:19065223

  16. Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

    Science.gov (United States)

    Bancelin, Stéphane; Lynch, Barbara; Bonod-Bidaud, Christelle; Ducourthial, Guillaume; Psilodimitrakopoulos, Sotiris; Dokládal, Petr; Allain, Jean-Marc; Schanne-Klein, Marie-Claire; Ruggiero, Florence

    2015-12-01

    Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues.

  17. Delivery of cyclodextrin polymers to bacterial biofilms - An exploratory study using rhodamine labelled cyclodextrins and multiphoton microscopy.

    Science.gov (United States)

    Thomsen, Hanna; Benkovics, Gábor; Fenyvesi, Éva; Farewell, Anne; Malanga, Milo; Ericson, Marica B

    2017-10-15

    Cyclodextrin (CD) polymers are interesting nanoparticulate systems for pharmaceutical delivery; however, knowledge regarding their applications towards delivery into complex microbial biofilm structures is so far limited. The challenge is to demonstrate penetration and transport through the biofilm and its exopolysaccharide matrix. The ideal functionalization for penetration into mature biofilms is unexplored. In this paper, we present a novel set of rhodamine labelled βCD-polymers, with different charge moieties, i.e., neutral, anionic, and cationic, and explore their potential delivery into mature Staphylococcus epidermidis biofilms using multiphoton laser scanning microscopy (MPM). The S. epidermidis biofilms, being a medically relevant model organism, were stained with SYTO9. By using MPM, three-dimensional imaging and spectral investigation of the distribution of the βCD-polymers could be obtained. It was found that the cationic βCD-polymers showed significantly higher integration into the biofilms, compared to neutral and anionic functionalized βCDs. None of the carriers presented any inherent toxicity to the biofilms, meaning that the addition of rhodamine moiety does not affect the inertness of the delivery system. Taken together, this study demonstrates a novel approach by which delivery of fluorescently labelled CD nanoparticles to bacterial biofilms can be explored using MPM. Future studies should be undertaken investigating the potential in using cationic functionalization of CD based delivery systems for targeting anti-microbial effects in biofilms. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. In-vivo multiphoton microscopy (MPM) of laser-induced optical breakdown (LIOB) in human skin (Conference Presentation)

    Science.gov (United States)

    Balu, Mihaela; Lentsch, Griffin; Korta, Dorota; Konig, Karsten; Kelly, Kristen M.; Tromberg, Bruce J.; Zachary, Christopher B.

    2017-02-01

    We use a multiphoton microscopy (MPM)-based clinical microscope (MPTflex, JenLab, Germany) to describe changes in human skin following treatment with a fractional non-ablative laser (PicoWay, Candela). The treatment was based on a fractionated picosecond Nd:YAG laser (1064 and 532nm, 3mJ and 1.5mJ (no attenuation), respectively maximum energy/pulse, 100 microbeams/6mmx6mm). Improvements in skin appearance resulting from treatment with this laser have been noted but optimizing the efficacy depends on a thorough understanding of the specific skin response to treatment. MPM is a nonlinear laser scanning microscopy technique that features sub-cellular resolution and label-free molecular contrast. MPM contrast in skin is derived from second-harmonic generation of collagen and two-photon excited fluorescence of NADH/FAD+, elastin, keratin, melanin. In this pilot study, two areas on the arm of a volunteer (skin type II) were treated with the picoWay laser (1064nm, 3mJ; 532nm, 1.5mJ; 1pass). The skin response to treatment was imaged in-vivo at 8 time points over the following 4 weeks. MPM revealed micro-injuries present in epidermis. Damaged individual cells were distinguished after 3h and 24h from treatment with both wavelengths. Pigmented cells were particularly damaged in the process, suggesting that melanin is the main absorber and the primary target for laser induced optical breakdown. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. These results represent the groundwork for future longitudinal studies on expanded number of subjects to understand the response to treatment in different skin types at different laser parameters, critical factors in optimizing treatment outcomes.

  19. Label-free structural characterization of mitomycin C-modulated wound healing after photorefractive keratectomy by the use of multiphoton microscopy

    Science.gov (United States)

    Lo, Wen; Wang, Tsung-Jen; Chen, Wei-Liang; Hsueh, Chiu-Mei; Chen, Shean-Jen; Chen, Yang-Fang; Chou, Hsiu-Chu; Lin, Pi-Jung; Hu, Fung-Rong; Dong, Chen-Yuan

    2010-05-01

    We applied multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) microscopy to monitor corneal wound healing after photorefractive keratectomy (PRK). Our results show that keratocyte activation can be observed by an increase in its MAF, while SHG imaging of corneal stroma can show the depletion of Bowman's layer after PRK and the reticular collagen deposition in the wound healing stage. Furthermore, quantification of the keratocyte activation and collagen deposition in conjunction with immunohistochemistry and histological images demonstrate the effectiveness of mitomycin C (MMC) in suppressing myofibroblast proliferation and collagen regeneration in the post-PRK wound healing process.

  20. USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EARLY LIFE STAGES

    Science.gov (United States)

    Multiphoton laser scanning micrsocopy holds promise as a tool to study the tissue distribution of environmental chemical contaminants during fish early life stage development. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polyaromatic hydrocarbon that a...

  1. Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy

    Science.gov (United States)

    Prieto, Sandra P.; Greening, Gage J.; Lai, Keith K.; Muldoon, Timothy J.

    2016-03-01

    Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns. The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.

  2. Functional cardiac imaging by random access microscopy

    Directory of Open Access Journals (Sweden)

    Claudia eCrocini

    2014-10-01

    Full Text Available Advances in the development of voltage sensitive dyes and Ca2+ sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca2+ release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca2+ release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca2+ or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.

  3. A custom image-based analysis tool for quantifying elastin and collagen micro-architecture in the wall of the human aorta from multi-photon microscopy.

    Science.gov (United States)

    Koch, Ryan G; Tsamis, Alkiviadis; D'Amore, Antonio; Wagner, William R; Watkins, Simon C; Gleason, Thomas G; Vorp, David A

    2014-03-21

    The aorta possesses a micro-architecture that imparts and supports a high degree of compliance and mechanical strength. Alteration of the quantity and/or arrangement of the main load-bearing components of this micro-architecture--the elastin and collagen fibers--leads to mechanical, and hence functional, changes associated with aortic disease and aging. Therefore, in the future, the ability to rigorously characterize the wall fiber micro-architecture could provide insight into the complicated mechanisms of aortic wall remodeling in aging and disease. Elastin and collagen fibers can be observed using state-of-the-art multi-photon microscopy. Image-analysis algorithms have been effective at characterizing fibrous constructs using various microscopy modalities. The objective of this study was to develop a custom MATLAB-language automated image-based analysis tool to describe multiple parameters of elastin and collagen micro-architecture in human soft fibrous tissue samples using multi-photon microscopy images. Human aortic tissue samples were used to develop the code. The tool smooths, cleans and equalizes fiber intensities in the image before segmenting the fibers into a binary image. The binary image is cleaned and thinned to a fiber skeleton representation of the image. The developed software analyzes the fiber skeleton to obtain intersections, fiber orientation, concentration, porosity, diameter distribution, segment length and tortuosity. In the future, the developed custom image-based analysis tool can be used to describe the micro-architecture of aortic wall samples in a variety of conditions. While this work targeted the aorta, the software has the potential to describe the architecture of other fibrous materials, tube-like networks and connective tissues.

  4. Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 µm wavelength range.

    Directory of Open Access Journals (Sweden)

    Delphine Débarre

    Full Text Available Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0-1.2 µm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2- and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG imaging of development until hatching.

  5. Accessible microscopy workstation for students and scientists with mobility impairments.

    Science.gov (United States)

    Duerstock, Bradley S

    2006-01-01

    An integrated accessible microscopy workstation was designed and developed to allow persons with mobility impairments to control all aspects of light microscopy with minimal human assistance. This system, named AccessScope, is capable of performing brightfield and fluorescence microscopy, image analysis, and tissue morphometry requisite for undergraduate science courses to graduate-level research. An accessible microscope is necessary for students and scientists with mobility impairments to be able to use a microscope independently to better understand microscopical imaging concepts and cell biology. This knowledge is not always apparent by simply viewing a catalog of histological images. The ability to operate a microscope independently eliminates the need to hire an assistant or rely on a classmate and permits one to take practical laboratory examinations by oneself. Independent microscope handling is also crucial for graduate students and scientists with disabilities to perform scientific research. By making a personal computer as the user interface for controlling AccessScope functions, different upper limb mobility impairments could be accommodated by using various computer input devices and assistive technology software. Participants with a range of upper limb mobility impairments evaluated the prototype microscopy workstation. They were able to control all microscopy functions including loading different slides without assistance.

  6. Flexible digital signal processing architecture for narrowband and spread-spectrum lock-in detection in multiphoton microscopy and time-resolved spectroscopy.

    Science.gov (United States)

    Wilson, Jesse W; Park, Jong Kang; Warren, Warren S; Fischer, Martin C

    2015-03-01

    The lock-in amplifier is a critical component in many different types of experiments, because of its ability to reduce spurious or environmental noise components by restricting detection to a single frequency and phase. One example application is pump-probe microscopy, a multiphoton technique that leverages excited-state dynamics for imaging contrast. With this application in mind, we present here the design and implementation of a high-speed lock-in amplifier on the field-programmable gate array (FPGA) coprocessor of a data acquisition board. The most important advantage is the inherent ability to filter signals based on more complex modulation patterns. As an example, we use the flexibility of the FPGA approach to enable a novel pump-probe detection scheme based on spread-spectrum communications techniques.

  7. Multiphoton microscopy based cryo-imaging of inflated frozen human lung sections at -60°C in healthy and COPD lungs

    Science.gov (United States)

    Abraham, Thomas; Kayra, Damian; Zhang, Angela; Suzuki, Masaru; McDonough, John; Elliott, W. M.; Cooper, Joel D.; Hogg, James C.

    2013-02-01

    Lung is a complex gas exchanger with interfacial area (where the gas exchange takes place) is about the size of a tennis court. Respiratory function is linked to the biomechanical stability of the gas exchange or alveolar regions which directly depends on the spatial distributions of the extracellular matrix fibers such fibrillar collagens and elastin fibers. It is very important to visualize and quantify these fibers at their native and inflated conditions to have correct morphometric information on differences between control and diseased states. This can be only achieved in the ex vivo states by imaging directly frozen lung specimens inflated to total lung capacity. Multiphoton microscopy, which uses ultra-short infrared laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from endogenously fluorescent proteins (e.g. elastin) and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens in fresh human lung tissues [J. Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D image data obtained directly from thick frozen inflated lung specimens (~0.7- 1.0 millimeter thick) visualized at -60°C without prior fixation or staining in healthy and diseased states. Lung specimens donated for transplantation and released for research when no appropriate recipient was identified served as controls, and diseased lung specimens donated for research by patients receiving lung transplantation for very severe COPD (n=4) were prepared as previously described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced between apex and base were examined using multiphoton microscopy while maintained at -60°C using a temperature controlled cold stage with a temperature resolution of 0.1°C. Infrared femto-second laser pulses tuned to 880nm, dry microscopic objectives, and non-de-scanned detectors/spectrophotometer located in the reflection geometry were

  8. Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy.

    Science.gov (United States)

    Wright, Amanda J; Burns, David; Patterson, Brett A; Poland, Simon P; Valentine, Gareth J; Girkin, John M

    2005-05-01

    We report on the introduction of active optical elements into confocal and multiphoton microscopes in order to reduce the sample-induced aberration. Using a flexible membrane mirror as the active element, the beam entering the rear of the microscope objective is altered to produce the smallest point spread function once it is brought to a focus inside the sample. The conventional approach to adaptive optics, commonly used in astronomy, is to utilise a wavefront sensor to determine the required mirror shape. We have developed a technique that uses optimisation algorithms to improve the returned signal without the use of a wavefront sensor. We have investigated a number of possible optimisation methods, covering hill climbing, genetic algorithms, and more random search methods. The system has demonstrated a significant enhancement in the axial resolution of a confocal microscope when imaging at depth within a sample. We discuss the trade-offs of the various approaches adopted, comparing speed with resolution enhancement.

  9. High speed multiphoton imaging

    Science.gov (United States)

    Li, Yongxiao; Brustle, Anne; Gautam, Vini; Cockburn, Ian; Gillespie, Cathy; Gaus, Katharina; Lee, Woei Ming

    2016-12-01

    Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.

  10. Measurement of absorption spectrum of deuterium oxide (D{sub 2}O) and its application to signal enhancement in multiphoton microscopy at the 1700-nm window

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yuxin; Wen, Wenhui; Wang, Kai; Wang, Ke, E-mail: kewangfs@szu.edu.cn [Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060 (China); Zhai, Peng [Key Laboratory of Biomedical Engineering of Shenzhen, College of Medicine, Shenzhen University, Shenzhen 518060 (China); Qiu, Ping, E-mail: pingqiu@szu.edu.cn [College of Physics Science and Technology, Shenzhen University, Shenzhen 518060 (China)

    2016-01-11

    1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy (MPM). Long working-distance water immersion objective lenses are typically used for deep-tissue imaging. However, absorption due to immersion water at 1700 nm is still high and leads to dramatic decrease in signals. In this paper, we demonstrate measurement of absorption spectrum of deuterium oxide (D{sub 2}O) from 1200 nm to 2600 nm, covering the three low water-absorption windows potentially applicable for deep-tissue imaging (1300 nm, 1700 nm, and 2200 nm). We apply this measured result to signal enhancement in MPM at the 1700-nm window. Compared with water immersion, D{sub 2}O immersion enhances signal levels in second-harmonic generation imaging, 3-photon fluorescence imaging, and third-harmonic generation imaging by 8.1, 24.8, and 24.7 times with 1662-nm excitation, in good agreement with theoretical calculation based on our absorption measurement. This suggests D{sub 2}O a promising immersion medium for deep-tissue imaging.

  11. Measurement of absorption spectrum of deuterium oxide (D2O) and its application to signal enhancement in multiphoton microscopy at the 1700-nm window

    Science.gov (United States)

    Wang, Yuxin; Wen, Wenhui; Wang, Kai; Zhai, Peng; Qiu, Ping; Wang, Ke

    2016-01-01

    1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy (MPM). Long working-distance water immersion objective lenses are typically used for deep-tissue imaging. However, absorption due to immersion water at 1700 nm is still high and leads to dramatic decrease in signals. In this paper, we demonstrate measurement of absorption spectrum of deuterium oxide (D2O) from 1200 nm to 2600 nm, covering the three low water-absorption windows potentially applicable for deep-tissue imaging (1300 nm, 1700 nm, and 2200 nm). We apply this measured result to signal enhancement in MPM at the 1700-nm window. Compared with water immersion, D2O immersion enhances signal levels in second-harmonic generation imaging, 3-photon fluorescence imaging, and third-harmonic generation imaging by 8.1, 24.8, and 24.7 times with 1662-nm excitation, in good agreement with theoretical calculation based on our absorption measurement. This suggests D2O a promising immersion medium for deep-tissue imaging.

  12. Carcinogenic damage to deoxyribonucleic acid is induced by near-infrared laser pulses in multiphoton microscopy via combination of two- and three-photon absorption

    Science.gov (United States)

    Nadiarnykh, Oleg; Thomas, Giju; Van Voskuilen, Johan; Sterenborg, Henricus J. C. M.; Gerritsen, Hans C.

    2012-11-01

    Nonlinear optical imaging modalities (multiphoton excited fluorescence, second and third harmonic generation) applied in vivo are increasingly promising for clinical diagnostics and the monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited resolution at near-infrared (IR) wavelengths. However, high peak intensity of femtosecond laser pulses required for two-photon processes causes formation of cyclobutane-pyrimidine-dimers (CPDs) in cellular deoxyribonucleic acid (DNA) similar to damage from exposure to solar ultraviolet (UV) light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis. In this study, we investigate CPD damage that results in Chinese hamster ovary cells in vitro from imaging them with two-photon excited autofluorescence. The CPD levels are quantified by immunofluorescent staining. We further evaluate the extent of CPD damage with respect to varied wavelength, pulse width at focal plane, and pixel dwell time as compared with more pronounced damage from UV sources. While CPD damage has been expected to result from three-photon absorption, our results reveal that CPDs are induced by competing two- and three-photon absorption processes, where the former accesses UVA absorption band. This finding is independently confirmed by nonlinear dependencies of damage on laser power, wavelength, and pulse width.

  13. Biaxial loading of arterial tissues with 3D in situ observations of adventitia fibrous microstructure: A method coupling multi-photon confocal microscopy and bulge inflation test.

    Science.gov (United States)

    Cavinato, Cristina; Helfenstein-Didier, Clementine; Olivier, Thomas; du Roscoat, Sabine Rolland; Laroche, Norbert; Badel, Pierre

    2017-10-01

    Disorders in the wall microstructure underlie all forms of vascular disease, such as the aortic aneurysm, the rupture of which is necessarily triggered at the microscopic level. In this context, we developed an original experimental approach, coupling a bulge inflation test to multiphoton confocal microscopy, for visualizing the 3D micro-structure of porcine, human non-aneurysmal and aneurysmal aortic adventitial collagen under increasing pressurization. The experiment complexity on such tissues led to deeply address the acquisition major hurdles. The important innovative features of the methodology are presented, especially regarding region-of-interest tracking, definition of a stabilization period prior to imaging and correction of z-motion, z being the objective's axis. Such corrections ensured consistent 3D qualitative and quantitative analyses without z-motion. Qualitative analyses of the stable 3D images showed dense undulated collagen fiber bundles in the unloaded state which tended to progressive straightening and separation into a network of thinner bundles at high pressures. Quantitative analyses were made using a combination of weighted 2D structure tensors and fitting of 4 independent Gaussian functions to measure parameters related to straightening and orientation of the fibers. They denoted 3 principal fibers directions, approximately 45°, 135° and 90° with respect to the circumferential axis in the circumferential-axial plane without any evident reorientation of the fibers under pressurization. Results also showed that fibers at zero-pressure state were straighter and less dispersed in orientation for human samples - especially aneurysms - than for pigs. Progressive straightening and decrease in dispersion were quantified during the inflation. These findings provide further insight into the micro-architectural changes within the arterial wall. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Multiphoton processes: conference proceedings

    Energy Technology Data Exchange (ETDEWEB)

    Lambropoulos, P.; Smith, S.J. (eds.)

    1984-01-01

    The chapters of this volume represent the invited papers delivered at the conference. They are arranged according to thermatic proximity beginning with atoms and continuing with molecules and surfaces. Section headings include multiphoton processes in atoms, field fluctuations and collisions in multiphoton process, and multiphoton processes in molecules and surfaces. Abstracts of individual items from the conference were prepared separately for the data base. (GHT)

  15. A pragmatic guide to multiphoton microscope design.

    Science.gov (United States)

    Young, Michael D; Field, Jeffrey J; Sheetz, Kraig E; Bartels, Randy A; Squier, Jeff

    2015-06-30

    Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope.

  16. 3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation.

    Science.gov (United States)

    Miller, Sean J; Rothstein, Jeffrey D

    2017-01-01

    Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining. Using 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer. With our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier. Taken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.

  17. Multiphoton imaging of renal tissues in vitro.

    Science.gov (United States)

    Peti-Peterdi, János

    2005-06-01

    The highly inhomogeneous and light-scattering structure of living renal tissue makes the application of conventional imaging techniques more difficult compared with other parenchymal organs. On the other hand, key physiological processes of the kidney, such as regulation of glomerular filtration, hemodynamics, concentration, and dilution, involve complex interactions between multiple cell types and otherwise inaccessible structures that necessitate visual approaches. An ideal solution is multiphoton excitation fluorescence microscopy, a state-of-the-art imaging technique superior for deep optical sectioning of living tissue samples. Here, we review the basics and advantages of multiphoton microscopy and provide examples for its application in renal physiology using dissected cortical and medullary tissues in vitro. In combination with microperfusion techniques, the major functions of the juxtaglomerular apparatus, tubuloglomerular feedback and renin release, can be studied with high spatial and temporal resolution. Salt-dependent changes in macula densa cell volume, vasoconstriction of the afferent arteriole, and activity of an intraglomerular precapillary sphincter composed of renin granular cells are visualized in real time. Release and tissue activity of renin can be studied on the individual granule level. Imaging of the living inner medulla shows how interstitial cells interconnect cells of the vasa recta, loop of Henle, and collecting duct. In summary, multiphoton microscopy is an exciting new optical sectioning technique that has great potential for numerous future developments and is ideal for applications that require deep optical sectioning of living tissue samples.

  18. Multiphoton coherent population oscillation

    CERN Document Server

    Sharypov, A V

    2014-01-01

    We study the bichromatic driving of a two-level system which displays long-lived coherent population oscillations (CPO). We show that under certain conditions, multiphoton parametric interaction leads to the appearance of CPO resonances at the subharmonic frequencies. In addition, in the region of the CPO resonances, there is strong parametric interaction between the weak sideband components of the electromagnetic field.

  19. Multiphoton imaging with a novel compact diode-pumped Ti:sapphire oscillator

    DEFF Research Database (Denmark)

    König, Karsten; Andersen, Peter E.; Le, Tuan;

    2015-01-01

    Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its...

  20. Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

    Science.gov (United States)

    Mouras, Rabah; Bagnaninchi, Pierre O.; Downes, Andrew R.; Elfick, Alistair P. D.

    2012-11-01

    Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell's morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

  1. Multiphoton imaging of renal regulatory mechanisms.

    Science.gov (United States)

    Peti-Peterdi, János; Toma, Ildikó; Sipos, Arnold; Vargas, Sarah L

    2009-04-01

    Most physiological functions of the kidneys, including the clearance of metabolic waste products, maintenance of body fluid, electrolyte homeostasis, and blood pressure, are achieved by complex interactions between multiple renal cell types and previously inaccessible structures in many organ parts that have been difficult to study. Multiphoton fluorescence microscopy offers a state-of-the-art imaging technique for deep optical sectioning of living tissues and organs with minimal deleterious effects. Dynamic regulatory processes and multiple functions in the intact kidney can be quantitatively visualized in real time, noninvasively, and with submicron resolution. This article reviews innovative multiphoton imaging technologies and their applications that provided the most complex, immediate, and dynamic portrayal of renal function-clearly depicting as well as analyzing the components and mechanisms involved in renal (patho)physiology.

  2. Assessing and benchmarking multiphoton microscopes for biologists.

    Science.gov (United States)

    Corbin, Kaitlin; Pinkard, Henry; Peck, Sebastian; Beemiller, Peter; Krummel, Matthew F

    2014-01-01

    Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.

  3. Clinical multiphoton FLIM tomography

    Science.gov (United States)

    König, Karsten

    2012-03-01

    This paper gives an overview on current clinical high resolution multiphoton fluorescence lifetime imaging in volunteers and patients. Fluorescence lifetime imaging (FLIM) in Life Sciences was introduced in Jena/Germany in 1988/89 based on a ZEISS confocal picosecond dye laser scanning microscope equipped with a single photon counting unit. The porphyrin distribution in living cells and living tumor-bearing mice was studied with high spatial, temporal, and spectral resolution. Ten years later, time-gated cameras were employed to detect dental caries in volunteers based on one-photon excitation of autofluorescent bacteria with long fluorescence lifetimes. Nowadays, one-photon FLIM based on picosecond VIS laser diodes are used to study ocular diseases in humans. Already one decade ago, first clinical twophoton FLIM images in humans were taken with the certified clinical multiphoton femtosecond laser tomograph DermaInspectTM. Multiphoton tomographs with FLIM modules are now operating in hospitals at Brisbane, Tokyo, Berlin, Paris, London, Modena and other European cities. Multiple FLIM detectors allow spectral FLIM with a temporal resolution down to 20 ps (MCP) / 250 ps (PMT) and a spectral resolution of 10 nm. Major FLIM applications include the detection of intradermal sunscreen and tattoo nanoparticles, the detection of different melanin types, the early diagnosis of dermatitis and malignant melanoma, as well as the measurement of therapeutic effects in pateints suffering from dermatitis. So far, more than 1,000 patients and volunteers have been investigated with the clinical multiphoton FLIM tomographs DermaInspectTM and MPTflexTM.

  4. Accessing the third dimension in localization-based super-resolution microscopy.

    Science.gov (United States)

    Hajj, Bassam; El Beheiry, Mohamed; Izeddin, Ignacio; Darzacq, Xavier; Dahan, Maxime

    2014-08-21

    Only a few years after its inception, localization-based super-resolution microscopy has become widely employed in biological studies. Yet, it is primarily used in two-dimensional imaging and accessing the organization of cellular structures at the nanoscale in three dimensions (3D) still poses important challenges. Here, we review optical and computational techniques that enable the 3D localization of individual emitters and the reconstruction of 3D super-resolution images. These techniques are grouped into three main categories: PSF engineering, multiple plane imaging and interferometric approaches. We provide an overview of their technical implementation as well as commentary on their applicability. Finally, we discuss future trends in 3D localization-based super-resolution microscopy.

  5. Correlated Multiphoton Holes

    CERN Document Server

    Afek, Itai; Silberberg, Yaron

    2010-01-01

    We generate bipartite states of light which exhibit an absence of multiphoton coincidence events between two modes amid a constant background flux. These `correlated photon holes' are produced by mixing a coherent state and relatively weak spontaneous parametric down-conversion using a balanced beamsplitter. Correlated holes with arbitrarily high photon numbers may be obtained by adjusting the relative phase and amplitude of the inputs. We measure states of up to five photons and verify their nonclassicality. The scheme provides a route for observation of high-photon-number nonclassical correlations without requiring intense quantum resources.

  6. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

    Science.gov (United States)

    Cua, Michelle; Wahl, Daniel J.; Zhao, Yuan; Lee, Sujin; Bonora, Stefano; Zawadzki, Robert J.; Jian, Yifan; Sarunic, Marinko V.

    2016-09-01

    Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.

  7. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging.

    Science.gov (United States)

    Cua, Michelle; Wahl, Daniel J; Zhao, Yuan; Lee, Sujin; Bonora, Stefano; Zawadzki, Robert J; Jian, Yifan; Sarunic, Marinko V

    2016-09-07

    Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.

  8. 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.

  9. Multiphoton ionization of Uracil

    Science.gov (United States)

    Prieto, Eladio; Martinez, Denhi; Guerrero, Alfonso; Alvarez, Ignacio; Cisneros, Carmen

    2016-05-01

    Multiphoton ionization and dissociation of Uracil using a Reflectron time of flight spectrometer was performed along with radiation from the second harmonic of a Nd:YAG laser. Uracil is one of the four nitrogen bases that belong to RNA. The last years special interest has been concentrated on the study of the effects under UV radiation in nucleic acids1 and also in the role that this molecule could have played in the origin and development of life on our planet.2 The MPI mass spectra show that the presence and intensity of the resulting ions strongly depend on the density power. The identification of the ions in the mass spectra is presented. The results are compared with those obtained in other laboratories under different experimental conditions and some of them show partial agreement.3 The present work was supported by CONACYT-Mexico Grant 165410 and DGAPA UNAM Grant IN101215 and IN102613.

  10. A novel flexible clinical multiphoton tomograph for early melanoma detection, skin analysis, testing of anti-age products, and in situ nanoparticle tracking

    Science.gov (United States)

    Weinigel, Martin; Breunig, Hans Georg; Gregory, Axel; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; König, Karsten

    2010-02-01

    High-resolution 3D microscopy based on multiphoton induced autofluorescence and second harmonic generation have been introduced in 1990. 13 years later, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have first been launched by JenLab company with the tomography DermaInspect®. This year, the second generation of clinical multiphoton tomographs was introduced. The novel multiphoton tomograph MPTflex, equipped with a flexible articulated optical arm, provides an increased flexibility and accessibility especially for clinical and cosmetical examinations. Improved image quality and signal to noise ratio (SNR) are achieved by a very short source-drain spacing, by larger active areas of the detectors and by single photon counting (SPC) technology. Shorter image acquisition time due to improved image quality reduces artifacts and simplifies the operation of the system. The compact folded optical design and the light-weight structure of the optical head eases the handling. Dual channel detectors enable to distinguish between intratissue elastic fibers and collagenous structures simultaneously. Through the use of piezo-driven optics a stack of optical cross-sections (optical sectioning) can be acquired and 3D imaging can be performed. The multiphoton excitation of biomolecules like NAD(P)H, flavins, porphyrins, elastin, and melanin is done by picojoule femtosecond laser pulses from an tunable turn-key femtosescond near infrared laser system. The ability for rapid high-quality image acquisition, the user-friendly operation of the system and the compact and flexible design qualifies this system to be used for melanoma detection, diagnostics of dermatological disorders, cosmetic research and skin aging measurements as well as in situ drug monitoring and animal research.

  11. Multiphoton Processes and Attosecond Physics

    CERN Document Server

    Midorikawa, Katsumi; 12th International Conference on Multiphoton Processes; 3rd International Conference on Attosecond Physics

    2012-01-01

    Recent advances in ultrashort pulsed laser technology have opened new frontiers in atomic, molecular and optical sciences. The 12th International Conference on Multiphoton Processes (ICOMP12) and the 3rd International Conference on Attosecond Physics (ATTO3), held jointly in Sapporo, Japan, during July 3-8, showcased studies at the forefront of research on multiphoton processes and attosecond physics. This book summarizes presentations and discussions from these two conferences.

  12. In vivo multiphoton imaging of the cornea: polarization-resolved second harmonic generation from stromal collagen

    Science.gov (United States)

    Latour, G.; Gusachenko, I.; Kowalczuk, L.; Lamarre, I.; Schanne-Klein, M.-C.

    2012-03-01

    Multiphoton microscopy provides specific and contrasted images of unstained collagenous tissues such as tendons or corneas. Polarization-resolved second harmonic generation (SHG) measurements have been implemented in a laserscanning multiphoton microscope. Distortion of the polarimetric response due to birefringence and diattenuation during propagation of the laser excitation has been shown in rat-tail tendons. A model has been developed to account for these effects and correct polarization-resolved SHG images in thick tissues. This new modality is then used in unstained human corneas to access two quantitative parameters: the fibrils orientation within the collagen lamellae and the ratio of the main second-order nonlinear tensorial components. Orientation maps obtained from polarization resolution of the trans-detected SHG images are in good agreement with the striated features observed in the raw images. Most importantly, polarization analysis of the epi-detected SHG images also enables to map the fibrils orientation within the collagen lamellae while epi-detected SHG images of corneal stroma are spatially homogenous and do not enable direct visualization of the fibrils orientation. Depth profiles of the polarimetric SHG response are also measured and compared to models accounting for orientation changes of the collagen lamellae within the focal volume. Finally, in vivo polarization-resolved SHG is performed in rat corneas and structural organization of corneal stroma is determined using epi-detected signals.

  13. Multiphoton gonioscopy to image the trabecular meshwork of porcine eyes

    Science.gov (United States)

    Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

    2013-03-01

    The aqueous outflow system (AOS), including the trabecular meshwork (TM), the collector channels (CC) and the Schlemm's canal (SC), regulates intraocular pressure (IOP) through the drainage of the aqueous humor (AH). Abnormal IOP elevation leads to increased pressure stress to retinal ganglion cells, resulting in cell loss that can ultimately lead to complete loss of eyesight. Therefore, development of imaging tools to detect abnormal structural and functional changes of the AOS is important in early diagnosis and prevention of glaucoma. Multiphoton microscopy (MPM), including twophoton autofluorescence (TPAF) and second harmonic generation (SHG), is a label-free microscopic technique that allows molecular specific imaging of biological tissues like the TM. Since the TM and other AOS structures are located behind the highly scattering scleral tissue, transscleral imaging of the TM does not provide enough optical resolution. In this work, a gonioscopic lens is used to allow direct optical access of the TM through the cornea for MPM imaging. Compared to transscleral imaging, the acquired MPM images show improved resolution as individual collagen fiber bundles of the TM can be observed. MPM gonioscopy may have the potential to be developed as a future clinical imaging tool for glaucoma diagnostics.

  14. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    Science.gov (United States)

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  15. Multiphoton tomography of astronauts

    Science.gov (United States)

    König, Karsten; Weinigel, Martin; Pietruszka, Anna; Bückle, Rainer; Gerlach, Nicole; Heinrich, Ulrike

    2015-03-01

    Weightlessness may impair the astronaut's health conditions. Skin impairments belong to the most frequent health problems during space missions. Within the Skin B project, skin physiological changes during long duration space flights are currently investigated on three European astronauts that work for nearly half a year at the ISS. Measurements on the hydration, the transepidermal water loss, the surface structure, elasticity and the tissue density by ultrasound are conducted. Furthermore, high-resolution in vivo histology is performed by multiphoton tomography with 300 nm spatial and 200 ps temporal resolution. The mobile certified medical tomograph with a flexible 360° scan head attached to a mechano-optical arm is employed to measure two-photon autofluorescence and SHG in the volar forearm of the astronauts. Modification of the tissue architecture and of the fluorescent biomolecules NAD(P)H, keratin, melanin and elastin are detected as well as of SHG-active collagen. Thinning of the vital epidermis, a decrease of the autofluoresence intensity, an increase in the long fluorescence lifetime, and a reduced skin ageing index SAAID based on an increased collagen level in the upper dermis have been found. Current studies focus on recovery effects.

  16. Quantitative multiphoton imaging

    Science.gov (United States)

    König, Karsten; Weinigel, Martin; Breunig, Hans Georg; Uchugonova, Aisada

    2014-02-01

    Certified clinical multiphoton tomographs for label-free multidimensional high-resolution in vivo imaging have been introduced to the market several years ago. Novel tomographs include a flexible 360° scan head attached to a mechanooptical arm for autofluorescence and SHG imaging as well as a CARS module. Non-fluorescent lipids and water, mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged in vivo with submicron resolution in human skin. Sensitive and rapid detectors allow single photon counting and the construction of 3D maps where the number of detected photons per voxel is depicted. Intratissue concentration profiles from endogenous as well exogenous substances can be generated when the number of detected photons can be correlated with the number of molecules with respect to binding and scattering behavior. Furthermore, the skin ageing index SAAID based on the ratio elastin/collagen as well as the epidermis depth based on the onset of SHG generation can be determined.

  17. In vivo real-time multiphoton imaging of T lymphocytes in the mouse brain after experimental stroke

    DEFF Research Database (Denmark)

    Fumagalli, Stefano; Coles, Jonathan A; Ejlerskov, Patrick;

    2011-01-01

    To gain a better understanding of T cell behavior after stroke, we have developed real-time in vivo brain imaging of T cells by multiphoton microscopy after middle cerebral artery occlusion.......To gain a better understanding of T cell behavior after stroke, we have developed real-time in vivo brain imaging of T cells by multiphoton microscopy after middle cerebral artery occlusion....

  18. Super-resolving multi-photon interferences with independent light sources

    CERN Document Server

    Oppel, Steffen; Kok, Pieter; von Zanthier, Joachim

    2012-01-01

    Multi-photon interferences with indistinguishable photons from independent light sources are at the focus of current research owing to their potential in optical quantum computing, creating remote entanglement for quantum computation and communication, and quantum metrology. The paradigmatic states for multi-photon interference are the highly entangled NOON states, which can be used to achieve increased resolution in spectroscopy, interferometry, lithography, and microscopy. Multi-photon interferences from independent, uncorrelated emitters can also lead to enhanced resolution in metrology and imaging. So far, such interferences have been observed with maximally two independent emitters. Here, we report multi-photon interferences with up to five independent emitters, displaying interference patterns equivalent to those of NOON states. Experimental results with independent thermal light sources confirm this NOON-like modulation. The experiment is an extension of the landmark measurement by Hanbury Brown and Tw...

  19. Multiphoton tomography for tissue engineering

    Science.gov (United States)

    König, Karsten

    2008-02-01

    Femtosecond laser multiphoton tomography has been employed in the field of tissue engineering to perform 3D high-resolution imaging of the extracellular matrix proteins elastin and collagen as well as of living cells without any fixation, slicing, and staining. Near infrared 80 MHz picojoule femtosecond laser pulses are able to excite the endogenous fluorophores NAD(P)H, flavoproteins, melanin, and elastin via a non-resonant two-photon excitation process. In addition, collagen can be imaged by second harmonic generation. Using a two-PMT detection system, the ratio of elastin to collagen was determined during optical sectioning. A high submicron spatial resolution and 50 picosecond temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics as well as a time-correlated single photon counting module with a fast microchannel plate detector and fast photomultipliers. Multiphoton tomography has been used to optimize the tissue engineering of heart valves and vessels in bioincubators as well as to characterize artificial skin. Stem cell characterization and manipulation are of major interest for the field of tissue engineering. Using the novel sub-20 femtosecond multiphoton nanoprocessing laser microscope FemtOgene, the differentiation of human stem cells within spheroids has been in vivo monitored with submicron resolution. In addition, the efficient targeted transfection has been demonstrated. Clinical studies on the interaction of tissue-engineered products with the natural tissue environment can be performed with in vivo multiphoton tomograph DermaInspect.

  20. Coherent Control of Multiphoton Transitions in the Gas and Condensed Phases with Shaped Ultrashort Pulses

    Energy Technology Data Exchange (ETDEWEB)

    Marcos Dantus

    2008-09-23

    Controlling laser-molecule interactions has become an integral part of developing devices and applications in spectroscopy, microscopy, optical switching, micromachining and photochemistry. Coherent control of multiphoton transitions could bring a significant improvement of these methods. In microscopy, multi-photon transitions are used to activate different contrast agents and suppress background fluorescence; coherent control could generate selective probe excitation. In photochemistry, different dissociative states are accessed through two, three, or more photon transitions; coherent control could be used to select the reaction pathway and therefore the yield-specific products. For micromachining and processing a wide variety of materials, femtosecond lasers are now used routinely. Understanding the interactions between the intense femtosecond pulse and the material could lead to technologically important advances. Pulse shaping could then be used to optimize the desired outcome. The scope of our research program is to develop robust and efficient strategies to control nonlinear laser-matter interactions using ultrashort shaped pulses in gas and condensed phases. Our systematic research has led to significant developments in a number of areas relevant to the AMO Physics group at DOE, among them: generation of ultrashort phase shaped pulses, coherent control and manipulation of quantum mechanical states in gas and condensed phases, behavior of isolated molecules under intense laser fields, behavior of condensed phase matter under intense laser field and implications on micromachining with ultrashort pulses, coherent control of nanoparticles their surface plasmon waves and their nonlinear optical behavior, and observation of coherent Coulomb explosion processes at 10^16 W/cm^2. In all, the research has resulted in 36 publications (five journal covers) and nine invention disclosures, five of which have continued on to patenting

  1. Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

    Science.gov (United States)

    Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

    2016-06-01

    Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

  2. In vivo microscopy.

    Science.gov (United States)

    Peti-Peterdi, János

    2016-04-01

    This article summarizes the past, present, and future promise of multiphoton excitation fluorescence microscopy for intravital kidney imaging. During the past 15years, several high-power visual research approaches have been developed using multiphoton imaging to study the normal functions of the healthy, intact, living kidney, and the various molecular and cellular mechanisms of the development of kidney diseases. In this review, the main focus will be on intravital multiphoton imaging of the glomerulus, the structure and function of the glomerular filtration barrier, especially the podocyte. Examples will be given for the combination of two powerful research tools, in vivo multiphoton imaging and mouse genetics using commercially available whole animal models for the detailed characterization of glomerular cell types, their function and fate, and for the better understanding of the molecular mechanisms of glomerular pathologies. One of the new modalities of multiphoton imaging, serial imaging of the same glomerulus in the same animal over several days will be emphasized for its potential for further advancing the field of nephrology research.

  3. 48-channel coincidence counting system for multiphoton experiment

    Science.gov (United States)

    Zhang, Chen; Li, Wei; Hu, Yi; Yang, Tao; Jin, Ge; Jiang, Xiao

    2016-11-01

    In this paper, we demonstrate a coincidence counting system with 48 input channels which is aimed to count all coincidence events, up to 531 441 kinds, in a multiphoton experiment. Using the dynamic delay adjusting inside the Field Programmable Gate Array, the alignment of photon signals of 48 channels is achieved. After the alignment, clock phase shifting is used to sample signal pulses. Logic constraints are used to stabilize the pulse width. The coincidence counting data stored in a 1G bit external random access memory will be sent to the computer to analyze the amount of 2-, 3-, 4-, 5-, and 6-fold coincidence events. This system is designed for multiphoton entanglement experiments with multiple degrees of freedom of photons.

  4. In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation

    Science.gov (United States)

    Ait El Madani, Hassan; Tancrède-Bohin, Emmanuelle; Bensussan, Armand; Colonna, Anne; Dupuy, Alain; Bagot, Martine; Pena, Ana-Maria

    2012-02-01

    Multiphoton microscopy has emerged in the past decade as a promising tool for noninvasive skin imaging. Our aim was to evaluate the potential of multiphoton microscopy to detect topical corticosteroids side effects within the epidermis and to provide new insights into their dynamics. Healthy volunteers were topically treated with clobetasol propionate on a small region of their forearms under overnight occlusion for three weeks. The treated region of each patient was investigated at D0, D7, D15, D22 (end of the treatment), and D60. Our study shows that multiphoton microscopy allows for the detection of corticoid-induced epidermis modifications: thinning of stratum corneum compactum and epidermis, decrease of keratinocytes size, and changes in their morphology from D7 to D22. We also show that multiphoton microscopy enables in vivo three-dimensional (3-D) quantitative assessment of melanin content. We observe that melanin density decreases during treatment and almost completely disappears at D22. Moreover, these alterations are reversible as they are no longer present at D60. Our study demonstrates that multiphoton microscopy is a convenient and powerful tool for noninvasive 3-D dynamical studies of skin integrity and pigmentation.

  5. Multiphoton Imaging of Rabbit Cornea Treated with Mitomycin C after Photorefractive Keratectomy

    Science.gov (United States)

    Hsueh, Chiu-Mei; Lo, Wen; Wang, Tsung-Jen; Hu, Fung-Rong; Dong, Chen-Yuan

    2007-07-01

    In this work we use multiphoton microscopy to observe the post surgery structure variation of rabbit cornea after photorefractive keratectomy (PRK). In addition, we added mitomycin C (MMC) to the post surgery rabbit cornea in order to investigate the effect of MMC treatment on the postoperative regeneration.

  6. Phase-imprinted multiphoton subradiant states

    Science.gov (United States)

    Jen, H. H.

    2017-08-01

    We propose to generate the multiphoton subradiant states and investigate their fluorescences in an array of two-level atoms. These multiphoton states are created initially from the timed Dicke states. Then we can use either a Zeeman or Stark field gradient pulse to imprint linearly increasing phases on the atoms, and this phase-imprinting process unitarily evolves the system to the multiphoton subradiant states. The fluorescence engages a long-range dipole-dipole interaction which originates from a system-reservoir coupling in the dissipation. We locate some of the subradiant multiphoton states from the eigenmodes and show that an optically thick atomic array is best for the preparation of the state with the most reduced decay rate. This phase-imprinting process enables quantum-state engineering of the multiphoton subradiant states and realizes a potential quantum storage of the photonic qubits in the two-level atoms.

  7. Accessing nuclear structure for field emission, in lens, scanning electron microscopy (FEISEM).

    Science.gov (United States)

    Allen, T D; Bennion, G R; Rutherford, S A; Reipert, S; Ramalho, A; Kiseleva, E; Goldberg, M W

    1996-01-01

    Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding, its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces.

  8. Multiphoton quantum optics and quantum state engineering

    Energy Technology Data Exchange (ETDEWEB)

    Dell' Anno, Fabio [Dipartimento di Fisica ' E. R. Caianiello' , Universita degli Studi di Salerno, CNISM and CNR-INFM Coherentia, and INFN Sezione di Napoli, Gruppo Collegato di Salerno, Via S. Allende, I-84081 Baronissi (Saudi Arabia) (Italy)]. E-mail: dellanno@sa.infn.it; De Siena, Silvio [Dipartimento di Fisica ' E. R. Caianiello' , Universita degli Studi di Salerno, CNISM and CNR-INFM Coherentia, and INFN Sezione di Napoli, Gruppo Collegato di Salerno, Via S. Allende, I-84081 Baronissi (SA) (Italy)]. E-mail: desiena@sa.infn.it; Illuminati, Fabrizio [Dipartimento di Fisica ' E. R. Caianiello' , Universita degli Studi di Salerno, CNISM and CNR-INFM Coherentia, and INFN Sezione di Napoli, Gruppo Collegato di Salerno, Via S. Allende, I-84081 Baronissi (SA) (Italy)]. E-mail: illuminati@sa.infn.it

    2006-05-15

    We present a review of theoretical and experimental aspects of multiphoton quantum optics. Multiphoton processes occur and are important for many aspects of matter-radiation interactions that include the efficient ionization of atoms and molecules, and, more generally, atomic transition mechanisms; system-environment couplings and dissipative quantum dynamics; laser physics, optical parametric processes, and interferometry. A single review cannot account for all aspects of such an enormously vast subject. Here we choose to concentrate our attention on parametric processes in nonlinear media, with special emphasis on the engineering of nonclassical states of photons and atoms that are relevant for the conceptual investigations as well as for the practical applications of forefront aspects of modern quantum mechanics. We present a detailed analysis of the methods and techniques for the production of genuinely quantum multiphoton processes in nonlinear media, and the corresponding models of multiphoton effective interactions. We review existing proposals for the classification, engineering, and manipulation of nonclassical states, including Fock states, macroscopic superposition states, and multiphoton generalized coherent states. We introduce and discuss the structure of canonical multiphoton quantum optics and the associated one- and two-mode canonical multiphoton squeezed states. This framework provides a consistent multiphoton generalization of two-photon quantum optics and a consistent Hamiltonian description of multiphoton processes associated to higher-order nonlinearities. Finally, we discuss very recent advances that by combining linear and nonlinear optical devices allow to realize multiphoton entangled states of the electromagnetic field, either in discrete or in continuous variables, that are relevant for applications to efficient quantum computation, quantum teleportation, and related problems in quantum communication and information.

  9. Access to residual carrier concentration in ZnO nanowires by calibrated scanning spreading resistance microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wang, L., E-mail: lin.wang@insa-lyon.fr; Brémond, G. [Institut des Nanotechnologies de Lyon (INL), Université de Lyon, CNRS UMR 5270, INSA Lyon, Bat. Blaise Pascal, 7 Avenue Jean Capelle, 69621 Villeurbanne (France); Chauveau, J. M. [Centre de Recherche sur l' Hétéro-Epitaxie et ses Applications (CRHEA), CNRS UPR10, rue Bernard Grégory 06560 Valbonne Sophia Antipolis (France); Physics Department, University of Nice Sophia Antipolis (UNS), Parc Valrose, 06103 Nice (France); Brenier, R. [Institut Lumière Matière (ILM), Université de Lyon, CNRS UMR 5306, Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne (France); Sallet, V.; Jomard, F.; Sartel, C. [Groupe d' Étude de la Matière Condensée (GEMaC), CNRS-Université de Versailles St Quentin en Yvelines, Université Paris-Saclay, 45 Avenue des Etats-Unis, 78035 Versailles (France)

    2016-03-28

    Scanning spreading resistance microscopy (SSRM) was performed on non-intentionally doped (nid) ZnO nanowires (NWs) grown by metal-organic chemical vapor deposition in order to measure their residual carrier concentration. For this purpose, an SSRM calibration profile has been developed on homoepitaxial ZnO:Ga multilayer staircase structures grown by molecular beam epitaxy. The Ga density measured by SIMS varies in the 1.7 × 10{sup 17 }cm{sup −3} to 3 × 10{sup 20 }cm{sup −3} range. From measurements on such Ga doped multi-layers, a monotonic decrease in SSRM resistance with increasing Ga density was established, indicating SSRM being a well-adapted technique for two dimensional dopant/carrier profiling on ZnO at nanoscale. Finally, relevant SSRM signal contrasts were detected on nid ZnO NWs, and the residual carrier concentration is estimated in the 1–3 × 10{sup 18 }cm{sup −3} range, in agreement with the result from four-probe measurements.

  10. Multiphoton fluorescence lifetime imaging of human hair.

    Science.gov (United States)

    Ehlers, Alexander; Riemann, Iris; Stark, Martin; König, Karsten

    2007-02-01

    In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.

  11. Multiphoton Quantum Optics and Quantum State Engineering

    CERN Document Server

    Dell'Anno, F; Illuminati, F; 10.1016/j.physrep.2006.01.004

    2009-01-01

    We present a review of theoretical and experimental aspects of multiphoton quantum optics. Multiphoton processes occur and are important for many aspects of matter-radiation interactions that include the efficient ionization of atoms and molecules, and, more generally, atomic transition mechanisms; system-environment couplings and dissipative quantum dynamics; laser physics, optical parametric processes, and interferometry. A single review cannot account for all aspects of such an enormously vast subject. Here we choose to concentrate our attention on parametric processes in nonlinear media, with special emphasis on the engineering of nonclassical states of photons and atoms. We present a detailed analysis of the methods and techniques for the production of genuinely quantum multiphoton processes in nonlinear media, and the corresponding models of multiphoton effective interactions. We review existing proposals for the classification, engineering, and manipulation of nonclassical states, including Fock states...

  12. MULTI-PHOTON PHOSPHOR FEASIBILITY RESEARCH

    Energy Technology Data Exchange (ETDEWEB)

    R. Graham; W. Chow

    2003-05-01

    Development of multi-photon phosphor materials for discharge lamps represents a goal that would achieve up to a doubling of discharge (fluorescent) lamp efficacy. This report reviews the existing literature on multi-photon phosphors, identifies obstacles in developing such phosphors, and recommends directions for future research to address these obstacles. To critically examine issues involved in developing a multi-photon phosphor, the project brought together a team of experts from universities, national laboratories, and an industrial lamp manufacturer. Results and findings are organized into three categories: (1) Multi-Photon Systems and Processes, (2) Chemistry and Materials Issues, and (3) Concepts and Models. Multi-Photon Systems and Processes: This category focuses on how to use our current understanding of multi-photon phosphor systems to design new phosphor systems for application in fluorescent lamps. The quickest way to develop multi-photon lamp phosphors lies in finding sensitizer ions for Gd{sup 3+} and identifying activator ions to red shift the blue emission from Pr{sup 3+} due to the {sup 1}S{sub 0} {yields} {sup 1}I{sub 6} transition associated with the first cascading step. Success in either of these developments would lead to more efficient fluorescent lamps. Chemistry and Materials Issues: The most promising multi-photon phosphors are found in fluoride hosts. However, stability of fluorides in environments typically found in fluorescent lamps needs to be greatly improved. Experimental investigation of fluorides in actual lamp environments needs to be undertaken while working on oxide and oxyfluoride alternative systems for backup. Concepts and Models: Successful design of a multi-photon phosphor system based on cascading transitions of Gd{sup 3+} and Pr{sup 3+} depends critically on how the former can be sensitized and the latter can sensitize an activator ion. Methods to predict energy level diagrams and Judd-Ofelt parameters of multi-photon

  13. Pressure effects in multiple resonant multiphoton transitions

    Science.gov (United States)

    Maïnos, C.; Le Duff, Y.; Castex, M. C.; Boursey, E.

    1989-02-01

    Although the rotational structure of a multiphoton process generally remains unaltered over a large range of gas pressure, this is not the case when multiple resonances are present. The rotational structure observed through intermediate rotational levels in a multiphoton process depends strongly on the resonance conditions. We show, for the NO molecule, that this structure changes drastically when the resonance conditions are modified by intermolecular collisions.

  14. Multiphoton entanglement concentration and quantum cryptography.

    Science.gov (United States)

    Durkin, Gabriel A; Simon, Christoph; Bouwmeester, Dik

    2002-05-01

    Multiphoton states from parametric down-conversion can be entangled both in polarization and photon number. Maximal high-dimensional entanglement can be concentrated postselectively from these states via photon counting. This makes them natural candidates for quantum key distribution, where the presence of more than one photon per detection interval has up to now been considered undesirable. We propose a simple multiphoton cryptography protocol for the case of low losses.

  15. Invited Review Article: Pump-probe microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Martin C., E-mail: Martin.Fischer@duke.edu; Wilson, Jesse W.; Robles, Francisco E. [Department of Chemistry, Duke University, Durham, North Carolina 27708 (United States); Warren, Warren S. [Departments of Chemistry, Biomedical Engineering, Physics, and Radiology, Duke University, Durham, North Carolina 27708 (United States)

    2016-03-15

    Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

  16. In vivo multiphoton imaging of the eyelid skin

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; König, Karsten

    2017-02-01

    Multiphoton tomography (MPT) has become an important imaging method for non-invasive and high-resolution imaging of the skin in vivo. Due to the nonlinear excitation, by using near-infrared (NIR) light, 3D information is intrinsically provided. In combination with fluorescence lifetime imaging (FLIM), it is possible to obtain both structural and metabolic data. Human in vivo measurements are usually limited to easily accessible regions. However, often imaging of specific body parts such as the eyelid are of interest for cosmetic reasons. By using the clinically certified multiphoton imaging tomograph MPTflex this demand can be fulfilled. An articulated mirror arm and scan-detector head enable imaging at otherwise difficult-to-access areas. We show the characterization of the epidermal and upper dermal layers of the eyelid skin of human volunteers in vivo based on endogenous autofluorescence intensity, lifetime, and second-harmonic generation signals. Skin properties such as the epidermal thickness were also assessed. Furthermore, the influence of an anti-aging cream on the eyelid and forearm skin was investigated. Changes of the skin epidermis autofluorescence lifetime were observed after two-weeks long application of an anti-aging cream. The SHG-to-AF aging index of dermis (SAAID) increased during that time.

  17. Using multiphoton fluorescence lifetime imaging to characterize liver damage and fluorescein disposition in liver in vivo

    Science.gov (United States)

    Thorling, Camilla A.; Studier, Hauke; Crawford, Darrell; Roberts, Michael S.

    2016-03-01

    Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.

  18. Multi-focal multiphoton lithography.

    Science.gov (United States)

    Ritschdorff, Eric T; Nielson, Rex; Shear, Jason B

    2012-03-01

    Multiphoton lithography (MPL) provides unparalleled capabilities for creating high-resolution, three-dimensional (3D) materials from a broad spectrum of building blocks and with few limitations on geometry, qualities that have been key to the design of chemically, mechanically, and biologically functional microforms. Unfortunately, the reliance of MPL on laser scanning limits the speed at which fabrication can be performed, making it impractical in many instances to produce large-scale, high-resolution objects such as complex micromachines, 3D microfluidics, etc. Previously, others have demonstrated the possibility of using multiple laser foci to simultaneously perform MPL at numerous sites in parallel, but use of a stage-scanning system to specify fabrication coordinates resulted in the production of identical features at each focal position. As a more general solution to the bottleneck problem, we demonstrate here the feasibility for performing multi-focal MPL using a dynamic mask to differentially modulate foci, an approach that enables each fabrication site to create independent (uncorrelated) features within a larger, integrated microform. In this proof-of-concept study, two simultaneously scanned foci produced the expected two-fold decrease in fabrication time, and this approach could be readily extended to many scanning foci by using a more powerful laser. Finally, we show that use of multiple foci in MPL can be exploited to assign heterogeneous properties (such as differential swelling) to micromaterials at distinct positions within a fabrication zone.

  19. Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

    Science.gov (United States)

    Lai, Zhenhua; Lian, Christine; Ma, Jie; Yu, Jingyi; Gu, Zetong; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2014-02-01

    Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

  20. New developments in multimodal clinical multiphoton tomography

    Science.gov (United States)

    König, Karsten

    2011-03-01

    80 years ago, the PhD student Maria Goeppert predicted in her thesis in Goettingen, Germany, two-photon effects. It took 30 years to prove her theory, and another three decades to realize the first two-photon microscope. With the beginning of this millennium, first clinical multiphoton tomographs started operation in research institutions, hospitals, and in the cosmetic industry. The multiphoton tomograph MPTflexTM with its miniaturized flexible scan head became the Prism-Award 2010 winner in the category Life Sciences. Multiphoton tomographs with its superior submicron spatial resolution can be upgraded to 5D imaging tools by adding spectral time-correlated single photon counting units. Furthermore, multimodal hybrid tomographs provide chemical fingerprinting and fast wide-field imaging. The world's first clinical CARS studies have been performed with a hybrid multimodal multiphoton tomograph in spring 2010. In particular, nonfluorescent lipids and water as well as mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen have been imaged in patients with dermatological disorders. Further multimodal approaches include the combination of multiphoton tomographs with low-resolution imaging tools such as ultrasound, optoacoustic, OCT, and dermoscopy systems. Multiphoton tomographs are currently employed in Australia, Japan, the US, and in several European countries for early diagnosis of skin cancer (malignant melanoma), optimization of treatment strategies (wound healing, dermatitis), and cosmetic research including long-term biosafety tests of ZnO sunscreen nanoparticles and the measurement of the stimulated biosynthesis of collagen by anti-ageing products.

  1. Multiphoton polymerization using optical trap assisted nanopatterning

    Science.gov (United States)

    Leitz, Karl-Heinz; Tsai, Yu-Cheng; Flad, Florian; Schäffer, Eike; Quentin, Ulf; Alexeev, Ilya; Fardel, Romain; Arnold, Craig B.; Schmidt, Michael

    2013-06-01

    In this letter, we show the combination of multiphoton polymerization and optical trap assisted nanopatterning (OTAN) for the additive manufacturing of structures with nanometer resolution. User-defined patterns of polymer nanostructures are deposited on a glass substrate by a 3.5 μm polystyrene sphere focusing IR femtosecond laser pulses, showing minimum feature sizes of λ/10. Feature size depends on the applied laser fluence and the bead surface spacing. A finite element model describes the intensity enhancement in the microbead focus. The results presented suggest that OTAN in combination with multiphoton processing is a viable technique for additive nanomanufacturing with sub-diffraction-limited resolution.

  2. Multiphoton fluorescence imaging of NADH to quantify metabolic changes in epileptic tissue in vitro

    Science.gov (United States)

    Chia, Thomas H.; Zinter, Joseph; Spencer, Dennis D.; Williamson, Anne; Levene, Michael J.

    2007-02-01

    A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD +; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

  3. In vivo multiphoton imaging of bile duct ligation

    Science.gov (United States)

    Liu, Yuan; Li, Feng-Chieh; Chen, Hsiao-Chin; Chang, Po-shou; Yang, Shu-Mei; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2008-02-01

    Bile is the exocrine secretion of liver and synthesized by hepatocytes. It is drained into duodenum for the function of digestion or drained into gallbladder for of storage. Bile duct obstruction is a blockage in the tubes that carry bile to the gallbladder and small intestine. However, Bile duct ligation results in the changes of bile acids in serum, liver, urine, and feces1, 2. In this work, we demonstrate a novel technique to image this pathological condition by using a newly developed in vivo imaging system, which includes multiphoton microscopy and intravital hepatic imaging chamber. The images we acquired demonstrate the uptake, processing of 6-CFDA in hepatocytes and excretion of CF in the bile canaliculi. In addition to imaging, we can also measure kinetics of the green fluorescence intensity.

  4. Route to direct multiphoton multiple ionization

    NARCIS (Netherlands)

    Lambropoulos, P.; Nikolopoulos, G. M.; Papamihail, K. G.

    2011-01-01

    We address the concept of direct multiphoton multiple ionization in atoms exposed to intense, short-wavelength radiation and explore the conditions under which such processes dominate over the sequential. Their contribution is shown to be quite robust, even under intensity fluctuations and interacti

  5. Observation of amyloid precursor protein cleavage and Aβ generation in living cells by using multiphoton laser scanning microscopy%多光子激光扫描成像技术对活细胞内淀粉样前体蛋白裂解和β-淀粉样蛋白生成的观察

    Institute of Scientific and Technical Information of China (English)

    李晓晴; 张苏明; 杨华静; 张智红

    2007-01-01

    Objective To investigate the proteolytic mechanism of amyloid precursor protein (APP) and to explore amyloidbeta (Aβ) generation in living neurons. Methods DNA fragments were amplified by PCR or synthesized. The four fragments- CFP- 54bp- YFP and C99 were ligated into pcDNAS.O vector to construct the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99. The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99.The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99.The expression of fusion gene was examined under a multiphoton laser scanning microscope.Fluorescence resonance energy transfer (FRET) was used to measure the p cleavage and y cleavage of APP.Aβ generation was confirmed by immunocytochemistry and multiphoton laser scanning microscopy.Cell viability was tested by MTT assay at different time points.Results (1) The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99.(2) Blue and yellow fluorescences were detected in the transfected cells.(3) FRET occurred in pcDNA3.0-CFP-54bp-YFP-transfected cells but not in pcDNA3.0-CFP-54bp-YFP-C99-transfected cells.(4) Aβ was produced in the pcDNA3.0-CFP-54bp-YFP-C99 transfected cells.(5) Aβ-deposition was widespread in the cell.(6) Cell viability decreased along with the intracellular Aβ deposition.Conclusion C99 is important for the APP β cleavage.Aβ may be generated and deposited in cells at the early stage of Alzheimer's disease.Intracellular Aβ accumulation brings deleterious effects on cells.%目的 在活细胞内探究淀粉样前体蛋白(amyloid precursor protein,APP)的裂解和β-淀粉样蛋白(amyloid beta,Aβ)的生成机制.方法 利用PCR扩增CFP(编码蓝色荧光蛋白),YFP(编码黄色荧光蛋白)和C99(编码APP最后99个氨基酸)三片段.含有54

  6. Enabling Multiphoton and Second Harmonic Generation Imaging in Paraffin-Embedded and Histologically Stained Sections.

    Science.gov (United States)

    Monaghan, Michael G; Kroll, Sebastian; Brucker, Sara Y; Schenke-Layland, Katja

    2016-06-01

    Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples.

  7. Multiphoton imaging for assessing renal disposition in acute kidney injury

    Science.gov (United States)

    Liu, Xin; Liang, Xiaowen; Wang, Haolu; Roberts, Darren M.; Roberts, Michael S.

    2016-11-01

    Estimation of renal function and drug renal disposition in acute kidney injury (AKI), is important for appropriate dosing of drugs and adjustment of therapeutic strategies, but is challenging due to fluctuations in kidney function. Multiphoton microscopy has been shown to be a useful tool in studying drug disposition in liver and can reflect dynamic changes of liver function. We extend this imaging technique to investigate glomerular filtration rate (GFR) and tubular transporter functional change in various animal models of AKI, which mimic a broad range of causes of AKI such as hypoxia (renal ischemia- reperfusion), therapeutic drugs (e.g. cisplatin), rhabdomyolysis (e.g. glycerol-induced) and sepsis (e.g. LPSinduced). The MPM images revealed acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation in AKI groups compared to control group. In control animal, systemically injected FITC-labelled inulin was rapidly cleared from glomerulus, while the clearance of FITC-inulin was significantly delayed in most of animals in AKI group, which may reflect the reduced GFR in AKI. Following intravenous injection, rhodamine 123, a fluorescent substrate of p-glycoprotein (one of tubular transporter), was excreted into urine in proximal tubule via p-glycoprotein; in response to AKI, rhodamine 123 was retained in tubular cells as revealed by slower decay of fluorescence intensity, indicating P-gp transporter dysfunction in AKI. Thus, real-time changes in GFR and transporter function can be imaged in rodent kidney with AKI using multiphoton excitation of exogenously injected fluorescent markers.

  8. Multiphoton tomography to detect chemo- and biohazards

    Science.gov (United States)

    König, Karsten

    2015-03-01

    In vivo high-resolution multiphoton/CARS tomography provides optical biopsies with 300 nm lateral resolution with chemical fingerprints. Thousands of volunteers and patients have been investigated for early cancer diagnosis, evaluation of anti-ageing cosmetic products, and changes of cellular metabolism by UV exposure and decreased oxygen supply. The skin as the outermost and largest organ is also the major target of CB agents. Current UV-based sensors are useful for bio-aerosol sensing but not for evaluating exposed in vivo skin. Here we evaluate the use of 4D multiphoton/CARS tomographs based on near infrared femtosecond laser radiation, time-correlated single photon counting (FLIM) and white light generation by photonic crystal fibers to detect bio- and chemohazards in human in vivo skin using twophoton fluorescence, SHG, and Raman signals.

  9. Collective multiphoton blockade in cavity quantum electrodynamics

    Science.gov (United States)

    Zhu, C. J.; Yang, Y. P.; Agarwal, G. S.

    2017-06-01

    We present a study of collective multiphoton blockade in coherently driven atoms in a single-mode cavity. Considering two atoms strongly coupled to an optical cavity, we show that the two-photon blockade with two-photon antibunching, and the three-photon blockade with three-photon antibunching can be observed simultaneously. The three-photon blockade probes both dressed states in the two-photon and three-photon spaces. The two-photon and three-photon blockades strongly depend on the location of the two atoms in the strong-coupling regime. The asymmetry in the atom-cavity coupling constants opens pathways for multiphoton blockade which is also shown to be sensitive to the atomic decay and pumping strengths. The work presented here predicts many quantum statistical features due to the collective behavior of atoms and can be useful to generate nonclassical photon pairs.

  10. Multiphoton tomography of intratissue tattoo nanoparticles

    Science.gov (United States)

    König, Karsten

    2012-02-01

    Most of today's intratissue tattoo pigments are unknown nanoparticles. So far, there was no real control of their use due to the absence of regulations. Some of the tattoo pigments contain carcinogenic amines e.g. azo pigment Red 22. Nowadays, the European Union starts to control the administration of tattoo pigments. There is an interest to obtain information on the intratissue distribution, their interaction with living cells and the extracellular matrix, and the mechanisms behind laser tattoo removal. Multiphoton tomographs are novel biosafety and imaging tools that can provide such information non-invasively and without further labeling. When using the spectral FLIM module, spatially-resolved emission spectra, excitation spectra, and fluorescence lifetimes can pr provided. Multiphoton tomographs are used by all major cosmetic comapanies to test the biosafety of sunscreen nanoparticles.

  11. Multiphoton spectroscopy of human skin in vivo

    Science.gov (United States)

    Breunig, Hans G.; Weinigel, Martin; König, Karsten

    2012-03-01

    In vivo multiphoton-intensity images and emission spectra of human skin are reported. Optical sections from different depths of the epidermis and dermis have been measured with near-infrared laser-pulse excitation. While the intensity images reveal information on the morphology, the spectra show emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, melanin, elastin and collagen as well as of second harmonic generation induced by the excitation-light interaction with the dermal collagen network.

  12. First multiphoton tomography of brain in man

    Science.gov (United States)

    König, Karsten; Kantelhardt, Sven R.; Kalasauskas, Darius; Kim, Ella; Giese, Alf

    2016-03-01

    We report on the first two-photon in vivo brain tissue imaging study in man. High resolution in vivo histology by multiphoton tomography (MPT) including two-photon FLIM was performed in the operation theatre during neurosurgery to evaluate the feasibility to detect label-free tumor borders with subcellular resolution. This feasibility study demonstrates, that MPT has the potential to identify tumor borders on a cellular level in nearly real-time.

  13. Medium-induced multi-photon radiation

    CERN Document Server

    Ma, Hao; Tywoniuk, Konrad

    2011-01-01

    We study the spectrum of multi-photon radiation off a fast quark in medium in the BDMPS/ASW approach. We reproduce the medium-induced one-photon radiation spectrum in dipole approximation, and go on to calculate the two-photon radiation in the Moli\\`{e}re limit. We find that in this limit the LPM effect holds for medium-induced two-photon ladder emission.

  14. Intravital Multiphoton Imaging of the Kidney: Tubular Structure and Metabolism.

    Science.gov (United States)

    Small, David M; Sanchez, Washington Y; Gobe, Glenda C

    2016-01-01

    Multiphoton microscopy (MPM) allows the visualization of dynamic pathophysiological events in real time in live animals. Intravital imaging can be applied to investigate novel mechanisms and treatments of different forms of kidney disease as well as improve our understanding of normal kidney physiology. Using rodent models, in conjunction with endogenous fluorescence and infused exogenous fluorescent dyes, measurement can be made of renal processes such as glomerular permeability, juxtaglomerular apparatus function, interactions of the tubulointerstitium, tubulovascular interactions, vascular flow rate, and the renin-angiotensin-aldosterone system. Subcellular processes including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes of apoptosis and necrosis can also be seen and measured by MPM. The current methods chapter presents an overview of MPM with a focus on techniques for intravital kidney imaging and gives examples of instances where intravital MPM has been utilized to study renal pathophysiology. Suggestions are provided for MPM methods within the confines of intravital microscopy and selected kidney structure. MPM is undoubtedly a powerful new technique for application in experimental nephrology, and we believe it will continue to create new paradigms for understanding and treating kidney disease.

  15. Clinical optical coherence tomography combined with multiphoton tomography for evaluation of several skin disorders

    Science.gov (United States)

    König, Karsten; Speicher, Marco; Bückle, Rainer; Reckfort, Julia; McKenzie, Gordon; Welzel, Julia; Koehler, Martin J.; Elsner, Peter; Kaatz, Martin

    2010-02-01

    The first clinical trial of optical coherence tomography (OCT) combined with multiphoton tomography (MPT) and dermoscopy is reported. State-of-the-art (i) OCT systems for dermatology (e.g. multibeam swept source OCT), (ii) the femtosecond laser multiphoton tomograph DermaInspectTM, and (iii) digital dermoscopes were applied to 47 patients with a diversity of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases. Dermoscopy, also called 'epiluminescent microscopy', provides two-dimensional color images of the skin surface. OCT imaging is based on the detection of optical reflections within the tissue measured interferometrically whereas nonlinear excitation of endogenous fluorophores and the second harmonic generation are the bases of MPT images. OCT cross sectional "wide field" image provides a typical field of view of 5 x 2 mm2 and offers fast information on the depth and the volume of the investigated lesion. In comparison, multiphoton tomography presents 0.36 x 0.36 mm2 horizontal or diagonal sections of the region of interest within seconds with submicron resolution and down to a tissue depth of 200 μm. The combination of OCT and MPT provides a synergistic optical imaging modality for early detection of skin cancer and other skin diseases.

  16. Optical investigation of the intergrowth structure and accessibility of Brønsted acid sites in etched SSZ-13 zeolite crystals by confocal fluorescence microscopy.

    Science.gov (United States)

    Sommer, Linn; Svelle, Stian; Lillerud, Karl Petter; Stöcker, Michael; Weckhuysen, Bert M; Olsbye, Unni

    2010-11-02

    Template decomposition followed by confocal fluorescence microscopy reveals a tetragonal-pyramidal intergrowth of subunits in micrometer-sized nearly cubic SSZ-13 zeolite crystals. In order to accentuate intergrowth boundaries and defect-rich areas within the individual large zeolite crystals, a treatment with an etching NaOH solution is applied. The defective areas are visualized by monitoring the spatial distribution of fluorescent tracer molecules within the individual SSZ-13 crystals by confocal fluorescence microscopy. These fluorescent tracer molecules are formed at the inner and outer crystal surfaces by utilizing the catalytic activity of the zeolite in the oligomerization reaction of styrene derivatives. This approach reveals various types of etching patterns that are an indication for the defectiveness of the studied crystals. We can show that specially one type of crystals, denoted as core-shell type, is highly accessible to the styrene molecules after etching. Despite the large crystal dimensions, the whole core-shell type SSZ-13 crystal is utilized for catalytic reaction. Furthermore, the confocal fluorescence microscopy measurements indicate a nonuniform distribution of the catalytically important Brønsted acid sites underlining the importance of space-resolved measurements.

  17. In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope

    Science.gov (United States)

    Brown, Christopher M.; Rivera, David R.; Pavlova, Ina; Ouzounov, Dimitre G.; Williams, Wendy O.; Mohanan, Sunish; Webb, Watt W.; Xu, Chris

    2012-04-01

    We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 μm by 115 μm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ~75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.

  18. Multiphoton imaging to distinguish grana and starch inside an intact leaf

    Science.gov (United States)

    Chen, Mei-Yu; Zhuo, Guan-Yu; Chen, Po-Fu; Wu, Pei-Chun; Liu, Tzu-Ming; Chu, Shi-Wei

    2013-02-01

    We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

  19. Correlative transmission electron microscopy and electrical properties study of switchable phase-change random access memory line cells

    Energy Technology Data Exchange (ETDEWEB)

    Oosthoek, J. L. M.; Kooi, B. J., E-mail: B.J.Kooi@rug.nl [Zernike Institute for Advanced Materials and Materials innovation institute M2i, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Voogt, F. C.; Attenborough, K. [NXP, Gerstweg 2, 6534 AE Nijmegen (Netherlands); Verheijen, M. A. [Department of Applied Physics, Eindhoven University of Technology, NL-5600 MB Eindhoven (Netherlands); Philips Innovation Services Eindhoven, High Tech Campus 11, NL-5656 AE Eindhoven (Netherlands); Hurkx, G. A. M. [NXP Semiconductors, High Tech Campus 60, 5656 AE Eindhoven (Netherlands); Gravesteijn, D. J. [NXP Semiconductors, Kapeldreef 75, B 3001 Leuven (Belgium)

    2015-02-14

    Phase-change memory line cells, where the active material has a thickness of 15 nm, were prepared for transmission electron microscopy (TEM) observation such that they still could be switched and characterized electrically after the preparation. The result of these observations in comparison with detailed electrical characterization showed (i) normal behavior for relatively long amorphous marks, resulting in a hyperbolic dependence between SET resistance and SET current, indicating a switching mechanism based on initially long and thin nanoscale crystalline filaments which thicken gradually, and (ii) anomalous behavior, which holds for relatively short amorphous marks, where initially directly a massive crystalline filament is formed that consumes most of the width of the amorphous mark only leaving minor residual amorphous regions at its edges. The present results demonstrate that even in (purposely) thick TEM samples, the TEM sample preparation hampers the probability to observe normal behavior and it can be debated whether it is possible to produce electrically switchable TEM specimen in which the memory cells behave the same as in their original bulk embedded state.

  20. Ultrafast Multiphoton Thermionic Photoemission from Graphite

    Science.gov (United States)

    Tan, Shijing; Argondizzo, Adam; Wang, Cong; Cui, Xuefeng; Petek, Hrvoje

    2017-01-01

    Electronic heating of cold crystal lattices in nonlinear multiphoton excitation can transiently alter their physical and chemical properties. In metals where free electron densities are high and the relative fraction of photoexcited hot electrons is low, the effects are small, but in semimetals, where the free electron densities are low and the photoexcited densities can overwhelm them, the intense femtosecond laser excitation can induce profound changes. In semimetal graphite and its derivatives, strong optical absorption, weak screening of the Coulomb potential, and high cohesive energy enable extreme hot electron generation and thermalization to be realized under femtosecond laser excitation. We investigate the nonlinear interactions within a hot electron gas in graphite through multiphoton-induced thermionic emission. Unlike the conventional photoelectric effect, within about 25 fs, the memory of the excitation process, where resonant dipole transitions absorb up to eight quanta of light, is erased to produce statistical Boltzmann electron distributions with temperatures exceeding 5000 K; this ultrafast electronic heating causes thermionic emission to occur from the interlayer band of graphite. The nearly instantaneous thermalization of the photoexcited carriers through Coulomb scattering to extreme electronic temperatures characterized by separate electron and hole chemical potentials can enhance hot electron surface femtochemistry, photovoltaic energy conversion, and incandescence, and drive graphite-to-diamond electronic phase transition.

  1. Multiphoton ionization of large water clusters.

    Science.gov (United States)

    Apicella, B; Li, X; Passaro, M; Spinelli, N; Wang, X

    2014-05-28

    Water clusters are multimers of water molecules held together by hydrogen bonds. In the present work, multiphoton ionization in the UV range coupled with time of flight mass spectrometry has been applied to water clusters with up to 160 molecules in order to obtain information on the electronic states of clusters of different sizes up to dimensions that can approximate the bulk phase. The dependence of ion intensities of water clusters and their metastable fragments produced by laser ionization at 355 nm on laser power density indicates a (3+1)-photon resonance-enhanced multiphoton ionization process. It also explains the large increase of ionization efficiency at 355 nm compared to that at 266 nm. Indeed, it was found, by applying both nanosecond and picosecond laser ionization with the two different UV wavelengths, that no water cluster sequences after n = 9 could be observed at 266 nm, whereas water clusters up to m/z 2000 Th in reflectron mode and m/z 3000 Th in linear mode were detected at 355 nm. The agreement between our findings on clusters of water, especially true in the range with n > 10, and reported data for liquid water supports the hypothesis that clusters above a critical dimension can approximate the liquid phase. It should thus be possible to study clusters just above 10 water molecules, for getting information on the bulk phase structure.

  2. Multiphoton tomography of the human eye

    Science.gov (United States)

    König, Karsten; Batista, Ana; Hager, Tobias; Seitz, Berthold

    2017-02-01

    Multiphoton tomography (MPT) is a novel label-free clinical imaging method for non-invasive tissue imaging with high spatial (300 nm) and temporal (100 ps) resolutions. In vivo optical histology can be realized due to the nonlinear excitation of endogenous fluorophores and second-harmonic generation (SHG) of collagen. Furthermore, optical metabolic imaging (OMI) is performed by two-photon autofluorescence lifetime imaging (FLIM). So far, applications of the multiphoton tomographs DermaInspect and MPTflex were limited to dermatology. Novel applications include intraoperative brain tumor imaging as well as cornea imaging. In this work we describe two-photon imaging of ex vivo human corneas unsuitable for transplantation. Furthermore, the cross-linking (CXL) process of corneal collagen based on UVA exposure and 0.1 % riboflavin was studied. The pharmacokinetics of the photosensitizer could be detected with high spatial resolution. Interestingly, an increase in the stromal autofluorescence intensity and modifications of the autofluorescence lifetimes were observed in the human corneal samples within a few days following CXL.

  3. Multiphoton imaging of the glomerular permeability of angiotensinogen.

    Science.gov (United States)

    Nakano, Daisuke; Kobori, Hiroyuki; Burford, James L; Gevorgyan, Haykanush; Seidel, Saskia; Hitomi, Hirofumi; Nishiyama, Akira; Peti-Peterdi, Janos

    2012-11-01

    Patients and animals with renal injury exhibit increased urinary excretion of angiotensinogen. Although increased tubular synthesis of angiotensinogen contributes to the increased excretion, we do not know to what degree glomerular filtration of systemic angiotensinogen, especially through an abnormal glomerular filtration barrier, contributes to the increase in urinary levels. Here, we used multiphoton microscopy to visualize and quantify the glomerular permeability of angiotensinogen in the intact mouse and rat kidney. In healthy mice and Munich-Wistar-Frömter rats at the early stage of glomerulosclerosis, the glomerular sieving coefficient of systemically infused Atto565-labeled human angiotensinogen (Atto565-hAGT), which rodent renin cannot cleave, was only 25% of the glomerular sieving coefficient of albumin, and its urinary excretion was undetectable. In a more advanced phase of kidney disease, the glomerular permeability of Atto565-hAGT was slightly higher but still very low. Furthermore, unlike urinary albumin, the significantly higher urinary excretion of endogenous rat angiotensinogen did not correlate with either the Atto565-hAGT or Atto565-albumin glomerular sieving coefficients. These results strongly suggest that the vast majority of urinary angiotensinogen originates from the tubules rather than glomerular filtration.

  4. Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs

    Science.gov (United States)

    Rowlands, Christopher J.; Bruns, Oliver T.; Bawendi, Moungi G.; So, Peter T. C.

    2015-06-01

    Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue.

  5. Stepwise multiphoton activation fluorescence reveals a new method of melanin detection

    Science.gov (United States)

    Lai, Zhenhua; Kerimo, Josef; Mega, Yair; DiMarzio, Charles A.

    2013-06-01

    The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

  6. Stepwise multiphoton activation fluorescence reveals a new method of melanin detection.

    Science.gov (United States)

    Lai, Zhenhua; Kerimo, Josef; Mega, Yair; Dimarzio, Charles A

    2013-06-01

    The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

  7. Multiphoton imaging of biological samples during freezing and heating

    Science.gov (United States)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2014-02-01

    We applied multiphoton microscopic imaging to observe freezing and heating effects in plant- and animal cell samples. The experimental setups consisted of a multiphoton imaging system and a heating and cooling stage which allows for precise temperature control from liquid nitrogen temperature (-196°C 77 K) up to +600°C (873 K) with heating/freezing rates between 0.01 K/min and 150 K/min. Two multiphoton imaging systems were used: a system based on a modified optical microscope and a flexible mobile system. To illustrate the imaging capabilities, plant leafs as well as animal cells were microscopically imaged in vivo during freezing based on autofluorescence lifetime and intensity of intrinsic molecules. The measurements illustrate the usefulness of multiphoton imaging to investigate freezing effects on animal and plant cells.

  8. Engineering multiphoton states for linear optics computation

    CERN Document Server

    Aniello, P; Napolitano, M; Paris, M G A; Aniello, Paolo; Lupo, Cosmo; Napolitano, Mario; Paris, Matteo G.A.

    2006-01-01

    Transformations achievable by linear optical components allow to generate the whole unitary group only when restricted to the one-photon subspace of a multimode Fock space. In this paper, we address the more general problem of encoding quantum information by multiphoton states, and elaborating it via ancillary extensions, linear optical passive devices and photodetection. Our scheme stems in a natural way from the mathematical structures underlying the physics of linear optical passive devices. In particular, we analyze an economical procedure for mapping a fiducial 2-photon 2-mode state into an arbitrary 2-photon 2-mode state using ancillary resources and linear optical passive N-ports assisted by post-selection. We found that adding a single ancilla mode is enough to generate any desired target state. The effect of imperfect photodetection in post-selection is considered and a simple trade-off between success probability and fidelity is derived.

  9. Soliton dynamics in the multiphoton plasma regime

    CERN Document Server

    Husko, Chad A; Colman, Pierre; Zheng, Jiangjun; De Rossi, Alfredo; Wong, Chee Wei; 10.1038/srep01100

    2013-01-01

    Solitary waves have consistently captured the imagination of scientists, ranging from fundamental breakthroughs in spectroscopy and metrology enabled by supercontinuum light, to gap solitons for dispersionless slow-light, and discrete spatial solitons in lattices, amongst others. Recent progress in strong-field atomic physics include impressive demonstrations of attosecond pulses and high-harmonic generation via photoionization of free-electrons in gases at extreme intensities of 1014 Wcm2. Here we report the first phase-resolved observations of femtosecond optical solitons in a semiconductor microchip, with multiphoton ionization at picojoule energies and 1010 Wcm2 intensities. The dramatic nonlinearity leads to picojoule observations of free-electron-induced blue-shift at 1016 cm3 carrier densities and self-chirped femtosecond soliton acceleration. Furthermore, we evidence the time-gated dynamics of soliton splitting on-chip, and the suppression of soliton recurrence due to fast free-electron dynamics. Thes...

  10. Rotational averaging of multiphoton absorption cross sections

    Energy Technology Data Exchange (ETDEWEB)

    Friese, Daniel H., E-mail: daniel.h.friese@uit.no; Beerepoot, Maarten T. P.; Ruud, Kenneth [Centre for Theoretical and Computational Chemistry, University of Tromsø — The Arctic University of Norway, N-9037 Tromsø (Norway)

    2014-11-28

    Rotational averaging of tensors is a crucial step in the calculation of molecular properties in isotropic media. We present a scheme for the rotational averaging of multiphoton absorption cross sections. We extend existing literature on rotational averaging to even-rank tensors of arbitrary order and derive equations that require only the number of photons as input. In particular, we derive the first explicit expressions for the rotational average of five-, six-, and seven-photon absorption cross sections. This work is one of the required steps in making the calculation of these higher-order absorption properties possible. The results can be applied to any even-rank tensor provided linearly polarized light is used.

  11. Rotational averaging of multiphoton absorption cross sections

    Science.gov (United States)

    Friese, Daniel H.; Beerepoot, Maarten T. P.; Ruud, Kenneth

    2014-11-01

    Rotational averaging of tensors is a crucial step in the calculation of molecular properties in isotropic media. We present a scheme for the rotational averaging of multiphoton absorption cross sections. We extend existing literature on rotational averaging to even-rank tensors of arbitrary order and derive equations that require only the number of photons as input. In particular, we derive the first explicit expressions for the rotational average of five-, six-, and seven-photon absorption cross sections. This work is one of the required steps in making the calculation of these higher-order absorption properties possible. The results can be applied to any even-rank tensor provided linearly polarized light is used.

  12. Multi-photon entanglement in high dimensions

    CERN Document Server

    Malik, Mehul; Huber, Marcus; Krenn, Mario; Fickler, Robert; Zeilinger, Anton

    2015-01-01

    Entanglement lies at the heart of quantum mechanics $-$ as a fundamental tool for testing its deep rift with classical physics, while also providing a key resource for quantum technologies such as quantum computation and cryptography. In 1987 Greenberger, Horne, and Zeilinger realized that the entanglement of more than two particles implies a non-statistical conflict between local realism and quantum mechanics. The resulting predictions were experimentally confirmed by entangling three photons in their polarization. Experimental efforts since have singularly focused on increasing the number of particles entangled, while remaining in a two-dimensional space for each particle. Here we show the experimental generation of the first multi-photon entangled state where both $-$ the number of particles and the number of dimensions $-$ are greater than two. Interestingly, our state exhibits an asymmetric entanglement structure that is only possible when one considers multi-particle entangled states in high dimensions....

  13. Scanning transmission X-ray microscopy probe for in situ mechanism study of graphene-oxide-based resistive random access memory.

    Science.gov (United States)

    Nho, Hyun Woo; Kim, Jong Yun; Wang, Jian; Shin, Hyun-Joon; Choi, Sung-Yool; Yoon, Tae Hyun

    2014-01-01

    Here, an in situ probe for scanning transmission X-ray microscopy (STXM) has been developed and applied to the study of the bipolar resistive switching (BRS) mechanism in an Al/graphene oxide (GO)/Al resistive random access memory (RRAM) device. To perform in situ STXM studies at the C K- and O K-edges, both the RRAM junctions and the I0 junction were fabricated on a single Si3N4 membrane to obtain local XANES spectra at these absorption edges with more delicate I0 normalization. Using this probe combined with the synchrotron-based STXM technique, it was possible to observe unique chemical changes involved in the BRS process of the Al/GO/Al RRAM device. Reversible oxidation and reduction of GO induced by the externally applied bias voltages were observed at the O K-edge XANES feature located at 538.2 eV, which strongly supported the oxygen ion drift model that was recently proposed from ex situ transmission electron microscope studies.

  14. Multiphoton dissociation and ionization of nickelocene

    Science.gov (United States)

    Leutwyler, Samuel; Even, Uzi; Jortner, Joshua

    1981-01-01

    In this paper we report the results of an experimental study of collision-free molecular multiphoton dissociation (MPD) and molecular multiphoton ionization (MPI) of nickelocene (NiC 10H 10), induced by the light of a tunable dye laser in the wavelength region 3750-5200 A. The spectral dependence of the ion signal reveals a multitude of narrow (fwhm from <0.5 cm -1 to 1.5 cm -1) intense peaks superimposed on a very weak background (relative amplitude ratio for peaks/background ≈ 10 3). The sharp resonances in the ion signal are attributed, on the basis of spectroscopic analysis, to two-photon resonant three-photon ionization of Ni(I) and to one-photon resonant three-photon ionization of Ni(I), the Ni(I) being produced by MPD of nickelocene. The ion signal in the spectral range 3750-3950 A reveals enhanced continuous background due to MPI of nickelocene. This ion signal spectra, together with studies of the intensity dependence of the overall (nickelocene MPD) - (Ni(I) MPI) processes, as well as the (nickelocene molecular MPI) reaction, reveal four reactive processes. (a) Two-photon molecular MPI for hω ⩾ 3.10 eV photons. (b) Three-photon molecular MPI for hω = 3.10-2.10 eV. (c) Two-photon MPD at hω ⩾ 2.86 eV. (d) Three-photon MPD for hω = 2.8-1.9 eV. The overall dissociation energy of nickelocene (Nicp 2) to give Ni + 2cp was determined to be 5.76 ± 0.60 eV and the two-photon ionization potential of this molecule is 6.29 ± 0.015 eV. Our results provide dynamic evidence concerning a simultaneous "explosive" photodissociation mechanism of Nicp 2 by process (c) and for the dominating role of the dissociative channel, characterized by a branching ratio of ⩾50 in favor of predissociation over autoionization, for process (c) at 6.3-6.6 eV. The MPD processes (c) and (d) are expected to exhibit intramolecular erosion of phase coherence effects. Processes (c) and (d) are of high efficiency ≈0.01 ions/molecule at saturation exhibited at laser power of ≈ 10

  15. Continuous-variable entanglement via multiphoton catalysis

    Science.gov (United States)

    Hu, Liyun; Liao, Zeyang; Zubairy, M. Suhail

    2017-01-01

    We theoretically investigate the performance of multiphoton catalysis applied on the two-mode squeezed state by examining the entropy of entanglement, logarithmic negativity, Eistein-Podolsky-Rosen (EPR), and Hillery-Zubairy (HZ) correlations, and the fidelity of teleportation. It is found that the entanglement increases with the number of catalysis operations if the squeezing parameter is low initially. Our comparisons show that the HZ correlation presents a better performance than the EPR correlation for detecting the entanglement, and the improvement of HZ correlation definitely results in the improvement of entropy of entanglement rather than negativity; the region of enhanced EPR correlation is a subregion of all other entanglement properties. In addition, we consider the performances of the fidelity by comparing such operations applied before or after the amplitude damping channel. It is shown that the catalysis operation of m =n =1 before the channel presents the best performance in the initial-low squeezing regime. This may provide a useful insight for a long-distance quantum communication.

  16. Multiphoton microscopic imaging of esophagus during the early phase of tumor progression.

    Science.gov (United States)

    Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

    2013-01-01

    Esophageal cancer is one of the most common cancer and leading cause of cancer death worldwide. Multiphoton microscopy (MPM) has become a novel optical tool of choice for imaging tissue architecture and cellular morphology based on two-photon excited fluorescence and second harmonic generation. In this study, we used MPM to image microstructure of human normal esophagus, carcinoma in situ, and early invasive carcinoma in order to investigate the morphological change of tissue structure during the early phase of tumor progression. The diagnostic features such as the appearance of cancerous cells, the absence of the basement membrane were extracted to distinguish between normal and cancerous esophagus tissue. The infiltration depth during tumor progression was determined by the appearance of cancerous cells. The significant change of layer structure between cancerous tissue and normal esophagus was described. We also quantitatively described the differences of morphology between normal and cancerous cells. These results correlated well with the corresponding histological findings. With the advancement of clinically miniaturized MPM and the multi-photon probe, combining MPM with standard endoscopy will therefore allow us to make a real-time in vivo diagnosis of early esophageal cancer at the cellular level. © Wiley Periodicals, Inc.

  17. Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

    Science.gov (United States)

    Lien, Chi-Hsiang; Abrigo, Gerald; Chen, Pei-Hsuan; Chien, Fan-Ching

    2017-02-01

    Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore-labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0±0.7 μm and the shifting distance of the temporal focal plane was less than 0.95 μm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

  18. High-resolution multiphoton optical tomography of tissues: an in vitro and in vivo study

    Science.gov (United States)

    Riemann, Iris; Schenke-Layland, Katja; Ehlers, Alexander; Dimitrow, Enrico; Kaatz, Martin; Elsner, Peter; Martin, Sven; König, Karsten

    2006-03-01

    Multiphoton optical tomography based on NIR (near-infrared) femtosecond laser pulses provides non-invasive optical sectioning of skin with high spatial intracellular resolution and high tissue penetration. The imaging system DermaInspect was used to perform this technology in clinical studies in vivo on patients with suspicious melanoma. Pigmented cell clusters based on non-linear luminescence were clearly distinguished from non-pigmented cells in the epidermis using the autofluorescence of endogenous fluorophores like NAD(P)H, flavins, keratin, elastin, collagen and melanin. Some of the investigated tissues showed differences in the structure of the epidermal layers and the presence of dendritic cells compared to normal skin. Multiphoton laser microscopy was used to visualize extracellular matrix (ECM) structures of native and tissueengineered heart valves. The quality of the resulting 3-D images allowed an exact differentiation between collagenous and elastic fibers. The analysis of heart valve tissues of patients with cardiomyopathy revealed a dramatic loss of its capability to generate SH (second harmonic), indicating a structural deformation of the collagenous fibers, which was virtually impossible to obtain by routine histological or immunohistological staining. These results indicate that NIR femtosecond laser scanning systems can be employed as novel non-invasive optical technology for 3-D resolved ECM component imaging and in vitro and in vivo tissue diagnosis.

  19. Multiphoton microscopic imaging of fibrotic focus in invasive ductal carcinoma of the breast

    Science.gov (United States)

    Chen, Sijia; Nie, Yuting; Lian, Yuane; Wu, Yan; Fu, Fangmeng; Wang, Chuan; Zhuo, Shuangmu; Chen, Jianxin

    2014-11-01

    During the proliferation of breast cancer, the desmoplastic can evoke a fibrosis response by invading healthy tissue. Fibrotic focus (FF) in invasive ductal carcinoma (IDC) of the breast had been reported to be associated with significantly poorer survival rate than IDC without FF. As an important prognosis indicator, it's difficult to obtain the exact fibrotic information from traditional detection method such as mammography. Multiphoton imaging based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) has been recently employed for microscopic examination of unstained tissue. In this study, multiphoton microscopy (MPM) was used to image the fibrotic focus in invasive ductal carcinoma tissue. The morphology and distribution of collagen in fibrotic focus can be demonstrated by the SHG signal. Variation of collagen between IDC with and without FF will be examined and further characterized, which may be greatly related to the metastasis of breast cancer. Our result suggested that the MPM can be efficient in identifying and locating the fibrotic focus in IDC. Combining with the pathology analysis and other detecting methods, MPM owns potential in becoming an advanced histological tool for detecting the fibrotic focus in IDC and collecting prognosis information, which may guide the subsequent surgery option and therapy procedure for patients.

  20. Multiphoton imaging of low grade, high grade intraepithelial neoplasia and intramucosal invasive cancer of esophagus

    Science.gov (United States)

    Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

    2017-04-01

    Esophageal squamous cell carcinoma (ESCC) is devastating because of its aggressive lymphatic spread and clinical course. It is believed to occur through low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and intramucosal invasive cancer (IMC) before transforming to submucosal cancer. In particular, these early lesions (LGIN, HGIN and IMC), which involve no lymph node nor distant metastasis, can be cured by endoscopic treatment. Therefore, early identification of these lesions is important so as to offer a curative endoscopic resection, thus slowing down the development of ESCC. In this work, spectral information and morphological features of the normal esophageal mucosa are first studied. Then, the morphological changes of LGIN, HGIN and IMC are described. Lastly, quantitative parameters are also extracted by calculating the nuclear-to-cytoplasmic ratio of epithelial cells and the pixel density of collagen in the lamina propria. These results show that multiphoton microscopy (MPM) has the ability to identify normal esophageal mucosa, LGIN, HGIN and IMC. With the development of multiphoton endoscope systems for in vivo imaging, combined with a laser ablation system, MPM has the potential to provide immediate pathologic diagnosis and curative treatment of ESCC before the transformation to submucosal cancer in the future.

  1. High-resolution multimodal clinical multiphoton tomography of skin

    Science.gov (United States)

    König, Karsten

    2011-03-01

    This review focuses on multimodal multiphoton tomography based on near infrared femtosecond lasers. Clinical multiphoton tomographs for 3D high-resolution in vivo imaging have been placed into the market several years ago. The second generation of this Prism-Award winning High-Tech skin imaging tool (MPTflex) was introduced in 2010. The same year, the world's first clinical CARS studies have been performed with a hybrid multimodal multiphoton tomograph. In particular, non-fluorescent lipids and water as well as mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen has been imaged with submicron resolution in patients suffering from psoriasis. Further multimodal approaches include the combination of multiphoton tomographs with low-resolution wide-field systems such as ultrasound, optoacoustical, OCT, and dermoscopy systems. Multiphoton tomographs are currently employed in Australia, Japan, the US, and in several European countries for early diagnosis of skin cancer, optimization of treatment strategies, and cosmetic research including long-term testing of sunscreen nanoparticles as well as anti-aging products.

  2. Selective enhancement of resonant multiphoton ionization with strong laser fields

    CERN Document Server

    Li, Min; Luo, Siqiang; Zhou, Yueming; Zhang, Qingbin; Lan, Pengfei; Lu, Peixiang

    2015-01-01

    High-resolution photoelectron momentum distributions of Xe atom ionized by 800-nm linearly polarized laser fields have been traced at intensities from 1.1*1013 W/cm2 to 3.5*1013 W/cm2 using velocity-map imaging techniques. At certain laser intensities, the momentum spectrum exhibits a distinct double-ring structure for low-order above-threshold ionization, which appears to be absent at lower or higher laser intensities. By investigating intensity-resolved photoelectron energy spectrum, we find that this double-ring structure originates from resonant multiphoton ionization involving multiple Rydberg states of atoms. Varying the laser intensity, we can selectively enhance multiphoton excitation of atomic Rydberg populations. The photoelectron angular distributions of multiphoton resonance are also investigated for the low-order above threshold ionization.

  3. Multi-photon creation and single-photon annihilation of electron-positron pairs

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Huayu

    2011-04-27

    In this thesis we study multi-photon e{sup +}e{sup -} pair production in a trident process, and singlephoton e{sup +}e{sup -} pair annihilation in a triple interaction. The pair production is considered in the collision of a relativistic electron with a strong laser beam, and calculated within the theory of laser-dressed quantum electrodynamics. A regularization method is developed systematically for the resonance problem arising in the multi-photon process. Total production rates, positron spectra, and relative contributions of different reaction channels are obtained in various interaction regimes. Our calculation shows good agreement with existing experimental data from SLAC, and adds further insights into the experimental findings. Besides, we study the process in a manifestly nonperturbative domain, whose accessibility to future all-optical experiments based on laser acceleration is shown. In the single-photon e{sup +}e{sup -} pair annihilation, the recoil momentum is absorbed by a spectator particle. Various kinematic configurations of the three incoming particles are examined. Under certain conditions, the emitted photon exhibits distinct angular and polarization distributions which could facilitate the detection of the process. Considering an equilibrium relativistic e{sup +}e{sup -} plasma, it is found that the single-photon process becomes the dominant annihilation channel for plasma temperatures above 3 MeV. Multi-particle correlation effects are therefore essential for the e{sup +}e{sup -} dynamics at very high density. (orig.)

  4. In vivo multiphoton endoscopy of endogenous skin fluorophores

    Science.gov (United States)

    Ehlers, Alexander; Schenkl, Selma; Riemann, Iris; Messerschmidt, Bernhard; Kaatz, Martin; Bückle, Rainer; König, Karsten

    2007-02-01

    Multiphoton tomography offers a painless method to examine patients under natural physiological conditions in vivo. Multiphoton excitation induces a weak autofluorescence of naturally endogenous fluorescent bio-molecules, such as flavines, NAD(P)H, metal-free porphyrines, components of lipofuscin, elastin and keratin. Additionally, collagen can be detected by second harmonic generation (SHG). Due to the nonlinearity, the effects occur only in a very tight focus, where the photon density is high enough. This leads to high axial and lateral resolution of elastin and collagen (SHG) in the dermal layer of human skin are presented.

  5. Observation of detection-dependent multi-photon coherence times

    DEFF Research Database (Denmark)

    Ra, Young-Sik; Tichy, Malte C.; Lim, Hyang-Tag;

    2013-01-01

    in which the multi-photon coherence time, defined by the width of the interference signal, depends on the number of interfering photons and on the measurement scheme chosen to detect the particles. A theoretical analysis reveals that all multi-photon interferences with more than two particles feature...... this dependence, which can be attributed to higher-order effects in the mutual indistinguishability of the particles. As a striking consequence, a single, well-defined many-particle quantum state can exhibit qualitatively different degrees of interference, depending on the chosen observable. Therefore, optimal...... sensitivity in many-particle quantum interferometry can only be achieved by choosing a suitable detection scheme....

  6. Multiphoton autofluorescence spectral analysis for fungus imaging and identification

    Science.gov (United States)

    Lin, Sung-Jan; Tan, Hsin-Yuan; Kuo, Chien-Jui; Wu, Ruei-Jr; Wang, Shiou-Han; Chen, Wei-Liang; Jee, Shiou-Hwa; Dong, Chen-Yuan

    2009-07-01

    We performed multiphoton imaging on fungi of medical significance. Fungal hyphae and spores of Aspergillus flavus, Micosporum gypseum, Micosoprum canis, Trichophyton rubrum, and Trichophyton tonsurans were found to be strongly autofluorescent but generate less prominent second harmonic signal. The cell wall and septum of fungal hyphae can be easily identified by autofluorescence imaging. We found that fungi of various species have distinct autofluorescence characteristics. Our result shows that the combination of multiphoton imaging and spectral analysis can be used to visualize and identify fungal species. This approach may be developed into an effective diagnostic tool for fungal identification.

  7. New insights and system designs for temporally focused multiphoton optogenetics

    Science.gov (United States)

    Mayblum, Tom; Schejter, Adi; Dana, Hod; Shoham, Shy

    2015-03-01

    Temporal focusing (TF) multiphoton systems constitute a powerful solution for cellular resolution optogenetic stimulation and recording in three-dimensional, scattering tissue. Here, we address two fundamental aspects in the design of such systems: first, we examine the design of TF systems with specific optical sectioning by comparatively analyzing previously published results. Next, we develop a solution for obtaining TF in a flexible three-dimensional pattern of cellmatched focal spots. Our solution employs spatio-temporal focusing (SSTF) in a unique optical system design that can be integrated before essentially any multiphoton imaging or stimulation system.

  8. Multiphoton above threshold effects in strong-field fragmentation

    DEFF Research Database (Denmark)

    B Madsen, C; Anis, F; B Madsen, L

    2012-01-01

    We present a study of multiphoton dissociative ionization from molecules. By solving the time-dependent Schrödinger equation for H2+ and projecting the solution onto double continuum scattering states, we observe the correlated electron-nuclear ionization dynamics in detail. We show—for the first...... time—how multiphoton structure prevails as long as one accounts for the energies of all the fragments. Our current work provides a new avenue to analyze strong-field fragmentation that leads to a deeper understanding of the correlated molecular dynamics....

  9. Quantification of scar margin in keloid different from atrophic scar by multiphoton microscopic imaging.

    Science.gov (United States)

    Zhu, Xiaoqin; Zhuo, Shuangmu; Zheng, Liqin; Jiang, Xingshan; Chen, Jianxin; Lin, Bifang

    2011-01-01

    Multiphoton microscopy (MPM) was applied to examine the marginal region at dermis of keloid compared with atrophic scar. High-resolution large-area image showed an obvious boundary at the scar margin and different morphological patterns of elastin and collagen on the two sides, further visualized by the focused three-dimensional images. Content alteration of elastin or collagen between the two sides of boundary was quantified to show significant difference between keloid and atrophic scar. Owing to the raised property of keloid with overproduced collagen on the scar side, the content alteration was positive for elastin and negative for collagen. On the contrary, the content alteration was negative for elastin and positive for collagen in the atrophic scar case due to the atrophic collagen on the scar side. It indicated that examination of the scar margin by MPM may lead a new way to discriminate different types of scars and better understand the scarring mechanisms.

  10. Monitoring UF/sub 6/ photodissociation via laser multiphoton ionization

    Energy Technology Data Exchange (ETDEWEB)

    Stuke, M.; Reisler, H.; Wittig, C.

    1981-08-01

    Laser multiphoton ionization (MPI) is used to detect nascent photoproducts following the UV photodissociation of UF/sub 6/. Sensitivity is high, and there is no measurable background due to the MPI of parent UF/sub 6/. The technique is very well suited for monitoring isotopically selective photodissociation on a ''single shot'' basis.

  11. Time resolved multiphoton excited fluorescence probes in model membranes

    CERN Document Server

    Bai, Y

    2000-01-01

    Using the time-correlated single-photon counting technique, this thesis reports on a time-resolved fluorescence study of several fluorescent probes successfully employed in membrane research. Concentration and temperature effects on fluorescence anisotropy parameters are demonstrated by DPH, p-terphenyl, alpha-NPO and PPO in DPPC lipid bilayers. Fluorescence anisotropy has shown that trans-stilbene and Rhd 800 have a two-site location in membranes. Multiphoton induced fluorescence of DPH, p-terphenyl, alpha-NPO and v-biphenyl in liposomes was measured using 800nm excitation with a femtosecond Ti:Sapphire laser. P-terphenyl, alpha-NPO and v-biphenyl are new probes for membranes. Comparison of one and multiphoton excitation results has demonstrated higher initial anisotropy with multiphoton excitation than with one-photon excitation. The rotational times were identical for one and multiphoton excitation, indicating the absence of significant local heating or sample perturbation. Excimer formation of alpha-NPO w...

  12. Multi-photon entanglement and applications in quantum information

    Energy Technology Data Exchange (ETDEWEB)

    Schmid, Christian I.T.

    2008-05-30

    In this thesis, two new linear optics networks are introduced and their application for several quantum information tasks is presented. Spontaneous parametric down conversion, is used in different configurations to provide the input states for the networks. The first network is a new design of a controlled phase gate which is particularly interesting for applications in multi-photon experiments as it constitutes an improvement of former realizations with respect to stability and reliability. This is explicitly demonstrated by employing the gate in four-photon experiments. In this context, a teleportation and entanglement swapping protocol is performed in which all four Bell states are distinguished by means of the phase gate. A similar type of measurement applied to the subsystem parts of two copies of a quantum state, allows further the direct estimation of the state's entanglement in terms of its concurrence. Finally, starting from two Bell states, the controlled phase gate is applied for the observation of a four photon cluster state. The analysis of the results focuses on measurement based quantum computation, the main usage of cluster states. The second network, fed with the second order emission of non-collinear type ii spontaneous parametric down conversion, constitutes a tunable source of a whole family of states. Up to now the observation of one particular state required one individually tailored setup. With the network introduced here many different states can be obtained within the same arrangement by tuning a single, easily accessible experimental parameter. These states exhibit many useful properties and play a central role in several applications of quantum information. Here, they are used for the solution of a four-player quantum Minority game. It is shown that, by employing four-qubit entanglement, the quantum version of the game clearly outperforms its classical counterpart. Experimental data obtained with both networks are utilized to

  13. Influence of Vacuum Cooling on Escherichia coli O157:H7 Infiltration in Fresh Leafy Greens via a Multiphoton-Imaging Approach

    Science.gov (United States)

    Vonasek, Erica

    2015-01-01

    Microbial pathogen infiltration in fresh leafy greens is a significant food safety risk factor. In various postharvest operations, vacuum cooling is a critical process for maintaining the quality of fresh produce. The overall goal of this study was to evaluate the risk of vacuum cooling-induced infiltration of Escherichia coli O157:H7 into lettuce using multiphoton microscopy. Multiphoton imaging was chosen as the method to locate E. coli O157:H7 within an intact lettuce leaf due to its high spatial resolution, low background fluorescence, and near-infrared (NIR) excitation source compared to those of conventional confocal microscopy. The variables vacuum cooling, surface moisture, and leaf side were evaluated in a three-way factorial study with E. coli O157:H7 on lettuce. A total of 188 image stacks were collected. The images were analyzed for E. coli O157:H7 association with stomata and E. coli O157:H7 infiltration. The quantitative imaging data were statistically analyzed using analysis of variance (ANOVA). The results indicate that the low-moisture condition led to an increased risk of microbial association with stomata (P vacuum cooling levels and moisture levels led to an increased risk of infiltration (P < 0.05). This study also demonstrates the potential of multiphoton imaging for improving sensitivity and resolution of imaging-based measurements of microbial interactions with intact leaf structures, including infiltration. PMID:26475109

  14. Endotoxemia increases the clearance of mPEGylated 5000-MW quantum dots as revealed by multiphoton microvascular imaging.

    Science.gov (United States)

    Bateman, Ryon M; Hodgson, Kevin C; Kohli, Kapil; Knight, Darryl; Walley, Keith R

    2007-01-01

    Imaging the microcirculation is becoming increasingly important in assessing life-threatening disease states. To address this issue in a highly light absorbing and light scattering tissue, we use laser scanning multiphoton microscopy and fluorescent 655-nm 5000-MW methoxy-PEGylated quantum dots to image the functional microcirculation deep in mouse hind limb skeletal muscle. Using this approach, we are able to minimize in vivo background tissue autofluorescence and visualize complete 3-D microvascular units, including feeding arterioles, capillary networks, and collecting venules to depths of 150 to 200 microm. In CD1 mice treated with lipopolysaccharide to model an endotoxemic response to bacterial infection, we find that these quantum dots accumulate at microvascular bifurcations and extravasate from the microcirculation in addition to accumulating in organs (liver, spleen, lung, and kidney). The quantum dots are cleared from the circulation with a first-order elimination rate constant seven times greater than under normal conditions, 1.6+/-0.06 compared to 0.23+/-0.05 h(-1), Pimaging time window. In vitro experiments using TNFalpha treated isolated leukocytes suggest that circulating monocytes (phagocytes) increased their nonspecific uptake of quantum dots when activated. In combination with multiphoton microscopy, quantum dots provide excellent in vivo imaging contrast of deep microvascular structures.

  15. Dual/differential coherent anti-Stokes Raman scattering module for multiphoton microscopes with a femtosecond Ti:sapphire oscillator

    Science.gov (United States)

    Li, Bei; Borri, Paola; Langbein, Wolfgang

    2013-06-01

    In the last decade, coherent anti-Stokes Raman scattering (CARS) microscopy has emerged as a powerful multiphoton imaging technique offering label-free chemical sensitivity and high three-dimensional resolution. However, its widespread application in the life sciences has been hampered by the use of costly pulsed lasers, the existence of a nonresonant background requiring involved technical solutions for its efficient suppression, and the limited acquisition speed of multiplex techniques addressing several vibrational resonances, if improved chemical specificity is needed. We have recently reported a differential CARS technique (D-CARS), which simultaneously measures two vibrational frequencies, enhancing the chemical selectivity and sensitivity without introducing costly hardware, while maintaining fast acquisition. In this study, we demonstrate a compact, fully automated, cost-effective module, which integrates on hardware and software level with a commercial multiphoton microscope based on a single 100 fs Ti:Sapphire oscillator and enables D-CARS microscopy in a user-friendly format for applications in the life sciences.

  16. From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

    Science.gov (United States)

    Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

    2017-02-01

    Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

  17. Ultralow-threshold multiphoton-pumped lasing from colloidal nanoplatelets in solution

    OpenAIRE

    Li, Mingjie; Zhi, Min; Zhu, Hai; Wu, Wen-Ya; Xu, Qing-Hua; Jhon, Mark Hyunpong; Chan, Yinthai

    2015-01-01

    Although multiphoton-pumped lasing from a solution of chromophores is important in the emerging fields of nonlinear optofluidics and bio-photonics, conventionally used organic dyes are often rendered unsuitable because of relatively small multiphoton absorption cross-sections and low photostability. Here, we demonstrate highly photostable, ultralow-threshold multiphoton-pumped biexcitonic lasing from a solution of colloidal CdSe/CdS nanoplatelets within a cuvette-based Fabry–Pérot optical res...

  18. Phase matching alters spatial multiphoton processes in dense atomic ensembles

    CERN Document Server

    Leszczyński, Adam; Wasilewski, Wojciech

    2016-01-01

    Multiphoton processes in dense atomic vapors such as four-wave mixing or coherent blue light generation are typically viewed from single-atom perspective. Here we study the surprisingly important effect of phase matching near two-photon resonances that arises due to spatial extent of the atomic medium within which the multiphoton process occurs. The non-unit refractive index of the atomic vapor may inhibit generation of light in nonlinear processes, significantly shift the efficiency maxima in frequencies and redirect emitted beam. We present these effects on an example of four-wave mixing in dense rubidium vapors in a double-ladder configuration. By deriving a simple theory that takes into account essential spatial properties of the process, we give precise predictions and confirm their validity in the experiment. The model allows us to improve on the geometry of the experiment and engineer more efficient four-wave mixing.

  19. A simple model of multiphoton micromachining in silk hydrogels

    Science.gov (United States)

    Applegate, Matthew B.; Alonzo, Carlo; Georgakoudi, Irene; Kaplan, David L.; Omenetto, Fiorenzo G.

    2016-06-01

    High resolution three-dimensional voids can be directly written into transparent silk fibroin hydrogels using ultrashort pulses of near-infrared (NIR) light. Here, we propose a simple finite-element model that can be used to predict the size and shape of individual features under various exposure conditions. We compare predicted and measured feature volumes for a wide range of parameters and use the model to determine optimum conditions for maximum material removal. The simplicity of the model implies that the mechanism of multiphoton induced void creation in silk is due to direct absorption of light energy rather than diffusion of heat or other photoproducts, and confirms that multiphoton absorption of NIR light in silk is purely a 3-photon process.

  20. Phase matching alters spatial multiphoton processes in dense atomic ensembles.

    Science.gov (United States)

    Leszczyński, Adam; Parniak, Michał; Wasilewski, Wojciech

    2017-01-09

    Multiphoton processes in dense atomic vapors such as four-wave mixing or coherent blue light generation are typically viewed from single-atom perspective. Here we study the surprisingly important effect of phase matching near two-photon resonances that arises due to spatial extent of the atomic medium within which the multiphoton process occurs. The non-unit refractive index of the atomic vapor may inhibit generation of light in nonlinear processes, significantly shift the efficiency maxima in frequencies and redirect emitted beam. We present these effects on an example of four-wave mixing in dense rubidium vapors in a double-ladder configuration. By deriving a simple theory that takes into account essential spatial properties of the process, we give precise predictions and confirm their validity in the experiment. The model allows us to improve on the geometry of the experiment and engineer more efficient four-wave mixing.

  1. Optical Investigation of the Intergrowth Structure and Accessibility of Brønsted Acid Sites in Etched SSZ-13 Zeolite Crystals by Confocal Fluorescence Microscopy

    NARCIS (Netherlands)

    Sommer, L.; Svelle, S.; Lillerud, K.-P.; Stöcker, M; Weckhuysen, B.M.; Olsbye, U.

    2013-01-01

    Template decomposition followed by confocal fluorescence microscopy reveals a tetragonal-pyramidal intergrowth of subunits in micrometer-sized nearly cubic SSZ-13 zeolite crystals. In order to accentuate intergrowth boundaries and defect-rich areas within the individual large zeolite crystals, a tre

  2. Lasers, lenses and light curves : adaptive optics microscopy and peculiar transiting exoplanets

    NARCIS (Netherlands)

    Werkhoven, Theodorus Isaak Mattheus van

    2014-01-01

    In the first part of this thesis, we present an adaptive optics implementation for multi-photon microscopy correcting sample-induced wavefront aberrations using either direct wavefront sensing to run a close-loop adaptive optics system (Chapter 3), or use a model-based sensorless approach to iterati

  3. Relaxation channels of multi-photon excited xenon clusters

    Energy Technology Data Exchange (ETDEWEB)

    Serdobintsev, P. Yu.; Melnikov, A. S. [Institute of Nanobiotechnologies, Peter the Great St.Petersburg Polytechnic University, Saint Petersburg 195251 (Russian Federation); Department of Physics, St. Petersburg State University, Saint Petersburg 198904 (Russian Federation); Rakcheeva, L. P., E-mail: lida@nanobio.spbstu.ru; Murashov, S. V.; Khodorkovskii, M. A. [Institute of Nanobiotechnologies, Peter the Great St.Petersburg Polytechnic University, Saint Petersburg 195251 (Russian Federation); Lyubchik, S. [REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica 2829-516 (Portugal); Timofeev, N. A.; Pastor, A. A. [Department of Physics, St. Petersburg State University, Saint Petersburg 198904 (Russian Federation)

    2015-09-21

    The relaxation processes of the xenon clusters subjected to multi-photon excitation by laser radiation with quantum energies significantly lower than the thresholds of excitation of atoms and ionization of clusters were studied. Results obtained by means of the photoelectron spectroscopy method showed that desorption processes of excited atoms play a significant role in the decay of two-photon excited xenon clusters. A number of excited states of xenon atoms formed during this process were discovered and identified.

  4. Multi-photon excitation in ZnO materials

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A brief introduction on the advance in the fabrication technology of ZnO materials was given.Related research on the multi-photon excitation processes in several kinds of ZnO materials under intense pump conditions by fs pulses were reviewed.Stimulated emission properties in ZnO microtubes and nanowires have also been dealt with.Possible nonlinear effects that emerged under the extremely intense field were discussed.

  5. Dynamics of multi-photon processes in semiconductor heterostructures

    OpenAIRE

    Marti, Daniel

    2003-01-01

    The present work is devoted to the study of the dynamics of multi-photon processes in semiconductor heterostructures. A time-dependent description is important for understanding in detail the transient response of semiconductors excited by ultrashort optical pulses. In the first part of this thesis, we set up a phenomenological model based on rate equations, in order to investigate the possibility of measuring degenerate two-photon gain in a semiconductor microcavity. The amplification predic...

  6. Multiphoton imaging in the detection of malignancy in the oesophagus

    Science.gov (United States)

    Kendall, Catherine A.; McCloskey, Karen; Stone, Nicholas; Barr, Hugh; Greenhalgh, Douglas

    2003-10-01

    A preliminary investigation of tissue autofluorescence and uptake of the photosensitiser protoporphyrin IX (PpIX) has been investigated using multiphoton imaging of excised oesophageal tissue. The technique has indicated that changes in collagen structure may be a potential marker for high-grade dysplasia and adenocarcinoma. Changes in the localisation of PpIX with the development of malignancy in oesophageal tissue were also visualised.

  7. 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.

  8. Record Multiphoton Absorption Cross-Sections by Dendrimer Organometalation.

    Science.gov (United States)

    Simpson, Peter V; Watson, Laurance A; Barlow, Adam; Wang, Genmiao; Cifuentes, Marie P; Humphrey, Mark G

    2016-02-12

    Large increases in molecular two-photon absorption, the onset of measurable molecular three-photon absorption, and record molecular four-photon absorption in organic π-delocalizable frameworks are achieved by incorporation of bis(diphosphine)ruthenium units with alkynyl linkages. The resultant ruthenium alkynyl-containing dendrimers exhibit strong multiphoton absorption activity through the biological and telecommunications windows in the near-infrared region. The ligated ruthenium units significantly enhance solubility and introduce fully reversible redox switchability to the optical properties. Increasing the ruthenium content leads to substantial increases in multiphoton absorption properties without any loss of optical transparency. This significant improvement in multiphoton absorption performance by incorporation of the organometallic units into the organic π-framework is maintained when the relevant parameters are scaled by molecular weights or number of delocalizable π-electrons. The four-photon absorption cross-section of the most metal-rich dendrimer is an order of magnitude greater than the previous record value.

  9. Electron Microscopy.

    Science.gov (United States)

    Beer, Michael

    1980-01-01

    Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

  10. Electron Microscopy.

    Science.gov (United States)

    Beer, Michael

    1980-01-01

    Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

  11. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

    Science.gov (United States)

    Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

    2016-05-01

    High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

  12. Multi-Photon Fluorescence Spectroscopy of Fluorescent Bio-Probes and Bio-Molecules

    Science.gov (United States)

    2000-07-01

    the set-up of a multi-photon fluorescence microscope. The information can also be useful in the detection of multi-photon fluorescence in bio -chip...technology. In addition, we have investigated a few highly fluorescent bio -molecules commonly found in plant cells.

  13. Anomalous multiphoton photoelectric effect in ultrashort time scales.

    Science.gov (United States)

    Kupersztych, J; Raynaud, M

    2005-09-30

    In a multiphoton photoelectric process, an electron needs to absorb a given number of photons to escape the surface of a metal. It is shown for the first time that this number is not a constant depending only on the characteristics of the metal and light, but varies with the interaction duration in ultrashort time scales. The phenomenon occurs when electromagnetic energy is transferred, via ultrafast excitation of electron collective modes, to conduction electrons in a duration less than the electron energy damping time. It manifests itself through a dramatic increase of electron production.

  14. The nature of multiphoton fluorescence from red blood cells

    Science.gov (United States)

    Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

    2016-03-01

    We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

  15. Quantum radiation reaction effects in multiphoton Compton scattering.

    Science.gov (United States)

    Di Piazza, A; Hatsagortsyan, K Z; Keitel, C H

    2010-11-26

    Radiation reaction effects in the interaction of an electron and a strong laser field are investigated in the realm of quantum electrodynamics. We identify the quantum radiation reaction with the multiple photon recoils experienced by the laser-driven electron due to consecutive incoherent photon emissions. After determining a quantum radiation dominated regime, we demonstrate how in this regime quantum signatures of the radiation reaction strongly affect multiphoton Compton scattering spectra and that they could be measurable in principle with presently available laser technology.

  16. Resilience of multi-photon entanglement under losses

    CERN Document Server

    Durkin, G A; Eisert, J; Bouwmeester, D

    2004-01-01

    We analyze the resilience under photon loss of the bi-partite entanglement present in multi-photon states produced by parametric down-conversion. The quantification of the entanglement is made possible by a symmetry of the states that persists even under polarization-independent losses. We examine the approach of the states to the set of states with a positive partial transpose as losses increase, and calculate the relative entropy of entanglement. We find that some bi-partite distillable entanglement persists for arbitrarily high losses.

  17. Circular Dichroism in Multiphoton Ionization of Resonantly Excited He+ Ions

    Science.gov (United States)

    Ilchen, M.; Douguet, N.; Mazza, T.; Rafipoor, A. J.; Callegari, C.; Finetti, P.; Plekan, O.; Prince, K. C.; Demidovich, A.; Grazioli, C.; Avaldi, L.; Bolognesi, P.; Coreno, M.; Di Fraia, M.; Devetta, M.; Ovcharenko, Y.; Düsterer, S.; Ueda, K.; Bartschat, K.; Grum-Grzhimailo, A. N.; Bozhevolnov, A. V.; Kazansky, A. K.; Kabachnik, N. M.; Meyer, M.

    2017-01-01

    Intense, circularly polarized extreme-ultraviolet and near-infrared (NIR) laser pulses are combined to double ionize atomic helium via the oriented intermediate He+(3 p ) resonance state. Applying angle-resolved electron spectroscopy, we find a large photon helicity dependence of the spectrum and the angular distribution of the electrons ejected from the resonance by NIR multiphoton absorption. The measured circular dichroism is unexpectedly found to vary strongly as a function of the NIR intensity. The experimental data are well described by theoretical modeling and possible mechanisms are discussed.

  18. Observation of detection-dependent multi-photon coherence times

    DEFF Research Database (Denmark)

    Ra, Young-Sik; Tichy, Malte C.; Lim, Hyang-Tag

    2013-01-01

    The coherence time constitutes one of the most critical parameters that determines whether or not interference is observed in an experiment. For photons, it is traditionally determined by the effective spectral bandwidth of the photon. Here we report on multi-photon interference experiments...... this dependence, which can be attributed to higher-order effects in the mutual indistinguishability of the particles. As a striking consequence, a single, well-defined many-particle quantum state can exhibit qualitatively different degrees of interference, depending on the chosen observable. Therefore, optimal...

  19. Analytical Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    2006-06-01

    In the Analytical Microscopy group, within the National Center for Photovoltaic's Measurements and Characterization Division, we combine two complementary areas of analytical microscopy--electron microscopy and proximal-probe techniques--and use a variety of state-of-the-art imaging and analytical tools. We also design and build custom instrumentation and develop novel techniques that provide unique capabilities for studying materials and devices. In our work, we collaborate with you to solve materials- and device-related R&D problems. This sheet summarizes the uses and features of four major tools: transmission electron microscopy, scanning electron microscopy, the dual-beam focused-ion-beam workstation, and scanning probe microscopy.

  20. 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.

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

    Science.gov (United States)

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

    2008-01-01

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

  2. Multiphoton microscopic imaging of histological sections without hematoxylin and eosin staining differentiates carcinoma in situ lesion from normal oesophagus

    Science.gov (United States)

    Chen, Jianxin; Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Jiang, Xingshan

    2013-10-01

    Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffin embedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

  3. Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy.

    Science.gov (United States)

    Winter, Peter W; Chandris, Panagiotis; Fischer, Robert S; Wu, Yicong; Waterman, Clare M; Shroff, Hari

    2015-02-23

    Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.

  4. Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy.

    Science.gov (United States)

    Anisha Thayil, K N; Gualda, E J; Psilodimitrakopoulos, S; Cormack, I G; Amat-Roldán, I; Mathew, M; Artigas, D; Loza-Alvarez, P

    2008-04-01

    We report a simple methodology to provide complete pulse characterization at the sample plane of a two-photon excited fluorescence (TPEF) microscope. This is achieved by using backward propagating second-harmonic generation (SHG) from starch granules. Without any modification to the microscope, SHG-autocorrelation traces were obtained by using a single starch granule that was placed alongside the biological specimen being imaged. A spectrally resolved SHG autocorrelation was acquired by placing a spectrometer at the output port of the microscope. Complete in situ pulse information is then directly retrieved in an analytical way using the measurement of electric filed by interferometric spectral trace observation (MEFISTO) technique.

  5. Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence.

    Science.gov (United States)

    Sinefeld, David; Paudel, Hari P; Ouzounov, Dimitre G; Bifano, Thomas G; Xu, Chris

    2015-11-30

    We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity.

  6. Multiphoton ionization and stabilization of helium in superintense xuv fields

    CERN Document Server

    Sørngård, S A; Nepstad, R; Førre, M

    2011-01-01

    Multiphoton ionization of helium is investigated in the superintense field regime, with particular emphasis on the role of the electron-electron interaction in the ionization and stabilization dynamics. To accomplish this, we solve ab initio the time-dependent Schr\\"odinger equation with the full electron-electron interaction included. By comparing the ionization yields obtained from the full calculations with corresponding results of an independent-electron model, we come to the somewhat counterintuitive conclusion that the single-particle picture breaks down at superstrong field strengths. We explain this finding from the perspective of the so-called Kramers-Henneberger frame, the reference frame of a free (classical) electron moving in the field. The breakdown is tied to the fact that shake-up and shake-off processes cannot be properly accounted for in commonly used independent-electron models. In addition, we see evidence of a change from the multiphoton to the shake-off ionization regime in the energy di...

  7. The effect of radial polarization in multiphoton lithography

    Science.gov (United States)

    Lin, Le; Zheng, Mei-Ling; Dong, Xian-Zi; Duan, Xuan-Ming; Zhao, Zhen-Sheng

    2015-10-01

    Considering the axially symmetric polarization and intensity distribution, radially polarized (RP) laser beam has comparatively higher axial component of electric field and smaller size of focal spot compared to linearly polarized (LP) laser. In this study, the effect of radial polarization on multiphoton fabrication has been studied, and polymer spots and lines are chosen as the study objects of 2D micro/nano structures of multiphoton lithography. These structures were fabricated with IP-L, a commercial negative photoresist, by RP fs-pulse laser beam which was tightly focused by an objective lens with high numerical aperture. Multiple experimental conditions, such as fabrication power, exposure time and scanning velocity, were verified in order to observe the structural variation of these polymer structures. On the basis of measurement from images of the scanning electron microscope, the transverse and longitudinal sizes of polymer spots and lines could be analyzed, and the relationship between the aspect ratio (AR) and the above experimental conditions could be acquired. The statistical results agree with our predictions that the RP laser beam can significantly reduce the AR, and the AR in RP laser fabrication has little correlation with conditions besides fabrication power, such as exposure time and scanning velocity.

  8. Clinical studies of pigmented lesions in human skin by using a multiphoton tomograph

    Science.gov (United States)

    Balu, Mihaela; Kelly, Kristen M.; Zachary, Christopher B.; Harris, Ronald M.; Krasieva, Tatiana B.; König, Karsten; Tromberg, Bruce J.

    2013-02-01

    In vivo imaging of pigmented lesions in human skin was performed with a clinical multiphoton microscopy (MPM)-based tomograph (MPTflex, JenLab, Germany). Two-photon excited fluorescence was used for visualizing endogenous fluorophores such as NADH/FAD, keratin, melanin in the epidermal cells and elastin fibers in the dermis. Collagen fibers were imaged by second harmonic generation. Our study involved in vivo imaging of benign melanocytic nevi, atypical nevi and melanoma. The goal of this preliminary study was to identify in vivo the characteristic features and their frequency in pigmented lesions at different stages (benign, atypical and malignant) and to evaluate the ability of in vivo MPM to distinguish atypical nevi from melanoma. Comparison with histopathology was performed for the biopsied lesions. Benign melanocytic nevi were characterized by the presence of nevus cell nests at the epidermal-dermal junction. In atypical nevi, features such as lentiginous hyperplasia, acanthosis and architectural disorder were imaged. Cytological atypia was present in all the melanoma lesions imaged, showing the strongest correlation with malignancy. The MPM images demonstrated very good correlation with corresponding histological images, suggesting that MPM could be a promising tool for in vivo non-invasive pigmented lesion diagnosis, particularly distinguishing atypical nevi from melanoma.

  9. A phasor approach analysis of multiphoton FLIM measurements of three-dimensional cell culture models

    Science.gov (United States)

    Lakner, P. H.; Möller, Y.; Olayioye, M. A.; Brucker, S. Y.; Schenke-Layland, K.; Monaghan, M. G.

    2016-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is a useful approach to obtain information regarding the endogenous fluorophores present in biological samples. The concise evaluation of FLIM data requires the use of robust mathematical algorithms. In this study, we developed a user-friendly phasor approach for analyzing FLIM data and applied this method on three-dimensional (3D) Caco-2 models of polarized epithelial luminal cysts in a supporting extracellular matrix environment. These Caco-2 based models were treated with epidermal growth factor (EGF), to stimulate proliferation in order to determine if FLIM could detect such a change in cell behavior. Autofluorescence from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in luminal Caco-2 cysts was stimulated by 2-photon laser excitation. Using a phasor approach, the lifetimes of involved fluorophores and their contribution were calculated with fewer initial assumptions when compared to multiexponential decay fitting. The phasor approach simplified FLIM data analysis, making it an interesting tool for non-experts in numerical data analysis. We observed that an increased proliferation stimulated by EGF led to a significant shift in fluorescence lifetime and a significant alteration of the phasor data shape. Our data demonstrates that multiphoton FLIM analysis with the phasor approach is a suitable method for the non-invasive analysis of 3D in vitro cell culture models qualifying this method for monitoring basic cellular features and the effect of external factors.

  10. Optical-resolution photoacoustic microscopy of amyloid-β deposits in vivo

    Science.gov (United States)

    Hu, Song; Yan, Ping; Maslov, Konstantin; Lee, Jin-Moo; Wang, Lihong V.

    2010-02-01

    Advances in high-resolution imaging have permitted microscopic observations within the brains of living animals. Applied to Alzheimer's disease (AD) mouse models, multiphoton microscopy has opened a new window to study the real-time appearance and growth of amyloid plaques. Here, we report an alternative technology-optical-resolution photoacoustic microscopy (OR-PAM)-for in vivo imaging of amyloid plaques in a transgenic AD mouse model. In vivo validation using multiphoton microscopy shows that OR-PAM has sufficient sensitivity and spatial resolution to identify amyloid plaques in living brains. In addition, with dual-wavelength OR-PAM, the three-dimensional morphology of amyloid plaques and the surrounding microvasculature are imaged simultaneously through a cranial window. In vivo transcranial OR-PAM imaging of amyloid plaques is highly likely once the imaging parameters are optimized.

  11. Novel microfabrication stage allowing for one-photon and multi-photon light assisted molecular immobilization and for multi-photon microscope

    Science.gov (United States)

    Gonçalves, Odete; Snider, Scott; Zadoyan, Ruben; Nguyen, Quoc-Thang; Vorum, Henrik; Petersen, Steffen B.; Neves-Petersen, Maria Teresa

    2017-02-01

    Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.

  12. Generating Nanostructures with Multiphoton Absorption Polymerization using Optical Trap Assisted Nanopatterning

    Science.gov (United States)

    Tsai, Yu-Cheng; Leitz, Karl-Heinz; Fardel, Romain; Schmidt, Michael; Arnold, Craig B.

    The need to generate sub 100 nm features is of interest for a variety of applications including optics, optoelectronics, and plasmonics. To address this requirement, several advanced optical lithography techniques have been developed based on either multiphoton absorption polymerization or near-field effects. In this paper, we combine strengths from multiphoton absorption and near field using optical trap assisted nanopatterning (OTAN). A Gaussian beam is used to position a microsphere in a polymer precursor fluid near a substrate. An ultrafast laser is focused by that microsphere to induce multiphoton polymerization in the near field, leading additive direct-write nanoscale processing.

  13. Hamiltonian models of multiphoton processes and four--photon squeezed states via nonlinear canonical transformations

    CERN Document Server

    De Siena, S; Illuminati, F; Siena, Silvio De; Lisi, Antonio Di; Illuminati, Fabrizio

    2002-01-01

    We introduce nonlinear canonical transformations that yield effective Hamiltonians of multiphoton down conversion processes, and we define the associated non-Gaussian multiphoton squeezed states as the coherent states of the multiphoton Hamiltonians. We study in detail the four-photon processes and the associated non-Gaussian four-photon squeezed states. The realization of squeezing, the behavior of the field statistics, and the structure of the phase space distributions show that these states realize a natural four-photon generalization of the two-photon squeezed states.

  14. The cross-over from tunnelling to multiphoton ionization of atoms

    CERN Document Server

    Klaiber, Michael

    2016-01-01

    We present a theory illuminating the cross-over from strong-field tunnelling ionization to weak-field multiphoton ionization in the interaction of a classical laser field with a hydrogen atom. A simple formula is derived in which the ionization amplitude appears as a product of two separate amplitudes. The first describes the initial polarization of the atom by virtual multiphoton absorption and the second the subsequent tunnelling out of the polarized atom. Tunnelling directly from the ground state and multiphoton absorption without tunnelling appear naturally as the limits of the theory.

  15. Resonance Enhanced Multi-photon Spectroscopy of DNA

    Science.gov (United States)

    Ligare, Marshall Robert

    For over 50 years DNA has been studied to better understand its connection to life and evolution. These past experiments have led to our understanding of its structure and function in the biological environment but the interaction of DNA with UV radiation at the molecular level is still not very well understood. Unique mechanisms in nucleobase chromaphores protect us from adverse chemical reactions after UV absorption. Studying these processes can help develop theories for prebiotic chemistry and the possibility of alternative forms of DNA. Using resonance enhanced multi-photon spectroscopic techniques in the gas phase allow for the structure and dynamics of individual nucleobases to be studied in detail. Experiments studying different levels of structure/complexity with relation to their biological function are presented. Resonant IR multiphoton dissociation spectroscopy in conjunction with molecular mechanics and DFT calculations are used to determine gas phase structures of anionic nucleotide clusters. A comparison of the identified structures with known biological function shows how the hydrogen bonding of the nucleotides and their clusters free of solvent create favorable structures for quick incorporation into enzymes such as DNA polymerase. Resonance enhanced multi-photon ionization (REMPI) spectroscopy techniques such as resonant two photon ionization (R2PI) and IR-UV double resonance are used to further elucidate the structure and excited state dynamics of the bare nucleobases thymine and uracil. Both exhibit long lived excited electronic states that have been implicated in DNA photolesions which can ultimately lead to melanoma and carcinoma. Our experimental data in comparison with many quantum chemical calculations suggest a new picture for the dynamics of thymine and uracil in the gas phase. A high probability of UV absorption from a vibrationally hot ground state to the excited electronic state shows that the stability of thymine and uracil comes from

  16. Adaptive nonlinear microscopy for whole tissue imaging

    Science.gov (United States)

    Müllenbroich, M. Caroline; McGhee, Ewan J.; Wright, Amanda J.; Anderson, Kurt I.; Mathieson, Keith

    2013-02-01

    Nonlinear microscopy is capable of imaging biological tissue non-invasively with sub-cellular resolution in three dimensions. For efficient multiphoton signal generation, it is necessary to focus high power, ultra-fast laser pulses into a volume of femtolitres. Aberrations introduced either by the system's optical setup or the sample under investigation cause a broadening of the diffraction limited focal spot which leads to loss of image intensity and resolution. Adaptive optics provides a means to compensate for these aberrations and is capable of restoring resolution and signal strength when imaging at depth. We describe the use of a micro-electro-mechanical systems (MEMS) deformable membrane mirror in a multiphoton adaptive microscope. The aberration correction is determined in a wavefront sensorless approach by rapidly altering the mirror shape with a random search algorithm until the fluorescence or second harmonic signal intensity is improved. We demonstrate the benefits of wavefront correction in a wide-variety of samples, including urea crystals, convallaria and organotypic tissue cultures. We show how the optimization algorithm can be adjusted, for example by including a bleaching compensation, to allow the user to switch between different imaging modalities, producing a versatile approach to aberration correction.

  17. Evaluation of Elastin/Collagen Content in Human Dermis in-Vivo by Multiphoton Tomography—Variation with Depth and Correlation with Aging

    Directory of Open Access Journals (Sweden)

    Jean-Christophe Pittet

    2014-08-01

    Full Text Available The aim of this study was to evaluate the influence of the depth of the dermis on the measured collagen and elastin levels and to establish the correlation between the amount of these two extracellular matrix (ECM components and age. Multiphoton Microscopy (MPM that measures the autofluorescence (AF and second harmonic generation (SHG was used to quantify the levels of elastin and collagen and to determine the SAAID (SHG-to-AF Aging Index of Dermis at two different skin depths. A 50 MHz ultrasound scanner was used for the calculation of the Sub Epidermal Non Echogenic Band (SENEB. The measurements of the skin mechanical properties were done with a cutometer. All measurements were performed on two groups of 30 healthy female volunteers. The MPM showed a decrease of the quantity of collagen and elastin as a function of depth of the dermis as well as age. The SAAID was lower for the older skin in the deeper dermis. Ultrasound imaging revealed a significant decrease of SENEB as a function of aging. The mechanical properties confirmed a loss of cutaneous elasticity and firmness. Although multiphoton microscopy is a powerful technique to study the characteristics of the dermis and its age-related damage, the location of the measurements (depth remains very important for the validation of these variations. These variations do not seem to be homogeneous according to the part of the dermis that is studied.

  18. Properties of Differential Scattering Section Based on Multi-photon Nonlinear Compton Effect

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Properties of damping electrons in collision with photons based on multi-photon nonlinear Compton effect are investigated. The expressions of the differential scattering section are derived. Several useful conclusions are drawn.

  19. Structure of multiphoton quantum optics. I. Canonical formalism and homodyne squeezed states

    CERN Document Server

    Dell'Anno, F; Illuminati, F; Anno, Fabio Dell'; Siena, Silvio De; Illuminati, Fabrizio

    2004-01-01

    We introduce a formalism of nonlinear canonical transformations for general systems of multiphoton quantum optics. For single-mode systems the transformations depend on a tunable free parameter, the homodyne local oscillator angle; for n-mode systems they depend on n heterodyne mixing angles. The canonical formalism realizes nontrivial mixings of pairs of conjugate quadratures of the electromagnetic field in terms of homodyne variables for single-mode systems; and in terms of heterodyne variables for multimode systems. In the first instance the transformations yield nonquadratic model Hamiltonians of degenerate multiphoton processes and define a class of non Gaussian, nonclassical multiphoton states that exhibit properties of coherence and squeezing. We show that such homodyne multiphoton squeezed states are generated by unitary operators with a nonlinear time evolution that realizes the homodyne mixing of a pair of conjugate quadratures. Tuning of the local oscillator angle allows to vary at will the statist...

  20. Multiphoton ionization of jet-cooled nickelocene with tunable nanosecond laser pulses

    Science.gov (United States)

    Ketkov, Sergey Yu.; Selzle, Heinrich L.; Schlag, Edward W.; Titova, Sofia N.

    2003-08-01

    Efficient multiphoton ionization of nickelocene molecules in a supersonically cooled molecular beam has been performed for the first time with a nanosecond tunable dye laser operating in the 35,000-cm -1 region which corresponds to the lowest Rydberg transition observed in the one-photon absorption spectrum. The time-of-flight mass spectra obtained show strong signals of intact molecular ions Cp 2Ni + (Cp=η 5-C 5H 5) and weaker peaks of fragment ions CpNi +. The conditions have been found for generation of Cp 2Ni + as the only ionic product of multiphoton excitation. The ion signal dependence on the laser intensity and the resonance-enhanced multiphoton ionization spectrum measured at the mass of Cp 2Ni + testify for saturation of absorption and/or ionization steps at the laser pulse intensities used (2-6 MW cm -2). Possible mechanisms of multiphoton processes resulting in formation of the ions observed are discussed.

  1. 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.

  2. Multi-Photon Absorption in Metal-Organic Frameworks.

    Science.gov (United States)

    Medishetty, Raghavender; Nemec, Lydia; Nalla, Venkatram; Henke, Sebastian; Samoc, Marek; Reuter, Karsten; Fischer, Roland A

    2017-09-12

    Multi-photon absorption (MPA) is among the most prominent nonlinear optical (NLO) effects and has applications, for example in telecommunications, defense, photonics and bio-medicines. Established MPA materials include dyes, quantum dots, organometallics and conjugated polymers, most often dispersed in solution. We demonstrate how metal-organic frameworks (MOFs), a novel NLO solid-state materials class, can be designed for exceptionally strong MPA behavior. MOFs consisting of zirconium- and hafnium-oxo-clusters and featuring a chromophore linker based on the tetraphenylethene (TPE) molecule exhibit record high two-photon absorption (2PA) cross section values, up to 3600 GM. The unique modular building-block principle of MOFs allows enhancing and optimizing their MPA properties in a theory guided approach by combining tailored charge polarization, conformational strain, three-dimensional arrangement and alignment of the chromophore linkers in the crystal. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Monitoring wound healing by multiphoton tomography/endoscopy

    Science.gov (United States)

    König, Karsten; Weinigel, Martin; Bückle, Rainer; Kaatz, Martin; Hipler, Christina; Zens, Katharina; Schneider, Stefan W.; Huck, Volker

    2015-02-01

    Certified clinical multiphoton tomographs are employed to perform rapid label-free high-resolution in vivo histology. Novel tomographs include a flexible 360° scan head attached to a mechano-optical arm for autofluorescence and SHG imaging as well as rigid two-photon GRIN microendoscope. Mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged with submicron resolution in human skin. The system was employed to study the healing of chronic wounds (venous leg ulcer) and acute wounds (curettage of actinic or seborrheic keratosis) on a subcellular level. Furthermore, a flexible sterile foil as interface between wound and focusing optic was tested.

  4. Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

    Science.gov (United States)

    Uchugonova, Aisada

    2017-06-01

    The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

  5. Intravital multiphoton photoconversion with a cell membrane dye.

    Science.gov (United States)

    Turcotte, Raphaël; Wu, Juwell W; Lin, Charles P

    2017-02-01

    Photoconversion, an irreversible shift in a fluorophore emission spectrum after light exposure, is a powerful tool for marking cellular and subcellular compartments and tracking their dynamics in vivo. This paper reports on the photoconversion properties of Di-8-ANEPPS, a commercially available membrane dye. When illuminated with near-infrared femtosecond laser pulses, Di-8-ANEPPS undergoes multiphoton photoconversion as indicated by the supralinear dependence of the conversion rate ρpc on the incident power (ρpc∝Iexc2.27), and by the ability to photoconvert a thin optical section in a three-dimensional matrix. The characteristic emission spectrum changed from red to blue, and ratiometric analysis on single cells in vitro revealed a 65-fold increase in the blue to red wavelength ratio after photoconversion. The spectral shift is preserved in vivo for hours, making Di-8-ANEPPS a useful dye for intravital cell marking and tracking applications.

  6. Features of multiphoton-stimulated bremsstrahlung in a quantized field

    Science.gov (United States)

    Burenkov, Ivan A.; Tikhonova, Olga V.

    2010-12-01

    The process of absorption and emission of external field quanta by a free electron during the scattering on a potential centre is investigated in the case of interaction with a quantized electromagnetic field. The analytical expression for differential cross-sections and probabilities of different multiphoton channels are obtained. We demonstrate that in the case of a non-classical 'squeezed vacuum' initial field state the probability for the electron to absorb a large number of photons appears to be larger by several orders of magnitude in comparison to the classical field and leads to the formation of the high-energy plateau in the electron energy spectrum. The generalization of the Marcuse effect to the case of the quantized field is worked out. The total probability of energy absorption by electron from the non-classical light is analysed.

  7. Features of multiphoton-stimulated bremsstrahlung in a quantized field

    Energy Technology Data Exchange (ETDEWEB)

    Burenkov, Ivan A; Tikhonova, Olga V, E-mail: ovtikhonova@mail.r [Institute of Nuclear Physics, Moscow State University, Leninskie Gory 1, Moscow, 119991 (Russian Federation)

    2010-12-14

    The process of absorption and emission of external field quanta by a free electron during the scattering on a potential centre is investigated in the case of interaction with a quantized electromagnetic field. The analytical expression for differential cross-sections and probabilities of different multiphoton channels are obtained. We demonstrate that in the case of a non-classical 'squeezed vacuum' initial field state the probability for the electron to absorb a large number of photons appears to be larger by several orders of magnitude in comparison to the classical field and leads to the formation of the high-energy plateau in the electron energy spectrum. The generalization of the Marcuse effect to the case of the quantized field is worked out. The total probability of energy absorption by electron from the non-classical light is analysed.

  8. Dynamics of multi-photon photoluminescence in gold nanoantennas

    CERN Document Server

    Biagioni, P; Huang, J -S; Kern, J; Duò, L; Hecht, B; Finazzi, M; Cerullo, G

    2011-01-01

    We perform a combined study of the degree of nonlinearity and the temporal dynamics of multiphoton-excited photoluminescence (MPPL) in gold nanoantennas. At variance with standard gold two-photon photoluminescence (TPPL), the large photoluminescence enhancement in resonant nanostructures is sometimes found to involve more than two absorbed photons per emitted photon. By two-pulse correlation measurements of TPPL and MPPL we are able to address the particular dynamics of these mechanisms. Our data give direct evidence for the two-step model that has been proposed for gold TPPL and suggest that MPPL is characterized by very similar dynamics. Interestingly, for resonant antennas we observe a reduced MPPL relaxation time compared to off-resonant antennas.

  9. Search for Multiphoton Signatures of a Higgs Boson

    Energy Technology Data Exchange (ETDEWEB)

    Atramentov, Oleksiy Vladimirovich [Iowa State Univ., Ames, IA (United States)

    2006-07-01

    In this thesis we describe a search for a fermiophobic Higgs boson in 3γ+X events. The study has been performed on 0.83 fb-1 of data collected with the D0 detector that resides at one of the interaction regions of the Tevatron collider, the world's highest energy accelerator. This study was motivated by a fairly recent phenomenological paper [33] where it was noticed that in certain class of models (2HDM Type I and THM) the multi-photon final states like this one become detectable at the luminosity that has been collected by the D0 experiment by 2006. The mechanism that permits such final state becomes available when the conventional higgs production mechanism (higgs strahlung) are suppressed. This leads to the fact that Higgs boson with mass (mhf < 90 GeV/c2) lower than the current limit has not been excluded.

  10. In vivo multiphoton tomography in skin aging studies

    Science.gov (United States)

    König, Karsten; Bückle, Rainer; Weinigel, Martin; Köhler, Johannes; Elsner, Peter; Kaatz, Martin

    2009-02-01

    High-resolution clinical multiphoton tomography based on the femtosecond laser system DermaInspect has been performed on hundreds of patients and volunteers in Australia, Asia, and Europe. The system enables the in vivo detection of the elastin and the collagen network as well as the imaging of melanin clusters in aging spots. The epidermis-dermis junction can be detected with submicron resolution. One major applications of this novel HighTech imaging tool is the determination of the skin aging index SAAID as well as the study of the effects of anti-aging products. In particular, the stimulated biosynthesis of collagen can be investigated over long periods of time. The system with its sub-500 nm lateral resolution is able to image age-related modifications of the extracellular matrix on the level of a single elastin fiber.

  11. Quadrature-dependent Bogoliubov transformations and multiphoton squeezed states

    CERN Document Server

    De Siena, S; Illuminati, F; Siena, Silvio De; Lisi, Antonio Di; Illuminati, Fabrizio

    2001-01-01

    We introduce a linear, canonical transformation of the fundamental single--mode field operators $a$ and $a^{\\dagger}$ that generalizes the linear Bogoliubov transformation familiar in the construction of the harmonic oscillator squeezed states. This generalization is obtained by adding to the linear transformation a nonlinear function of any of the fundamental quadrature operators $X_{1}$ and $X_{2}$, making the original Bogoliubov transformation quadrature--dependent. Remarkably, the conditions of canonicity do not impose any constraint on the form of the nonlinear function, and lead to a set of nontrivial algebraic relations between the $c$--number coefficients of the transformation. We examine in detail the structure and the properties of the new quantum states defined as eigenvectors of the transformed annihilation operator $b$. These eigenvectors define a class of multiphoton squeezed states. The structure of the uncertainty products and of the quasiprobability distributions in phase space shows that bes...

  12. Multiphoton processes in KrF-laser induced gas breakdown

    Energy Technology Data Exchange (ETDEWEB)

    Poprawe, R.; Herziger, G.

    1986-05-01

    Ionization by multiphoton processes is the dominant generation mechanism of first electrons in the UV-laser-induced gas breakdown. A strongly simplified analytical model for the ion generation rate is used to give an estimate of the threshold intensity I/sub TH/. The result is compared to descriptions by Keldish and Bebb to check its limits of applicability. Solving the kinetic conservation equations for the focus volume gives an estimation of the breakdown intensity where the Debye criterion has been used in the sense of a plasma definition. As an example, breakdown experiments with a KrF-escimer laser have been carried out at different pressures in an argon atmosphere. The pressure dependency of the breakdown threshold intensity and its order of magnitude are predicted by the model.

  13. Hund's coupling case sequences in resonant multiphoton transitions

    Science.gov (United States)

    Maïnos, C.; Castex, M. C.; Nkwawo, H.

    1990-10-01

    Different Hund's coupling case sequences are considered for the n1+n2 near resonant multiphoton rovibronic process in electric dipole allowed transitions of any spin multiplicity. The transitional path interferences strength tensor is introduced. This tensor involves a polarization and rotational dependence as well as a transitional path dependence which couples the electronic vibrational motion with the rotational structure. The intensity of a rotational line may decompose in terms of the matrix element of this tensor and a pure electronic vibrational tensor. The specificity of the coupling case sequence is found condensed in the rotational line factors which are explicitly determined for all the coupling case sequences obtained from the case (a) and case (b) coupling.

  14. High-resolution multiphoton imaging of tumors in vivo.

    Science.gov (United States)

    Wyckoff, Jeffrey; Gligorijevic, Bojana; Entenberg, David; Segall, Jeffrey; Condeelis, John

    2011-10-01

    Analysis of the individual steps in metastasis is crucial if insights at the molecular level are to be linked to the cell biology of cancer. A technical hurdle to achieving the analysis of the individual steps of metastasis is the fact that, at the gross level, tumors are heterogeneous in both animal models and patients. Human primary tumors show extensive variation in all properties ranging from growth and morphology of the tumor through tumor-cell density in the blood and formation and growth of metastases. Methods capable of the direct visualization and analysis of tumor-cell behavior at single-cell resolution in vivo have become crucial in advancing the understanding of mechanisms of metastasis, the definition of microenvironment, and the markers related to both. This article discusses the use of high-resolution multiphoton imaging of tumors (specifically breast tumors in mice) in vivo.

  15. Multiphoton ionization of acetone-water clusters at 355 nm

    Institute of Scientific and Technical Information of China (English)

    WANG Reng; KONG Xiang-he; ZHANG Shu-dong; ZHANG Xia; FAN Xing-yan; ZHAO Shu-yan

    2006-01-01

    @@ The multiphoton ionization of acetone-water clusters were detected at 355 nm laser wavelength by using the time of flight mass spectrometer(TOF-MS).The experiments show that all products are protonated.Three main products such as (CH3COCH3)n-(H2O)n-2H+,(CH3COCH3)n-(H2O)n-1H+ and (CH3COCH3)n-(H2O)nH+ are concluded from the results.In order to study the equilibrium structures of the (CH3COCH3)n-(H2O)n-2H+,the ab-initio calculation is used on them.The experiment is even done when the volume rate of acetone to water is 1:2.

  16. Photoacoustic microscopy: superdepth, superresolution, and superb contrast.

    Science.gov (United States)

    Yao, Junjie; Song, Liang; Wang, Lihing V

    2015-01-01

    Since its invention in the 17th century, optical microscopy has revolutionized biomedical studies by scrutinizing the biological realm on cellular levels, taking advantage of its excellent light-focusing capability. However, most biological tissues scatter light highly. As light travels in tissue, cumulative scattering events cause the photons to lose their original propagation direction and, thus, their ability to be focused, which has largely limited the penetration depth of optical microscopy. Conventional planar optical microscopy can provide penetration of only ~100 ?m before photons begin to be scattered. The penetration of modern optical microcopy, such as confocal microscopy and multiphoton microscopy, is still limited to approximately the optical diffusion limit (~1 mm in the skin as approximated by one optical transport mean free path), where scattered photons retain a strong memory of the original propagation direction. So far, it still remains a challenge for pure optical methods to achieve high-resolution in vivo imaging beyond the diffusion limit (i.e., superdepth imaging).

  17. Quantum secure communication using a multi-photon tolerant protocol

    Science.gov (United States)

    El Rifai, Mayssaa; Verma, Pramode K.

    2015-03-01

    This paper proposes a quantum secure communication protocol using multiple photons to represent each bit of a message to be shared. The multi-photon tolerant approach to quantum cryptography provides a quantum level security while using more than a single photon per transmission. The protocol proposed is a multi-stage protocol; an explanation of its operation and implementation are provided. The multi-stage protocol is based on the use of unitary transformations known only to Alice and Bob. This paper studies the security aspects of the multi-stage protocol by assessing its vulnerability to different attacks. It is well known that as the number of photons increases, the level of vulnerability of the multi-stage protocol increases. This paper sets a limit on the number of photons that can be used while keeping the multi-stage protocol a multi-photon tolerant quantum secure method for communication. The analysis of the number of photons to be used is based on the probability of success of a Helstrom discrimination done by an eavesdropper on the channel. Limiting the number of photons up to certain threshold per stage makes it impossible for an eavesdropper to decipher the message sent over the channel. The proposed protocol obviates the disadvantages associated with single photon implementations, such as limited data rates and distances along with the need to have no more than a single photon per time slot. The multi-stage protocol is a step toward direct quantum communication rather than quantum key distribution associated with single photon approaches.

  18. Multi-Photon Absorption Spectra: A Comparison Between Transmittance Change and Fluorescence Methods

    Science.gov (United States)

    2015-05-21

    AFRL-OSR-VA-TR-2015-0134 multi-photon absorption spectra Cleber Mendonca INSTITUTO DE FISICA DE SAO CARLOS Final Report 05/21/2015 DISTRIBUTION A...5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Instituto de Fisica de Sao Carlos - Universidade de Sao Paulo Av...of this project is to determine the multi-photon absorption spectrum of organic compounds, in order to establish its correlation with the molecular

  19. Excitation-wavelength dependence of multiphoton excitation of fluorophores of human skin in vivo

    Science.gov (United States)

    Breunig, Hans Georg; Studier, Hauke; König, Karsten

    2010-02-01

    We present in vivo measurements of the excitation-wavelength dependence of the autofluorescence of major endogenous fluorophores of human skin with a multiphoton tomograph. For the investigation high-resolution multiphoton images at different depths inside the skin were recorded and the main fluorophores identified. In particular, for the autofluorescence of the fluorophores keratin, NAD(P)H, elastin and for the second-harmonic-generation light induced by collagen fibers clear trends are shown.

  20. Electron emission from insulator and semiconductor surfaces by multiphoton excitation below the optical damage threshold

    Science.gov (United States)

    Siekhaus, W. J.; Kinney, J. H.; Milam, D.; Chase, L. L.

    1986-03-01

    Electron emission due to 1.06 and 0.35 μm laser excitation has been measured at several pulse lengths for a number of wide bandgap semiconductors and insulators. The dependence on fluence and wavelength is consistent with multiphoton excitation across the bandgap. The work functions of the materials investigated do not appear to influence the rate-limiting multiphoton excitation process.

  1. Quantum random walks with multiphoton interference and high order correlation functions

    CERN Document Server

    Gard, Bryan T; Anisimov, Petr M; Lee, Hwang; Dowling, Jonathan P

    2011-01-01

    We show a simulation of quantum random walks with multiple photons using a staggered array of 50/50 beam splitters with a bank of detectors at any desired level. We discuss the multiphoton interference effects that are inherent to this setup, and introduce one, two, and threefold coincidence detection schemes. The use of Feynman diagrams are used to intuitively explain the unique multiphoton interference effects of these quantum random walks.

  2. Label-free in vivo in situ diagnostic imaging by cellular metabolism quantification with a flexible multiphoton endomicroscope (Conference Presentation)

    Science.gov (United States)

    Leclerc, Pierre; Hage, Charles-Henri; Fabert, Marc; Brevier, Julien; O'Connor, Rodney P.; Bardet-Coste, Sylvia M.; Habert, Rémi; Braud, Flavie; Kudlinski, Alexandre; Louradour, Frederic

    2017-02-01

    Multiphoton microscopy is a cutting edge imaging modality leading to increasing advances in biology and also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there have been several developments of fiber-based multiphoton microendoscopes. The application for those probes is now limited by few major restrictions, such as the difficulty to collect autofluorescence signals from tissues and cells theses being inherently weak (e.g. the ones from intracellular NADH or FAD metabolites). This limitation reduces the usefulness of microendoscopy in general, effectively restraining it to morphological imaging modality requiring staining of the tissues. Our aim is to go beyond this limitation, showing for the first time label-free cellular metabolism monitoring, in vivo in situ in real time. The experimental setup is an upgrade of a recently published one (Ducourthial et.al, Scientific Reports, 2016) where femtosecond pulse fiber delivery is further optimized thank's to a new transmissive-GRISM-based pulse stretcher permitting high energy throughput and wide bandwidth. This device allows fast sequential operation with two different excitation wavelengths for efficient two-photon excited NADH and FAD autofluorescence endoscopic detection (i.e. 860 nm for FAD and 760 nm for NADH), enabling cellular optical redox ratio quantification at 8 frames/s. The obtained results on cell models in vitro and also on animal models in vivo (e.g. neurons of a living mouse) prove that we accurately assess the level of NADH and FAD at subcellular resolution through a 3-meters-long fiber with our miniaturized probe (O.D. =2.2 mm).

  3. Recent developments in fluorescence-based microscopy applied in biomedical sciences

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The present short review aims to give an overview of the most recent de velopments in fluorescence microscopy and its applications in biomedical science s. Apart from improvements in well-established methods based on conventional fl u orescence microscopy and confocal microscopy (fluorescence in situ hybridisa tion (FISH), tyramide signal amplification (TSA) in immunocytochemistry, new fluorop hores), more recently introduced techniques like fluorescence resonance energy t ransfer (FRET), fluorescence recovery after photobleaching (FRAP), multiphoton m icroscopy and fluorescence correlation spectroscopy (FCS) will be discussed.

  4. In vivo non-invasive multiphoton tomography of human skin

    Science.gov (United States)

    König, Karsten; Riemann, Iris; Ehlers, Alexander; Le Harzic, Ronan

    2005-10-01

    High resolution non-invasive 3D imaging devices are required to detect pathogenic microorganisms such as Anthrax spores, bacteria, viruses, fungi and chemical agents entering biological tissues such as the epidermis. Due to the low light penetration depth and the biodamage potential, ultraviolet light sources can not be employed to realize intratissue imaging of bio- and chemohazards. We report on the novel near infrared laser technology multiphoton tomography and the high resolution 4D imaging tool DermaInspect for non-invasive detection of intratissue agents and their influence on cellular metabolism based on multiphoton autofluorescence imaging (MAI) and second harmonic generation (SHG). Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence of both, skin tissues and microorganisms, originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Bacteria emit in the blue/green spectral range due to NAD(P)H and flavoproteins and, in certain cases, in the red spectral range due to the biosynthesis of Zn-porphyrins, coproporphyrin and protoporphyrin. Collagen and exogenous non-centrosymmetric molecules can be detected by SHG signals. The system DermaInspect consists of a wavelength-tunable compact 80/90 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezo-driven objective, fast photon detector and time-resolved single photon counting unit. It can be used to perform optical sectioning and 3D autofluorescence lifetime imaging (τ-mapping) with 1 μm spatial resolution and 270 ps temporal resolution. The parameter fluorescence lifetime depends on the type of fluorophore and its microenvironment and can be used to distinguish bio- and chemohazards from cellular background and to gain information for pathogen

  5. Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique

    Science.gov (United States)

    Lin, Jian; Lu, Fake; Zheng, Wei; Xu, Shuoyu; Tai, Dean; Yu, Hanry; Huang, Zhiwei

    2011-11-01

    We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.

  6. Multiphoton photochemical crosslinking-based fabrication of protein micropatterns with controllable mechanical properties for single cell traction force measurements

    Science.gov (United States)

    Tong, Ming Hui; Huang, Nan; Zhang, Wei; Zhou, Zhuo Long; Ngan, Alfonso Hing Wan; Du, Yanan; Chan, Barbara Pui

    2016-01-01

    Engineering 3D microstructures with predetermined properties is critical for stem cell niche studies. We have developed a multiphoton femtosecond laser-based 3D printing platform, which generates complex protein microstructures in minutes. Here, we used the platform to test a series of fabrication and reagent parameters in precisely controlling the mechanical properties of protein micropillars. Atomic force microscopy was utilized to measure the reduced elastic modulus of the micropillars, and transmission electron microscopy was used to visualize the porosity of the structures. The reduced elastic modulus of the micropillars associated positively and linearly with the scanning power. On the other hand, the porosity and pore size of the micropillars associated inversely and linearly with the scanning power and reagent concentrations. While keeping the elastic modulus constant, the stiffness of the micropillars was controlled by varying their height. Subsequently, the single cell traction forces of rabbit chondrocytes, human dermal fibroblasts, human mesenchymal stem cells, and bovine nucleus pulposus cells (bNPCs) were successfully measured by culturing the cells on micropillar arrays of different stiffness. Our results showed that the traction forces of all groups showed positive relationship with stiffness, and that the chondrocytes and bNPCs generated the highest and lowest traction forces, respectively.

  7. Real-time high dynamic range laser scanning microscopy

    Science.gov (United States)

    Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

    2016-04-01

    In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

  8. Real-time high dynamic range laser scanning microscopy.

    Science.gov (United States)

    Vinegoni, C; Leon Swisher, C; Fumene Feruglio, P; Giedt, R J; Rousso, D L; Stapleton, S; Weissleder, R

    2016-04-01

    In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

  9. Endoscopic Microscopy

    Science.gov (United States)

    Sokolov, Konstantin; Sung, Kung-Bin; Collier, Tom; Clark, Anne; Arifler, Dizem; Lacy, Alicia; Descour, Michael; Richards-Kortum, Rebecca

    2002-01-01

    In vivo endoscopic optical microscopy provides a tool to assess tissue architecture and morphology with contrast and resolution similar to that provided by standard histopathology – without need for physical tissue removal. In this article, we focus on optical imaging technologies that have the potential to dramatically improve the detection, prevention, and therapy of epithelial cancers. Epithelial pre-cancers and cancers are associated with a variety of morphologic, architectural, and molecular changes, which currently can be assessed only through invasive, painful biopsy. Optical imaging is ideally suited to detecting cancer-related alterations because it can detect biochemical and morphologic alterations with sub-cellular resolution throughout the entire epithelial thickness. Optical techniques can be implemented non-invasively, in real time, and at low cost to survey the tissue surface at risk. Our manuscript focuses primarily on modalities that currently are the most developed: reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). However, recent advances in fluorescence-based endoscopic microscopy also are reviewed briefly. We discuss the basic principles of these emerging technologies and their current and potential applications in early cancer detection. We also present research activities focused on development of exogenous contrast agents that can enhance the morphological features important for cancer detection and that have the potential to allow vital molecular imaging of cancer-related biomarkers. In conclusion, we discuss future improvements to the technology needed to develop robust clinical devices. PMID:14646041

  10. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy

    Science.gov (United States)

    Hayashi, Shinichi

    2016-08-01

    Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy with a 2-fold higher lateral resolution than conventional wide-field fluorescence (WF) microscopy. Confocal fluorescence (CF) microscopy has approximately the same optical cutoff frequency as SIM; however, the maximum theoretical increase in lateral resolution over that of WF is 1.4-fold with an infinitesimal pinhole diameter. Quantitative comparisons based on an analytical imaging formula revealed that modulation transfer functions (MTFs) of SIM reconstructed images before postprocessing are nearly identical to those of CF images recorded with an infinitesimal pinhole diameter. Here, we propose a new method using an adequate pinhole diameter combined with the use of an apodized Fourier inverse filter to increase the lateral resolution of CF images to as much as that SIM images without significant noise degradation in practice. Furthermore, the proposed method does not require a posteriori parameterization and has reproducibility. This approach can be easily applied to conventional laser scanning CF, spinning disk CF, and multiphoton microscopies.

  11. Multiphoton catalysis with coherent state input: nonclassicality and decoherence

    Science.gov (United States)

    Hu, Li-Yun; Wu, Jia-Ni; Liao, Zeyang; Zubairy, M. Suhail

    2016-09-01

    We propose a scheme to generate a new kind of non-Gaussian state—the Laguerre polynomial excited coherent state (LPECS)—by using multiphoton catalysis with coherent state input. The nonclassical properties of the LPECS are studied in terms of nonclassical depth, Mandel’s parameter, second-order correlation, quadrature squeezing, and the negativity of the Wigner function (WF). It is found that the LPECS is highly nonclassical and its nonclassicality depends on the amplitude of the coherent state, the catalysis photon number, and the parameters of the unbalanced beam splitter (BS). In particular, the maximum degree of squeezing can be enhanced by increasing the catalysis photon number. In addition, we examine the effect of decoherence using the WF, which shows that the negative region, the characteristic time of decoherence, and the structure of the WF are affected by catalysis photon number and the parameters of the unbalanced BS. Our work provides general analysis on how to prepare polynomial quantum states, which may be useful in the fields of quantum information and quantum computation.

  12. Multiphoton ionization of pyrrole-water mixed clusters

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Multiphoton ionization of the hydrogen-bonded pyrrole-water clusters ( C4H5 N)n H2O)m is studied with a reflectron—time of flight mass spectrometer at 355 nm. With increasing partial concentration of pyrrole in a gas mixture source, a series of poly-pyrrole-water binary-mixed cluster ions can be observed, including unprotonated cluster ions [(C4H5N)x (H2O)y]* , protonated cluster ions [(C4H5N)x (H2O)yH]* and dehydrogenated cluster ions [ ( C4 H4 N) ( C4 H5 N) x ( H2O) y ] + . Ab initio calculations of their structures, bond strengths, charge distributions and re action energies are carried out. Stable structures of these clusters are obtained from the calculations. A probable forma tion mechanism of the cluster ions [(C4H5N)x(H2O)y] + , [(C4H5N)x (H2O)y]H+ and [(C4H4N) (C4H5N)x (H2O)y]-is supposed to be the ionization of clusters followed by dissociation.

  13. Multiphoton ionization photoelectron spectroscopy of xenon: Experiment and theory

    Energy Technology Data Exchange (ETDEWEB)

    Bajic, S.J.; Compton, R.N.; Tang, X.; L' Huiller, A.; Lambropoulos, P.

    1988-11-01

    Photoelectron energy and angular distributions for resonantly enhanced multiphoton ionization (REMPI) of xenon via the three-photon-allowed 7s(3/2)/sub 1//sup 0/ and 5d(3/2)/sub 1//sup 0/ states have been studied both experimentally and theoretically. The electron kinetic energy spectra give the probability of leaving Xe/sup +/ in either the /sup 2/P/sub 1/2/ or /sup 2/P/sub 3/2/ core. The measured branching ratio for leaving each ionic core is used to test the theoretical description of the REMPI process. Measurements of both the angular distributions and the (3+1) REMPI via the 5d state are adequately reproduced by multichannel quantum defect theory. However, measurements of angular distributions for the electrons resulting from (3+1) via the 7s(3/2)/sub 1//sup 0/ state into Xe/sup +/ /sup 2/P/sub 3/2/ (core preserving) or Xe/sup +/ /sup 2/P/sub 1/2/ (core changing) are in striking disagreement with theory. 1 ref., 2 figs.

  14. Multi-photon Resonance Phenomena Using Laguerre-Gaussian Beams

    CERN Document Server

    Kazemi, Seyedeh Hamideh

    2016-01-01

    We study the influence of Laguerre-Gaussian (LG) and Gaussian fields on the linewidth of the optical spectrum of multi-photon resonance phenomena. First, we investigate the dependence of the steady-state coherence on the laser profile in a two-level system. Thanks to the LG field, the linewidth of the one-photon optical pumping peak is explicitly narrower than for a Gaussian field. We then investigate the atomic coherence in a two-level pump-probe atomic system and show that using the LG fields, a narrower two-photon absorption peak can be obtained compared to the usual Gaussian ones. In next section, we investigate the effect of the laser profiles on the coherent population trapping in the $\\Lambda$-type molecular open systems. It is shown that, comparing with the the Gaussian fields, the LG fields reduce the linewidth of the optical spectrum. In addition, for a laser-driven four-level atomic system we study the effect of laser profiles on the Autler-Townes doublet structure in the absorption spectrum. We al...

  15. Multi-photon processes in alkali metal vapors

    Science.gov (United States)

    Gai, Baodong; Hu, Shu; Li, Hui; Shi, Zhe; Cai, Xianglong; Guo, Jingwei; Tan, Yannan; Liu, Wanfa; Jin, Yuqi; Sang, Fengting

    2015-02-01

    Achieving population inversion through multi-photon cascade pumping is almost always difficult, and most laser medium work under 1-photon excitation mechanism. But for alkali atoms such as cesium, relatively large absorption cross sections of several low, cascading energy levels enable them properties such as up conversion. Here we carried out research on two-photon excitation alkali fluorescence. Two photons of near infrared region are used to excite alkali atoms to n 2 D5/2, n 2 D3/2 or higher energy levels, then the blue fluorescence of (n+1) 2 P3/2,(n+1) 2 P1/2-->n 2 S1/2 are observed. Different pumping paths are tried and by the recorded spectra, transition routes of cesium are deducted and concluded. Finally the possibility of two-photon style DPALs (diode pumped alkali laser) are discussed, such alkali lasers can give output wavelengths in the shorter end of visual spectroscopy (400-460 nm) and are expected to get application in underwater communication and material laser processing.

  16. Quantum Decoherence for Multi-Photon Entangled States

    Institute of Scientific and Technical Information of China (English)

    SUN Yan-Hua; ZHU Xia; KUANG Le-Man

    2005-01-01

    @@ We investigate quantum decoherence of the multi-photon entangled state |ψNm> = Nm[cosγ|N- m>1|m>2 +eiθm sinγ|m>1|N - m>2]. When the entangled channel |ψNm> is embedded in an environment, the channel decoheres and becomes a mixed state governed by a master equation. We calculate thelinear entropy and the relative entropy of entanglement, which describe the mixedness and the amount of entanglement for the mixed state, respectively. We show that quantum decoherence weakens the amount of entanglement and enhances the mixedness with the time evolution. It is indicated that the relative entropy of entanglement depends on not only the initial entanglement angle and the decohering parameter, but also the number of photons in each mode. In particular, we find that the decohering speed depends on the number-difference of photons in the two modes. The larger the number-difference of photons is, the higher the decohering speed.

  17. The polarization effect of a laser in multiphoton Compton scattering

    Science.gov (United States)

    Liang, Guo-Hua; Lü, Qing-Zheng; Teng, Ai-Ping; Li, Ying-Jun

    2014-05-01

    The multiphoton Compton scattering in a high-intensity laser beam is studied by using the laser-dressed quantum electrodynamics (QED) method, which is a non-perturbative theory for the interaction between a plane electromagnetic field and a charged particle. In order to analyze in the real experimental condition, a Lorentz transformation for the cross section of this process is derived between the laboratory frame and the initial rest frame of electrons. The energy of the scattered photon is analyzed, as well as the cross sections for different laser intensities and polarizations and different electron velocities. The angular distribution of the emitted photon is investigated in a special velocity of the electron, in which for a fixed number of absorbed photons, the electron energy will not change after the scattering in the lab frame. We obtain the conclusion that higher laser intensities suppress few-laser-photon absorption and enhance more-laser-photon absorption. A comparison between different polarizations is also made, and we find that the linearly polarized laser is more suitable to generate nonlinear Compton scattering.

  18. Characterization of multiphoton emission from aggregated gold nano particles

    Science.gov (United States)

    Eguchi, Akira; Lu, Phat; Kim, Youngsik; Milster, Tom D.

    2016-09-01

    Although gold nanoparticles (GNPs) are promising probes for biological imaging because of their attracting optical properties and bio-friendly nature, properties of the multi-photon (MP) emission from GNP aggregates produced by a short-wave infrared (SWIR) laser have not been examined. In this paper, characterization of MP emission from aggregated 50 nm GNPs excited by a femtosecond (fs) laser at 1560 nm is discussed with respect to aggregate structures. The key technique in this work is single particle spectroscopy. A pattern matching technique is applied to correlate MP emission and SEM images, which includes an optimization processes to maximize cross correlation coefficients between a binary microscope image and a binary SEM image with respect to xy displacement, image rotation angle, and image magnification. Once optimization is completed, emission spots are matched to the SEM image, which clarifies GNP ordering and emission properties of each aggregate. Correlation results showed that GNP aggregates have stronger MP emission than single GNPs. By combining the pattern matching technique with spectroscopy, MP emission spectrum is characterized for each GNP aggregate. A broad spectrum in the visible region and near infrared (NIR) region is obtained from GNP dimers, unlike previously reported surface plasmon enhanced emission spectrum.

  19. Multiphoton Ionization Detection in Collinear Laser Spectroscopy of Isolde Beams

    CERN Multimedia

    2002-01-01

    The experiments using the multiphoton ionization technique have been continued in the beginning of 1990 with stable beam tests on the modified apparatus and with another radioactive beam time on Yb. Higher laser power and an increased vacuum in the ionization region (see figure) yielded a further gain in sensitivity, mainly due to the better suppression of the background ions produced in rest gas collisions. For even Yb isotopes we have now reached a detection efficiency of $\\epsilon$~=~1~x~10$^{-5}$ ions per incoming atom at a background count rate of 30~ions from a beam of 5~x~10$^9$. This sensitivity was high enough for spectroscopy on $^{157}$Yb, where the typical ISOLDE yield of 5~x~10$^7$Yb ions is covered by an isobaric contamination of more than 10$^{10}$ ions. Measurements have also been performed on $^{175}$Yb. These give the first precise value for the magnetic moment of this isotope, $\\mu$~=~0.766(8)$ mu _{N} $, which agrees rather well with the magnetic moment of the isotone $^{177}$Hf. The isoto...

  20. Structure of multiphoton quantum optics. I. Canonical formalism and homodyne squeezed states

    Science.gov (United States)

    dell'Anno, Fabio; de Siena, Silvio; Illuminati, Fabrizio

    2004-03-01

    We introduce a formalism of nonlinear canonical transformations for general systems of multiphoton quantum optics. For single-mode systems the transformations depend on a tunable free parameter, the homodyne local-oscillator angle; for n -mode systems they depend on n heterodyne mixing angles. The canonical formalism realizes nontrivial mixing of pairs of conjugate quadratures of the electromagnetic field in terms of homodyne variables for single-mode systems, and in terms of heterodyne variables for multimode systems. In the first instance the transformations yield nonquadratic model Hamiltonians of degenerate multiphoton processes and define a class of non-Gaussian, nonclassical multiphoton states that exhibit properties of coherence and squeezing. We show that such homodyne multiphoton squeezed states are generated by unitary operators with a nonlinear time evolution that realizes the homodyne mixing of a pair of conjugate quadratures. Tuning of the local-oscillator angle allows us to vary at will the statistical properties of such states. We discuss the relevance of the formalism for the study of degenerate (up-)down-conversion processes. In a companion paper [ F. Dell’Anno, S. De Siena, and F. Illuminati, 69, 033813 (2004) ], we provide the extension of the nonlinear canonical formalism to multimode systems, we introduce the associated heterodyne multiphoton squeezed states, and we discuss their possible experimental realization.

  1. Kerr nonlinearity and multi-photon absorption in germanium at mid-infrared wavelengths

    Science.gov (United States)

    Sohn, B.-U.; Monmeyran, C.; Kimerling, L. C.; Agarwal, A. M.; Tan, D. T. H.

    2017-08-01

    Multiphoton absorption coefficients and nonlinear refractive indices of germanium in the near and mid-infrared (2-5 μm) are reported. The nonlinear coefficients are measured by open and closed aperture Z-scan with 150 fs pulses at a repetition rate of 1 kHz. The nonlinear refractive index of Ge has a peak value of 9.1 ×10-5cm2/GW at a wavelength of 3 μm. The effect of free electrons generated by multiphoton absorption is discussed by investigating the variation of multiphoton absorption coefficients at different input powers. Kramers-Kronig relations are also discussed with regard to the relationship between nonlinear refractive index and two photon absorption coefficient.

  2. Structure of multiphoton quantum optics. II. Bipartite systems, physical processes, and heterodyne squeezed states

    CERN Document Server

    Dell'Anno, F; Illuminati, F; Anno, Fabio Dell'; Siena, Silvio De; Illuminati, Fabrizio

    2004-01-01

    Extending the scheme developed for a single mode of the electromagnetic field in the preceding paper ``Structure of multiphoton quantum optics. I. Canonical formalism and homodyne squeezed states'', we introduce two-mode nonlinear canonical transformations depending on two heterodyne mixing angles. They are defined in terms of hermitian nonlinear functions that realize heterodyne superpositions of conjugate quadratures of bipartite systems. The canonical transformations diagonalize a class of Hamiltonians describing non degenerate and degenerate multiphoton processes. We determine the coherent states associated to the canonical transformations, which generalize the non degenerate two--photon squeezed states. Such heterodyne multiphoton squeezed are defined as the simultaneous eigenstates of the transformed, coupled annihilation operators. They are generated by nonlinear unitary evolutions acting on two-mode squeezed states. They are non Gaussian, highly non classical, entangled states. For a quadratic nonline...

  3. Coherent Phase Control of Multiphoton Ionization in Three-Level Ladder-Type System

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shi-An; CHEN Yu-Ting; WANG Zu-Geng; SUN Zhen-Rong

    2009-01-01

    We present the theoretical investigation of photoelectron spectroscopy resulting from the strong field induced multiphoton ionization in a typical three-level ladder-style system.Our theoretical results show that the photoelectron spectral structure can be alternatively steered by spectral phase modulation.This physical mechanism for strong field quantum control is explicitly exploited by the time-dependent dressed state population.It is concluded that the phase-shaped laser pulses can be used to selectively manipulate the multiphoton ionization process in complicated quantum systems.

  4. Effect of Multiphoton Processes on Geometric Quantum Computation in Superconducting Circuit QED

    Institute of Scientific and Technical Information of China (English)

    CHEN Chang-Yong

    2012-01-01

    We study the influence of multi-photon processes on the geometric quantum computation in the systems of superconducting qubits based on the displacement-like and the general squeezed operator methods. As an example, we focus on the question about how to implement a two-qubit geometric phase gate using superconducting circuit quantum electrodynamics with both single- and two-photon interaction between the qubits and the cavity modes. We find that the multiphoton processes are not only controllable but also improve the gating speed. The comparison with other physical systems and experimental feasibility are discussed in detail.

  5. Multiphoton excitation characteristics of cellular fluorophores of human skin in vivo.

    Science.gov (United States)

    Breunig, Hans Georg; Studier, Hauke; König, Karsten

    2010-04-12

    In vivo multiphoton tomography with a wavelength-tunable femtosecond laser has been performed to investigate the autofluorescence intensity of major endogenous fluorophores of human skin in dependence on the excitation wavelength. In high-resolution multiphoton images of different skin layers, clear trends were found for fluorophores like keratin, NAD(P)H, melanin as well as for the elastin and collagen networks. The analysis of the measurements is supplemented by additional measurements of fluorescence lifetime imaging and signal-decay curves by time-correlated single-photon counting.

  6. Multi-photon resonance phenomena using Laguerre-Gaussian beams

    Science.gov (United States)

    Hamideh Kazemi, Seyedeh; Mahmoudi, Mohammad

    2016-12-01

    We study the influence of laser profile on the linewidth of the optical spectrum of multi-photon resonance phenomena. First, we investigate the dependence of the absorption spectrum on the laser profile in a two-level system. Thanks to the Laguerre-Gaussian field, the linewidth of the one-photon optical pumping and two-photon absorption peaks are explicitly narrower than that obtained with a Gaussian field. In the next section, it is shown that, compared to the Gaussian fields, the Laguerre-Gaussian ones reduce the linewidth of the optical spectrum in the coherent population trapping. Interestingly, it turns out that the use of a Laguerre-Gaussian beam makes the linewidth of the spectrum narrower as compared with a Gaussian one in Doppler-broadened electromagnetically induced transparency. Moreover, we study the effect of the laser profile on the Autler-Townes doublet structure in the absorption spectrum for a laser-driven four-level atomic system. We also consider the different values of the Laguerre-Gaussian mode beam waist, and, perhaps more remarkably, we find that for the small waist values, the Autler-Townes doublet can be removed and a prominent narrow central peak appears in the absorption spectrum. Finally, we investigate the effect of the laser profile on the linewidth of the sub-natural three-photon absorption peak of double dark resonance. The differences in the linewidth are quite large, offering potential applications in metrology and isotope separation methods. Our results can be used for super ultra-high resolution laser spectroscopy and to improve the resolution of the technology of isotope/isomer separation and photo-biology even at essential overlap of the spectra of the different particles.

  7. Particle modeling of microplasma generated by resonance enhanced multiphoton ionization

    Science.gov (United States)

    Tholeti, Siva Sashank

    Resonance-enhanced multiphoton ionization (REMPI) is a technique applied to the spectroscopy of atoms. The REMPI technique typically involves a resonant single or multiple photon absorption to an electronically excited intermediate state followed by another photon which ionizes the atom. Rayleigh scattering of REMPI plasma has given rise to a non-intrusive, time accurate measurement of electron formation and loss, which lead to many applications viz. trace species detection and micro-plasma diagnostics. It is very important to quantify the expansion process and the evolution of energy of electrons and ions. The operation scale of this process is in microns and non continuum nature of the process lead to the use of PIC/MCC scheme to compu- tationally model REMPI technique. This work attempts to understand and analyze the processes taking place during the expansion of REMPI plasma computationally using the PIC/MCC scheme. One dimensional and two dimensional approximations are considered to analyze the REMPI plasma expansion in Argon gas generated by a laser with a focal shape of a prolate ellipsoid. The expansion of the plasma is found to be very sensitive to the initial velocity distribution of the electrons. REMPI plasma expansion is shown to be ambipolar in nature, with the radial expansion more predominant than axial expansion, hence requiring the 2D model. Electron energy distribution functions(EEDFs) are found at various radial locations along with the corresponding mean energies. The deviation of the EEDFs from that of equilibrium Maxwell-Boltzmann energy distribution is presented both qualitatively and quanti- tatively, indicating the predominant processes at various instances in time.

  8. Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags.

    Science.gov (United States)

    Hackl, Matthias J; Burford, James L; Villanueva, Karie; Lam, Lisa; Suszták, Katalin; Schermer, Bernhard; Benzing, Thomas; Peti-Peterdi, János

    2013-12-01

    Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PECs) in vivo. In podocin-GFP mice, podocytes formed sporadic multicellular clusters after unilateral ureteral ligation and migrated into the parietal Bowman's capsule. The tracking of single cells in podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP or RFP revealed the simultaneous migration of multiple podocytes. In phosphoenolpyruvate carboxykinase (PEPCK)-GFP mice, serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support a highly dynamic rather than a static nature of the glomerular environment and cellular composition. Future application of this new approach should advance our understanding of the mechanisms of glomerular injury and regeneration.

  9. Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

    Science.gov (United States)

    Patsialou, Antonia; Bravo-Cordero, Jose Javier; Wang, Yarong; Entenberg, David; Liu, Huiping; Clarke, Michael; Condeelis, John S.

    2014-01-01

    Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: a. single cells and b. multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor. PMID:25013744

  10. Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

    Science.gov (United States)

    Duan, Yubo; Chen, Nanguang

    2015-11-15

    Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

  11. Fundamentals of fluorescence microscopy exploring life with light

    CERN Document Server

    Mondal, Partha Pratim

    2014-01-01

    This book starts at an introductory level and leads reader to the most advanced developments in fluorescence imaging and super-resolution techniques that have enabled the emergence of new disciplines such as nanobioimaging, multiphoton microscopy, photodynamic therapy, nanometrology and nanosensors. The interdisciplinary subject of fluorescence microscopy and imaging requires complete knowledge of imaging optics and molecular physics. So, this book approaches the subject by introducing optical imaging concepts before going deep into the advanced imaging systems and their applications. Molecular orbital theory forms the basis for understanding fluorescent molecules and thereby facilitates complete explanation of light-matter interaction at the geometrical focus. The two disciplines have some overlap since light controls the states of molecules and conversely, molecular states control the emitted light. These two mechanisms together determine essential fluorescence  factors and phenomena such as, molecular cro...

  12. Two-colour multiphoton ionization of hydrogen by an intense laser field and its third harmonic

    Energy Technology Data Exchange (ETDEWEB)

    Potvliege, R.M.; Smith, P.H.G. (Durham Univ. (United Kingdom). Dept. of Physics)

    1991-12-28

    We present results of ab initio non-perturbative Floquet calculations of rates for multiphoton ionization of atomic hydrogen by an intense laser field and its third harmonic, discussing in particular resonant ionization and the dependence of the ionization rate on the relative phase of the two fields. (author).

  13. The Multiphoton Interaction of Lambda Model Atom and Two-Mode Fields

    Science.gov (United States)

    Liu, Tang-Kun

    1996-01-01

    The system of two-mode fields interacting with atom by means of multiphotons is addressed, and the non-classical statistic quality of two-mode fields with interaction is discussed. Through mathematical calculation, some new rules of non-classical effects of two-mode fields which evolue with time, are established.

  14. Multiphoton fluorescence spectra and lifetimes of biliverdins and their protein-associated complex

    Science.gov (United States)

    Huang, Chin-Jie; Wu, Cheng-Ham; Liu, Tzu-Ming

    2012-03-01

    To investigate whether endogenous biliverdins can serve as a fluorescence metabolic marker in cancer diagnosis, we measured their multiphoton fluorescence spectra and lifetimes with femtosecond Cr:forsterite laser. Excited at 1230nm, the two-photon fluorescence of biliverdins peaks around 670nm. The corresponding lifetime (catabolism in human cells or tissues.

  15. Structure of multiphoton quantum optics. II. Bipartite systems, physical processes, and heterodyne squeezed states

    Science.gov (United States)

    dell'Anno, Fabio; de Siena, Silvio; Illuminati, Fabrizio

    2004-03-01

    Extending the scheme developed for a single mode of the electromagnetic field in the preceding paper [F. Dell’Anno, S. De Siena, and F. Illuminati, Phys. Rev. A 69, 033812 (2004)], we introduce two-mode nonlinear canonical transformations depending on two heterodyne mixing angles. They are defined in terms of Hermitian nonlinear functions that realize heterodyne superpositions of conjugate quadratures of bipartite systems. The canonical transformations diagonalize a class of Hamiltonians describing nondegenerate and degenerate multiphoton processes. We determine the coherent states associated with the canonical transformations, which generalize the nondegenerate two-photon squeezed states. Such heterodyne multiphoton squeezed states are defined as the simultaneous eigenstates of the transformed, coupled annihilation operators. They are generated by nonlinear unitary evolutions acting on two-mode squeezed states. They are non-Gaussian, highly nonclassical, entangled states. For a quadratic nonlinearity the heterodyne multiphoton squeezed states define two-mode cubic phase states. The statistical properties of these states can be widely adjusted by tuning the heterodyne mixing angles, the phases of the nonlinear couplings, as well as the strength of the nonlinearity. For quadratic nonlinearity, we study the higher-order contributions to the susceptibility in nonlinear media and we suggest possible experimental realizations of multiphoton conversion processes generating the cubic-phase heterodyne squeezed states.

  16. Multiphoton ionization of jet-cooled nickelocene with tunable nanosecond laser pulses

    Energy Technology Data Exchange (ETDEWEB)

    Ketkov, Sergey Yu.; Selzle, Heinrich L.; Schlag, Edward W.; Titova, Sofia

    2003-08-15

    Efficient multiphoton ionization of nickelocene molecules in a supersonically cooled molecular beam has been performed for the first time with a nanosecond tunable dye laser operating in the 35,000-cm{sup -1} region which corresponds to the lowest Rydberg transition observed in the one-photon absorption spectrum. The time-of-flight mass spectra obtained show strong signals of intact molecular ions Cp{sub 2}Ni{sup +} (Cp={eta}{sup 5}-C{sub 5}H{sub 5}) and weaker peaks of fragment ions CpNi{sup +}. The conditions have been found for generation of Cp{sub 2}Ni{sup +} as the only ionic product of multiphoton excitation. The ion signal dependence on the laser intensity and the resonance-enhanced multiphoton ionization spectrum measured at the mass of Cp{sub 2}Ni{sup +} testify for saturation of absorption and/or ionization steps at the laser pulse intensities used (2-6 MW cm{sup -2}). Possible mechanisms of multiphoton processes resulting in formation of the ions observed are discussed.

  17. Study of Microwave Multiphoton Transition of Rydberg Potassium Atom by Using B-Spline

    Institute of Scientific and Technical Information of China (English)

    JIN Cheng; ZHOU Xiao-Xin; ZHAO Song-Feng

    2005-01-01

    The B-spline expansion technique and time-dependent two-level approach are applied to study the interaction between the microwave field and potassium atoms in a static electric field. We obtain theoretical multiphoton resonance spectra that can be compared with the experimental data. We also obtain the time evolution of the final state in different microwave fields.

  18. Electron-Nuclear Energy Sharing in Above-Threshold Multiphoton Dissociative Ionization of H2

    DEFF Research Database (Denmark)

    Wu, J.; Kunitski, M.; Pitzer, M.

    2013-01-01

    We report experimental observation of the energy sharing between electron and nuclei in above-threshold multiphoton dissociative ionization of H2 by strong laser fields. The absorbed photon energy is shared between the ejected electron and nuclei in a correlated fashion, resulting in multiple...... diagonal lines in their joint energy spectrum governed by the energy conservation of all fragment particles....

  19. Electron-nuclear energy sharing in above-threshold multiphoton dissociative ionization of H2.

    Science.gov (United States)

    Wu, J; Kunitski, M; Pitzer, M; Trinter, F; Schmidt, L Ph H; Jahnke, T; Magrakvelidze, M; Madsen, C B; Madsen, L B; Thumm, U; Dörner, R

    2013-07-12

    We report experimental observation of the energy sharing between electron and nuclei in above-threshold multiphoton dissociative ionization of H2 by strong laser fields. The absorbed photon energy is shared between the ejected electron and nuclei in a correlated fashion, resulting in multiple diagonal lines in their joint energy spectrum governed by the energy conservation of all fragment particles.

  20. Exploring the potential of Multiphoton Laser Ablation Lithography (MP-LAL) as a reliable technique for sub-50nm patterning

    Science.gov (United States)

    Manouras, Theodoros; Angelakos, Evangelos; Vamvakaki, Maria; Argitis, Panagiotis

    2016-03-01

    In this work, direct-write, high-resolution multiphoton photolithography using doped random methacrylic co-polymer thin films is demonstrated, using a continuous wave ultraviolet (UV) 375 nm diode laser source. The random copolymers are specifically designed for enhancing resolution and addressing issues arising from laser ablation processes, such as the berm-formation around the created holes in the film, which can be accessed by tuning the polymeric material properties including Tg, surface adhesion etc. The methacrylic copolymer is composed of monomers, each of them especially selected to improve individual properties. The material formulations comprise perylene molecules absorbing at the exposure wavelength where the polymeric matrix is transparent. It was found that if the radiation intensity exceeds a certain threshold, the perylene molecules transfer the absorbed light energy to the acrylate polymer matrix leading to polymer degradation and ablation of the exposed areas. The non-linear nature of the light absorption and energy transfer processes resulted in the creation of holes with critical dimensions well below the used wavelength reaching the sub 50 nm domain. Arrays of holes having various dimensions were fabricated in the laser ablation experiments using a directwrite laser system developed specifically for the purposes of this project.

  1. Multi-photon microscope driven by novel green laser pump

    DEFF Research Database (Denmark)

    Marti, Dominik; Djurhuus, Martin; Jensen, Ole Bjarlin

    2016-01-01

    system. However, translation is hindered due to the high cost, high training demand and large footprint of a standard setup. We show in this article that minification of the setup, while also reducing cost and complexity, is indeed possible without compromising on image quality, by using a novel diode......Multi-photon microscopy is extensively used in research due to its superior possibilities when compared to other microscopy modalities. The technique also has the possibility to advance diagnostics in clinical applications, due to its capabilities complementing existing technology in a multimodal...

  2. The multiphoton ultraviolet and visible upconversion luminescence of ZBLAN material

    Science.gov (United States)

    Chen, Xiaobo; Song, Zengfu; Wang, Yafei; Xiong, Jun; Yang, Guojiian; Li, Song; Zhou, Jing; Peng, Fanglin; Zhou, Gu

    2008-03-01

    The multiphoton ultraviolet and visible upconversion luminescence of Tm 3+Yb 3+ codoped ZBLAN fluoride glass as excited by a 975nm diode laser was studied. Two typical ultraviolet 290.6nm 1I 6 -> 3H 6 and 362.0nm 1D II -> 3H 6 upconversion luminescence lines were found. The careful measurement of the variation of upconversion luminescence intensity F as a function of the 975nm pumping laser power P has proven that the 290.6nm 1I 6 -> 3H 6 and 362.0nm 1D II -> 3H 6 upconversion luminescences are a six-photon and a five-photon upconversion luminescence respectively. Several visible upconversion luminescence lines at 450.5nm, 473.9nm, 648.5nm, (687.3nm, 696.2nm) and (793.5nm, 800.7nm) were found also, which result from the fluorescence transitions of 1D II -> 3F 4, 1G 4 -> 3H6, 1G 4 -> 3F 4, 3F 3 -> 3H 6 and 3H 4 -> 3H 6 of Tm 3+ ion respectively. It has been proved that the upconversion luminescence of 1G 4 state is a three-photon upconversion process, while that of 3F 3 or 3H 4 state is a two-photon upconversion process. The theoretical analysis suggests that the upconversion mechanism of the 362.0nm 1D II -> Tm 3+ upconversion luminescence is the cross energy transfer of { 3H 4(Tm 3+) -> 3F 4(Tm 3+), 1G 4(Tm 3+) -> 1D II(Tm 3+)} and {1G 4(Tm 3+) -> 3F 4(Tm 3+), 3H 4(Tm 3+) -> 1D II(Tm 3+)} between Tm 3+ ions, whereas the mechanism of the 290.6nm 1I 6 -> 3H 6 upconversion luminescence is the sequential energy transfer of {2F 5/2(Yb 3+) -> 2F 7/2(Yb 3+), 1D II(Tm 3+) -> 3P I(Tm 3+)} and {2F 5/2(Yb3+) -> 2F 7/2(Yb 3+), 1D II(Tm 3+) -> 3P II(Tm 3+)} from Yb 3+ ions to Tm 3+ ions. In addition, the upconversion luminescence of G 4 and 3H 4 state also results from the sequential energy transfer {2F 5/2(Yb 3+) -> 2F 7/2(Yb 3+), 3H 4(Tm 3+) -> 1G 4(Tm 3+)} and {2F 5/2(Yb 3+) -> 2F 7/2(Yb 3+), 3F 4(Tm 3+) -> 3F II(Tm 3+)} from Yb 3+ ions to Tm 3+ ions respectively.

  3. ImageJ for microscopy.

    Science.gov (United States)

    Collins, Tony J

    2007-07-01

    ImageJ is an essential tool for us that fulfills most of our routine image processing and analysis requirements. The near-comprehensive range of import filters that allow easy access to image and meta-data, a broad suite processing and analysis routine, and enthusiastic support from a friendly mailing list are invaluable for all microscopy labs and facilities-not just those on a budget.

  4. Compact Coding Using Multi-Photon Tolerant Quantum Protocols for Quantum Communication

    CERN Document Server

    Hajj, Rasha El; Chan, Kam Wai Clifford

    2016-01-01

    This paper presents a new encryption scheme called Compact Coding that encodes information in time, phase, and intensity domains, simultaneously. While these approaches have previously been used one at a time, the proposed scheme brings to bear for the first time their strengths simultaneously leading to an increase in the secure information transfer rate. The proposed scheme is applicable to both optical fibers and free space optics, and can be considered as an alternative to polarization coding. This paper applies the proposed compact coding scheme to multi-photon tolerant quantum protocols in order to produce quantum-level security during information transfer. We present the structure of the proposed coding scheme in a multi-photon environment and address its operation.

  5. Partial indistinguishability theory for multi-photon experiments in multiport devices

    CERN Document Server

    Shchesnovich, V S

    2014-01-01

    We develop a simple approach for description of multi-photon experiments with multi-port unitary linear optical devices for arbitrary (multi-photon) input and arbitrary photon detectors. The probabilities at the output of a multi-port device are expressed as a quadratic form with a non-negative definite Hermitian matrix describing partial indistinguishability of photons. In the case of input consisting of single photon or vacuum per input mode the output probabilities are expressed as an integral of the absolute value squared of the matrix permanent of a Hadamard product of network matrix and a matrix depending on spectral state of photons and spectral sensitivities of detectors. For example, zero coincidence probability condition for dissimilar detectors formulated in Phys. Rev. Lett. 110, 113603 (2013) using the matrix immanants is given in a much simpler form. We analyze a model of the Boson-Sampling computer with only partially indistinguishable single photons having Gaussian spectral function and Gaussia...

  6. Multi-photon dressing of an anharmonic superconducting many-level quantum circuit

    Energy Technology Data Exchange (ETDEWEB)

    Braumueller, Jochen; Cramer, Joel; Schloer, Steffen; Rotzinger, Hannes; Radtke, Lucas; Lukashenko, Alexander; Yang, Ping; Skacel, Sebastian; Probst, Sebastian; Weides, Martin [Karlsruhe Institute of Technology (KIT), Physikalisches Institut, 76131 Karlsruhe (Germany); Marthaler, Michael; Guo, Lingzhen [Karlsruhe Institute of Technology (KIT), Institut fuer Theoretische Festkoerperphysik, 76131 Karlsruhe (Germany); Ustinov, Alexey V. [Karlsruhe Institute of Technology (KIT), Physikalisches Institut, 76131 Karlsruhe (Germany); National University of Science and Technology MISIS, Moscow 119049 (Russian Federation)

    2015-07-01

    We report on the investigation of a superconducting anharmonic multi-level circuit that is coupled to a harmonic readout resonator. We observe multi-photon transitions via virtual energy levels of our system up to the fifth excited state. The back-action of these higher-order excitations on our readout device is analyzed quantitatively and demonstrated to be in accordance with theoretical expectation. By applying a strong microwave drive we achieve multi-photon dressing of our system which is dynamically coupled by a weak probe tone. The emerging higher-order Rabi sidebands and associated Autler-Townes splittings involving up to five levels of the investigated anharmonic circuit are observed. Experimental results are in good agreement with master equation simulations.

  7. Density functional theory for molecular multiphoton ionization in the perturbative regime.

    Science.gov (United States)

    Toffoli, Daniele; Decleva, Piero

    2012-10-07

    A general implementation of the lowest nonvanishing order perturbation theory for the calculation of molecular multiphoton ionization cross sections is proposed in the framework of density functional theory. Bound and scattering wave functions are expanded in a multicentric basis set and advantage is taken of the full molecular point group symmetry, thus enabling the application of the formalism to medium-size molecules. Multiphoton ionization cross sections and angular asymmetry parameters have been calculated for the two- and four-photon ionization of the H(2) (+) molecule, for linear and circular light polarizations. Both fixed and random orientations of the target molecule have been considered. To demonstrate the efficiency of the proposed methodology, the two-photon cross section and angular asymmetry parameters for the HOMO and HOMO-1 orbital ionization of benzene are also presented.

  8. High-fidelity spatially resolved multiphoton counting for quantum imaging applications

    CERN Document Server

    Chrapkiewicz, Radoslaw; Banaszek, Konrad

    2015-01-01

    We present a method for spatially resolved multiphoton counting based on an intensified camera with the retrieval of multimode photon statistics fully accounting for non-linearities in the detection process. The scheme relies on one-time quantum tomographic calibration of the detector. Faithful, high-fidelity reconstruction of single- and two-mode statistics of multiphoton states is demonstrated for coherent states and their statistical mixtures. The results consistently exhibit classical values of Mandel and Fano parameters in contrast to raw statistics of camera photo-events. Detector operation is reliable for illumination levels up to the average of one photon per an event area, substantially higher than in previous approaches to characterize quantum statistical properties of light with spatial resolution.

  9. Influence of the ac Stark effect on multiphoton transitions in molecules

    Science.gov (United States)

    Meerts, W. Leo; Ozier, Irving; Hougen, Jon T.

    1989-05-01

    A multiphoton mechanism for molecular beam transitions is presented which relies on a large first-order ac Stark effect to modulate the energy separation of the initial and final states of the multiphoton transition, but which does not require the presence of any intermediate level(s). The theoretical formalism uses ideas from the laser multiphoton literature for a two-level system interacting with a monochromatic electromagnetic radiation field, together with a close analog of the rotating wave approximation. The diagonal matrix elements of the Hamiltonian operator corresponding to the large ac Stark effect are removed by a mathematical substitution which in effect transforms appropriate differences of these diagonal elements into transition moments involving higher harmonics of the frequency of the monochromatic radiation field. The electric field strength of the true monochromatic radiation field is ``distributed'' among the higher harmonics of the effective field according to an expression involving Bessel functions. Because these Bessel functions are bounded, there exists for a given time t of exposure to the radiation, a threshold for the magnitude of the transition dipole matrix element coupling the two levels: Below this threshold, the transition probability in a traditional one-photon molecular beam electric resonance experiment cannot be made unity simply by increasing the amplitude of the radiation field. In fact, if the coupling matrix element is small enough, the molecular beam electric resonance signal cannot be detected within exposure time t. The algebraic formalism described above is checked by computer solution of an initial value problem involving four real coupled linear differential equations. It is then used to explain the multiphoton transitions previously observed in molecular beam electric resonance studies on the two symmetric top molecules OPF3 and CH3 CF3, where the number of photons involved in a given transition varies from 1

  10. Exact Eigenstates for a Class of Model Describing Interactions Among Five Bosonic Modes with Multiphoton Process

    Institute of Scientific and Technical Information of China (English)

    YANGWen-Xing; LIJia-Hua; LIWei-Bin; LUOJin-Ming; XIEXiao-Tao; WEIHua

    2004-01-01

    We present an efficient approach to studying the spectra and eigenstates for the model describing interactions among five bosonic modes without using the assumption of the Bethe ansatz. The exact analytical results of all the eigenstates and eigenvalues are in terms of a parameter A for a class of models describing five-mode multiphoton process. The parameter is determined by the roots of a polynomial and is solvable analytically or numerically.

  11. Clinical optical coherence tomography combined with multiphoton tomography of patients with skin diseases.

    Science.gov (United States)

    König, Karsten; Speicher, Marco; Bückle, Rainer; Reckfort, Julia; McKenzie, Gordon; Welzel, Julia; Koehler, Martin J; Elsner, Peter; Kaatz, Martin

    2009-07-01

    We report on the first clinical study based on optical coherence tomography (OCT) in combination with multiphoton tomography (MPT) and dermoscopy. 47 patients with a variety of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art OCT systems for dermatology including multibeam swept source OCT, (ii) the femtosecond laser multiphoton tomograph, and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface. OCT images reflect modifications of the intratissue refractive index whereas MPT is based on nonlinear excitation of endogenous fluorophores and second harmonic generation. A stack of cross-sectional OCT "wide field" images with a typical field of view of 5 x 2 mm(2) gave fast information on the depth and the volume of the lesion. Multiphoton tomography provided 0.36 x 0.36 mm(2) horizontal/diagonal optical sections within seconds of a particular region of interest with superior submicron resolution down to a tissue depth of 200 mum. The combination of OCT and MPT provides a unique powerful optical imaging modality for early detection of skin cancer and other skin diseases as well as for the evaluation of the efficiency of treatments.

  12. Infrared multiphoton dissociation of unsubstituted metal carbonyls at 5 μm

    Science.gov (United States)

    Au, Mei-Kuen; Hackett, P. A.; Humphries, M.; John, P.

    1984-01-01

    A frequency-doubled carbon dioxide laser of modest output energy (1 mJ) has been used to study, for the first time, the infrared multiphoton absorption by, and dissociation of, the unsubstituted carbonyls of vanadium, chromium, iron, nickel, molybdenum, and tungsten. The multiphoton absorption cross-sections measured for Ni(CO)4, Fe(CO)5, Cr(CO)6, Mo(CO)6, and V(CO)6 are high (σ˜2×10-17) and ensure facile multiphoton dissociation. In focussed beams a pressure independent reaction yield proportional to the 1.5 power of the beam energy is observed for Fe(CO)5, Cr(CO)6, and Mo(CO)6 implying threshold fluences of only 32, 25, and 26 mJ cm-2, respectively. The stoichiometry of the reaction, observed by a pressure measurement technique, is consistent with production of metal atoms and carbon monoxide as final products for Ni(CO)4, Fe(CO)5, Cr(CO)6, and Mo(CO)6. This extensive decarbonylation along the ground state surface is consistent with recent studies of the photochemistry of these molecules from excited electronic states.

  13. Characterization of the second- and third-order nonlinear optical susceptibilities of monolayer MoS2 using multiphoton microscopy

    Science.gov (United States)

    Woodward, R. I.; Murray, R. T.; Phelan, C. F.; de Oliveira, R. E. P.; Runcorn, T. H.; Kelleher, E. J. R.; Li, S.; de Oliveira, E. C.; Fechine, G. J. M.; Eda, G.; de Matos, C. J. S.

    2017-03-01

    We report second- and third-harmonic generation in monolayer MoS2 as a tool for imaging and accurately characterizing the material’s nonlinear optical properties under 1560 nm excitation. Using a surface nonlinear optics treatment, we derive expressions relating experimental measurements to second- and third-order nonlinear sheet susceptibility magnitudes, obtaining values of | {χ }{{s}}(2)| =2.0× {10}-20 m2 V-1 and, for the first time for monolayer MoS2, | {χ }{{s}}(3)| =1.7× {10}-28 m3 V-2. These sheet susceptibilities correspond to effective bulk nonlinear susceptibility values of | {χ }{{b}}(2)| =2.9 × {10}-11 m V-1 and | {χ }{{b}}(3)| =2.4× {10}-19 m2 V-2, accounting for the sheet thickness. Experimental comparisons between MoS2 and graphene are also performed, demonstrating ˜3.4 times stronger third-order sheet nonlinearity in monolayer MoS2, highlighting the material’s potential for nonlinear photonics in the telecommunications C band.

  14. Characterization of the second- and third-order nonlinear optical susceptibilities of monolayer MoS$_2$ using multiphoton microscopy

    CERN Document Server

    Woodward, R I; Phelan, C F; de Oliveira, R E P; Runcorn, T H; Kelleher, E J R; Li, S; de Oliveira, E C; Fechine, G J M; Eda, G; de Matos, C J S

    2016-01-01

    We report second- and third-harmonic generation in monolayer MoS$_2$ as a tool for imaging and accurately characterizing the material's nonlinear optical properties under 1560 nm excitation. Using a surface nonlinear optics treatment, we derive expressions relating experimental measurements to second- and third-order nonlinear sheet susceptibility magnitudes, obtaining values of $|\\chi_s^{(2)}|=2\\times10^{-20}$ m$^2$ V$^{-1}$ and for the first time for monolayer MoS$_2$, $|\\chi_s^{(3)}|=2\\times10^{-28}$ m$^3$ V$^{-2}$. Experimental comparisons between MoS$_2$ and graphene are also performed, demonstrating $\\sim$4 times stronger third-order nonlinearity in monolayer MoS$_2$, highlighting the material's potential for nonlinear photonics in the telecommunications C band.

  15. A new method using multiphoton imaging and morphometric analysis for differentiating chromophobe renal cell carcinoma and oncocytoma kidney tumors

    Science.gov (United States)

    Wu, Binlin; Mukherjee, Sushmita; Jain, Manu

    2016-03-01

    Distinguishing chromophobe renal cell carcinoma (chRCC) from oncocytoma on hematoxylin and eosin images may be difficult and require time-consuming ancillary procedures. Multiphoton microscopy (MPM), an optical imaging modality, was used to rapidly generate sub-cellular histological resolution images from formalin-fixed unstained tissue sections from chRCC and oncocytoma.Tissues were excited using 780nm wavelength and emission signals (including second harmonic generation and autofluorescence) were collected in different channels between 390 nm and 650 nm. Granular structure in the cell cytoplasm was observed in both chRCC and oncocytoma. Quantitative morphometric analysis was conducted to distinguish chRCC and oncocytoma. To perform the analysis, cytoplasm and granules in tumor cells were segmented from the images. Their area and fluorescence intensity were found in different channels. Multiple features were measured to quantify the morphological and fluorescence properties. Linear support vector machine (SVM) was used for classification. Re-substitution validation, cross validation and receiver operating characteristic (ROC) curve were implemented to evaluate the efficacy of the SVM classifier. A wrapper feature algorithm was used to select the optimal features which provided the best predictive performance in separating the two tissue types (classes). Statistical measures such as sensitivity, specificity, accuracy and area under curve (AUC) of ROC were calculated to evaluate the efficacy of the classification. Over 80% accuracy was achieved as the predictive performance. This method, if validated on a larger and more diverse sample set, may serve as an automated rapid diagnostic tool to differentiate between chRCC and oncocytoma. An advantage of such automated methods are that they are free from investigator bias and variability.

  16. Nanostructures based on quantum dots for application in promising methods of single- and multiphoton imaging and diagnostics

    Science.gov (United States)

    Nabiev, I. R.

    2017-01-01

    Molecules recognizing biomarkers of diseases (monoclonal antibodies (monoABs)) are often too large for biomedical applications, and the conditions that are used to bind them with nanolabels lead to disordered orientation of monoABs with respect to the nanoparticle surface. Extremely small nanoprobes, designed via oriented conjugation of quantum dots (QDs) with single-domain antibodies (sdABs) derived from the immunoglobulin of llama and produced in the E. coli culture, have a hydrodynamic diameter less than 12 nm and contain equally oriented sdAB molecules on the surface of each QD. These nanoprobes exhibit excellent specificity and sensitivity in quantitative determination of a small number of cells expressing biomarkers. In addition, the higher diffusion coefficient of sdABs makes it possible to perform immunohistochemical analysis in bulk tissue, inaccessible for conventional monoABs. The necessary conditions for implementing high-quality immunofluorescence diagnostics are a high specificity of labeling and clear differences between the fluorescence of nanoprobes and the autofluorescence of tissues. Multiphoton micros-copy with excitation in the near-IR spectral range, which is remote from the range of tissue autofluorescence excitation, makes it possible to solve this problem and image deep layers in biological tissues. The two-photon absorption cross sections of CdSe/ZnS QDs conjugated with sdABs exceed the corresponding values for organic fluorophores by several orders of magnitude. These nanoprobes provide clear discrimination between the regions of tumor and normal tissues with a ratio of the sdAB fluorescence to the tissue autofluorescence upon two-photon excitation exceeding that in the case of single-photon excitation by a factor of more than 40. The data obtained indicate that the sdAB-QD conjugates used as labels provide the same, or even better, quality as the "gold standard" of immunohistochemical diagnostics. The developed nanoprobes are expected to

  17. Annular pupil filter under shot-noise condition for linear and non linear microscopy.

    Science.gov (United States)

    Ronzitti, Emiliano; Vicidomini, Giuseppe; Caorsi, Valentina; Diaspro, Alberto

    2009-04-13

    The imaging performances of multiphoton excitation and confocal laser scanning microscopy are herby considered: in typical experimental imaging conditions, a small finite amount of photon reaches the detector giving shot-noise fluctuations which affects the signal acquired. A significant detriment in the high frequencies transmission capability is obtained. In order to partially recover the high frequencies information lost, the insertion of a pupil plane filter in the microscope illumination light pathway on the objective lens is proposed. We demonstrate high-frequency and resolution enhancement in the case of linear and non linear fluorescence microscope approach under shot-noise condition.

  18. Simplified method for ultra high-resolution photoacoustic microscopy via transient absorption.

    Science.gov (United States)

    Mattison, Scott P; Applegate, Brian E

    2014-08-01

    Photoacoustic microscopy (PAM) is a hybrid imaging modality that combines optical illumination with ultrasonic detection to achieve absorption contrast imaging of endogenous and exogenous chromophores. Optical resolution PAM achieves high lateral-resolution by tightly focusing the excitation light; however the axial resolution is still dependent upon the bandwidth of the ultrasonic transducer. As a result, PAM images have highly asymmetric voxels with submicron lateral resolution and axial resolution typically limited to tens of microns. We have previously reported on a resonant multiphoton approach to PAM called transient absorption ultrasonic microscopy (TAUM), which enables high axial resolution by frequency encoding the photoacoustic signal at the overlap of a pump and a probe beam. This approach enables photoacoustic imaging with subcellular resolution on par with other multiphoton microscopy techniques. Here, we report on an innovation that enables TAUM imaging with a much less sophisticated optical system than previously reported. If we allow the time delay between the pump and probe to collapse to zero, the pump and probe optical paths can be combined. An amplitude modulator in the single beam path is sufficient to encode the TAUM signal at the second harmonic of the modulation frequency. The resulting system is essentially a standard optical resolution PAM system that incorporates an amplitude modulator and utilizes a Fourier post processing algorithm to improve the axial resolution by approximately an order of magnitude. A prototype system based on this approach has been assembled and tested on fixed bovine erythrocytes.

  19. Correlated Light Microscopy and Electron Microscopy

    NARCIS (Netherlands)

    Sjollema, Klaas A.; Schnell, Ulrike; Kuipers, Jeroen; Kalicharan, Ruby; Giepmans, Ben N. G.; MullerReichert, T; Verkade, P

    2012-01-01

    Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals

  20. Correlated Light Microscopy and Electron Microscopy

    NARCIS (Netherlands)

    Sjollema, Klaas A.; Schnell, Ulrike; Kuipers, Jeroen; Kalicharan, Ruby; Giepmans, Ben N. G.; MullerReichert, T; Verkade, P

    2012-01-01

    Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals sel

  1. Automated motion artifact removal for intravital microscopy, without a priori information

    Science.gov (United States)

    Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

    2014-03-01

    Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

  2. Differential responses of vanilla accessions to root rot and colonization by Fusarium oxysporum f. sp. radicis-vanillae.

    Directory of Open Access Journals (Sweden)

    Sayuj eKoyyappurath

    2015-12-01

    Full Text Available Root and stem rot (RSR disease caused by Fusarium oxysporum f. sp. radicis-vanillae (Forv is the most damaging disease of vanilla (Vanilla planifolia and V. ×tahitensis, Orchidaceae. Breeding programs aimed at developing resistant vanilla varieties are hampered by the scarcity of sources of resistance to RSR and insufficient knowledge about the histopathology of Forv. In this work we have i identified new genetic resources resistant to RSR including V. planifolia inbreds and vanilla relatives, ii thoroughly described the colonization pattern of Forv into selected vanilla accessions, confirming its necrotic non-vascular behavior in roots, and iii evidenced the key role played by hypodermis, and particularly lignin deposition onto hypodermal cell walls, for resistance to Forv in two highly resistant vanilla accessions.Two hundred and fifty-four vanilla accessions were evaluated in the field under natural conditions of infection and in controlled conditions using in-vitro plants root-dip inoculated by the highly pathogenic isolate Fo072. For the 26 accessions evaluated in both conditions, a high correlation was observed between field evaluation and in-vitro assay.The root infection process and plant response of one susceptible and two resistant accessions challenged with Fo072 were studied using wide field and multiphoton microscopy. In susceptible V. planifolia, hyphae penetrated directly into the rhizodermis in the hairy root region then invaded the cortex through the passage cells where it induced plasmolysis, but never reached the vascular region. In the case of the resistant accessions, the penetration was stopped at the hypodermal layer. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to Forv. The thickness of lignin constitutively deposited onto outer cell walls of hypodermis was highly correlated with the level of resistance for 21

  3. First-principles calculation of multiphoton absorption cross section of α-quartz under femtosecond laser irradiation

    Science.gov (United States)

    Yu, Dong; Jiang, Lan; Wang, Feng; Qu, Liangti; Lu, Yongfeng

    2016-05-01

    Time-dependent density functional theory-based first-principles calculations have been used to study the ionization process and electron excitation. The results show that the number of excited electrons follows the power law σ k I k at peak intensities of I key role. The multiphoton absorption cross section of α-quartz σ k is further calculated to be 3.54 × 1011 cm-3 ps-1 (cm2/TW)6. Using the plasma model, the theoretical results of the damage threshold fluences are consistent with the experimental data, which validates the calculated value of multiphoton absorption cross section. By employing the calculated cross section value in the plasma model, the damage threshold fluences are theoretically estimated, being consistent with the experimental data, which validates the calculated value of multiphoton absorption cross section. The preliminary multiscale model shows great potential in the simulation of laser processing.

  4. Open access

    NARCIS (Netherlands)

    Valkenburg, P.M.

    2015-01-01

    Open access week Van 19 tot en met 25 oktober 2015 vond wereldwijd de Open Access Week plaats. Tijdens deze week werden er over de hele wereld evenementen georganiseerd waar open access een rol speelt. Ook in Nederland zijn er diverse symposia, workshops en debatten georganiseerd zoals het debat in

  5. Membranes and Fluorescence microscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2009-01-01

    be provided by microscopy-related techniques. In this chapter, I will attempt to summarize representative examples concerning how microscopy (which provides information on membrane lateral organization by direct visualization) and spectroscopy techniques (which provides information about molecular interaction...

  6. Multi-photon Decays of the Higgs Boson in Standard Model: Leading Terms from Heisenberg-Euler Effective Lagrangian

    CERN Document Server

    Liao, Yi

    2012-01-01

    We calculate the multi-photon decay widths of the Higgs boson from an effective Lagrangian for a system of electromagnetic and Higgs fields. We utilize a low-energy theorem to connect the above Lagrangian to the Heisenberg-Euler effective Lagrangian induced by charged particles that gain mass from interactions with the Higgs boson. In the standard model only the W^\\pm gauge bosons and the top quark are relevant, and we compute their contributions to the effective couplings and the multi-photon decay widths of the Higgs boson.

  7. Design, Fabrication and Computational Characterization of a 3D Micro-Valve Built by Multi-Photon Polymerization

    Directory of Open Access Journals (Sweden)

    Stratos Galanopoulos

    2014-08-01

    Full Text Available We report on the design, modeling and fabrication by multi-photon polymerization of a complex medical fluidic device. The physical dimensions of the built micro-valve prototype are compared to those of its computer-designed model. Important fabrication issues such as achieving high dimensional resolution and ability to control distortion due to shrinkage are presented and discussed. The operational performance of both multi-photon and CAD-created models under steady blood flow conditions was evaluated and compared through computational fluid dynamics analysis.

  8. Influence of Multi-Photon Process on Entanglement of Interacting System of the Qubit and Thermal Field

    Institute of Scientific and Technical Information of China (English)

    LI Wei-Jun; LI Shang-Bin; XU Jing-Bo

    2004-01-01

    @@ We study the system of a single qubit couples to a single mode thermal field according to a multi-photon JaynesCummings-type interaction with phase decoherence. Both the time evolving entanglement and the stationary state entanglement are calculated by adopting the log-negativity as a measure. It is found that the multi-photon process can enhance the stationary state entanglement of this system and can enlarge the range of the parameter△/g and the mean photon number of initial thermal field in which the stationary state is distillable.

  9. Determination of multiphoton absorption of silk fibroin using the Z-scan technique.

    Science.gov (United States)

    Applegate, Matthew B; Marelli, Benedetto; Kaplan, David L; Omenetto, Fiorenzo G

    2013-12-02

    The multiphoton absorption of silk fibroin at 810 nm was determined by open-aperture Z-scan. Three-photon absorption was confirmed at this wavelength and the three-photon cross section of silk fibroin was measured. Silk fibroin of varying molecular weight was tested and the cross section was found to increase exponentially with increasing molecular weight. The confirmation of a relatively large three-photon absorption cross section in silk will help lay the groundwork for future investigation of direct laser writing of three-dimensional structures within a silk matrix.

  10. Experimental study of multi-photon contamination on the measurement of fluorescent decay time

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In the measurement of fluorescent lifetime based on time correlation-single photon counting technique by means of TAC, due to the contamination of multi-photons a deviation of fluorescent lifetime measured from the expected value is experimentally studied. A correction function instead of a simple exponential function is used to fit the experiment data. The validation of the correction function is checked using the experimental data of several test samples: YAP, NaI(T1) and LSO. The results show that the correction function well fits the data and the reasonable fluorescent lifetimes are obtained.

  11. Multiphoton resonant ionization of hydrogen atom exposed to two-colour laser pulses

    Institute of Scientific and Technical Information of China (English)

    Wang Pei-Jie; Fang Yan

    2008-01-01

    This paper studies the multiphoton resonant ionization by two-colour laser pulses in the hydrogen atom by solving the time-dependent Schr(o)dinger equation.By fixing the parameters of fundamental laser field and scanning the frequency of second laser field,it finds that the ionization probability shows several resonance peaks and is also much larger than the linear superposition of probabilities by applying two lasers separately.The enhancement of the ionization happens when the system is resonantly pumped to the excited states by absorbing two or more colour photons non-sequentially.

  12. Optical-optical double-resonant multiphoton ionization spectra of Rydberg states of nitrogen dioxide

    Institute of Scientific and Technical Information of China (English)

    Zhang Gui-Yin; Zhang Lian-Shui; Sun Bo; Han Xiao-Feng; Yu Wei

    2005-01-01

    The optical-optical double-resonant multiphoton ionization(OODR-MPI) technique has been applied to the study of the Rydberg states of nitrogen dioxide. The results show that ,althougy the OODR-MPI spectra of NO2 are composed of regular progression bands at different pump laser intensities, their ionization pathways are different.The NO2 mollecule is ionized through the (3+1+1)double-resonant process as the pump laser intensity is in a high value, or else it is through the (1+2+1)rpocess.The final resonant states in the two ionizing processes have been attributed to different Rydberg states.

  13. Highly efficient avalanche multiphoton luminescence from coupled Au nanowires in the visible region

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    We report highly efficient avalanche multiphoton luminescence(MPL)from ordered-arrayed gold nanowires(NWs).The time-average excitation intensity I_(exc) is as low as 5.0-9.1 kW/cm~2.The intensity of avalanche MPL I_(MPL) is about 10~4 times larger than that of three-photon luminescence,the slope ■logI_(MPL)/■logI_(exc) of avalanche MPL reaches as high as 18.3 and the corresponding polarization dependence of I_(MPL) has a form of cos~(50)■_p.The emission dynamics of avalanche MPL and three-photon luminesc...

  14. Non-linear image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Gregor, Ingo; Ros, Robert; Enderlein, Jörg

    2017-02-01

    Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates. We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging. 1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101. 2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53-54.

  15. Open-Ended Recursive Approach for the Calculation of Multiphoton Absorption Matrix Elements.

    Science.gov (United States)

    Friese, Daniel H; Beerepoot, Maarten T P; Ringholm, Magnus; Ruud, Kenneth

    2015-03-10

    We present an implementation of single residues for response functions to arbitrary order using a recursive approach. Explicit expressions in terms of density-matrix-based response theory for the single residues of the linear, quadratic, cubic, and quartic response functions are also presented. These residues correspond to one-, two-, three- and four-photon transition matrix elements. The newly developed code is used to calculate the one-, two-, three- and four-photon absorption cross sections of para-nitroaniline and para-nitroaminostilbene, making this the first treatment of four-photon absorption in the framework of response theory. We find that the calculated multiphoton absorption cross sections are not very sensitive to the size of the basis set as long as a reasonably large basis set with diffuse functions is used. The choice of exchange-correlation functional, however, significantly affects the calculated cross sections of both charge-transfer transitions and other transitions, in particular, for the larger para-nitroaminostilbene molecule. We therefore recommend the use of a range-separated exchange-correlation functional in combination with the augmented correlation-consistent double-ζ basis set aug-cc-pVDZ for the calculation of multiphoton absorption properties.

  16. Role of quantum trajectory in high-order harmonic generation in the Keldysh multiphoton regime.

    Science.gov (United States)

    Li, Peng-Cheng; Jiao, Yuan-Xiang; Zhou, Xiao-Xin; Chu, Shih-I

    2016-06-27

    We present a systematic study of spectral and temporal structure of high-order harmonic generation (HHG) by solving accurately the time-dependent Schrödinger equation for a hydrogen atom in the multiphoton regime where the Keldysh parameter is greater unity. Combining with a time-frequency transform and an extended semiclassical analysis, we explore the role of quantum trajectory in HHG. We find that the time-frequency spectra of the HHG plateau near cutoff exhibit a decrease in intensity associated with the short- and long-trajectories when the ionization process is pushed from the multiphoton regime into the tunneling regime. This implies that the harmonic emission spectra in the region of the HHG plateau near and before the cutoff are suppressed. To see the generality of this prediction, we also present a time-dependent density-functional theoretical study of the effect of correlated multi-electron responses on the spectral and temporal structure of the HHG plateau of the Ar atom.

  17. Multiphoton crosslinking for biocompatible 3D printing of type I collagen.

    Science.gov (United States)

    Bell, Alex; Kofron, Matthew; Nistor, Vasile

    2015-09-03

    Multiphoton fabrication is a powerful technique for three-dimensional (3D) printing of structures at the microscale. Many polymers and proteins have been successfully structured and patterned using this method. Type I collagen comprises a large part of the extracellular matrix for most tissue types and is a widely used cellular scaffold material for tissue engineering. Current methods for creating collagen tissue scaffolds do not allow control of local geometry on a cellular scale. This means the environment experienced by cells may be made up of the native material but unrelated to native cellular-scale structure. In this study, we present a novel method to allow multiphoton crosslinking of type I collagen with flavin mononucleotide photosensitizer. The method detailed allows full 3D printing of crosslinked structures made from unmodified type I collagen and uses only demonstrated biocompatible materials. Resolution of 1 μm for both standing lines and high-aspect ratio gaps between structures is demonstrated and complex 3D structures are fabricated. This study demonstrates a means for 3D printing with one of the most widely used tissue scaffold materials. High-resolution, 3D control of the fabrication of collagen scaffolds will facilitate higher fidelity recreation of the native extracellular environment for engineered tissues.

  18. Single- and multiphoton events with missing energy in $e^{+} e^{-}$ 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, G J; Böhm, A; Boldizsar, L; Borgia, B; Bottai, S; Bourilkov, D; Bourquin, M; Braccini, S; Branson, J G; Brochu, F; Burger, J D; Burger, W J; Cai, X D; Capell, M; Carlino, G; Cartacci, A; 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, L; Cindolo, F; Clare, I; Clare, R; Coignet, G; Colino, N; Costantini, S; Cucciarelli, S; De Salvo, A; De Notaristefani, F; Debreczeni, J; Deglon, P; Degré, A; Dehmelt, K; Deiters, K; Delmeire, E; Denes, P; Diemoz, M; Dierckxsens, M; Dionisi, C; Dittmar, M; Doria, A; Dova, M T; Duchesneau, D; Duda, M; Echenard, B; El-Hage, A; El-Mamouni, H; Eline, A; Engler, A; Eppling, F J; Extermann, P; Falagán, M A; Falciano, S; Favara, A; Fay, J; Fedin, O; Felcini, M; Ferguson, T; Fesefeldt, H S; Fiandrini, E; Field, J H; Filthaut, F; 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; Gupta, V K; Gurtu, A; Gutay, L J; Haas, D; 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; 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; Kraber, M; Krämer, R W; Krüger, A; Kunin, A; Laktineh, I; Landi, G; Le Coultre, P; Le Goff, J M; Lebeau, M; Lebedev, A; Lebrun, P; Lecomte, P; Lecoq, P; Leiste, R; Levtchenko, M; Levchenko, P M; Li, C; Likhoded, S; Lin, C H; Lin, W T; Linde, Frank L; Lista, L; Liu, Z A; Lohmann, W; Longo, E; Lü, Y S; Luci, C; Luminari, L; Lustermann, W; Ma W G; Malgeri, L; Malinin, A; Maña, C; 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; Napolitano, M; Natale, S; Nessi-Tedaldi, F; Newman, H; Nisati, A; Novák, T; Nowak, H; Ofierzynski, R A; Organtini, G; Pal, I; Palomares, C; Paolucci, P; Paramatti, R; Passaleva, G; Patricelli, S; Paul, T; Pauluzzi, M; Paus, C; Pauss, F; 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; Prokofev, D; Prokofiev, D O; Quartieri, J; Rahal-Callot, G; Rahaman, M A; Raics, P; Raja, N; Ramelli, R; Rancoita, P G; Ranieri, R; Raspereza, A V; Razis, P; Ren, D; Rescigno, M; Reucroft, S; Riemann, S; Riles, K; Roe, B P; Romeo, G C; Romero, L; Rosca, A; Rosenbleck, C; Rosier-Lees, S; Roth, S; Rubio, J A; Ruggiero, G; Rykaczewski, H; Sakharov, A; Salicio, J; Sánchez, E; Saremi, S; Sarkar, S; Schäfer, C; Shchegelskii, V; Schopper, Herwig Franz; Schotanus, D J; Sciacca, C; Servoli, L; Shevchenko, S; Shivarov, N; Shoutko, V; Shumilov, E; Shvorob, A; Son, D; Souga, C; Spillantini, P; Steuer, M; Stickland, D P; Stoyanov, B; Strässner, A; Sudhakar, K; Sultanov, G G; Sun, L Z; Sushkov, S; Suter, K; Swain, J D; Szillási, Z; Tang, X W; Tarjan, P; Tauscher, L; 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; Vásquez, R; Veszpremi, V; Vesztergombi, G; Vetlitskii, I; Vicinanza, D; Viertel, G; Villa, S; Vivargent, M; Vlachos, S; Vodopyanov, I; Vogel, H; Vogt, H; Vorobev, I; Vorobyov, A A; Wadhwa, M; Wang, Q; 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; Zimmermann, B; Zöller, M; De Asmundis, R; De Guevara, P L; de Jong, P; de la Cruz, B; Della Volpe, D; van Dalen, J A; van Gulik, R

    2004-01-01

    Single- and multiphoton events with missing energy are selected in 619 pb/sup -1/ of data collected by the L3 detector at LEP at centre- of-mass energies between 189 and 209 GeV. The cross sections of the process e^{+}e^{-} to nu nu gamma ( gamma ) are found to be in agreement with the standard model expectations, and the number of light neutrino species is determined, including lower energy data, to be N/sub nu /=2.98+or-0.05+or-0.04. Selection results are given in the form of tables which can be used to test future models involving single- and multiphoton signatures at LEP. These final states are also predicted by models with large extra dimensions and by several supersymmetric models. No evidence for such models is found. Among others, lower limits between 1.5 and 0.65 TeV are set, at 95% confidence level, on the new scale of gravity for the number of extra dimensions between 2 and 6.

  19. Single- and Multi-Photon Events with Missing Energy in $e^+ e^-$ 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; Dehmelt, K; Deiters, K; Della Volpe, D; Delmeire, E; Denes, P; De Notaristefani, F; De Salvo, A; Diemoz, M; Dierckxsens, M; Dionisi, C; Dittmar, M; Doria, A; Dova, M T; Duchesneau, D; Duda, M; Echenard, B; Eline, A; El-Hage, A; El-Mamouni, H; Engler, A; Eppling, F J; Extermann, P; Falagán, M A; Falciano, S; Favara, A; Fay, J; Fedin, O; Felcini, M; Ferguson, T; Fesefeldt, H S; Fiandrini, E; Field, J H; Filthaut, F; 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; 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; 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; Lin, C H; Lin, W T; Linde, Frank L; Lista, L; Liu, Z A; Lohmann, W; Longo, E; Lü, Y S; Luci, C; Luminari, L; Lustermann, W; Ma Wen Gan; Malgeri, L; Malinin, A; Maña, C; 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; Nisati, A; Novák, T; Nowak, H; Ofierzynski, R A; Organtini, G; Pal, I; Palomares, C; 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; Prokofev, D; Prokofiev, D O; 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; Rosenbleck, C; Rosier-Lees, S; Roth, S; Rubio, J A; Ruggiero, G; Rykaczewski, H; Sakharov, A; Saremi, S; Sarkar, S; Salicio, J; Sánchez, E; Schäfer, C; Shchegelskii, V; Schopper, Herwig Franz; Schotanus, D J; Sciacca, C; Servoli, L; Shevchenko, S; Shivarov, N; Shoutko, V; Shumilov, E; Shvorob, A V; Son, D; Souga, C; Spillantini, P; Steuer, M; Stickland, D P; Stoyanov, B; Strässner, A; Sudhakar, K; Sultanov, G G; Sun, L Z; Sushkov, S; 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; Vásquez, R; 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; Wang, Q; 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; Zimmermann, B; Zöller, M

    2004-01-01

    Single- and multi-photon events with missing energy are selected in 619/pb of data collected by the L3 detector at LEP at centre-of-mass energies between 189GeV and 209GeV. The cross sections of the process e^+e^- -> nu nu gamma (gamma) are found to be in agreement with the Standard Model expectations, and the number of light neutrino species is determined, including lower energy data, to be N_nu = 2.98 +/- 0.05 +/- 0.04. Selection results are also given in the form of tables which can be used to test future models involving single- and multi-photon signatures at LEP. These final states are also predicted by models with large extra dimensions and by several supersymmetric models. No evidence for such models is found. Among others, lower limits between 1.5TeV and 0.65TeV are set, at 95% confidence level, on the new scale of gravity for the number of extra dimensions between 2 and 8.

  20. Simultaneous Spatial and Temporal Focusing in Nonlinear Microscopy.

    Science.gov (United States)

    Durst, M E; Zhu, G; Xu, C

    2008-04-01

    Simultaneous spatial and temporal focusing (SSTF), when combined with nonlinear microscopy, can improve the axial excitation confinement of wide-field and line-scanning imaging. Because two-photon excited fluorescence depends inversely on the pulse width of the excitation beam, SSTF decreases the background excitation of the sample outside of the focal volume by broadening the pulse width everywhere but at the geometric focus of the objective lens. This review theoretically describes the beam propagation within the sample using Fresnel diffraction in the frequency domain, deriving an analytical expression for the pulse evolution. SSTF can scan the temporal focal plane axially by adjusting the GVD in the excitation beam path. We theoretically define the axial confinement for line-scanning SSTF imaging using a time-domain understanding and conclude that line-scanning SSTF is similar to the temporally-decorrelated multifocal multiphoton imaging technique. Recent experiments on the temporal focusing effect and its axial confinement, as well as the axial scanning of the temporal focus by tuning the GVD, are presented. We further discuss this technique for axial-scanning multiphoton fluorescence fiber probes without any moving parts at the distal end. The temporal focusing effect in SSTF essentially replaces the focusing of one spatial dimension in conventional wide-field and line-scanning imaging. Although the best axial confinement achieved by SSTF cannot surpass that of a regular point-scanning system, this trade-off between spatial and temporal focusing can provide significant advantages in applications such as high-speed imaging and remote axial scanning in an endoscopic fiber probe.

  1. Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in novel mouse models with fluorescent lineage tags

    Science.gov (United States)

    Hackl, Matthias J.; Burford, James L.; Villanueva, Karie; Lam, Lisa; Suszták, Katalin; Schermer, Bernhard; Benzing, Thomas; Peti-Peterdi, János

    2014-01-01

    Podocytes are critical in the maintenance of a healthy glomerular filter, however they have been difficult to study in the intact kidney due to technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PEC) in vivo. In Podocin-GFP mice podocytes formed sporadic multi-cellular clusters after unilateral ureteral ligation (UUO) and migrated into the parietal Bowman’s capsule. The tracking of single cells in Podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP, or RFP revealed the simultaneous migration of multiple podocytes. In PEPCK-GFP mice serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support the highly dynamic rather than static nature of the glomerular environment and cellular composition. Future application of this new approach promises to advance our understanding of the mechanisms of glomerular injury and regeneration. PMID:24270544

  2. Comparison of analytical and Monte Carlo calculations of multi-photon effects in bremsstrahlung emission by high-energy electrons

    DEFF Research Database (Denmark)

    Mangiarotti, Alessio; Sona, Pietro; Ballestrero, Sergio

    2012-01-01

    Approximate analytical calculations of multi-photon effects in the spectrum of total radiated energy by high-energy electrons crossing thin targets are compared to the results of Monte Carlo type simulations. The limits of validity of the analytical expressions found in the literature are establi...

  3. Comparison of analytical and Monte Carlo calculations of multi-photon effects in bremsstrahlung emission by high-energy electrons

    Energy Technology Data Exchange (ETDEWEB)

    Mangiarotti, A. [Laboratorio de Instrumentacao e Fisica Experimental de Particulas, Coimbra (Portugal); Departamento de Fisica, Faculdade de Ciencias e Tecnologia da Universidade de Coimbra, Coimbra (Portugal); Sona, P., E-mail: pietro.sona@fi.infn.it [Dipartimento di Fisica e Astronomia, Universita degli Studi di Firenze, Polo Scientifico, Via G. Sansone 1, 50019 Sesto Fiorentino (Italy); INFN, Sezione di Firenze (Italy); Ballestrero, S. [Department of Physics University of Johannesburg, Johannesburg (South Africa); CERN PH/ADT, Geneve (Switzerland); Uggerhoj, U.I.; Andersen, K.K. [Department of Physics and Astronomy, University of Aarhus, Aarhus (Denmark)

    2012-10-15

    Approximate analytical calculations of multi-photon effects in the spectrum of total radiated energy by high-energy electrons crossing thin targets are compared to the results of Monte Carlo type simulations. The limits of validity of the analytical expressions found in the literature are established. The separate contributions to spectral distortion of electromagnetic processes other than bremsstrahlung are also studied in detail.

  4. New microscopy for nanoimaging

    CERN Document Server

    Kinjo, Y; Watanabe, M

    2002-01-01

    Two types of new microscopy, namely, X-ray contact microscopy (XRCM) in combination with atomic force microscopy (AFM) and X-ray projection microscopy (XRPM) using synchrotron radiation and zone plate optics were used to image the fine structures of human chromosomes. In the XRCM plus AFM system, location of X-ray images on a photoresist has become far easier than that with our previous method using transmission electron microscopy coupled with the replica method. In addition, the images obtained suggested that the conformation of chromatin fiber differs from the current textbook model regarding the architecture of a eukaryotic chromosome. X-ray images with high contrast of the specimens could be obtained with XRPM. The resolution of each microscopy was about 30 and 200-300 nm for XRCM plus AFM and XRPM, respectively. (author)

  5. Photothermal Single Particle Microscopy

    OpenAIRE

    Selmke, Markus; Braun, Marco; Cichos, Frank

    2011-01-01

    Photothermal microscopy has recently complemented single molecule fluorescence microscopy by the detection of individual nano-objects in absorption. Photothermal techniques gain their superior sensitivity by exploiting a heat induced refractive index change around the absorbing nano-object. Numerous new applications to nanoparticles, nanorods and even single molecules have been reported all refering to the fact that photothermal microscopy is an extinction measurement on a heat induced refrac...

  6. Access Denied

    Science.gov (United States)

    Villano, Matt

    2008-01-01

    Building access control (BAC)--a catchall phrase to describe the systems that control access to facilities across campus--has traditionally been handled with remarkably low-tech solutions: (1) manual locks; (2) electronic locks; and (3) ID cards with magnetic strips. Recent improvements have included smart cards and keyless solutions that make use…

  7. Open Access

    Science.gov (United States)

    Suber, Peter

    2012-01-01

    The Internet lets us share perfect copies of our work with a worldwide audience at virtually no cost. We take advantage of this revolutionary opportunity when we make our work "open access": digital, online, free of charge, and free of most copyright and licensing restrictions. Open access is made possible by the Internet and copyright-holder…

  8. Open Access

    Science.gov (United States)

    Suber, Peter

    2012-01-01

    The Internet lets us share perfect copies of our work with a worldwide audience at virtually no cost. We take advantage of this revolutionary opportunity when we make our work "open access": digital, online, free of charge, and free of most copyright and licensing restrictions. Open access is made possible by the Internet and copyright-holder…

  9. Electron Microscopy Center (EMC)

    Data.gov (United States)

    Federal Laboratory Consortium — The Electron Microscopy Center (EMC) at Argonne National Laboratory develops and maintains unique capabilities for electron beam characterization and applies those...

  10. [Artefacts of confocal microscopy].

    Science.gov (United States)

    Vekshin, N L; Frolov, M S

    2014-01-01

    Typical artefacts caused by using confocal fluorescent microscopy while studying living cells are considered. The role of light scattering, mobility, staining, local concentrations, etc. is discussed.

  11. Coherent light microscopy

    CERN Document Server

    Ferraro, Pietro; Zalevsky, Zeev

    2011-01-01

    This book deals with the latest achievements in the field of optical coherent microscopy. While many other books exist on microscopy and imaging, this book provides a unique resource dedicated solely to this subject. Similarly, many books describe applications of holography, interferometry and speckle to metrology but do not focus on their use for microscopy. The coherent light microscopy reference provided here does not focus on the experimental mechanics of such techniques but instead is meant to provide a users manual to illustrate the strengths and capabilities of developing techniques. Th

  12. Bridging fluorescence microscopy and electron microscopy

    NARCIS (Netherlands)

    Giepmans, Ben N. G.

    Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major

  13. Bridging fluorescence microscopy and electron microscopy

    NARCIS (Netherlands)

    Giepmans, Ben N. G.

    2008-01-01

    Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakth

  14. Photoemission electron microscopy and scanning electron microscopy of Magnetospirillum magnetotacticum's magnetosome chains.

    Science.gov (United States)

    Keutner, Christoph; von Bohlen, Alex; Berges, Ulf; Espeter, Philipp; Schneider, Claus M; Westphal, Carsten

    2014-10-01

    Magnetotactic bacteria are of great interdisciplinary interest, since a vast field of applications from magnetic recording media to medical nanorobots is conceivable. A key feature for a further understanding is the detailed knowledge about the magnetosome chain within the bacteria. We report on two preparation procedures suitable for UHV experiments in reflective geometry. Further, we present the results of scanning electron microscopy, as well as the first photoemission electron microscopy experiments, both accessing the magnetosomes within intact magnetotactic bacteria and compare these to scanning electron microscopy data from the literature. From the images, we can clearly identify individual magnetosomes within their chains.

  15. Imaging photoelectron circular dichroism of chiral molecules by femtosecond multiphoton coincidence detection.

    Science.gov (United States)

    Lehmann, C Stefan; Ram, N Bhargava; Powis, Ivan; Janssen, Maurice H M

    2013-12-21

    Here, we provide a detailed account of novel experiments employing electron-ion coincidence imaging to discriminate chiral molecules. The full three-dimensional angular scattering distribution of electrons is measured after photoexcitation with either left or right circular polarized light. The experiment is performed using a simplified photoelectron-photoion coincidence imaging setup employing only a single particle imaging detector. Results are reported applying this technique to enantiomers of the chiral molecule camphor after three-photon ionization by circularly polarized femtosecond laser pulses at 400 nm and 380 nm. The electron-ion coincidence imaging provides the photoelectron spectrum of mass-selected ions that are observed in the time-of-flight mass spectra. The coincident photoelectron spectra of the parent camphor ion and the various fragment ions are the same, so it can be concluded that fragmentation of camphor happens after ionization. We discuss the forward-backward asymmetry in the photoelectron angular distribution which is expressed in Legendre polynomials with moments up to order six. Furthermore, we present a method, similar to one-photon electron circular dichroism, to quantify the strength of the chiral electron asymmetry in a single parameter. The circular dichroism in the photoelectron angular distribution of camphor is measured to be 8% at 400 nm. The electron circular dichroism using femtosecond multiphoton excitation is of opposite sign and about 60% larger than the electron dichroism observed before in near-threshold one-photon ionization with synchrotron excitation. We interpret our multiphoton ionization as being resonant at the two-photon level with the 3s and 3p Rydberg states of camphor. Theoretical calculations are presented that model the photoelectron angular distribution from a prealigned camphor molecule using density functional theory and continuum multiple scattering X alpha photoelectron scattering calculations

  16. Lasers for nonlinear microscopy.

    Science.gov (United States)

    Wise, Frank

    2013-03-01

    Various versions of nonlinear microscopy are revolutionizing the life sciences, almost all of which are made possible because of the development of ultrafast lasers. In this article, the main properties and technical features of short-pulse lasers used in nonlinear microscopy are summarized. Recent research results on fiber lasers that will impact future instruments are also discussed.

  17. Fluorescence antibunching microscopy

    CERN Document Server

    Schwartz, Osip

    2011-01-01

    Breaking the diffraction limit in microscopy by utilizing quantum properties of light has been the goal of intense research in the recent years. We propose a quantum superresolution technique based on non-classical emission statistics of fluorescent markers, routinely used as contrast labels for bio-imaging. The technique can be readily implemented using standard fluorescence microscopy equipment.

  18. LEDs for fluorescence microscopy

    NARCIS (Netherlands)

    Young, I.T.; Garini, Y.; Dietrich, H.R.C.; Van Oel, W.; Liqui Lung, G.

    2004-01-01

    Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light

  19. Photothermal Single Particle Microscopy

    CERN Document Server

    Selmke, Markus; Cichos, Frank

    2011-01-01

    Photothermal microscopy has recently complemented single molecule fluorescence microscopy by the detection of individual nano-objects in absorption. Photothermal techniques gain their superior sensitivity by exploiting a heat induced refractive index change around the absorbing nano-object. Numerous new applications to nanoparticles, nanorods and even single molecules have been reported all refering to the fact that photothermal microscopy is an extinction measurement on a heat induced refractive index profile. Here, we show that the actual physical mechanism generating a photothermal signal from a single molecule/particle is fundamentally different from the assumed extinction measurement. Combining photothermal microscopy, light scattering microscopy as well as accurate Mie scattering calculations to single gold nanoparticles, we reveal that the detection mechanism is quantitatively explained by a nanolensing effect of the long range refractive index profile. Our results lay the foundation for future develop...

  20. Amplification of infrared multiphoton dissociation efficiency in a quadruple ion trap using IR-active ligands.

    Science.gov (United States)

    Pikulski, Michael; Wilson, Jeffrey J; Aguilar, Apolonio; Brodbelt, Jennifer S

    2006-12-15

    A strategy for increasing the efficiency of infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap (QIT) is described. IR-active ligands (IRALs) are incorporated into noncovalent complexes of the type [M2+(analyte) IRAL]+, where M is a transition metal such as copper or cobalt and IRAL is an auxiliary ligand with an IR-active phosphonate functional group. The complexes are formed via self-assembly in solution directly prior to ESI-MS analysis. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energy-variable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands.

  1. Time and spectrum-resolving multiphoton correlator for 300-900 nm

    Science.gov (United States)

    Johnsen, Kelsey D.; Kolenderski, Piotr; Scarcella, Carmelo; Thibault, Marilyne; Tosi, Alberto; Jennewein, Thomas

    2014-10-01

    We demonstrate a single-photon sensitive spectrometer in the visible range, which allows us to perform time-resolved and multi-photon spectral correlation measurements at room temperature. It is based on a monochromator composed of two gratings, collimation optics, and an array of single photon avalanche diodes. The time resolution can reach 110 ps and the spectral resolution is 2 nm/pixel, limited by the design of the monochromator. This technique can easily be combined with commercial monochromators and can be useful for joint spectrum measurements of two photons emitted in the process of parametric down conversion, as well as time-resolved spectrum measurements in optical coherence tomography or medical physics applications.

  2. Simultaneous measurement of neural spike recordings and multi-photon calcium imaging in neuroblastoma cells.

    Science.gov (United States)

    Kim, Suhwan; Jung, Unsang; Baek, Juyeong; Kang, Shinwon; Kim, Jeehyun

    2012-11-08

    This paper proposes the design and implementation of a micro-electrode array (MEA) for neuroblastoma cell culturing. It also explains the implementation of a multi-photon microscope (MPM) customized for neuroblastoma cell excitation and imaging under ambient light. Electrical signal and fluorescence images were simultaneously acquired from the neuroblastoma cells on the MEA. MPM calcium images of the cultured neuroblastoma cell on the MEA are presented and also the neural activity was acquired through the MEA recording. A calcium green-1 (CG-1) dextran conjugate of 10,000 D molecular weight was used in this experiment for calcium imaging. This study also evaluated the calcium oscillations and neural spike recording of neuroblastoma cells in an epileptic condition. Based on our observation of neural spikes in neuroblastoma cells with our proposed imaging modality, we report that neuroblastoma cells can be an important model for epileptic activity studies.

  3. Sub-15 fs multiphoton lithography of three-dimensional structures for live cell applications

    Science.gov (United States)

    Licht, Martin; Uchugonova, Aisada; König, Karsten; Straub, Martin

    2012-06-01

    Development, morphology and intratissue location of cells are influenced by the 3D nano- and microenvironment. In this paper we demonstrate multiphoton photopolymerization to generate three-dimensional structures for cell culture applications with micro- and nanotopographic features using SU-8 photoresist and mr-NIL 6000 nanoimprint resist. Moving the focal spot of high-repetition rate near-infrared sub-15 fs pulsed laser light by a galvanometric beam scanner in combination with a piezoelectric vertical stage, nearly arbitrary trajectories of polymerized photoresist were generated. This technique can be used to generate cage structures with submicron interior features for live cell applications. Preliminary experiments with PC-3 and HT-1080 cells indicate the influence of the structures on cell behavior.

  4. Time and spectrum-resolving multiphoton correlator for 300–900 nm

    Energy Technology Data Exchange (ETDEWEB)

    Johnsen, Kelsey D.; Thibault, Marilyne; Jennewein, Thomas [Institute for Quantum Computing and Department for Physics and Astronomy, University of Waterloo, 200 University Ave. West, Waterloo, Ontario N2L 3G1 (Canada); Kolenderski, Piotr, E-mail: kolenderski@fizyka.umk.pl [Institute for Quantum Computing and Department for Physics and Astronomy, University of Waterloo, 200 University Ave. West, Waterloo, Ontario N2L 3G1 (Canada); Faculty of Physics, Astronomy and Informatics, Institute of Physics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun (Poland); Scarcella, Carmelo; Tosi, Alberto [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 Milano (Italy)

    2014-10-14

    We demonstrate a single-photon sensitive spectrometer in the visible range, which allows us to perform time-resolved and multi-photon spectral correlation measurements at room temperature. It is based on a monochromator composed of two gratings, collimation optics, and an array of single photon avalanche diodes. The time resolution can reach 110 ps and the spectral resolution is 2 nm/pixel, limited by the design of the monochromator. This technique can easily be combined with commercial monochromators and can be useful for joint spectrum measurements of two photons emitted in the process of parametric down conversion, as well as time-resolved spectrum measurements in optical coherence tomography or medical physics applications.

  5. Entangler and analyzer for multiphotonGreenberger-Horne-Zeilinger states using weak nonlinearities

    Institute of Scientific and Technical Information of China (English)

    DING Dong; YAN FengLi; GAO Ting

    2014-01-01

    In the regime of weak nonlinearity we present two general,feasible schemes for manipulating photon states.One is an entangler for generating any one of the n-photon Greenberger-Home-Zeilinger (GHZ) states.Interactions of the incoming photons with crossKerr media followed by a phase shift gate and a measurement on a probe beam plus appropriate local operations using classical feed-forward of the measurement results allow one to obtain the desired states in a nearly deterministic manner.The second scheme discussed is an analyzer for multiphoton maximally entangled states,which is derived from the above entangler.In this scheme,all of the 2n n-photon GHZ states can,nearly deterministically,be discriminated.

  6. Sine wave gating silicon single-photon detectors for multiphoton entanglement experiments

    Science.gov (United States)

    Zhou, Nan; Jiang, Wen-Hao; Chen, Luo-Kan; Fang, Yu-Qiang; Li, Zheng-Da; Liang, Hao; Chen, Yu-Ao; Zhang, Jun; Pan, Jian-Wei

    2017-08-01

    Silicon single-photon detectors (SPDs) are the key devices for detecting single photons in the visible wavelength range. Here we present high detection efficiency silicon SPDs dedicated to the generation of multiphoton entanglement based on the technique of high-frequency sine wave gating. The silicon single-photon avalanche diode components are acquired by disassembling 6 commercial single-photon counting modules (SPCMs). Using the new quenching electronics, the average detection efficiency of SPDs is increased from 68.6% to 73.1% at a wavelength of 785 nm. These sine wave gating SPDs are then applied in a four-photon entanglement experiment, and the four-fold coincidence count rate is increased by 30% without degrading its visibility compared with the original SPCMs.

  7. Influence of Heat-radiating on Multi-photon Compton Scattering High-energy Electron

    Institute of Scientific and Technical Information of China (English)

    HAO Dong-shan; WANG Xin-min

    2007-01-01

    Using the model of the inverse Compton scattering between high-energy electrons and heat-radiation photons, the influence of heat-radiating photons on multi-photon Compton scattering high-energy electrons is studied . The results show that the energy loss, power loss, light resistance and light pressure of the high-energy electron formed by heat radiating are all proportional to the temperature T4 of the vacuum cavity of the electron,the Lorentz factor γ2 of the high-energy electrons, the scattering section of the electron and the number of photons acting at the same time with high-energy electrons. A good method for lessening the energy loss of the high-energy electron by using the one-photon Compton scattering between high-energy electrons and heat radiation photons is proposed.

  8. Dataset on coherent control of fields and induced currents in nonlinear multiphoton processes in a nanosphere.

    Science.gov (United States)

    McArthur, Duncan; Hourahine, Ben; Papoff, Francesco

    2015-11-24

    We model a scheme for the coherent control of light waves and currents in metallic nanospheres which applies independently of the nonlinear multiphoton processes at the origin of waves and currents. Using exact mathematical formulae, we calculate numerically with a custom fortran code the effect of an external control field which enable us to change the radiation pattern and suppress radiative losses or to reduce absorption, enabling the particle to behave as a perfect scatterer or as a perfect absorber. Data are provided in tabular, comma delimited value format and illustrate narrow features in the response of the particles that result in high sensitivity to small variations in the local environment, including subwavelength spatial shifts.

  9. Resonance-enhanced multiphoton ionization mass spectrometry (REMPI-MS): applications for process analysis.

    Science.gov (United States)

    Streibel, Thorsten; Zimmermann, Ralf

    2014-01-01

    Process analysis is an emerging discipline in analytical sciences that poses special requirements on analytical techniques, especially when conducted in an online manner. Mass spectrometric methods seem exceedingly suitable for this task, particularly if a soft ionization method is applied. Resonance-enhanced multiphoton ionization (REMPI) in combination with time-of-flight mass spectrometry (TOFMS) provides a selective and sensitive means for monitoring (poly)aromatic compounds in process flows. The properties of REMPI and various variations of the ionization process are presented. The potential of REMPI for process analysis is highlighted with several examples, and drawbacks of the method are also noted. Applications of REMPI-TOFMS for the detection and monitoring of aromatic species in a large variety of combustion processes comprising flames, vehicle exhaust, and incinerators are discussed. New trends in technical development and combination with other analytical methods are brought forward.

  10. The Application of Resonance-Enhanced Multiphoton Ionization Technique in Gas Chromatography Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Adan Li

    2014-01-01

    Full Text Available Gas chromatography resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (GC/REMPI-TOFMS using a nanosecond laser has been applied to analyze the 16 polycyclic aromatic hydrocarbons (PAHs. The excited-state lifetime, absorption characters, and energy of electronic states of the 16 PAHs were investigated to optimize the ionization yield. A river water sample pretreated by means of solid phase extraction was analyzed to evaluate the performance of the analytical instrument. The results suggested that REMPI is superior to electron impact ionization method for soft ionization and suppresses the background signal due to aliphatic hydrocarbons. Thus, GC/REMPI-TOFMS is a more reliable method for the determination of PAHs present in the environment.

  11. 2008 Multiphoton Processes Gordon Research Conferences - June 8-13, 2008

    Energy Technology Data Exchange (ETDEWEB)

    Mette B. Gaarde

    2009-08-28

    In 2008 the Gordon Research Conference on Multiphoton Processes is held for the 14th time. The meeting continues to evolve as it embraces both the rapid technological and intellectual growth in the field as well as the multi-disciplinary expertise of the participants. This time the sessions will focus on: (1) Attosecond Science; (2) Free-electron laser experiments and theory; (3) Ultrafast dynamics of molecules; (4) Laser control of molecules; (5) Ultrafast imaging; (6) Super-high intensity and x-rays; (7) Strong field processes in molecules; and (8) Control atoms with light and vice versa. The scientific program will blur traditional disciplinary boundaries as the presenters and discussion leaders involve chemists, physicists, and optical engineers, representing both experiment and theory. The broad range of expertise and different perspectives of attendees should provide a stimulating and unique environment for solving problems and developing new ideas in this rapidly evolving field.

  12. Nonadiabatic dynamics and multiphoton resonances in strong field molecular ionization with few cycle laser pulses

    CERN Document Server

    Tagliamonti, Vincent; Zhao, Arthur; Rozgonyi, Tamás; Marquetand, Philipp; Weinacht, Thomas

    2016-01-01

    We study strong field molecular ionization using few- (four to ten) cycle laser pulses. Employing a supercontinuum light source, we are able to tune the optical laser wavelength (photon energy) over a range of about $\\sim$200 nm (500 meV). We measure the photoelectron spectrum for a series of different molecules as a function of laser intensity, frequency, and bandwidth and illustrate how the ionization dynamics vary with these parameters. We find that multiphoton resonances and nonadiabatic dynamics (internal conversion) play an important role and result in ionization to different ionic continua. Interestingly, while nuclear dynamics can be "frozen" for sufficiently short laser pulses, we find that resonances strongly influence the photoelectron spectrum and final cationic state of the molecule regardless of pulse duration -- even for pulses that are less than four cycles in duration.

  13. 2010 MULTIPHOTON PROCESSES GORDON RESEARCH CONFERENCE, JUNE 6-11, 2010, TILTON, NH

    Energy Technology Data Exchange (ETDEWEB)

    Mette Gaarde

    2010-06-11

    The Gordon Research Conference on Multiphoton Processes will be held for the 15th time in 2010. The meeting continues to evolve as it embraces both the rapid technological and intellectual growth in the field as well as the multi-disciplinary expertise of the participants. This time the sessions will focus on: (1) Ultrafast coherent control; (2) Free-electron laser experiments and theory; (3) Generation of harmonics and attosecond pulses; (4) Ultrafast imaging; (5) Applications of very high intensity laser fields; (6) Strong-field processes in molecules and solids; (7) Attosecond science; and (8) Controlling light. The scientific program will blur traditional disciplinary boundaries as the presenters and discussion leaders involve chemists, physicists, and optical engineers, representing both experiment and theory. The broad range of expertise and different perspectives of attendees should provide a stimulating and unique environment for solving problems and developing new ideas in this rapidly evolving field.

  14. Dynamical Properties of Scaled Atomic Wehrl Entropy of Multiphoton JCM in the Presence of Atomic Damping

    Directory of Open Access Journals (Sweden)

    S. Abdel-Khalek

    2013-01-01

    Full Text Available We study the dynamics of the atomic inversion, scaled atomic Wehrl entropy, and marginal atomic Q-function for a single two-level atom interacting with a one-mode cavity field taking in the presence of atomic damping. We obtain the exact solution of the master equation in the interaction picture using specific initial conditions. We examine the effects of atomic damping parameter and number of multiphoton transition on the scaled atomic Wehrl entropy, atomic Q-function, and their marginal distribution. We observe an interesting monotonic relation between the different physical quantities in the case of different values of the number of photon transition during the time evolution.

  15. Circular dichroism in hydrogen multiphoton ionization by a bichromatic field of frequencies {omega} and 3{omega}

    Energy Technology Data Exchange (ETDEWEB)

    Fifirig, Magda [Department of Chemistry, University of Bucharest, Bucharest (Romania); Cionga, Aurelia [Institute of Space Sciences, Bucharest-Magurele (Romania)

    2002-02-28

    The dichroic effects in the multiphoton ionization of the ground state hydrogen atom by a coherent superposition of a laser beam and its third harmonic, are studied via perturbative calculations. The final state of the photoelectrons, which has the energy E=E{sub 1}+3(h/2{pi}){omega} (E{sub 1} the ground state energy and {omega} the laser frequency), is reached by two interfering quantum paths: (a) absorption of one harmonic photon and (b) absorption of three laser photons. In the chosen regime of field intensities, the radiative corrections to the absorption of one harmonic photon may be disregarded. Our numerical results illustrate the influence of the laser frequency, of the relative intensity and of the harmonic phase upon the dichroic signal. (author)

  16. Influence of laser intensity on the double-resonance multiphoton ionization process of NO molecule

    Institute of Scientific and Technical Information of China (English)

    Guiyin Zhang; Yidong Jin

    2008-01-01

    The analytic forrnula of the ionization efficiency in the process of double resonance enhanced multi-photon ionization (DREMPI) is derived from the dynamic rate equation about the interaction of photon and material. Based on rids formula, the ionization efficiency arid the laser power index versus laser intensity in the DREMPI process of NO molecule, via A2∑ and S2∑ intermediate resonant states, is numerically sinnllated. It is shown that the ionization efficiency of NO molecule increases with the laser intensity until getting saturation, while the laser power index decreases with the enhancement of the laser intensity arid changes to zero at last. The variation of the laser power index with the laser intensity indicates that the ionization efficiency reaches saturation in the one, two, and three excitation steps respectively. It is also found that the narrower the laser pulse duration is, the higher becomes the laser intensity for saturation.

  17. Morphology and size dependence of silver microstructures in fatty salts-assisted multiphoton photoreduction microfabrication

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Yao-Yu.; Dong, Xian-Zi [Chinese Academy of Sciences, Laboratory of Organic NanoPhotonics and Key Laboratory of Photochemical Conversion and Functional Materials, Technical Institute of Physics and Chemistry, Beijing (China); Graduate School of the Chinese Academy of Sciences, Beijing (China); Takeyasu, Nobuyuki [RIKEN (Institute of Physical and Chemical Research), Nanophotonics Laboratory, Wako, Saitama (Japan); Tanaka, Takuo [RIKEN (Institute of Physical and Chemical Research), Metamaterials Laboratory, Wako, Saitama (Japan); Zhao, Zhen-Sheng; Duan, Xuan-Ming [Chinese Academy of Sciences, Laboratory of Organic NanoPhotonics and Key Laboratory of Photochemical Conversion and Functional Materials, Technical Institute of Physics and Chemistry, Beijing (China); Kawata, Satoshi [RIKEN (Institute of Physical and Chemical Research), Nanophotonics Laboratory, Wako, Saitama (Japan); Osaka University, Department of Applied Physics, Graduate School of Engineering, Suita, Osaka (Japan)

    2009-08-15

    The morphology and size dependence of silver microstructures in a novel microfabrication process, fatty salts-assisted multiphoton photoreduction (MPR), were investigated by using the fatty salts with different carbon chain lengths (C{sub n}:n=4,5,7,9) under varied powers and irradiation times of a femtosecond near-infrared laser with the wavelength of 800 nm. Not only the feature size of the silver structures was reduced but also the surface smoothness was improved by increasing the chain length of the fatty salts. The highest resolution of a silver line was obtained to be 285 nm, which exceeded the diffraction limit. The fatty salts-assisted MPR microfabrication approach would provide an efficient protocol for fabricating metallic micro/nanostructures with fine morphology and size and could play an important role in the fabrication of the metallic micro/nanostructures for applications in photonics and electronics as well as in sensors. (orig.)

  18. Five-Photon Absorption and Selective Enhancement of Multiphoton Absorption Processes.

    Science.gov (United States)

    Friese, Daniel H; Bast, Radovan; Ruud, Kenneth

    2015-05-20

    We study one-, two-, three-, four-, and five-photon absorption of three centrosymmetric molecules using density functional theory. These calculations are the first ab initio calculations of five-photon absorption. Even- and odd-order absorption processes show different trends in the absorption cross sections. The behavior of all even- and odd-photon absorption properties shows a semiquantitative similarity, which can be explained using few-state models. This analysis shows that odd-photon absorption processes are largely determined by the one-photon absorption strength, whereas all even-photon absorption strengths are largely dominated by the two-photon absorption strength, in both cases modulated by powers of the polarizability of the final excited state. We demonstrate how to selectively enhance a specific multiphoton absorption process.

  19. Probing spin-orbit-interaction-induced electron dynamics in the carbon atom by multiphoton ionization

    Science.gov (United States)

    Rey, H. F.; van der Hart, H. W.

    2014-09-01

    We use R-matrix theory with time dependence (RMT) to investigate multiphoton ionization of ground-state atomic carbon with initial orbital magnetic quantum number ML=0 and ML=1 at a laser wavelength of 390 nm and peak intensity of 1014W/cm2. Significant differences in ionization yield and ejected-electron momentum distribution are observed between the two values for ML. We use our theoretical results to model how the spin-orbit interaction affects electron emission along the laser polarization axis. Under the assumption that an initial C atom is prepared at zero time delay with ML=0, the dynamics with respect to time delay of an ionizing probe pulse modeled by using RMT theory is found to be in good agreement with available experimental data.

  20. Three-Dimensional Organic Microlasers with Low Lasing Thresholds Fabricated by Multiphoton Lithography

    CERN Document Server

    Chen, Vincent W; Lafargue, Clement; Mansfield, Eric S; Yom, Jeannie; Johnstone, Luke; Hales, Joel M; Bittner, Stefan; Charpignon, Severin; Ulbricht, David; Lautru, Joseph; Denisyuk, Igor; Zyss, Joseph; Perry, Joseph W; Lebental, Melanie

    2014-01-01

    Cuboid-shaped organic microcavities containing a pyrromethene laser dye and supported upon a photonic crystal have been investigated as an approach to reducing the lasing threshold of the cavities. Multiphoton lithography facilitated fabrication of the cuboid cavities directly on the substrate or on the decoupling structure, while similar structures were fabricated on the substrate by UV lithography for comparison. Significant reduction of the lasing threshold by up to a factor of 4 has been observed for cavities supported by the photonic crystal relative to those fabricated on the substrate. The lasing mode spectra of the cuboid microresonators provide strong evidence showing that the lasing modes are localized in the horizontal plane, with the shape of an inscribed diamond.

  1. Single- and multi-photon final states with missing energy at e sup + e sup - colliders

    CERN Document Server

    Montagna, G; Nicrosini, O; Piccinini, F

    1999-01-01

    The search for new physics in single- and multi-photon final states with large missing energy at LEP and future e sup + e sup - colliders requires precise predictions for the Standard Model irreducible background. While at LEP1 the theoretical situation is under control, going to LEP2 (and beyond) some improvements are necessary. To approach the aimed O(1%) theoretical accuracy, the tree-level matrix elements for the processes e sup + e sup - -> nu nu-barn gamma, with n=1, 2, 3, are exactly computed in the Standard Model, including the possibility of anomalous couplings for single-photon production. Due to the presence of observed photons in the final state, particular attention is paid to the treatment of higher-order QED corrections. Comparisons with existing calculations are shown and commented on. An improved version of the event generator NUNUGPV is presented.

  2. High-order harmonic generation and multi-photon ionization of Na2 in laser fields

    Institute of Scientific and Technical Information of China (English)

    Zhang Yan-Ping; Zhang Feng-Shou; Meng Ke-Lai; Xiao Guo-Qing

    2007-01-01

    In this paper high-order harmonic generation (HHG) spectra and the ionization probabilities of various charge states of small cluster Na2 in the multiphoton regimes are calculated by using time-dependent local density approximation (TDLDA) for one-colour (1064 nm) and two-colour (1064 nm and 532 nm) ultrashort (25 fs) laser pulses. HHG spectra of Na2 have not the large extent of plateaus due to pronounced collective effects of electron dynamics. In addition, the two-colour laser field can result in the breaking of the symmetry and generation of the even order harmonic such as the second order harmonic. The results of ionization probabilities show that a two-colour laser field can increase the ionization probability of higher charge state.

  3. Non-dipole effects in multiphoton ionization of hydrogen atom in short superintense laser fields

    Energy Technology Data Exchange (ETDEWEB)

    Jobunga, Eric O. [AG Moderne Optik, Institut fuer Physik, Humboldt-Universitaet zu Berlin, Newtonstr. 15, D-12489 Berlin (Germany); Department of Mathematics and Physics, Technical University of Mombasa, P. O. Box 90420-80100, Mombasa (Kenya); Saenz, Alejandro [AG Moderne Optik, Institut fuer Physik, Humboldt-Universitaet zu Berlin, Newtonstr. 15, D-12489 Berlin (Germany)

    2014-07-01

    The development of novel light sources has enabled the realization of high-precision experiments investigating various non-linear processes in the dynamics of atomic, molecular, and ionic systems interacting with high intense laser pulses. At high intensities or short wavelengths, the analysis of these experiments would definitely require a reliable non-perturbative solution of the time-dependent Schroedinger or Dirac equation. These solutions should consider both the temporal and the spatial intensity variations of the laser pulse.We have solved the non-relativistic time dependent Schroedinger equation for a ground state hydrogen atom interacting with short intense spatially and temporally resolved laser fields corresponding to the multiphoton ATI regime for a monochromatic source with λ= 800 nm. We shall analyse the effects of the A{sup 2} term and the corresponding orders of the multipolar expansion of the transition matrix.

  4. Multi-Photon Multi-Channel Interferometry for Quantum Information Processing

    CERN Document Server

    Dhand, Ish

    2016-01-01

    This thesis reports advances in the theory of design, characterization and simulation of multi-photon multi-channel interferometers. I advance the design of interferometers through an algorithm to realize an arbitrary discrete unitary transformation on the combined spatial and internal degrees of freedom of light. This procedure effects an arbitrary $n_{s}n_{p}\\times n_{s}n_{p}$ unitary matrix on the state of light in $n_{s}$ spatial and $n_{p}$ internal modes. I devise an accurate and precise procedure for characterizing any multi-port linear optical interferometer using one- and two-photon interference. Accuracy is achieved by estimating and correcting systematic errors that arise due to spatiotemporal and polarization mode mismatch. Enhanced accuracy and precision are attained by fitting experimental coincidence data to a curve simulated using measured source spectra. The efficacy of our characterization procedure is verified by numerical simulations. I develop group-theoretic methods for the analysis and ...

  5. Spatiotemporal steering of photoelectron emission in multiphoton above-threshold ionization

    CERN Document Server

    Gong, Xiaochun; Liu, Mingming; Song, Qiying; Lin, Kang; Ji, Qinying; Zhang, Wenbin; Ma, Junyang; Lu, Peifen; Liu, Yunquan; He, Feng; Zeng, Heping; Yang, Weifeng; Wu, Jian

    2016-01-01

    We experimentally demonstrate spatiotemporal steering of photoelectron emission in multiphoton above-threshold single ionization of atoms exposed to a phase-controlled orthogonally polarized two-color (OTC) laser pulse. Spatial and energy resolved photoelectron angular distributions are measured as a function of the laser phase, allowing us to look into the fine structures and emission dynamics. The slow and fast photoelectrons, distinguished by the energy larger or smaller than 2Up with Up being the ponderomotive energy of a free electron in the laser field, have distinct spatiotemporal dependences of the laser waveform. The phase-of-phase of the slow electron oscillates as functions of both the energy and emission direction, however, the fast electron present rather flat phase structure which merely depends on its emission direction. Three-dimensional generalized quantum trajectory Monte Carlo simulations are performed to explore the sub-cycle dynamics of the electron emission in the phase-controlled OTC pu...

  6. Multiphoton dissociation/ionisation of dimethyl sulphide (CH3SCH3) at 355 and 532 nm

    Indian Academy of Sciences (India)

    Rajesh K Vatsa; Chiranjib Majumder; Onnattu D Jayakumar; Shailendra K Kulshreshtha; Jai P Mittal

    2001-04-01

    Multiphoton dissociation/ionization has been studied for CH3SCH3 at 355 and 532 nm using a time-of-flight mass spectrometer. The major ion signals observed at 355 nm are C+, CH$_{3}^{+}$, HCS+, CH2S+, CH3S+ and CH3SCH$_{3}^{+}$. Power dependence studies at 355 nm show a (2 + 1) REMPI process for the formation of parent ion. Peaks at / = 46, 47 and 61 show two-photon laser power dependence whereas / = 15 and 45 peaks show four-photon dependence. However, in 532 nm photoionisation, no parent ion signal is observed. A peak at / = 35 corresponding to SH$_{3}^{+}$ has been observed. SH$_{3}^{+}$ has been suggested to originate from CH3SCH$_{2}^{+}$ via a cyclic transition state. Photoionisation results of CH3SCH3 have been compared with those of CH3SSCH3, at these two wavelengths.

  7. Super-Resolved Traction Force Microscopy (STFM).

    Science.gov (United States)

    Colin-York, Huw; Shrestha, Dilip; Felce, James H; Waithe, Dominic; Moeendarbary, Emad; Davis, Simon J; Eggeling, Christian; Fritzsche, Marco

    2016-04-13

    Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.

  8. Determination of triacetone triperoxide using ultraviolet femtosecond multiphoton ionization time-of-flight mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Ezoe, Ryota [Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Imasaka, Tomoko [Laboratory of Chemistry, Graduate School of Design, Kyushu University, 4-9-1, Shiobaru, Minami-ku, Fukuoka 815-8540 (Japan); Imasaka, Totaro, E-mail: imasaka@cstf.kyushu-u.ac.jp [Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Division of Optoelectronics and Photonics, Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)

    2015-01-01

    Highlights: • A UV ultrashort laser pulse was useful for ionization of triacetone triperoxide. • A molecular ion was strongly enhanced in multiphoton ionization mass spectrometry. • Triacetone triperoxide in the human blood was measured without any interferences. • An organic compound of phorone was formed in the human blood from acetone. - Abstract: Triacetone triperoxide (TATP), an explosive compound, was measured using gas chromatography combined with multiphoton ionization time-of-flight mass spectrometry (GC/MPI-TOFMS). By decreasing the pulse width of a femtosecond laser from 80 to 35 fs, a molecular ion was drastically enhanced and was measured as one of the major ions in the mass spectrum. The detection limits obtained using the molecular (M·{sup +}) and fragment (C{sub 2}H{sub 3}O{sup +}) ions were similar or slightly superior to those obtained using conventional mass spectrometry based on electron and chemical ionization. In order to improve the reliability, an isotope of TATP, i.e., TATP-d18, was synthesized and used as an internal standard in the trace analysis of TATP in a sample of human blood. TATP could be identified in a two-dimensional display, even though numerous interfering compounds were present in the sample. Acetone, which is frequently used as a solvent in sampling TATP, produced a chemical species with a retention time nearly identical to that of TATP and provided a C{sub 2}H{sub 3}O{sup +} fragment ion that was employed for measuring a chromatogram of TATP in conventional MS. This compound, the structure of which was assigned as phorone, was clearly differentiated from TATP based on a molecular ion observable in MPI-TOFMS.

  9. Calculation of Multiphoton Transition in Li Atoms via Chirped Microwave Pulse

    Institute of Scientific and Technical Information of China (English)

    JIA Guang-Rui; ZHANG Xian-Zhou; LIU Yu-Fang; YU Kun; ZHAO Yue-Jin

    2011-01-01

    The position and width of avoided crossings of Li atom energy levels in a static electric field is presented by using the B-spline basis set method combined with the model potential.Using the time-dependent multilevel approach,the population of Li atoms is transferred to the target state completely by one-photon,two-photon or a single multiphoton adiabatic rapid passage,which requires only a small frequency sweep.The calculation results agree well with the experiment and novel explanations are given to understand the experimental results.It is well known that adiabatic rapid passage (ARP) works perfectly in the population transfer of an atomic system.[1-3] Coherent population transfer via ARP in atoms through one-photon[2] or twophoton[4] transitions using chirped pulses has been demonstrated.If the frequency of an external field is swept through the resonance at a rate lower than the square of the Rabi frequency,the population can be transferred through many levels by sequential ARPs with approximately 100% efficiency.%The position and width of avoided crossings of Li atom energy levels in a static electric field is presented by using the B-spline basis set method combined with the model potential Using the time-dependent multilevel approach, the population of Li atoms is transferred to the target state completely by one-photon, two-photon or a single multiphoton adiabatic rapid passage, which requires only a small frequency sweep. The calculation results agree well with the experiment and novel explanations are given to understand the experimental results.

  10. Multiphoton dissociation and thermal unimolecular reactions induced by infrared lasers. [REAMPA code

    Energy Technology Data Exchange (ETDEWEB)

    Dai, H.L.

    1981-04-01

    Multiphoton dissociation (MPD) of ethyl chloride was studied using a tunable 3.3 ..mu..m laser to excite CH stretches. The absorbed energy increases almost linearly with fluence, while for 10 ..mu..m excitation there is substantial saturation. Much higher dissociation yields were observed for 3.3 ..mu..m excitation than for 10 ..mu..m excitation, reflecting bottlenecking in the discrete region of 10 ..mu..m excitation. The resonant nature of the excitation allows the rate equations description for transitions in the quasicontinuum and continuum to be extended to the discrete levels. Absorption cross sections are estimated from ordinary ir spectra. A set of cross sections which is constant or slowly decreasing with increasing vibrational excitation gives good fits to both absorption and dissociation yield data. The rate equations model was also used to quantitatively calculate the pressure dependence of the MPD yield of SF/sub 6/ caused by vibrational self-quenching. Between 1000-3000 cm/sup -1/ of energy is removed from SF/sub 6/ excited to approx. > 60 kcal/mole by collision with a cold SF/sub 6/ molecule at gas kinetic rate. Calculation showed the fluence dependence of dissociation varies strongly with the gas pressure. Infrared multiphoton excitation was applied to study thermal unimolecular reactions. With SiF/sub 4/ as absorbing gas for the CO/sub 2/ laser pulse, transient high temperature pulses were generated in a gas mixture. IR fluorescence from the medium reflected the decay of the temperature. The activation energy and the preexponential factor of the reactant dissociation were obtained from a phenomenological model calculation. Results are presented in detail. (WHK)

  11. Diagnostic electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Dickersin, G.R.

    1988-01-01

    In this book the author presents a comprehensive reference text on diagnostic electron microscopy. Throughout the book he illustrates how ultrastructural identification can be helpful for the recognition of cell type and the identification of mechanisms of pathogenesis in various diseases. In addition to electron microscopy photographs, there are also numerous light microscopy photographs for comparison. This text presents the classification of neoplasms in the order and arrangement most familiar to the pathologist. Contents: Introduction; Diagram of a Normal Cell; Normal Cell Function; Embryology; Neoplasms; Infectious Agents; Metabolic Diseases; Renal Diseases; Skeletal Muscle and Peripheral Nerve Diseases; Index.

  12. Preferential Transport and Metabolism of Glucose in Bergmann Glia over Purkinje Cells: A Multiphoton Study of Cerebellar Slices

    Institute of Scientific and Technical Information of China (English)

    L.F.BARROS; R.COURJARET; P.JAKOBY; A.LOAIZA; C.LOHR; J.W.DEITMER

    2009-01-01

    了解不同类型的细胞如何处理葡萄糖有助于解释能量供应是如何是如何根据大脑能量需求来进行调整的.荧光追踪结合共聚焦显微镜技术已用于研究培养的脑细胞摄取葡萄糖的实时动态过程.本文采用这种技术利用多光子显微镜观察急性制备的大鼠小脑脑片.带荧光的葡萄糖类似物2NBDG和6NBDG在小脑皮质的分子层中的转运速度比其在蒲肯野细胞胞体和颗粒细胞中快若干倍.洗脱游离示踪剂后,可见大部分磷酸化示踪剂都位于Bergmann胶质细胞,用胶质细胞标记物sulforhodamine 101免疫染色后进一步确认这一结果.有效回收荧光光漂白后显示,2NBDG-P可通过Bergmann胶质细胞之间的缝隙连接沿着分子层水平扩散.本文的结果表明在急性小脑切片中,Bergmann胶质细胞对葡萄糖的转运能力和糖酵解率高于蒲肯野细胞若干倍.由于小脑主要由葡萄糖提供能量,蒲肯野神经元被认为比Bergmann胶质细胞更耗能量,这些结果表明,在胶质细胞和神经元之间存在类似乳酸的能量代谢物介导的环路.%Knowing how different cell types handle glucose should help to decipher how energy supply is adjusted to energy demand in the brain. Previously, the uptake of glucose by cultured brain cells was studied in real-time using fluorescent tracers and confocal microscopy. Here, we have adapted this technique to acute slices prepared from the rat cerebellum by means of multiphoton microscopy. The transport of the fluorescent glucose analogs 2NBDG and 6NBDG was several-fold faster in the molecular layer of the cerebellar cortex than in Purkinje cell somata and granule cells. After washout of free tracer, it became apparent that most phosphorylated tracer was located in Bergmann glia, which was confirmed by counterstaining with the glial marker sulforhodamine 101. The effective recovery of fluorescence after photobleaching showed that 2NBDG-P can diffuse

  13. Accessible Knowledge - Knowledge on Accessibility

    DEFF Research Database (Denmark)

    Kirkeby, Inge Mette

    2015-01-01

    Although serious efforts are made internationally and nationally, it is a slow process to make our physical environment accessible. In the actual design process, architects play a major role. But what kinds of knowledge, including research-based knowledge, do practicing architects make use of when...... designing accessible environments? The answer to the question is crucially important since it affects how knowledge is distributed and how accessibility can be ensured. In order to get first-hand knowledge about the design process and the sources from which they gain knowledge, 11 qualitative interviews...... were conducted with architects with experience of designing for accessibility. The analysis draws on two theoretical distinctions. The first is research-based knowledge versus knowledge used by architects. The second is context-independent knowledge versus context-dependent knowledge. The practitioners...

  14. Open access

    CERN Document Server

    Suber, Peter

    2012-01-01

    The Internet lets us share perfect copies of our work with a worldwide audience at virtually no cost. We take advantage of this revolutionary opportunity when we make our work "open access": digital, online, free of charge, and free of most copyright and licensing restrictions. Open access is made possible by the Internet and copyright-holder consent, and many authors, musicians, filmmakers, and other creators who depend on royalties are understandably unwilling to give their consent. But for 350 years, scholars have written peer-reviewed journal articles for impact, not for money, and are free to consent to open access without losing revenue. In this concise introduction, Peter Suber tells us what open access is and isn't, how it benefits authors and readers of research, how we pay for it, how it avoids copyright problems, how it has moved from the periphery to the mainstream, and what its future may hold. Distilling a decade of Suber's influential writing and thinking about open access, this is the indispe...

  15. Photothermal imaging scanning microscopy

    Science.gov (United States)

    Chinn, Diane; Stolz, Christopher J.; Wu, Zhouling; Huber, Robert; Weinzapfel, Carolyn

    2006-07-11

    Photothermal Imaging Scanning Microscopy produces a rapid, thermal-based, non-destructive characterization apparatus. Also, a photothermal characterization method of surface and subsurface features includes micron and nanoscale spatial resolution of meter-sized optical materials.

  16. International Multidisciplinary Microscopy Congress

    CERN Document Server

    Oral, Ahmet; Ozer, Mehmet; InterM; INTERM2013

    2014-01-01

    The International Multidisciplinary Microscopy Congress (INTERM2013) was organized on October 10-13, 2013. The aim of the congress was to bring together scientists from various branches to discuss the latest advances in the field of microscopy. The contents of the congress have been broadened to a more "interdisciplinary" scope, so as to allow all scientists working on related subjects to participate and present their work. These proceedings include 39 peer-reviewed technical papers, submitted by leading academic and research institutions from over 12 countries and representing some of the most cutting-edge research available. The 39 papers are grouped into the following sections: - Applications of Microscopy in the Physical Sciences - Applications of Microscopy in the Biological Sciences

  17. Clinical specular microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hirst, L.W.; Laing, R.A.

    1987-01-01

    This book provides the general ophthalmologist with a guide to the clinical applications of specular microscopy. Important material is included on laser injury, cataract surgery, corneal transplants, glaucoma, uveitis, and trauma.

  18. Pulse-Shaping-Based Nonlinear Microscopy: Development and Applications

    Science.gov (United States)

    Flynn, Daniel Christopher

    The combination of optical microscopy and ultrafast spectroscopy make the spatial characterization of chemical kinetics on the femtosecond time scale possible. Commercially available octave-spanning Ti:Sapphire oscillators with sub-8 fs pulse durations can drive a multitude of nonlinear transitions across a significant portion of the visible spectrum with minimal average power. Unfortunately, dispersion from microscope objectives broadens pulse durations, decreases temporal resolution and lowers the peak intensities required for driving nonlinear transitions. In this dissertation, pulse shaping is used to compress laser pulses after the microscope objective. By using a binary genetic algorithm, pulse-shapes are designed to enable selective two-photon excitation. The pulse-shapes are demonstrated in two-photon fluorescence of live COS-7 cells expressing GFP-variants mAmetrine and tdTomato. The pulse-shaping approach is applied to a new multiphoton fluorescence resonance energy transfer (FRET) stoichiometry method that quantifies donor and acceptor molecules in complex, as well as the ratio of total donor to acceptor molecules. Compared to conventional multi-photon imaging techniques that require laser tuning or multiple laser systems to selectively excite individual fluorophores, the pulse-shaping approach offers rapid selective multifluorphore imaging at biologically relevant time scales. By splitting the laser beam into two beams and building a second pulse shaper, a pulse-shaping-based pump-probe microscope is developed. The technique offers multiple imaging modalities, such as excited state absorption (ESA), ground state bleach (GSB), and stimulated emission (SE), enhancing contrast of structures via their unique quantum pathways without the addition of contrast agents. Pulse-shaping based pump-probe microscopy is demonstrated for endogenous chemical-contrast imaging of red blood cells. In the second section of this dissertation, ultrafast spectroscopic

  19. Tour de force microscopy

    OpenAIRE

    Mervyn Miles; Massimo Antognozzi; Heiko Haschke; Jamie Hobbs; Andrew Humphris; Terence McMaster

    2003-01-01

    Scanning probe microscopy (SPM) is capable of imaging synthetic polymers and biomolecular systems at sub-molecular resolution, without the need for staining or coating, in a range of environments including gas and liquid, so offering major advantages over other forms of microscopy. However, there are some limitations, which could be alleviated by (i) reducing the force interaction between the probe and specimen and (ii) increasing the rate of imaging. New developments in instrumentation from ...

  20. Access French

    CERN Document Server

    Grosz, Bernard

    2014-01-01

    Access is the major new language series designed with the needs of today's generation of students firmly in mind. Whether learning for leisure or business purposes or working towards a curriculum qualification, Access French is specially designed for adults of all ages and gives students a thorough grounding in all the skills required to understand, speak, read and write contemporary French from scratch. The coursebook consists of 10 units covering different topic areas, each of which includes Language Focus panels explaining the structures covered and a comprehensive glossary. Learning tips

  1. Theoretical description of circular dichroism in photoelectron angular distributions of randomly oriented chiral molecules after multi-photon photoionization

    CERN Document Server

    Goetz, R E; Nikoobakht, B; Berger, R; Koch, C P

    2016-01-01

    Photoelectron circular dichroism refers to the forward/backward asymmetry in the photoelectron angular distribution with respect to the propagation axis of circularly polarized light. It has recently been demonstrated in femtosecond multi-photon photoionization experiments with randomly oriented camphor and fenchone molecules [C. Lux et al., Angew. Chem. Int. Ed. 51, 5001 (2012);C. S. Lehmann et al., J. Chem. Phys. 139, 234307 (2013)]. A theoretical framework describing this process as (2+1) resonantly enhanced multi-photon ionization is constructed, which consists of two-photon photoselection from randomly oriented molecules and successive one-photon ionisation of the photoselected molecules. It combines perturbation theory for the light-matter interaction with ab initio calculations for the two-photon absorption and a single-center expansion of the photoelectron wavefunction in terms of hydrogenic continuum functions. It is verified that the model correctly reproduces the basic symmetry behavior expected un...

  2. Interband photorefractive effect in beta-BBO crystal due to multiphoton excitation by intense ultrashort optical pulses.

    Science.gov (United States)

    Xu, Shixiang; Cai, Hua; Zeng, Heping

    2007-08-20

    This paper presents the first experimental observation of interband photo- refractive (PR) effects in beta-BBO crystal due to multiphoton excitation with intense ultrashort pulses. In order to fully characterize the PR effects, a sensitive intracavity scheme is developed to magnify the dynamics of nonlinear lenses induced by the PR effects. The reproducible PR phenomena depend strongly on the power, wavelength, and spatial intensity profile of the intense laser pulses and the electro-optic coefficient of the optical materials. Its response time is from tens of seconds to several minutes. The results may be very helpful for us to find a solution to overcome the deleterious influence of multiphoton induced photo-charges on nonlinear optical frequency conversions, e.g. optical parametric chirped pulse amplification.

  3. Interplay between relativistic energy corrections and resonant excitations in x-ray multiphoton ionization dynamics of Xe atoms

    Science.gov (United States)

    Toyota, Koudai; Son, Sang-Kil; Santra, Robin

    2017-04-01

    In this paper, we theoretically study x-ray multiphoton ionization dynamics of heavy atoms taking into account relativistic and resonance effects. When an atom is exposed to an intense x-ray pulse generated by an x-ray free-electron laser (XFEL), it is ionized to a highly charged ion via a sequence of single-photon ionization and accompanying relaxation processes, and its final charge state is limited by the last ionic state that can be ionized by a single-photon ionization. If x-ray multiphoton ionization involves deep inner-shell electrons in heavy atoms, energy shifts by relativistic effects play an important role in ionization dynamics, as pointed out in Phys. Rev. Lett. 110, 173005 (2013), 10.1103/PhysRevLett.110.173005. On the other hand, if the x-ray beam has a broad energy bandwidth, the high-intensity x-ray pulse can drive resonant photoexcitations for a broad range of ionic states and ionize even beyond the direct one-photon ionization limit, as first proposed in Nat. Photon. 6, 858 (2012), 10.1038/nphoton.2012.261. To investigate both relativistic and resonance effects, we extend the xatom toolkit to incorporate relativistic energy corrections and resonant excitations in x-ray multiphoton ionization dynamics calculations. Charge-state distributions are calculated for Xe atoms interacting with intense XFEL pulses at a photon energy of 1.5 keV and 5.5 keV, respectively. For both photon energies, we demonstrate that the role of resonant excitations in ionization dynamics is altered due to significant shifts of orbital energy levels by relativistic effects. Therefore, it is necessary to take into account both effects to accurately simulate multiphoton multiple ionization dynamics at high x-ray intensity.

  4. Multiphoton absorption is probably not the primary threshold damage mechanism for femtosecond laser pulse exposures in the retinal pigment epithelium

    Science.gov (United States)

    Glickman, Randolph D.; Johnson, Thomas E.

    2004-07-01

    Laser induced breakdown has the lowest energy threshold in the femtosecond domain, and is responsible for production of threshold ocular lesions. It has been proposed that multiphoton absorption may also contribute to ultrashort-pulse tissue damage, based on the observation that 33 fs, 810 nm pulse laser exposures caused more DNA breakage in cultured, primary RPE cells, compared to CW laser exposures delivering the same average power. Subsequent studies, demonstrating two-photon excitation of fluorescence in isolated RPE melanosomes, appeared to support the role of multiphoton absorption, but mainly at suprathreshold irradiance. Additional experiments have not found a consistent difference in the DNA strand breakage produced by ultrashort and CW threshold exposures. DNA damage appears to be dependent on the amount of melanin pigmentation in the cells, rather than the pulsewidth of the laser; current studies have found that, at threshold, CW and ultrashort pulse laser exposures produce almost identical amounts of DNA breakage. A theoretical analysis suggest that the number of photons delivered to the RPE melanosome during a single 33-fsec pulse at the ED50 irradiance is insufficient to produce multiphoton excitation. This result appears to exclude the melanosome as a locus for two- or three-photon excitation; however, a structure with a larger effective absorption cross-section than the melanosome may interact with the laser pulses. One possibility is that the nuclear chromatin acts as a unit absorber of photons resulting in DNA damage, but this does not explain the near equivalence of ultrashort and CW exposures in the comet assay model. This equivalence indicated that multiphoton absorption is not a major contributor to the ultrashort pulse laser damage threshold in the near infrared.

  5. Controlling the transmitted information of a multi-photon interacting with a single-Cooper pair box

    Energy Technology Data Exchange (ETDEWEB)

    Kadry, Heba, E-mail: hkadry1@yahoo.com; Abdel-Aty, Abdel-Haleem, E-mail: hkadry1@yahoo.com; Zakaria, Nordin, E-mail: hkadry1@yahoo.com [Computer and Information Science Department, Universiti Teknologi Petronas, Seri Iskandar, 31750 Tronoh, Perak (Malaysia); Cheong, Lee Yen [Fundamental and Applied Science Department, Universiti Teknologi Petronas, Seri Iskandar, 31750 Tronoh, Perak (Malaysia)

    2014-10-24

    We study a model of a multi-photon interaction of a single Cooper pair box with a cavity field. The exchange of the information using this system is studied. We quantify the fidelity of the transmitted information. The effect of the system parameters (detuning parameter, field photons, state density and mean photon number) in the fidelity of the transmitted information is investigated. We found that the fidelity of the transmitted information can be controlled using the system parameters.

  6. S-matrix analysis of vibrational and alignment effects in intense-field multiphoton ionization of molecules

    Energy Technology Data Exchange (ETDEWEB)

    Requate, A.

    2007-03-15

    Theoretical analysis of the vibrational excitation of small molecules during multiphoton ionization in intense laser fields of optical and infrared frequencies. Analysis of the alignment dependence of the electron impact ionization of diatomic molecules in the presence of an intense laser field as the final step in the process of Nonsequential Double Ionization. Quantum mechanical description using S-matrix theory in Strong Field Approximation (SFA), i.e. beyond perturbation theory. (orig.)

  7. Hemodialysis access - self care

    Science.gov (United States)

    Kidney failure - chronic-hemodialysis access; Renal failure - chronic-hemodialysis access; Chronic renal insufficiency - hemodialysis access; Chronic kidney failure - hemodialysis access; Chronic renal failure - ...

  8. Investigation of the effect of hydration on dermal collagen in ex vivo human skin tissue using second harmonic generation microscopy

    Science.gov (United States)

    Samatham, Ravikant; Wang, Nicholas K.; Jacques, Steven L.

    2016-02-01

    Effect of hydration on the dermal collagen structure in human skin was investigated using second harmonic generation microscopy. Dog ears from the Mohs micrographic surgery department were procured for the study. Skin samples with subject aged between 58-90 years old were used in the study. Three dimensional Multiphoton (Two-photon and backward SHG) control data was acquired from the skin samples. After the control measurement, the skin tissue was either soaked in deionized water for 2 hours (Hydration) or kept at room temperature for 2 hours (Desiccation), and SHG data was acquired. The data was normalized for changes in laser power and detector gain. The collagen signal per unit volume from the dermis was calculated. The desiccated skin tissue gave higher backward SHG compared to respective control tissue, while hydration sample gave a lower backward SHG. The collagen signal decreased with increase in hydration of the dermal collagen. Hydration affected the packing of the collagen fibrils causing a change in the backward SHG signal. In this study, the use of multiphoton microscopy to study the effect of hydration on dermal structure was demonstrated in ex vivo tissue.

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

    Science.gov (United States)

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

    2006-09-15

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

  10. Clinical application of multiphoton tomography in combination with high-frequency ultrasound for evaluation of skin diseases.

    Science.gov (United States)

    König, Karsten; Speicher, Marco; Köhler, Martin J; Scharenberg, Rüdiger; Kaatz, Martin

    2010-12-01

    The first-ever application of high-frequency ultrasound combined with multiphoton tomography (MPT) and dermoscopy in a clinical trial is reported. 47 patients with different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond laser multiphoton tomograph and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface with a magnification up to 70 x. Depending on the ultrasonic frequencies from 7.5 MHz to 100 MHz, the signal depth varies from about 1 mm to 80 mm. Vertical ultrasound wide-field images provide fast information on depth and volume of the lesion. The 100 MHz ultrasound allows imaging with resolutions down to 16 μm (axial) and 32 μm (lateral). Multiphoton tomography provides 0.36 x 0.36 x 0.001 mm³ horizontal optical sections of a particular region of interest with submicron resolution down to 200 μm tissue depth. The autofluorescence of mitochondrial coenzymes, keratin, melanin, and elastin as well as the network of collagen structures can be imaged. The combination of ultrasound and MPT opens novel synergistic possibilities in diagnostics of skin diseases with a special focus on the early detection of skin cancer as well as the evaluation of treatments.

  11. Confocal Raman Microscopy

    CERN Document Server

    Dieing, Thomas; Toporski, Jan

    2011-01-01

    Confocal Raman Microscopy is a relatively new technique that allows chemical imaging without specific sample preparation. By integrating a sensitive Raman spectrometer within a state-of-the-art microscope, Raman microscopy with a spatial resolution down to 200nm laterally and 500nm vertically can be achieved using visible light excitation. Recent developments in detector and computer technology as well as optimized instrument design have reduced integration times of Raman spectra by orders of magnitude, so that complete images consisting of tens of thousands of Raman spectra can be acquired in seconds or minutes rather than hours, which used to be standard just one decade ago. The purpose of this book is to provide the reader a comprehensive overview of the rapidly developing field of Confocal Raman Microscopy and its applications.

  12. Optical imaging. Expansion microscopy.

    Science.gov (United States)

    Chen, Fei; Tillberg, Paul W; Boyden, Edward S

    2015-01-30

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~10(7) cubic micrometers of the mouse hippocampus with a conventional confocal microscope.

  13. Femtosecond laser pulse optimization for multiphoton cytometry and control of fluorescence

    Science.gov (United States)

    Tkaczyk, Eric Robert

    This body of work encompasses optimization of near infrared femtosecond laser pulses both for enhancement of flow cytometry as well as adaptive pulse shaping to control fluorescence. A two-photon system for in vivo flow cytometry is demonstrated, which allows noninvasive quantification of circulating cell populations in a single live mouse. We monitor fluorescently-labeled red blood cells for more than two weeks, and are also able to noninvasively measure circulation times of two distinct populations of breast cancer cells simultaneously in a single mouse. We build a custom laser excitation source in the form of an extended cavity mode-locked oscillator, which enables superior detection in whole blood or saline of cell lines expressing fluorescent proteins including the green fluorescent protein (GFP), tdTomato and mPlum. A mathematical model explains unique features of the signals. The ability to distinguish different fluorescent species is central to simultaneous measurement of multiple molecular targets in high throughput applications including the multiphoton flow cytometer. We demonstrate that two dyes which are not distinguishable to one-photon measurements can be differentiated and in fact quantified in mixture via phase-shaped two-photon excitation pulses found by a genetic algorithm. We also selectively enhance or suppress two-photon fluorescence of numerous common dyes with tailored pulse shapes. Using a multiplicative (rather than ratiometric) fitness parameter, we are able to control the fluorescence while maintaining a strong signal. With this method, we control the two-photon fluorescence of the blue fluorescent protein (BFP), which is of particular interest in investigations of protein-protein interactions, and has frustrated previous attempts of control. Implementing an acousto-optic interferometer, we use the same experimental setup to measure two-photon excitation cross-sections of dyes and prove that photon-photon interferences are the

  14. Conventional transmission electron microscopy.

    Science.gov (United States)

    Winey, Mark; Meehl, Janet B; O'Toole, Eileen T; Giddings, Thomas H

    2014-02-01

    Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.

  15. Second harmonic generation microscopy

    DEFF Research Database (Denmark)

    Brüggemann, Dagmar Adeline; Brewer, Jonathan R.; Risbo, Jens

    2010-01-01

    Myofibers and collagen show non-linear optical properties enabling imaging using second harmonic generation (SHG) microscopy. The technique is evaluated for use as a tool for real-time studies of thermally induced changes in thin samples of unfixed and unstained pork. The forward and the backward......-temperature endotherm peak observable in the differential scanning calorimetry (DSC) thermograms. DSC analysis of epimysium, the connective tissue layer that enfold skeletal muscles, produces one large endotherm starting at 57 °C and peaking at 59.5 °C. SHG microscopy of collagen fibers reveals a variability of thermal...

  16. Basic confocal microscopy

    Directory of Open Access Journals (Sweden)

    Manuela Monti

    2012-03-01

    Full Text Available This is an eleven chapter’s effort done by a bunch of Authors coordinated by Prof. R.L. Price and W.G. Jerome (who have personally written almost half of the book that with great skills are revealing us the secrets of confocal microscopy. Considering the significant progresses in different fields of biology, confocal microscopy is extremely important to dynamically see all the different molecules involved in the controlling networks build up by gene expressions in time and space. Necessary prerequisites to accomplish such goals are some fundamental microscopic technologies well and clearly presented in the first chapters....

  17. Confocal scanning microscopy

    DEFF Research Database (Denmark)

    Bariani, Paolo

    This report is based on a metrological investigation on confocal microscopy technique carried out by Uffe Rolf Arlø Theilade and Paolo Bariani. The purpose of the experimental activity was twofold a metrological instrument characterization and application to assessment of rough PP injection moulded...... replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

  18. Spectrally encoded confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tearney, G.J.; Webb, R.H.; Bouma, B.E. [Wellman Laboratories of Photomedicine, Massachusetts General Hospital, 50 Blossom Street, BAR 703, Boston, Massachusetts 02114 (United States)

    1998-08-01

    An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique. {copyright} {ital 1998} {ital Optical Society of America}

  19. Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

    OpenAIRE

    Doory Kim; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Xiaowei Zhuang

    2015-01-01

    Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and ima...

  20. Confocal scanning microscopy

    DEFF Research Database (Denmark)

    Bariani, Paolo

    replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

  1. Scanning ultrafast electron microscopy.

    Science.gov (United States)

    Yang, Ding-Shyue; Mohammed, Omar F; Zewail, Ahmed H

    2010-08-24

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.

  2. Single particle electron microscopy

    NARCIS (Netherlands)

    Boekema, Egbert J.; Folea, Mihaela; Kouril, Roman; Kouřil, Roman

    2009-01-01

    Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structures at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of EM

  3. Scanning ultrafast electron microscopy

    Science.gov (United States)

    Yang, Ding-Shyue; Mohammed, Omar F.; Zewail, Ahmed H.

    2010-01-01

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability. PMID:20696933

  4. Ballistic hole magnetic microscopy

    NARCIS (Netherlands)

    Haq, E.; Banerjee, T.; Siekman, M.H.; Lodder, J.C.; Jansen, R.

    2005-01-01

    A technique to study nanoscale spin transport of holes is presented: ballistic hole magnetic microscopy. The tip of a scanning tunneling microscope is used to inject hot electrons into a ferromagnetic heterostructure, where inelastic decay creates a distribution of electron-hole pairs. Spin-dependen

  5. Ballistic hole magnetic microscopy

    NARCIS (Netherlands)

    Haq, E.; Banerjee, T.; Siekman, M.H.; Lodder, J.C.; Jansen, R.

    2005-01-01

    A technique to study nanoscale spin transport of holes is presented: ballistic hole magnetic microscopy. The tip of a scanning tunneling microscope is used to inject hot electrons into a ferromagnetic heterostructure, where inelastic decay creates a distribution of electron-hole pairs.

  6. Automated control of optical polarization for nonlinear microscopy

    Science.gov (United States)

    Brideau, Craig; Stys, Peter K.

    2012-03-01

    Laser-scanning non-linear optical techniques such as multi-photon fluorescence excitation microscopy (MPM), Second/ Third Harmonic Generation (SHG/THG), and Coherent Anti-Stokes Raman Scattering (CARS) are being utilized in research laboratories worldwide. The efficiencies of these non-linear effects are dependent on the polarization state of the excitation light relative to the orientation of the sample being imaged. In highly ordered anisotropic biological samples this effect can become pronounced and the excitation polarization can have a dramatic impact on imaging experiments. Therefore, controlling the polarization state of the exciting light is important; however this is challenging when the excitation light passes through a complex optical system. In a typical laser-scanning microscope, components such as the dichroic filters, lenses, and even mirrors can alter the polarization state of a laser beam before it reaches the sample. We present an opto-mechanical solution to compensate for the polarization effects of an optical path, and to precisely program the polarization state of the exciting laser light. The device and accompanying procedures allow the delivery of precise laser polarization states at constant average power levels to a sample during an imaging experiment.

  7. Multimode fibres: a pathway towards deep-tissue fluorescence microscopy

    Science.gov (United States)

    Plöschner, Martin; Tyc, TomáÅ.¡; Čižmár, TomáÅ.¡

    2015-12-01

    Fluorescence microscopy has emerged as a pivotal platform for imaging in the life sciences. In recent years, the overwhelming success of its different modalities has been accompanied by various efforts to carry out imaging deeper inside living tissues. A key challenge of these efforts is to overcome scattering and absorption of light in such environments. Multiple strategies (e.g. multi-photon, wavefront correction techniques) extended the penetration depth to the current state-of-the-art of about 1000μm at the resolution of approximately 1μm. The only viable strategy for imaging deeper than this is by employing a fibre bundle based endoscope. However, such devices lack resolution and have a significant footprint (1mm in diameter), which prohibits their use in studies involving tissues deep in live animals. We have recently demonstrated a radically new approach that delivers the light in/out of place of interest through an extremely thin (tens of microns in diameter) cylindrical glass tube called a multimode optical fibre (MMF). Not only is this type of delivery much less invasive compared to fibre bundle technology, it also enables higher resolution and has the ability to image at any plane behind the fibre without any auxiliary optics. The two most important limitations of this exciting technology are (i) the lack of bending flexibility and (ii) high demands on computational power, making the performance of such systems slow. We will discuss how to overcome these limitations.

  8. Dynamic investigation of Drosophila myocytes with second harmonic generation microscopy

    Science.gov (United States)

    Greenhalgh, Catherine; Stewart, Bryan; Cisek, Richard; Prent, Nicole; Major, Arkady; Barzda, Virginijus

    2006-09-01

    The functional dynamics and structure of both larval and adult Drosophila melanogaster muscle were investigated with a nonlinear multimodal microscope. Imaging was carried out using a home built microscope capable of recording the multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation signals simultaneously at a scanning rate of up to ~12 frames/sec. The sample was excited by a home built femtosecond Ti:Sapphire laser at 840 nm, or by a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) crystal based oscillator at 1042 nm. There was no observable damage detected in the myocyte after prolonged scanning with either of the lasers. Microscopic second harmonic generation (SHG) appears particularly strong in the myocytes. This allows the fast contraction dynamics of the myocytes to be followed. The larger sarcomere size observed in the larvae myocytes is especially well suited for studying the contraction dynamics. Microscopic imaging of muscle contractions showed different relaxation and contraction rates. The SHG intensities were significantly higher in the relaxed state of the myocyte compared to the contracted state. The imaging also revealed disappearance of SHG signal in highly stretched sarcomeres, indicating that SHG diminishes in the disordered structures. The study illustrates that SHG microscopy, combined with other nonlinear contrast mechanisms, can help to elucidate physiological mechanisms of contraction. This study also provides further insight into the mechanisms of harmonic generation in biological tissue and shows that crystalline arrangement of macromolecules has a determining factor for the high efficiency second harmonic generation from the bulk structures.

  9. Photoemission Electron Microscopy for Analysis of Dielectric Structures and the Goos-Hanchen Shift

    Science.gov (United States)

    Stenmark, Theodore Axel

    Photoemission Electron Microscopy (PEEM) is a versatile tool that relies on the photoelectric effect to produce high-resolution electron images. Ultrafast pulse lasers allow for multi-photon PEEM where multiple visible or IR photons excite a single electron in a nonlinear process. The photoelectron yield in both cases is related to the near-field region of electromagnetic fields at the surface of the sample. We use this ability here to analyze wave propagation in a linear dielectric waveguide with wavelengths of 410 nm and 780 nm. The propagation constant of the waveguide can be extracted from interference patterns created by light propagating in the waveguide and incident light. Various properties like the polarization dependence of the propagation can be analyzed. The electromagnetic field interaction at the boundaries can then be deduced, which is essential to understand power flow in wave guiding structures. These results match well with simulations using finite element techniques as well as electromagnetic theory.

  10. Deformation mechanisms of human amnion: Quantitative studies based on second harmonic generation microscopy.

    Science.gov (United States)

    Mauri, Arabella; Ehret, Alexander E; Perrini, Michela; Maake, Caroline; Ochsenbein-Kölble, Nicole; Ehrbar, Martin; Oyen, Michelle L; Mazza, Edoardo

    2015-06-25

    Multiphoton microscopy has proven to be a versatile tool to analyze the three-dimensional microstructure of the fetal membrane and the mechanisms of deformation on the length scale of cells and the collagen network. In the present contribution, dedicated microscopic tools for in situ mechanical characterization of tissue under applied mechanical loads and the related methods for data interpretation are presented with emphasis on new stepwise monotonic uniaxial experiments. The resulting microscopic parameters are consistent with previous ones quantified for cyclic and relaxation tests, underlining the reliability of these techniques. The thickness reduction and the substantial alignment of collagen fiber bundles in the compact and fibroblast layer starting at very small loads are highlighted, which challenges the definition of a reference configuration in terms of a force threshold. The findings presented in this paper intend to inform the development of models towards a better understanding of fetal membrane deformation and failure, and thus of related problems in obstetrics and other clinical conditions.

  11. 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.

  12. Dissociation of benzylamine ions following infrared multiple photon absorption, electron impact ionization, and UV multiphoton ionization

    Energy Technology Data Exchange (ETDEWEB)

    Catanzarite, J.H.; Haas, Y.; Reisler, H.; Wittig, C.

    1983-05-01

    The dissociation of benzylamine ions following (i) electron impact (EI) ionization, (ii) multiphoton ionization (MPI) at 266 nm, and (iii) infrared multiple photon absorption (IRMPA) at 9.26 ..mu..m is reported. In the EI and MPI experiments, three competitive dissociation pathways are observed. In the IRMPA experiments, benzylamine ions prepared by MPI at low fluences are fragmented very efficiently following irradiation with the focused output from a pulsed CO/sub 2/ laser. However, in contrast to the EI and MPI results, the IRMPD experiments reveal only a single, lowest energy, dissociation pathway and the fragmentation pattern is consistent with a sequential mechanism in which daughter ions continue to absorb the IR radiation and dissociate. The differences are explained by the different natures of the excitation processes: in IRMPA, the relatively slow up-pumping rate and the long rise time of the CO/sub 2/ laser pulse restrict the levels of excitation in the dissociating parent ions and favor sequential processes along the lowest energy decomposition pathways.

  13. Ultrasensitive standoff chemical sensing based on nonlinear multi-photon laser wave-mixing spectroscopy

    Science.gov (United States)

    Gregerson, Marc; Hetu, Marcel; Iwabuchi, Manna; Jimenez, Jorge; Warren, Ashley; Tong, William G.

    2012-10-01

    Nonlinear multi-photon laser wave mixing is presented as an ultrasensitive optical detection method for chem/bio agents in thin films and gas- and liquid-phase samples. Laser wave mixing is an unusually sensitive optical absorption-based detection method that offers significant inherent advantages including excellent sensitivity, small sample requirements, short optical path lengths, high spatial resolution, high spectral resolution and standoff remote detection capability. Wave mixing can detect trace amounts of chemicals even when using micrometer-thin samples, and hence, it can be conveniently interfaced to fibers, microarrays, microfluidic systems, lab-on-a-chip, capillary electrophoresis and other capillary- or fiber-based chemical separation systems. The wave-mixing signal is generated instantaneously as the two input laser beams intersect inside the analyte of interest. Laser excitation wavelengths can be tuned to detect multiple chemicals in their native form since wave mixing can detect both fluorescing and non-fluorescing samples at parts-pertrillion or better detection sensitivity levels. The wave-mixing signal is a laser-like coherent beam, and hence, it allows reliable and effective remote sensing of chemicals. Sensitive wave-mixing detectors offer many potential applications including sensitive detection of biomarkers, early detection of diseases, sensitive monitoring of environmental samples, and reliable detection of hazardous chem/bio agents with a standoff detection capability.

  14. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

    Science.gov (United States)

    Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

    2013-02-01

    There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

  15. Biosensing and -imaging with enantiomeric luminescent conjugated polythiophenes using multiphoton excitation

    Science.gov (United States)

    Lindgren, Mikael; Stabo-Eeg, Frantz; Schnell, Edrun A.; Nilsson, K. Peter R.; Hammarstrom, Per; Inganaes, Olle

    2005-08-01

    We report on the initial time-resolved luminescense and nonlinear absorption properties of two polythiophenes 3-substituted with chiral charged amino acid-derivatized substituents, POWT and POMT. The photo-physical characterization yielded quantum efficiency typically in the range 0.01 - 0.1, however, with two-photon absorption cross-section better than or similar to a typical two-photon reference chromophore, such as fluorescein. They were tested as conformational sensitive optical probes for the recording of pH-induced conformational changes of synthetic peptides, proteins and samples of protein amyloid fibrils characteristic of amyloid related diseases. Particularly, the POMT polyelectrolyte with the L-enantiomeric side chains is shown to favor this induction of well defined structure as judged by the circular dichroic signal as well as a stronger enhancement of luminescense for the L-form over the D-form complex. Furthermore, time-resolved fluorescense and two-photon induced fluorescence both also showed a difference in the complexation with the D and L form. This shows that the multi-photon excitation path can be an efficient means for chiral recognition of biomolecular complexes. It is demonstrated how the conjugated polyelectrolyte L-POMT can be used to spectrally image the formation of amyloid fibrils of insulin using both one- and two-photon absorption based fluorescence imaging.

  16. Directed Self-Assembly of Block Copolymers in 3D Templates Fabricated by Multiphoton Lithography

    Science.gov (United States)

    Singer, Jonathan; Thomas, Edwin

    2012-02-01

    Confinement of block-copolymers (BCP) within physical templates has been used as a method to both enhance the microdomain order and manipulate the morphology. Previous investigations have focused on 1D or quasi-2D templates (e.g. trenches, cylindrical pores) patterned at a variety of length scales. We have sought to employ the nearly-arbitrary structural fabrication afforded by multiphoton direct write lithography (MPL) to extend these stereolithographic control techniques to 3D directed assembly. Complex architectures, for example those formed by large scale lithographic techniques such as interference lithography or self-assembly, can be broken down into simplifier component structures: e.g. multibranch junctions, bends, and symmetric or asymmetric connecting pores. By utilizing MPL, we can fabricate model geometries possessing these features written in photoresist at various scales. We then infiltrate these structures with PS-PDMS BCP and observe the resulting morphology by SEM from focused ion beam cross-sections, allowing development of design rules that may be applied towards progressively more complex templates and the fabrication of 3D hierarchical structures with highly ordered and novel domain features spanning from the micron to the 10 nm scale.

  17. Effects of dc electric fields on multiphoton ionization of rubidium atoms at low and high densities

    Science.gov (United States)

    Hammer, Nathan I.; Compton, Robert N.

    2001-08-01

    Multiphoton ionization (MPI) of rubidium atoms at both low (atomic beam) and high (heat pipe) densities is studied using a tunable OPO laser. At high Rb densities ionization of the laser excited ns, np, and nd states occurs both through photoionization and collisional ionization. Excitation of the np states is found to be induced by the external electric field at both low and high densities. In addition, np signal is also seen at very low (E→0) fields in the heat pipe, providing evidence for collision mixing as well as field mixing. At low densities, signal for the high np states initially increases with applied field, but soon saturates (i.e. becomes field independent) while the signal for high nd states decreases with increasing field. At low Rb densities strong resonance features are observed in the energy region between the zero field limit (IP) and the field ionization limit. These features, as well as the field ionization threshold, are found to be dependent upon the angle between the laser polarization and the direction of the applied dc field. Evidence for tunneling through the barrier created by the -e2/r-eEr potential is also presented for ns and nd states.

  18. Resonant multi-photon IR dissociation spectroscopy of a trapped and sympathetically cooled biomolecular ion species

    CERN Document Server

    Wellers, Ch; Vasilyev, S; Offenberg, D; Schiller, S

    2011-01-01

    In this work we demonstrate vibrational spectroscopy of polyatomic ions that are trapped and sympathetically cooled by laser-cooled atomic ions. We use the protonated dipeptide tryptophane-alanine (HTyrAla+) as a model system, cooled by Barium ions to less than 800mK secular temperature. The spectroscopy is performed on the fundamental vibrational transition of a local vibrational mode at 2.74 {\\mu}m using a continuous-wave optical parametric oscillator (OPO). Resonant multi-photon IR dissociation spectroscopy (without the use of a UV laser) generates charged molecular fragments, which are sympathetically cooled and trapped, and subsequently released from the trap and counted. We measured the cross section for R-IRMPD under conditions of low intensity, and found it to be approximately two orders smaller than the vibrational excitation cross section. The observed rotational bandwidth of the vibrational transition is larger than the one expected from the combined effects of 300 K black-body temperature, conform...

  19. Multiphoton reaction of DTTCI observed by femtosecond pump–probe and two-pulse correlation measurements

    Energy Technology Data Exchange (ETDEWEB)

    Furuta, Koich [Molecular Photoscience Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501 (Japan); Fuyuki, Masanori [Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501 (Japan); Wada, Akihide, E-mail: aki.wada@koala.kobe-u.ac.jp [Molecular Photoscience Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501 (Japan)

    2013-06-03

    Highlights: • Photoisomerization of DTTCI preferred two-photon process in red-tail excitation. • Photoisomerization time from the S{sub n} state was approximately 0.5 ns. • Two concurrent processes are involved in the two-photon excitation process. • Intermediate state in two-step excitation shows double-exponential decay behavior. • Relaxation of the intermediate state shows time-constants of 0.1 ps and 5 ps. - Abstract: To understand the elementary steps of chemical reactions, unimolecular reactions are important and considered to be model reactions. We carried out pump–probe and two-pulse correlation (2PC) measurements of the multiphoton-induced reaction of DTTCI (3,3′-diethyl-2,2′-thiatricarbocyanine iodide), a kind of cyanine dyes, under the red-tail excitation condition and found that both photoisomerization and photodegradation were caused by a two-photon process and the reaction time for photoisomerization was approximately 0.5 ns. Excitation dynamics was examined by 2PC measurements and the temporal character of the intermediate state was analyzed.

  20. Stellar Multi-Photon Absorption Materials: Beyond the Telecommunication Wavelength Band.

    Science.gov (United States)

    Schwich, Torsten; Barlow, Adam; Cifuentes, Marie P; Szeremeta, Janusz; Samoc, Marek; Humphrey, Mark G

    2017-06-22

    Very large molecular two- and three-photon absorption cross-sections are achieved by appending ligated bis(diphosphine)ruthenium units to oligo(p-phenyleneethynylene) (OPE)-based "stars" with arms up to 7 phenyleneethynylene (PE) units in length. Extremely large three- and four-photon absorption cross-sections, through the telecommunications wavelengths range and beyond, are obtained for these complexes upon optimizing OPE length and the ruthenium-coordinated peripheral ligand. Multi-photon absorption (MPA) cross-sections are optimized with stars possessing arms 2 PE units in length. Peripheral ligand variation modifies MPA merit and, in particular, 4-nitrophenylethynyl ligand incorporation enhances maximal MPA values and "switches on" four-photon absorption (4PA) in these low molecular-weight complexes. The 4-nitrophenylethynyl-ligated 2PE-armed star possesses a maximal four-photon absorption cross-section of 1.8×10(-108)  cm(8)  s(3) at 1750 nm, and significant MPA activity extending beyond 2000 nm. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Multiphoton and tunneling ionization of atoms in an intense laser field

    Institute of Scientific and Technical Information of China (English)

    Fu Yan-Zhuo; Zhao Song-Feng; Zhou Xiao-Xin

    2012-01-01

    We study the ionization probabilities of atoms by a short laser pulse with three different theoretical methods,i.e.,the numerical solution of the time-dependent Schr(o)dinger equation (TDSE),the Perelomov Popov-Terent'ev (PPT)theory,and the Ammosov-Delone-Krainov (ADK) theory.Our results show that laser intensity dependent ionization probabilities of several atoms (i.e.,H,He,and Ne) obtained from the PPT theory accord quite well with the TDSE results both in the multiphoton and tunneling ionization regimes,while the ADK results fit well to the TDSE data only in the tunneling ionization regime.Our calculations also show that laser intensity dependent ionization probabilities of a H atom at three different laser wavelengths of 600 nm,800 nm,and 1200 nm obtained from the PPT theory are also in good agreement with those from the TDSE,while the ADK theory fails to give the wavelength dependence of ionization probability.Only when the laser wavelength is long enough,will the results of ADK be close to those of TDSE.

  2. Open-ended response theory with polarizable embedding: multiphoton absorption in biomolecular systems.

    Science.gov (United States)

    Steindal, Arnfinn Hykkerud; Beerepoot, Maarten T P; Ringholm, Magnus; List, Nanna Holmgaard; Ruud, Kenneth; Kongsted, Jacob; Olsen, Jógvan Magnus Haugaard

    2016-10-12

    We present the theory and implementation of an open-ended framework for electric response properties at the level of Hartree-Fock and Kohn-Sham density functional theory that includes effects from the molecular environment modeled by the polarizable embedding (PE) model. With this new state-of-the-art multiscale functionality, electric response properties to any order can be calculated for molecules embedded in polarizable atomistic molecular environments ranging from solvents to complex heterogeneous macromolecules such as proteins. In addition, environmental effects on multiphoton absorption (MPA) properties can be studied by evaluating single residues of the response functions. The PE approach includes mutual polarization effects between the quantum and classical parts of the system through induced dipoles that are determined self-consistently with respect to the electronic density. The applicability of our approach is demonstrated by calculating MPA strengths up to four-photon absorption for the green fluorescent protein. We show how the size of the quantum region, as well as the treatment of the border between the quantum and classical regions, is crucial in order to obtain reliable MPA predictions.

  3. Aqueous multiphoton lithography with multifunctional silk-centred bio-resists

    Science.gov (United States)

    Sun, Yun-Lu; Li, Qi; Sun, Si-Ming; Huang, Jing-Chun; Zheng, Bo-Yuan; Chen, Qi-Dai; Shao, Zheng-Zhong; Sun, Hong-Bo

    2015-01-01

    Silk and silk fibroin, the biomaterial from nature, nowadays are being widely utilized in many cutting-edge micro/nanodevices/systems via advanced micro/nanofabrication techniques. Herein, for the first time to our knowledge, we report aqueous multiphoton lithography of diversiform-regenerated-silk-fibroin-centric inks using noncontact and maskless femtosecond laser direct writing (FsLDW). Initially, silk fibroin was FsLDW-crosslinked into arbitrary two/three-dimensional micro/nanostructures with good elastic properties merely using proper photosensitizers. More interestingly, silk/metal composite micro/nanodevices with multidimension-controllable metal content can be FsLDW-customized through laser-induced simultaneous fibroin oxidation/crosslinking and metal photoreduction using the simplest silk/Ag+ or silk/[AuCl4]− aqueous resists. Noticeably, during FsLDW, fibroin functions as biological reductant and matrix, while metal ions act as the oxidant. A FsLDW-fabricated prototyping silk/Ag microelectrode exhibited 104-Ω−1 m−1-scale adjustable electric conductivity. This work not only provides a powerful development to silk micro/nanoprocessing techniques but also creates a novel way to fabricate multifunctional metal/biomacromolecule complex micro/nanodevices for applications such as micro/nanoscale mechanical and electrical bioengineering and biosystems. PMID:26472600

  4. Dynamics mechanism of optical-optical double-resonant multiphoton ionization of nitrogen dioxide

    Institute of Scientific and Technical Information of China (English)

    Guiyin Zhang; Yidong Jin

    2007-01-01

    The optical-optical double-resonant multiphoton ionization (OODR-MPI) spectrum of NO2 molecule in the 460-605-nm wavelength region of the probe photon is presented. The mechanism of the OODR-MPI of NO2 molecule is analyzed. The results show that the resonant features can be assigned to the transitions from the first 3sσg Rydberg intermediate resonant state to the final npσu Rydberg series. The ionization pathway is NO2 (X2A1) 3hv1→ 3sσghv2→npσu hv2 or autoionization→NO2+ + e. It is found that the converging potential of the npσu Rydberg series and the quantum defect of np orbit about NO2 are (78803 ± 14) cm-1 and 0.652 ± 0.014, respectively. The bending vibration frequency of 5pσu state is determined also.

  5. Intravital multiphoton tomography as a novel tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis

    Science.gov (United States)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Niemeyer, Verena; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.

    2010-02-01

    Atopic Dermatitis (AD) is an inflammatory disease of human skin. Its pathogenesis is still unknown; however, dysfunctions of the epidermal barrier and the immune response are regarded as key factors for the development of AD. In our study we applied intravital multiphoton tomography (5D-IVT), equipped with a spectral-FLIM module for in-vivo and ex-vivo analysis of human skin affected with AD. In addition to the morphologic skin analysis, FLIM technology gain access to the metabolic status of the epidermal cells referring to the NADH specific fluorescence lifetime. We evaluated a characteristic 5D-IVT skin pattern of AD in comparison to histological sections and detected a correlation with the disease activity measured by SCORAD. FLIM analysis revealed a shift of the mean fluorescence lifetime (taum) of NADH, indicating an altered metabolic activity. Within an ex-vivo approach we have investigated cryo-sections of human skin with or without barrier defects. Spectral-FLIM allows the detection of autofluorescent signals that reflect the pathophysiological conditions of the defect skin barrier. In our study the taum value was shown to be different between healthy and affected skin. Application of the 5D-IVT allows non-invasive in-vivo imaging of human skin with a penetration depth of 150 μm. We could show that affected skin could be distinguished from healthy skin by morphological criteria, by FLIM and by spectral-FLIM. Further studies will evaluate the application of the 5D-IVT technology as a diagnostic tool and to monitor the therapeutic efficacy.

  6. Polarized Light Microscopy

    Science.gov (United States)

    Frandsen, Athela F.

    2016-01-01

    Polarized light microscopy (PLM) is a technique which employs the use of polarizing filters to obtain substantial optical property information about the material which is being observed. This information can be combined with other microscopy techniques to confirm or elucidate the identity of an unknown material, determine whether a particular contaminant is present (as with asbestos analysis), or to provide important information that can be used to refine a manufacturing or chemical process. PLM was the major microscopy technique in use for identification of materials for nearly a century since its introduction in 1834 by William Fox Talbot, as other techniques such as SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared spectroscopy), XPD (X-ray Powder Diffraction), and TEM (Transmission Electron Microscopy) had not yet been developed. Today, it is still the only technique approved by the Environmental Protection Agency (EPA) for asbestos analysis, and is often the technique first applied for identification of unknown materials. PLM uses different configurations in order to determine different material properties. With each configuration additional clues can be gathered, leading to a conclusion of material identity. With no polarizing filter, the microscope can be used just as a stereo optical microscope, and view qualities such as morphology, size, and number of phases. With a single polarizing filter (single polars), additional properties can be established, such as pleochroism, individual refractive indices, and dispersion staining. With two polarizing filters (crossed polars), even more can be deduced: isotropy vs. anisotropy, extinction angle, birefringence/degree of birefringence, sign of elongation, and anomalous polarization colors, among others. With the use of PLM many of these properties can be determined in a matter of seconds, even for those who are not highly trained. McCrone, a leader in the field of polarized light microscopy, often

  7. Photoemission electron microscopy, a tool for plasmonics

    Energy Technology Data Exchange (ETDEWEB)

    Douillard, L., E-mail: ludovic.douillard@cea.fr; Charra, F.

    2013-08-15

    Highlights: •Brief review of photoemission electron microscopy PEEM as a tool for plasmonics, •PEEM gives access to (tip free) near field maps of subwavelength length resolution, •PEEM makes use of a true optical excitation scheme, exhibiting strong connections to ultrafast optical spectrometries. -- Abstract: A key challenge to plasmonics is the development of experimental tools allowing access to the spatial distribution of the optical near field at the nanometre scale. A recent approach for mapping the near optical field is the use of the photoemission electron microscopy PEEM. Indeed, photoemission can be strongly enhanced upon excitation of surface plasmons. By collecting the photoemitted electrons, two-dimensional intensity maps reflecting the actual distribution of the optical near-field are obtained. In the following a brief overview of the possibilities of the photoemission electron microscopy as a tool for plasmonics is given. Main focuses will be set on experimental results regarding the mapping of the near optical field at nanometer scale, the investigation of the spatio-temporal dynamics of plasmon-polariton waves and the manipulation at will of the near field.

  8. Quantitative deconvolution microscopy.

    Science.gov (United States)

    Goodwin, Paul C

    2014-01-01

    The light microscope is an essential tool for the study of cells, organelles, biomolecules, and subcellular dynamics. A paradox exists in microscopy whereby the higher the needed lateral resolution, the more the image is degraded by out-of-focus information. This creates a significant need to generate axial contrast whenever high lateral resolution is required. One strategy for generating contrast is to measure or model the optical properties of the microscope and to use that model to algorithmically reverse some of the consequences of high-resolution imaging. Deconvolution microscopy implements model-based methods to enable the full diffraction-limited resolution of the microscope to be exploited even in complex and living specimens. © 2014 Elsevier Inc. All rights reserved.

  9. Two-colour coherent control of multiphoton ionization :a comparison between long-range and short-range potential model atoms

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Using the numerical solution of the time-dependent Schr(o)dinger equation of a one-dimensional model atom in a two-colour laser field, we have investigated the effects of the potential models on coherent control of atomic multiphoton ionization. It is found that the photoelectron spectra are obviously different for the long-range (Coulomb-like) and short-range (with no excited bound states) potential model atoms, which are produced by two-colour coherent control of atomic multiphoton ionization in a few laser cycles. Our results indicate that two-colour coherent control of atomic multiphoton ionization can be observed in simulations, depending on the choice of the model potentials.

  10. Design and commissioning of a directly coupled in-vivo multiphoton microscope for skin imaging in humans and large animals

    Science.gov (United States)

    Mulholland, William J.; Kendall, Mark A.

    2004-02-01

    The application of near infrared multiphoton excitation to the laser-scanning microscope was first conceived by Denk, Strickler and Webb in 1990. Since then, advances in design have seen the multiphoton laser scanning microscope (MPLSM) applied to a wide range of biological research areas, including skin imaging and vaccine delivery. The technique has the attributes of low phototoxicity, high-resolution functional imaging to depths in scattered tissues. These characteristics have encouraged engineers and scientists to develop in-vivo imaging systems. For these applications, laser excitation pulses can be delivered to the sample through optical fibers. Although this solution provides a number of advantages relating to movement and flexibility of the site of interest relative to the laser source, the peak powers that can be delivered down the fiber are limited. We report on the design and commissioning of a directly coupled in-vivo MPM system, optimised for the imaging of epidermal vaccines delivered to a range of biological models and humans. Specifically, we seek to apply the system to visualise in-vivo, the influence of hand-held, helium powered needle-free systems on skin cells. A standard Nikon E600FN microscope, dissected above the optical plane was cantilevered from a vibration isolated table using rigid support arms. The modified microscope was coupled to an infrared optimised Bio-Rad Radiance 2100MP, multiphoton dedicated laser scanning control and image acquisition system. Femtosecond laser pulses were provided by a 10W Verdi pumped Mira Ti:Sapphire laser, from Coherent Inc. The microscope was modified such that the transmission half may be selectively attached for conventional imaging with ex-vivo and cell culture samples, or removed for in-vivo imaging of skin sites on the body of humans and large animals. Optical performance of the system, and aspects of its design and commissioning are discussed in this paper.

  11. Strong-Field Breit-Wheeler Pair Production in Short Laser Pulses: Identifying Multiphoton Interference and Carrier-Envelope Phase Effects

    CERN Document Server

    Jansen, Martin J A

    2015-01-01

    The creation of electron-positron pairs by the strong-field Breit-Wheeler process in intense short laser pulses is investigated in the framework of laser-dressed quantum electrodynamics. Regarding laser field parameters in the multiphoton regime, special attention is brought to the energy spectrum of the created particles, which can be reproduced and explained by means of an intuitive model. The model is based on the probabilities of multiphoton events driven by the spectral components of the laser pulse. It allows, in particular, to identify interferences between different pair production channels which exhibit a characteristic dependence on the laser carrier-envelope phase.

  12. Strong-field Breit-Wheeler pair production in short laser pulses: Identifying multiphoton interference and carrier-envelope-phase effects

    Science.gov (United States)

    Jansen, Martin J. A.; Müller, Carsten

    2016-03-01

    The creation of electron-positron pairs by the strong-field Breit-Wheeler process in intense short laser pulses is investigated in the framework of laser-dressed quantum electrodynamics. Regarding laser field parameters in the multiphoton regime, special attention is brought to the energy spectrum of the created particles, which can be reproduced and explained by means of an intuitive model. The model is based on the probabilities of multiphoton events driven by the spectral components of the laser pulse. It allows us, in particular, to identify interferences between different pair production channels which exhibit a characteristic dependence on the laser carrier-envelope phase.

  13. Multiphoton Ionization as a clock to Reveal Molecular Dynamics with Intense Short X-ray Free Electron Laser Pulses

    CERN Document Server

    Fang, L; Murphy, B; Tarantelli, F; Kukk, E; Cryan, J P; Glownia, M; Bucksbaum, P H; Coffee, R N; Chen, M; Buth, C; Berrah, N

    2013-01-01

    We investigate molecular dynamics of multiple ionization in N2 through multiple core-level photoabsorption and subsequent Auger decay processes induced by intense, short X-ray free electron laser pulses. The timing dynamics of the photoabsorption and dissociation processes is mapped onto the kinetic energy of the fragments. Measurements of the latter allow us to map out the average internuclear separation for every molecular photoionization sequence step and obtain the average time interval between the photoabsorption events. Using multiphoton ionization as a tool of multiple-pulse pump-probe scheme, we demonstrate the modi?cation of the ionization dynamics as we vary the x-ray laser pulse duration.

  14. Infrared multiphoton dissociation of octafluorocyclobutane with a two-frequency Q-switched CO/sub 2/ laser

    Energy Technology Data Exchange (ETDEWEB)

    Pochon, E.; Weston, R.E. Jr.; Flynn, G.W.

    1985-01-03

    The infrared multiphoton dissociation of octafluorocyclobutane has been investigated by the use of two CO/sub 2/ laser pulses of different frequency, synchronized by the same Q-switching mirror. The dissociation yield has been determined as a function of laser pulse energy, frequency of the dissociating pulse, substrate pressure, and interpulse delay. Accompanying transport phenomena (expansion and diffusion) were taken into account by the use of appropriate models. The kinetic behavior of highly vibrationally excited c-C/sub 4/F/sub 8/ molecules indicates that collisional relaxation occurs at nearly gas kinetic rates. 47 references, 10 figures.

  15. Generation of highly vibrationally excited H2 and detection by 2+1 resonantly enhanced multiphoton ionization

    Science.gov (United States)

    Robie, Daniel C.; Jusinski, Leonard E.; Bischel, William K.

    1990-02-01

    We report the first detection by optical means of highly vibrationally excited H2 X1Σ+g(vx=6-11). Vibrationally excited H2 was generated using a recently discovered hot-wire effect in H2 gas, and was detected in 40 bands with 2+1 resonantly enhanced multiphoton ionization via the EF state (vEF=0-14). Rotational temperatures are in the range 200-650 K, well below that required for thermal excitation of the observed vibrational levels.

  16. Enhanced harmonic generation and wave-mixing via two-color multiphoton excitation of atoms/molecules

    CERN Document Server

    Avetissian, H K; Mkrtchian, G F

    2016-01-01

    We consider harmonics generation and wave-mixing by two-color multi photon resonant excitation of three-level atoms/molecules in strong laser fields. The coherent part of the spectra corresponding to multicolor harmonics generation is investigated. The obtained analytical results on the basis of generalized rotating wave approximation are in a good agreement with numerical calculations. The results applied to the hydrogen atom and homonuclear diatomic molecular ion show that one can achieve efficient generation of moderately high multicolor harmonics via multiphoton resonant excitation by appropriate laser pulses.

  17. UV multiphoton ionization and IR photodissociation of CF{sub 3}I cluster beams

    Energy Technology Data Exchange (ETDEWEB)

    Lokhman, V.N.; Ogurok, D.D. [Institute of Spectroscopy, Russian Academy of Sciences, 142 190 Troitsk, Moscow Region (Russian Federation); Ryabov, E.A. [Institute of Spectroscopy, Russian Academy of Sciences, 142 190 Troitsk, Moscow Region (Russian Federation)], E-mail: ryabov@isan.troitsk.ru

    2007-03-06

    This paper presents the results of studies into the ultraviolet multiphoton ionization (UV MPI) and infrared photodissociation of (CF{sub 3}I){sub n} clusters formed upon the ultrasonic outflow of CF{sub 3}I molecules from a pulsed jet nozzle. The clusters were found to undergo UV MPI under the action of the XeCl-laser radiation (308 nm) to yield I{sup +} and I{sub 2}{sup +} ions as final products. The UV MPI yield was measured as a function of the radiation intensity and stagnation pressure. The efficiency of the UV MPI process was found to depend on the size of the clusters. The IR photodissociation of the (CF{sub 3}I){sub n} clusters was investigated as a function of the energy fluence and frequency of the CO{sub 2}-laser radiation acting on the {nu}{sub 1} mode of the CF{sub 3}I molecule. The size of the clusters and their binding energy were estimated on the basis of the measurement results and model calculations. The main velocity characteristics of the (CF{sub 3}I){sub n} clusters in the beam, namely, the velocity of their directed motion and also the perpendicular and parallel velocity components of their thermal motion, were measured as a function of the CF{sub 3}I gas outflow conditions using their UV MPI and combined UV MPI + IR photodissociation. The thermal motion velocity distributions were demonstrated to be bimodal at the least.

  18. Ultrafast Multiphoton Pump-probe Photoemission Excitation Pathways in Rutile TiO2(110)

    Energy Technology Data Exchange (ETDEWEB)

    Argondizzo, Adam; Cui, Xuefeng; Wang, Cong; Sun, Huijuan; Shang, Honghui; Zhao, Jin; Petek, Hrvoje

    2015-04-27

    We investigate the spectroscopy and photoinduced electron dynamics within the conduction band of reduced rutile TiO2(110) surface by multiphoton photoemission (mPP) spectroscopy with wavelength tunable ultrafast (!20 fs) laser pulse excitation. Tuning the mPP photon excitation energy between 2.9 and 4.6 eV reveals a nearly degenerate pair of new unoccupied states located at 2.73 ± 0.05 and 2.85 ± 0.05 eV above the Fermi level, which can be analyzed through the polarization and sample azimuthal orientation dependence of the mPP spectra. Based on the calculated electronic structure and optical transition moments, as well as related spectroscopic evidence, we assign these resonances to transitions between Ti 3d bands of nominally t2g and eg symmetry, which are split by crystal field. The initial states for the optical transition are the reduced Ti3+ states of t2g symmetry populated by formation oxygen vacancy defects, which exist within the band gap of TiO2. Furthermore,we studied the electron dynamics within the conduction band of TiO2 by three-dimensional time-resolved pump-probe interferometric mPP measurements. The spectroscopic and time-resolved studies reveal competition between 2PP and 3PP processes where the t2g-eg transitions in the 2PP process saturate, and are overtaken by the 3PP process initiated by the band-gap excitation from the valence band of TiO2.

  19. Examination of wound healing after curettage by multiphoton tomography of human skin in vivo.

    Science.gov (United States)

    Springer, S; Zieger, M; Böttcher, A; Lademann, J; Kaatz, M

    2017-11-01

    The multiphoton tomography (MPT) has evolved into a useful tool for the non-invasive investigation of morphological and biophysical characteristics of human skin in vivo. Until now, changes of the skin have been evaluated mainly by using clinical and histological techniques. In this study, the progress of wound healing was investigated by MPT over 3 weeks with a final examination after 24 months. Especially, the collagen degradation, reepithelization and tissue formation were examined. As specific parameter for wound healing and its course the second-harmonic generation-to-autofluorescence aging index of dermis (SAAID) was used. About 10 volunteers aged between 25 and 58 years were examined. Acute wounds were scanned with three Z-stacks taken per visit. The stacks were taken up to a depth of 225 μm at increments of 5 μm and a scan time for 3 seconds per scan. Subsequently, the SAAID was evaluated as an indicator for wound healing. Furthermore, single scans were taken for morphological investigations. The evaluation revealed a distinct difference in the SAAID behavior between the Z-stacks taken at each visit. Furthermore, the degradation of collagen and cells and their reappearance could be shown in the course of the visits. Clear differences in the curve behavior of the SAAID at every visit were shown in this study. The SAAID curves and morphological images could be correlated with findings of the clinical examination of different wound healing phases. Therefore, SAAID curves and morphological MPT imaging could provide a non-invasive tool for the determination of wound healing phases in patients in vivo. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. nf Rydberg complexes of NO in a magnetic field, probed by double resonance multiphoton ionization

    Science.gov (United States)

    Guizard, S.; Shafizadeh, N.; Horani, M.; Gauyacq, D.

    1991-06-01

    nf (v=1) Rydberg states of NO have been probed by double resonance multiphoton ionization in a 1 T external magnetic field. Due to the nonpenetrating character of the f orbitals, these Rydberg states are very sensitive probes of any external perturbation. As n increases, a decoupling of the angular momentum l of the Rydberg electron from the molecular frame occurs gradually, as the magnetic interaction becomes more and more important with respect to intramolecular forces. Up to n≂15, only the linear Zeeman perturbation has been taken into account. The rotational-electronic structure of the 7f and 15f states has been interpreted theoretically by considering the linear Zeeman perturbation in addition to the Coulombic interaction and the long range interaction due to the quadrupole moment and the polarizability of the ion core. The intensities and line positions of the transitions from the intermediate A 2Σ+,v=1 level to the 7f and 15f levels have been calculated. The alignment of the N, MS, MN Zeeman sublevels of the A state by the two-photon pump excitation from the ground state as well as the polarization of both lasers have been taken into account in the calculations. A good agreement between the observed and the calculated transitions has been obtained. For the 7f levels, the electronic-rotational structure is well described in a coupled case (d) representation. For the 15f levels, the strong coupling of l to the field axis led to the first observation of the Paschen-Back effect in a molecule, within each rotational N+ series, with an accompanying drastic simplification of the spectra. This level is better described in a decoupled case (d) representation corresponding to a moderately strong field regime.