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Sample records for act intravital multiphoton

  1. Intravital multiphoton microscopy for imaging hepatobiliary function

    NASA Astrophysics Data System (ADS)

    Li, Feng-Chieh; Sun, Tzu-Lin; Lee, Hsuan-Shu; Yang, Shu-Mei; Dong, Chen-Yuan

    2007-07-01

    Liver is the chemical factory in body responsible for important functions such as metabolism and detoxification. When liver can not be regenerated in time to amend damages that has occurred, failure of hepatic functions can result. Traditionally, the study of liver pathology has depended on histological techniques, but such methods are limited to ex-vivo observation. In order to study hepatic metabolism in vivo, we have designed a hepatic imaging chamber made of biocompatible titanium alloy (6V4Al-Ti, ELI grade). In combination with multiphoton and second harmonic generation microscopy, our approach allows the intravital observation of hepatic intravital activities to be achieved. Processes such as hepatic metabolism and disease progression can be studied using this methodology.

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

    PubMed

    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.

  3. Multiphoton intravital microscopy setup to visualize the mouse mammary gland

    NASA Astrophysics Data System (ADS)

    Adur, Javier; Herrera Torres, Ana M.; Masedunskas, Andrius; Baratti, Mariana O.; de Thomaz, Andre A.; Pelegati, Vitor B.; Carvalho, Hernandes F.; Cesar, Carlos L.

    2013-06-01

    Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of the last century, its major advancements have occurred in the last twenty years with the development of non-linear microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological questions in basic research and is now a fundamental tool that provide information on tissues such as morphology, cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with multiphoton techniques.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27538357

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

    NASA Astrophysics Data System (ADS)

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

    PubMed

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

    2016-01-01

    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. PMID:27538357

  7. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging

    PubMed Central

    Entenberg, David; Wyckoff, Jeffrey; Gligorijevic, Bojana; Roussos, Evanthia T; Verkhusha, Vladislav V; Pollard, Jeffrey W; Condeelis, John

    2014-01-01

    Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via ‘over-clocking’ of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in ~24 h. PMID:21959234

  8. Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment.

    PubMed

    Kirkpatrick, Nathaniel D; Chung, Euiheon; Cook, Daniel C; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L; Padera, Timothy P; Fukumura, Dai; Jain, Rakesh K

    2012-01-01

    The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates-only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

  9. Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment.

    PubMed

    Kirkpatrick, Nathaniel D; Chung, Euiheon; Cook, Daniel C; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L; Padera, Timothy P; Fukumura, Dai; Jain, Rakesh K

    2012-01-01

    The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates-only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment. PMID:24353926

  10. Multiphoton Microscopy Applied for Real-Time Intravital Imaging of Bacterial Infections In Vivo

    PubMed Central

    Choong, Ferdinand X.; Sandoval, Ruben M.; Molitoris, Bruce A.; Richter-Dahlfors, Agneta

    2014-01-01

    To understand the underlying mechanisms of bacterial infections, researchers have for long addressed the molecular interactions occurring when the bacterium interacts with host target cells. In these studies, primarily based on in vitro systems, molecular details have been revealed along with increased knowledge regarding the general infection process. With the recent advancements in in vivo imaging techniques, we are now in a position to bridge a transition from classical minimalistic in vitro approaches to allow infections to be studied in its native complexity—the live organ. Techniques such as multiphoton microscopy (MPM) allow cellular-level visualization of the dynamic infection process in real time within the living host. Studies in which all interplaying factors, such as the influences of the immune, lymphatic, and vascular systems can be accounted for, are likely to provide new insights to our current understanding of the infection process. MPM imaging becomes extra powerful when combined with advanced surgical procedure, allowing studies of the illusive early hours of infection. In this chapter, our intention is to provide a general view on how to design and carry out intravital imaging of a bacterial infection. While exemplifying this using a spatiotemporally well-controlled uropathogenic Escherichia coli (UPEC) infection in rat kidneys, we hope to provide the reader with general considerations that can be adapted to other bacterial infections in organs other than the kidney. PMID:22341218

  11. Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.

    PubMed

    Heymann, Felix; Niemietz, Patricia M; Peusquens, Julia; Ergen, Can; Kohlhepp, Marlene; Mossanen, Jana C; Schneider, Carlo; Vogt, Michael; Tolba, Rene H; Trautwein, Christian; Martin, Christian; Tacke, Frank

    2015-01-01

    Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a

  12. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-03-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed 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. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

  13. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    PubMed Central

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-01-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed 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. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy. PMID:27004454

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

    PubMed

    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%.

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

    PubMed Central

    Muriello, Pamela A.; Dunn, Kenneth W.

    2008-01-01

    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%. PMID:19343075

  16. Intravital multiphoton tomography as a novel tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis

    NASA Astrophysics Data System (ADS)

    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.

  17. Intravital multiphoton imaging of the selective uptake of water-dispersible quantum dots into sinusoidal liver cells.

    PubMed

    Liang, Xiaowen; Grice, Jeffrey E; Zhu, Yian; Liu, David; Sanchez, Washington Y; Li, Zhen; Crawford, Darrell H G; Le Couteur, David G; Cogger, Victoria C; Liu, Xin; Xu, Zhi Ping; Roberts, Michael S

    2015-04-01

    Although many studies reporting the organ-level biodistribution of nanoparticles (NPs) in animals, very few have addressed the fate of NPs in organs at the cellular level. The liver appears to be the main organ for accumulation of NPs after intravenous injection. In this study, for the first time, the in vivo spatiotemporal disposition of recently developed mercaptosuccinic acid (MSA)-capped cadmium telluride/cadmium sulfide (CdTe/CdS) quantum dots (QDs) is explored in rat liver using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging (FLIM), with subcellular resolution (∼1 μm). With high fluorescence efficiency and largely improved stability in the biological environment, these QDs show a distinct distribution pattern in the liver compared to organic dyes, rhodamine 123 and fluorescein. After intravenous injection, fluorescent molecules are taken up by hepatocytes and excreted into the bile, while negatively charged QDs are retained in the sinusoids and selectively taken up by sinusoidal cells (Kupffer cells and liver sinusoidal endothelial cells), but not by hepatocytes within 3 h. The results could help design NPs targeting the specific types of liver cells and choose the fluorescent markers for appropriate cellular imaging.

  18. Deep insights: intravital imaging with two-photon microscopy.

    PubMed

    Schießl, Ina Maria; Castrop, Hayo

    2016-09-01

    Intravital multiphoton microscopy is widely used to assess the structure and function of organs in live animals. Although different tissues vary in their accessibility for intravital multiphoton imaging, considerable progress has been made in the imaging quality of all tissues due to substantial technical improvements in the relevant imaging components, such as optics, excitation laser, detectors, and signal analysis software. In this review, we provide an overview of the technical background of intravital multiphoton microscopy. Then, we note a few seminal findings that were made through the use of multiphoton microscopy. Finally, we address the technical limitations of the method and provide an outlook for how these limitations may be overcome through future technical developments. PMID:27352273

  19. Intravital multiphoton tomography as an appropriate tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Mess, Christian; Dimitrova, Valentina; Schwarz, Martin; Riemann, Iris; Niemeyer, Verena; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.

    2011-03-01

    Increasing incidence of inflammatory skin diseases such as Atopic Dermatitis (AD) has been noted in the past years. According to recent estimations around 15% of newborn subjects are affected with a disease severity that requires medical treatment. Although its pathogenesis is multifactorial, recent reports indicate that an impaired physical skin barrier predispose for the development of AD. The major part of this barrier is formed by the stratum corneum (SC) wherein corneocytes are embedded in a complex matrix of proteins and lipids. Its components were synthesized in the stratum granulosum (SG) and secreted via lamellar bodies at the SC/SG interface. Within a clinical in vivo study we focused on the skin metabolism at the SC/SG interface in AD affected patients in comparison to healthy subjects. Measurement of fluorescence life-time of NADH provides access to the metabolic state of skin. Due to the application of a 5D intravital tomographic skin analysis we facilitate the non-invasive investigation of human epidermis in the longitudinal course of AD therapy. We could ascertain by blinded analysis of 40 skin areas of 20 patients in a three month follow-up that the metabolic status at the SC/SG interface was altered in AD compromised skin even in non-lesional, apparent healthy skin regions. This illustrates an impaired skin barrier formation even at non-affected skin of AD subjects appearing promotive for the development of acute skin inflammation. Therefore, our findings allow a deeper understanding of the individual disease development and the improved management of the therapeutic intervention in clinical application.

  20. Early development of cutaneous cancer revealed by intravital nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Lin, Wei-Chou; Chen, Yang-Fang; Chen, Shean-Jen; Lin, Sung-Jan; Dong, Chen-Yuan

    2010-09-01

    We performed intravital multiphoton microscopy to image and analyze normal and carcinogen treated skin tissues of nude mice in vivo. Using intravital images and the quantitative pixel to pixel ratiometric processing of multiphoton autofluorescence to second harmonic generation index (MAFSI), we can visualize the interaction between epithelial cells and extracellular matrix. We found that as the imaging depth increases, MAFSI has different distribution in normal and treated cutaneous specimens. Since the treated skin eventually became squamous cell carcinoma, our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy.

  1. Shedding light on cutaneous innate immune responses: the intravital microscopy approach.

    PubMed

    Jain, Rohit; Weninger, Wolfgang

    2013-04-01

    The skin is under constant assault by environmental factors and microbes. Innate immune cells in epidermis and dermis regulate immune responses against pathogens while maintaining tolerance against commensal bacteria and autoantigens. The introduction of intravital imaging approaches, in particular multiphoton microscopy, has enabled studying the cellular and molecular regulation of cutaneous immunity in real time within intact skin. Here, we discuss recent advances in our understanding of innate immune cell behaviour in the skin, as unravelled by intravital microscopy, with emphasis on the function of myeloid cells, including dendritic cells, neutrophils and monocytes.

  2. Fluorescent Tobacco mosaic virus-Derived Bio-Nanoparticles for Intravital Two-Photon Imaging

    PubMed Central

    Niehl, Annette; Appaix, Florence; Boscá, Sonia; van der Sanden, Boudewijn; Nicoud, Jean-François; Bolze, Frédéric; Heinlein, Manfred

    2016-01-01

    Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature. PMID:26793221

  3. The Use of Fluorescent Proteins for Intravital Imaging of Cancer Cell Invasion

    PubMed Central

    Hulit, James; Kedrin, Dmitriy; Gligorijevic, Bojana; Entenberg, David; Wyckoff, Jeffrey; Condeelis, John; Segall, Jeffrey E.

    2014-01-01

    The analysis of cancer cell behavior in the primary tumor in living animals provides an opportunity to explore the process of invasion and intravasation in the complex microenvironment that is present in vivo. In this chapter, we describe the methods that we have developed for performing intravital imaging of mammary tumors. We provide procedures for generating tumors through injection of tumor cell lines, and multiphoton imaging using a skin-flap tumor dissection and a mammary imaging window. PMID:22700401

  4. MULTIPHOTON PROCESSES

    SciTech Connect

    2002-07-05

    The Gordon Research Conference (GRC) on MULTIPHOTON PROCESSES was held at Tilton School, Tilton, NH. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  5. Intravital Microscopic Methods to Evaluate Anti-inflammatory Effects and Signaling Mechanisms Evoked by Hydrogen Sulfide

    PubMed Central

    Zuidema, Mozow Y.; Korthuis, Ronald J.

    2016-01-01

    Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule with potent anti-inflammatory properties. Exogenous application of H2S donors, administered either acutely during an inflammatory response or as an antecedent preconditioning intervention that invokes the activation of anti-inflammatory cell survival programs, effectively limits leukocyte rolling, adhesion and emigration, generation of reactive oxygen species, chemokine and cell adhesion molecule expression, endothelial barrier disruption,capillary perfusion deficits, and parenchymal cell dysfunction and injury. This chapter focuses on intravital microscopic methods that can be used to assess the anti-inflammatory effects exerted by H2S, as well as to explore the cellular signaling mechanisms by which this gaseous molecule limits the aforementioned inflammatory responses. Recent advances include use of intravital multiphoton microscopy and optical biosensor technology to explore signaling mechanisms in vivo. PMID:25747477

  6. Automated motion artifact removal for intravital microscopy, without a priori information

    NASA Astrophysics Data System (ADS)

    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.

  7. Automated motion artifact removal for intravital microscopy, without a priori information.

    PubMed

    Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

    2014-03-28

    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.

  8. Live-Animal Imaging of Renal Function by Multiphoton Microscopy

    PubMed Central

    Dunn, Kenneth W.; Sutton, Timothy A.; Sandoval, Ruben M.

    2015-01-01

    Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. PMID:23042524

  9. Quantifying endocytosis in vivo using intravital two-photon microscopy.

    PubMed

    Sandoval, Ruben M; Molitoris, Bruce A

    2008-01-01

    The recent introduction of multiphoton microscopy coupled with advances in optics, computer sciences, designer fluorophores, molecular labeling, and previously developed physiologic approaches have empowered investigators to quantitatively study the cell-specific dynamic events, such as endocytosis, within a functioning organ with subcellular resolution. This rapidly emerging field of investigation, with superior spatial and temporal resolution and high sensitivity, enables investigators to track molecules and determine their mode of cellular uptake, intracellular trafficking, and metabolism in a cell-specific fashion in complex heterogeneous organs such as the kidney with repeated determinations possible over a prolonged period of time. This approach is enhanced by the ability to obtain and quantify volumetric data with using up to three different fluorophores simultaneously. We have utilized this intravital approach to understand and quantify kidney proximal tubule cell uptake and intracellular distribution and metabolism of fluorescently labeled molecules, including folic acid, gentamicin, and small interfering ribonucleic acid (siRNA). Limitations of this technique include tissue penetration, which is the major barrier to successful clinical utilization of this technology. However, its use in preclinical animal models offers new insight into physiologic processes and the pathophysiology and treatment of disease processes. PMID:18369960

  10. Multiphoton processes: conference proceedings

    SciTech Connect

    Lambropoulos, P.; Smith, S.J.

    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)

  11. Correlative intravital imaging of cGMP signals and vasodilation in mice

    PubMed Central

    Thunemann, Martin; Schmidt, Kjestine; de Wit, Cor; Han, Xiaoxing; Jain, Rakesh K.; Fukumura, Dai; Feil, Robert

    2014-01-01

    Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism. PMID:25352809

  12. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

    NASA Astrophysics Data System (ADS)

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-02-01

    Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

  13. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

    PubMed Central

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-01-01

    Abstract. Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe−/−Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages “dancing on the spot” and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells. PMID:25710308

  14. Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment.

    PubMed

    Harney, Allison S; Wang, Yarong; Condeelis, John S; Entenberg, David

    2016-01-01

    In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment. PMID:27341448

  15. Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

    PubMed Central

    Harney, Allison S.; Wang, Yarong; Condeelis, John S.; Entenberg, David

    2016-01-01

    In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment. PMID:27341448

  16. Substrate-Free Self-Assembled SiOx-Core Nanodots from Alkylalkoxysilane as a Multicolor Photoluminescence Source for Intravital Imaging

    PubMed Central

    Lin, Pei-Ying; Hsieh, Chiung-Wen; Kung, Mei-Lang; Hsieh, Shuchen

    2013-01-01

    Intravital fluorescence imaging has great potential in biological and biomedical research, as it provides the ability to directly observe biological structures and processes in their natural state. Contrast agents for intravital imaging applications should exhibit good biocompatibility, multiphoton fluorescence, and long emission. Carbon nanodots and semiconductor nanocrystals meet these requirements in most cases, with the added benefit that their properties can be ‘tuned' for specific applications by controlling the size and surface chemistry of the nanoparticles. Here, we report on a simple heat-assisted strategy to fabricate SiOx-core self-assembled nanodots using self-assembled monolayer (SAM) materials. Our results demonstrate that substrate-free self-assembled nanodots from alkylalkoxysilane exhibit controllable structural and chemical characteristics that are well suited for applications in biological, biomedical, and clinical research, and may find further use in optoelectronic and sensor devices. PMID:23609156

  17. Multiphoton electromagnetically induced transparency

    NASA Astrophysics Data System (ADS)

    Wen, Lingling; Kang, Hoonsoo; Zhu, Yifu; Wu, Ying

    2003-05-01

    We show that in multi-level atomic systems coupled by multiple laser fields, all linear and nonlinear absorptions may be completely suppressed, leading to the multiphoton electromagnetically induced transparency (EIT). Under suitable conditions, multiphoton EIT may be used to realize selective steady-state population inversion in coherently pumped atomic systems and achieve efficient nonlinear light generation at low light intensities. As examples, we will present studies of multiphoton EIT in five-level and six-level atomic systems, which demonstrate steady-state population inversion from selective nonlinear excitation. We will also present studies of resonant hyper-Raman and four-wave mixing processes that are enhanced via suppression of the lower-order linear and nonlinear absorptions, and are capable of generating short-wavelength, coherent light at low pump intensities.

  18. In vivo multiphoton imaging of bile duct ligation

    NASA Astrophysics Data System (ADS)

    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.

  19. Intravital Microscopic Interrogation of Peripheral Taste Sensation

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

    2015-03-01

    Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

  20. Intravital microscopic interrogation of peripheral taste sensation.

    PubMed

    Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

    2015-03-02

    Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

  1. Scanning microfluorometry in intravital microvascular research.

    PubMed

    Witte, S

    1989-01-01

    Our own development of fluorometric scanning techniques in intravital microscopy of the microcirculation is described. Very tiny amount of fluorometric substances are detected with a high temporal and locational resolution. The everted small intestinal mesentery of the rat serves as a model. We have given a detailed description of the microscopes used, the optical systems, the conditions of measurement of the microfluorometry, the scanning techniques and the evaluation of the measurement data. The present state of technical development detects 10(-12) g of a fluorochromed plasma protein in 8 ms in a measurement field of 2 microns 2. The four-digit measurement data of a scanning line of 200 microns length in 0.25 micron locational resolution are registered in about 2 s.

  2. Intravital two-photon imaging: a versatile tool for dissecting the immune system.

    PubMed

    Ishii, Taeko; Ishii, Masaru

    2011-03-01

    During the past decade, multi-photon or 'two-photon' excitation microscopy has launched a new era in the field of biological imaging. The near-infrared excitation laser for two-photon microscopy can penetrate thicker specimens, enabling the visualisation of living cell behaviour deep within tissues and organs without thin sectioning. The minimised photobleaching and toxicity enables the visualisation of live and intact specimens for extended periods. In this brief review, recent findings in intravital two-photon imaging for the physiology and pathology of the immune system are discussed. The immune system configures highly dynamic networks, where many cell types actively travel throughout the body and interact with each other in specific areas. Hence, real-time intravital imaging may be a powerful tool for dissecting the mechanisms of this dynamic system. The most unique characteristic of the immune system is its highly dynamic nature. A variety of cell types, such as lymphocytes, macrophages and dendritic cells (DCs), are continuously circulating throughout the body, migrating through the peripheral tissues and interacting with each other in their respective niches. Conventional methodologies in immunology, such as flow cytometry, cell or tissue culture, biochemistry and histology, have brought tremendous achievement within this field, although the dynamics of immune cells in an entire animal remain less clear. Technological progress of fluorescence microscopy has enabled us to visualise the intact biological phenomenon that has been uninvestigated. Among the advancements, the recent emergence and prevalence of two-photon, excitation-based, laser microscopy has revolutionised the research field, such that the dynamic behaviour of cells deep inside living organs can be visualised and analysed.

  3. 5D-intravital tomography as a novel tool for non-invasive in-vivo analysis of human skin

    NASA Astrophysics Data System (ADS)

    König, Karsten; Weinigel, Martin; Breunig, Hans G.; Gregory, Axel; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; Schwarz, Martin; Riemann, Iris; Stracke, Frank; Huck, Volker; Gorzelanny, Christian; Schneider, Stefan W.

    2010-02-01

    Some years ago, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have been launched. These tomographs provide optical biopsies with submicron resolution based on two-photon excited autofluorescence (NAD(P)H, flavoproteins, keratin, elastin, melanin, porphyrins) and second harmonic generation by collagen. The 3D tomograph was now transferred into a 5D imaging system by the additional detection of the emission spectrum and the fluorescence lifetime based on spatially and spectrally resolved time-resolved single photon counting. The novel 5D intravital tomograph (5D-IVT) was employed for the early detection of atopic dermatitis and the analysis of treatment effects.

  4. Clinical multiphoton FLIM tomography

    NASA Astrophysics Data System (ADS)

    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.

  5. Intravital Microscopy for THz-Bio Analysis

    NASA Astrophysics Data System (ADS)

    Kim, Pilhan

    Intravital microscopy is a high-resolution imaging technique to observe biological phenomena in living organisms. It often also stated as in vivo microscopy. Literal meaning of in vivo is "within the living" and there is another term, ex vivo of which literal meaning is "out of the living". Both terms are commonly used to describe the status of sample at the moment of biological manipulations or investigations are done. In vivo study is a form of research using whole living organism in experiment to investigate a certain biological phenomenon in its natural environment, whereas ex vivo study uses non-living subjects such as tissues or organs dissected from dead animal. In addition, in vitro of which literal meaning is "within the glass" is another commonly used term. In vitro study is a form of research using small living subject such as cell in a controlled environment such as petri dish or test tube. Cell culture, the process of growing cells in a petri dish, is the most common form of in vitro study. Figure 1 summarizes the status of samples for biological study categorized by in vivo, in vitro and ex vivo.

  6. Intravital microscopy of the lung: minimizing invasiveness.

    PubMed

    Fiole, Daniel; Tournier, Jean-Nicolas

    2016-09-01

    In vivo microscopy has recently become a gold standard in lung immunology studies involving small animals, largely benefiting from the democratization of multiphoton microscopy allowing for deep tissue imaging. This technology represents currently our only way of exploring the lungs and inferring what happens in human respiratory medicine. The interest of lung in vivo microscopy essentially relies upon its relevance as a study model, fulfilling physiological requirements in comparison with in vitro and ex vivo experiments. However, strategies developed in order to overcome movements of the thorax caused by breathing and heartbeats remain the chief drawback of the technique and a major source of invasiveness. In this context, minimizing invasiveness is an unavoidable prerequisite for any improvement of lung in vivo microscopy. This review puts into perspective the main techniques enabling lung in vivo microscopy, providing pros and cons regarding invasiveness. PMID:26846880

  7. Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment

    PubMed Central

    Ricard, Clément; Debarbieux, Franck Christian

    2014-01-01

    The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology. PMID:24605087

  8. Multiphoton ionization spectroscopy of the sodium dimer

    NASA Astrophysics Data System (ADS)

    Keller, John; Weiner, John

    1984-07-01

    We report an investigation of the role of molecular multiphoton ionization in the production of Na+2 when sodium vapor is subjected to intense optical radiation. Previous authors attribute the source of much of the ion dimer signal to laser-induced associative ionization of atom sodium. In this experiment, we distinguish the molecular process from atomic collisional mechanisms by producing an intense molecular beam created through free-jet expansion of the metal vapor. The beam of nearly 50% dimers cooled to their low rotational and vibrational states allow us to obtain a simplified three-photon ionization spectrum. We find that the spectrum displays two-photon resonances corresponding to known Rydberg level transitions and that the A state, acting as virtual intermediate, plays a crucial role in the large peak-to-peak intensity variations. We employ a simple model of multiphoton ionization which uses a rate-equation approach to generate a calculated spectrum. Based on the experimental results and the success of the model in reflecting them, we conclude that much of the highly structured component of the dimer ion signal reported previously under different experimental conditions is probably due to molecular multiphoton ionization but that this structure rides on a slowly varying broad signal envelope due to laser-induced associative ionization.

  9. Generalized Multiphoton Quantum Interference

    NASA Astrophysics Data System (ADS)

    Tillmann, Max; Tan, Si-Hui; Stoeckl, Sarah E.; Sanders, Barry C.; de Guise, Hubert; Heilmann, René; Nolte, Stefan; Szameit, Alexander; Walther, Philip

    2015-10-01

    Nonclassical interference of photons lies at the heart of optical quantum information processing. Here, we exploit tunable distinguishability to reveal the full spectrum of multiphoton nonclassical interference. We investigate this in theory and experiment by controlling the delay times of three photons injected into an integrated interferometric network. We derive the entire coincidence landscape and identify transition matrix immanants as ideally suited functions to describe the generalized case of input photons with arbitrary distinguishability. We introduce a compact description by utilizing a natural basis that decouples the input state from the interferometric network, thereby providing a useful tool for even larger photon numbers.

  10. Intravital Microscopy for Imaging the Tumor Microenvironment in Live Mice.

    PubMed

    Naumenko, Victor; Jenne, Craig; Mahoney, Douglas J

    2016-01-01

    The development of intravital microscopy has provided unprecedented capacity to study the tumor microenvironment in live mice. The dynamic behavior of cancer, stromal, vascular, and immune cells can be monitored in real time, in situ, in both primary tumors and metastatic lesions, allowing treatment responses to be observed at single cell resolution and therapies tracked in vivo. These features provide a unique opportunity to elucidate the cellular mechanisms underlying the biology and treatment of cancer. We describe here a method for imaging the microenvironment of subcutaneous tumors grown in mice using intravital microscopy. PMID:27581025

  11. Multiphoton ionization of Uracil

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Salomonnson, Emma; Mihalko, Laura Anne; Verkhusha, Vladislav V.; Luker, Kathryn E.; Luker, Gary D.

    2012-09-01

    Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760 nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.

  14. Intraoperative intravital microscopy permits the study of human tumour vessels

    PubMed Central

    Fisher, Daniel T.; Muhitch, Jason B.; Kim, Minhyung; Doyen, Kurt C.; Bogner, Paul N.; Evans, Sharon S.; Skitzki, Joseph J.

    2016-01-01

    Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment. PMID:26883450

  15. [Bone and Stem Cells. Intravital imaging of bone marrow microenvironment].

    PubMed

    Mizuno, Hiroki; Kikuta, Junichi; Ishii, Masaru

    2014-04-01

    Various kinds of cell types, such as osteoclasts, osteoblasts, hematopoietic cells, and mesenchymal cells, have been reported to exist in the bone marrow and communicate with each other. Although there have been many previous studies about bone marrow microenvironment, most of them were analyzed by conventional methods such as histological analysis and flow cytometry. These methods could not observe the dynamic cell movement in living bone marrow. Recently rapid development of fluorescent imaging techniques enables us to understand the cellular dynamics in vivo . That's why we have originally established an advanced imaging system for visualizing living bone tissues with intravital two-photon microscopy. Here we show the latest data and the detailed methodology of intravital imaging of bone marrow microenvironment, and also discuss its further application.

  16. Intraoperative intravital microscopy permits the study of human tumour vessels.

    PubMed

    Fisher, Daniel T; Muhitch, Jason B; Kim, Minhyung; Doyen, Kurt C; Bogner, Paul N; Evans, Sharon S; Skitzki, Joseph J

    2016-02-17

    Tumour vessels have been studied extensively as they are critical sites for drug delivery, anti-angiogenic therapies and immunotherapy. As a preclinical tool, intravital microscopy (IVM) allows for in vivo real-time direct observation of vessels at the cellular level. However, to date there are no reports of intravital high-resolution imaging of human tumours in the clinical setting. Here we report the feasibility of IVM examinations of human malignant disease with an emphasis on tumour vasculature as the major site of tumour-host interactions. Consistent with preclinical observations, we show that patient tumour vessels are disorganized, tortuous and ∼50% do not support blood flow. Human tumour vessel diameters are larger than predicted from immunohistochemistry or preclinical IVM, and thereby have lower wall shear stress, which influences delivery of drugs and cellular immunotherapies. Thus, real-time clinical imaging of living human tumours is feasible and allows for detection of characteristics within the tumour microenvironment.

  17. Multiphoton tomography of astronauts

    NASA Astrophysics Data System (ADS)

    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.

  18. Quantitative multiphoton imaging

    NASA Astrophysics Data System (ADS)

    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.

  19. Intravital microscopy to image membrane trafficking in live rats

    PubMed Central

    Masedunskas, Andrius; Sramkova, Monika; Parente, Laura; Weigert, Roberto

    2014-01-01

    Summary Intravital microscopy (IVM) is a powerful tool that enables imaging various biological processes in live animals. Here, we describe a series of procedures designed to image subcellular structures, such as endsosomes and secretory vesicles in the salivary glands (SGs) of live rats. To this aim, we used fluorescently labeled molecules and/or fluorescently-tagged proteins that were transiently transfected in the live animal. PMID:23027003

  20. Multiphoton microspectroscopy of biological specimens

    NASA Astrophysics Data System (ADS)

    Lin, Bai-Ling; Kao, Fu-Jen; Cheng, Ping C.; Sun, Chi-Kuang; Chen, RangWu; Wang, YiMin; Chen, JianCheng; Wang, Yung-Shun; Liu, Tzu-Ming; Huang, Mao-Kuo

    2000-07-01

    The non-linear nature of multi-photon fluorescence excitation restricts the fluorescing volume to the vicinity of the focal point. As a result, the technology has the capacity for micro- spectroscopy of biological specimen at high spatial resolution. Chloroplasts in mesophyll protoplast of Arabidopsis thaliana and maize stem sections were used to demonstrate the feasibility of multi-photon fluorescence micro-spectroscopy at subcellular compartments. Time-lapse spectral recording provides a means for studying the response of cell organelles to high intensity illumination.

  1. Multiphoton tomography for tissue engineering

    NASA Astrophysics Data System (ADS)

    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.

  2. Motion compensation using a suctioning stabilizer for intravital microscopy

    PubMed Central

    Vinegoni, Claudio; Lee, Sungon; Gorbatov, Rostic; Weissleder, Ralph

    2013-01-01

    Motion artifacts continue to present a major challenge to single cell imaging in cardiothoracic organs such as the beating heart, blood vessels, or lung. In this study, we present a new water-immersion suctioning stabilizer that enables minimally invasive intravital fluorescence microscopy using water-based stick objectives. The stabilizer works by reducing major motion excursions and can be used in conjunction with both prospective or retrospective gating approaches. We show that the new approach offers cellular resolution in the beating murine heart without perturbing normal physiology. In addition, because this technique allows multiple areas to be easily probed, it offers the opportunity for wide area coverage at high resolution. PMID:24086796

  3. Intravital confocal Raman microscopy with multiplexed SERS contrast agents

    NASA Astrophysics Data System (ADS)

    McVeigh, Patrick Z.; Wilson, Brian C.

    2012-03-01

    Intravital microscopy has been demonstrated to be a powerful technique for studying the delivery of contrast or therapeutic agents to tumours growing in a realistic 3D environment at high resolution. Surface enhanced Raman scattering (SERS)-active nanoparticle contrast agents provide the ability to improve in-vivo detection of tumour tissue through multiplex detection of their uniquely bright spectral lines. However, most work to date has been carried out in-vitro or in ex-vivo tissues. Here we present the results from confocal Raman microscopy in a dorsal skinfold window chamber in mice using SERS-active gold nanoparticle contrast agents directed towards an overexpressed tumour receptor tyrosine kinase.

  4. Intravital video microscopy measurements of retinal blood flow in mice.

    PubMed

    Harris, Norman R; Watts, Megan N; Leskova, Wendy

    2013-01-01

    Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats. PMID:24429840

  5. Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles

    PubMed Central

    Leong, Hon Sing; Steinmetz, Nicole F; Ablack, Amber; Destito, Giuseppe; Zijlstra, Andries; Stuhlmann, Heidi; Manchester, Marianne; Lewis, John D

    2011-01-01

    Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of cpMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application. PMID:20671724

  6. Stochastic scanning multiphoton multifocal microscopy.

    PubMed

    Jureller, Justin E; Kim, Hee Y; Scherer, Norbert F

    2006-04-17

    Multiparticle tracking with scanning confocal and multiphoton fluorescence imaging is increasingly important for elucidating biological function, as in the transport of intracellular cargo-carrying vesicles. We demonstrate a simple rapid-sampling stochastic scanning multifocal multiphoton microscopy (SS-MMM) fluorescence imaging technique that enables multiparticle tracking without specialized hardware at rates 1,000 times greater than conventional single point raster scanning. Stochastic scanning of a diffractive optic generated 10x10 hexagonal array of foci with a white noise driven galvanometer yields a scan pattern that is random yet space-filling. SS-MMM creates a more uniformly sampled image with fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. SS-MMM is verified by simulation and experimentally demonstrated by tracking microsphere diffusion in solution. PMID:19516485

  7. COMPACT NON-CONTACT TOTAL EMISSION DETECTION FOR IN-VIVO MULTI-PHOTON EXCITATION MICROSCOPY

    PubMed Central

    Glancy, Brian; Karamzadeh, Nader S.; Gandjbakhche, Amir H.; Redford, Glen; Kilborn, Karl; Knutson, Jay R.; Balaban, Robert S.

    2014-01-01

    Summary We describe a compact, non-contact design for a Total Emission Detection (c-TED) system for intra-vital multi-photon imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), while murine skeletal muscle and rat kidney showed gains of over two and just under two-fold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a two-fold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers (enabled by greater light collection efficiency) yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multi-photon imaging methods is discussed. PMID:24251437

  8. Multiphoton microscopy of atheroslcerotic plaques

    NASA Astrophysics Data System (ADS)

    Lilledahl, Magnus B.; de Lange Davies, Catharina; Haugen, Olav A.; Svaasand, Lars O.

    2007-02-01

    Multiphoton microscopy is a techniques that fascilitates three dimensional imaging of intact, unstained tissue. Especially connective tissue has a relatively strong nonlinear optical response and can easily be imaged. Atherosclerosis is a disease where lipids accumulate in the vessel wall and there is a thickening of the intima by growth of a cap of connective tissue. The mechanical strength of this fibrous cap is of clinically importance. If the cap ruptures a thrombosis forms which can block a coronary vessel and therby causing myocardial infarction. Multiphoton microscopy can be used to image the fibrous cap and thereby determine the thickness of the cap and the structure of the connective fibres. This could possibly be developed into a diagnostic and clincal tool to monitor the vulnerability of a plaque and also to better understand the development of a plaque and effects of treatment. We have collected multiphoton microscopy images from atherosclerotic plaque in human aorta, both two photon excited fluorescens and second harmonic generated signal. The feasability of using this technique to determine the state of the plaque is explored.

  9. Intravital imaging reveals new ancillary mechanisms co-opted by cancer cells to drive tumor progression

    PubMed Central

    Lucas, Morghan C.; Timpson, Paul

    2016-01-01

    Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression. PMID:27239290

  10. Advanced Motion Compensation Methods for Intravital Optical Microscopy.

    PubMed

    Vinegoni, Claudio; Lee, Sungon; Feruglio, Paolo Fumene; Weissleder, Ralph

    2014-03-01

    Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions.

  11. Advanced Motion Compensation Methods for Intravital Optical Microscopy

    PubMed Central

    Vinegoni, Claudio; Lee, Sungon; Feruglio, Paolo Fumene; Weissleder, Ralph

    2013-01-01

    Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions. PMID:24273405

  12. Multiphoton Microscopy for Ophthalmic Imaging

    PubMed Central

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

    2011-01-01

    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. PMID:21274261

  13. Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

    PubMed Central

    Bardet, Sylvia M.; Carr, Lynn; Soueid, Malak; Arnaud-Cormos, Delia; Leveque, Philippe; O’Connor, Rodney P.

    2016-01-01

    Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35–45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma. PMID:27698479

  14. Multiphoton cryo microscope with sample temperature control

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    We present a multiphoton microscope system which combines the advantages of multiphoton imaging with precise control of the sample temperature. The microscope provides online insight in temperature-induced changes and effects in plant tissue and animal cells with subcellular resolution during cooling and thawing processes. Image contrast is based on multiphoton fluorescence intensity or fluorescence lifetime in the range from liquid nitrogen temperature up to +600°C. In addition, micro spectra from the imaged regions can be recorded. We present measurement results from plant leaf samples as well as Chinese hamster ovary cells.

  15. MULTI-PHOTON PHOSPHOR FEASIBILITY RESEARCH

    SciTech Connect

    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

  16. Differential Multiphoton Laser Scanning Microscopy

    PubMed Central

    Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

    2016-01-01

    Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot. PMID:27390511

  17. Multiphoton dissociative ionization of CS+

    NASA Astrophysics Data System (ADS)

    Rajput, Jyoti; Jochim, Bethany; Zohrabi, M.; Betsch, K. J.; Ablikim, U.; Berry, Ben; Severt, T.; Summers, A. M.; Armstrong, G. S. J.; Esry, B. D.; Carnes, K. D.; Ben-Itzhak, I.

    2015-05-01

    We have studied the dissociative photoionization of a CS+ molecular ion beam in the strong-field regime using <50 fs IR laser pulses (λ ~ 790 nm) from a 10 kHz, ~2 mJ (per pulse) Ti:Sapphire laser system. A coincidence three-dimensional momentum imaging method was used to measure all ions and neutrals formed during this multiphoton process. Two prominent channels were observed: charge-symmetric dissociation, yielding C+ + S+, and charge-asymmetric dissociation, yielding C + S2+. The differences between these two channels with reference to their relative production probability, energetics, and angular distributions is the focus of this work. This work was supported by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy. BJ is also supported by DOE-SCGF (DE-AC05-06OR23100).

  18. Multiphoton entanglement concentration and quantum cryptography.

    PubMed

    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.

  19. Intravital imaging of intestinal lacteals unveils lipid drainage through contractility.

    PubMed

    Choe, Kibaek; Jang, Jeon Yeob; Park, Intae; Kim, Yeseul; Ahn, Soyeon; Park, Dae-Young; Hong, Young-Kwon; Alitalo, Kari; Koh, Gou Young; Kim, Pilhan

    2015-11-01

    Lacteals are lymphatic vessels located at the center of each intestinal villus and provide essential transport routes for lipids and other lipophilic molecules. However, it is unclear how absorbed molecules are transported through the lacteal. Here, we used reporter mice that express GFP under the control of the lymphatic-specific promoter Prox1 and a custom-built confocal microscope and performed intravital real-time visualization of the absorption and transport dynamics of fluorescence-tagged fatty acids (FAs) and various exogenous molecules in the intestinal villi in vivo. These analyses clearly revealed transepithelial absorption of these molecules via enterocytes, diffusive distribution over the lamina propria, and subsequent transport through lacteals. Moreover, we observed active contraction of lacteals, which seemed to be directly involved in dietary lipid drainage. Our analysis revealed that the smooth muscles that surround each lacteal are responsible for contractile dynamics and that lacteal contraction is ultimately controlled by the autonomic nervous system. These results indicate that the lacteal is a unique organ-specific lymphatic system and does not merely serve as a passive conduit but as an active pump that transports lipids. Collectively, using this efficient imaging method, we uncovered drainage of absorbed molecules in small intestinal villus lacteals and the involvement of lacteal contractibility. PMID:26436648

  20. Intravital leukocyte detection using the gradient inverse coefficient of variation.

    PubMed

    Dong, Gang; Ray, Nilanjan; Acton, Scott T

    2005-07-01

    The problem of identifying and counting rolling leukocytes within intravital microscopy is of both theoretical and practical interest. Currently, methods exist for tracking rolling leukocytes in vivo, but these methods rely on manual detection of the cells. In this paper we propose a technique for accurately detecting rolling leukocytes based on Bayesian classification. The classification depends on a feature score, the gradient inverse coefficient of variation (GICOV), which serves to discriminate rolling leukocytes from a cluttered environment. The leukocyte detection process consists of three sequential steps: the first step utilizes an ellipse matching algorithm to coarsely identify the leukocytes by finding the ellipses with a locally maximal GICOV. In the second step, starting from each of the ellipses found in the first step, a B-spline snake is evolved to refine the leukocytes boundaries by maximizing the associated GICOV score. The third and final step retains only the extracted contours that have a GICOV score above the analytically determined threshold. Experimental results using 327 rolling leukocytes were compared to those of human experts and currently used methods. The proposed GICOV method achieves 78.6% leukocyte detection accuracy with 13.1% false alarm rate.

  1. Intravital imaging of intestinal lacteals unveils lipid drainage through contractility

    PubMed Central

    Choe, Kibaek; Jang, Jeon Yeob; Park, Intae; Kim, Yeseul; Ahn, Soyeon; Park, Dae-Young; Hong, Young-Kwon; Alitalo, Kari; Koh, Gou Young; Kim, Pilhan

    2015-01-01

    Lacteals are lymphatic vessels located at the center of each intestinal villus and provide essential transport routes for lipids and other lipophilic molecules. However, it is unclear how absorbed molecules are transported through the lacteal. Here, we used reporter mice that express GFP under the control of the lymphatic-specific promoter Prox1 and a custom-built confocal microscope and performed intravital real-time visualization of the absorption and transport dynamics of fluorescence-tagged fatty acids (FAs) and various exogenous molecules in the intestinal villi in vivo. These analyses clearly revealed transepithelial absorption of these molecules via enterocytes, diffusive distribution over the lamina propria, and subsequent transport through lacteals. Moreover, we observed active contraction of lacteals, which seemed to be directly involved in dietary lipid drainage. Our analysis revealed that the smooth muscles that surround each lacteal are responsible for contractile dynamics and that lacteal contraction is ultimately controlled by the autonomic nervous system. These results indicate that the lacteal is a unique organ-specific lymphatic system and does not merely serve as a passive conduit but as an active pump that transports lipids. Collectively, using this efficient imaging method, we uncovered drainage of absorbed molecules in small intestinal villus lacteals and the involvement of lacteal contractibility. PMID:26436648

  2. Intravital microscopy as a tool to study drug delivery in preclinical studies

    PubMed Central

    Amornphimoltham, Panomwat; Masedunskas, Andrius; Weigert, Roberto

    2010-01-01

    The technical developments in the field of non-linear microscopy have made intravital microscopy one of the most successful techniques for studying physiological and pathological processes in live animals. Intravital microscopy has been utilized to address many biological questions in basic research and is now a fundamental tool for preclinical studies, with an enormous potential for clinical applications. The ability to dynamically image cellular and subcellular structures combined with the possibility to perform longitudinal studies have empowered investigators to use this discipline to study the mechanisms of action of therapeutic agents and assess the efficacy on their targets in vivo. The goal of this review is to provide a general overview of the recent advances in intravital microscopy and to discuss some of its applications in preclinical studies. PMID:20933026

  3. Current developments in clinical multiphoton tomography

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    Two-photon microscopy has been introduced in 1990 [1]. 13 years later, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have been launched by the JenLab company with the tomograph DermaInspectTM. In 2010, the second generation of clinical multiphoton tomographs was introduced. The novel mobile multiphoton tomograph MPTflexTM, equipped with a flexible articulated optical arm, provides an increased flexibility and accessibility especially for clinical and cosmetical examinations. The multiphoton excitation of fluorescent biomolecules like NAD(P)H, flavins, porphyrins, elastin, and melanin as well as the second harmonic generation of collagen is induced by picojoule femtosecond laser pulses from an tunable turn-key near infrared laser system. The ability for rapid highquality 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. So far, more than 1,000 patients and volunteers have been investigated with the multiphoton tomographs in Europe, Asia, and Australia.

  4. Multi-photon excitation microscopy

    PubMed Central

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-01-01

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments. PMID:16756664

  5. Multi-photon excitation microscopy.

    PubMed

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-01-01

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

  6. Multi-photon excitation microscopy.

    PubMed

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-01-01

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments. PMID:16756664

  7. Clinical multiphoton and CARS microscopy

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Weinigel, M.; Darvin, M. E.; Lademann, J.; König, K.

    2012-03-01

    We report on clinical CARS imaging of human skin in vivo with the certified hybrid multiphoton tomograph CARSDermaInspect. The CARS-DermaInspect provides simultaneous imaging of non-fluorescent intradermal lipid and water as well as imaging of two-photon excited fluorescence from intrinsic molecules. Two different excitation schemes for CARS imaging have been realized: In the first setup, a combination of fs oscillator and optical parametric oscillator provided fs-CARS pump and Stokes pulses, respectively. In the second setup a fs oscillator was combined with a photonic crystal fiber which provided a broadband spectrum. A spectral range out of the broadband-spectrum was selected and used for CARS excitation in combination with the residual fs-oscillator output. In both setups, in addition to CARS, single-beam excitation was used for imaging of two-photon excited fluorescence and second harmonic generation signals. Both CARS-excitation systems were successfully used for imaging of lipids inside the skin in vivo.

  8. Multi-focal multiphoton lithography.

    PubMed

    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.

  9. Multi-focal multiphoton lithography.

    PubMed

    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. PMID:22282105

  10. [Forensic medical diagnostics of intra-vitality of the strangulation mark by morphological methods].

    PubMed

    Bogomolov, D V; Zbrueva, Yu V; Putintsev, V A; Denisova, O P

    2016-01-01

    The objective of the present study WaS to overview the current domestic and foreign literature concerning the up-to-date methods employed for the expert evaluation of intra-vitality of the strangulation mark. The secondary objective was to propose the new approaches for addressing this problem. The methods of expert diagnostics with a view to determining the time of infliction of injuries as exemplified by mechanical asphyxia are discussed. It is concluded that immunohistochemical and morphometric studies provide the most promising tools for the evaluation of intra-vitality of the strangulation mark for the purpose of forensic medical expertise.

  11. [Forensic medical diagnostics of intra-vitality of the strangulation mark by morphological methods].

    PubMed

    Bogomolov, D V; Zbrueva, Yu V; Putintsev, V A; Denisova, O P

    2016-01-01

    The objective of the present study WaS to overview the current domestic and foreign literature concerning the up-to-date methods employed for the expert evaluation of intra-vitality of the strangulation mark. The secondary objective was to propose the new approaches for addressing this problem. The methods of expert diagnostics with a view to determining the time of infliction of injuries as exemplified by mechanical asphyxia are discussed. It is concluded that immunohistochemical and morphometric studies provide the most promising tools for the evaluation of intra-vitality of the strangulation mark for the purpose of forensic medical expertise. PMID:27358932

  12. Multiphoton microscopy in defining liver function

    NASA Astrophysics Data System (ADS)

    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.

  13. A pragmatic guide to multiphoton microscope design

    PubMed Central

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

    2016-01-01

    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. PMID:27182429

  14. RECENT PROGRESS IN MULTIFOCAL MULTIPHOTON MICROSCOPY

    PubMed Central

    LIU, LIXIN; SHAO, YONGHONG; NIU, HANBEN

    2013-01-01

    Multifocal multiphoton microscopy (MMM) has recently become an important tool in biomedicine for performing three-dimensional fast fluorescence imaging. Using various beamsplitting techniques, MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then records fluorescence signal from all foci simultaneously with an area array detector, which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light. In this paper, we discuss the features of several MMM setups using different beamsplitting devices, including a Nipkow spinning disk, a microlens array, a set of beamsplitting mirrors, or a diffractive optical element (DOE). In particular, we present our recent work on the development of an MMM using a spatial light modulator (SLM). PMID:24363782

  15. New developments in multimodal clinical multiphoton tomography

    NASA Astrophysics Data System (ADS)

    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.

  16. Repeatability of intravital capillaroscopic measurement of capillary density.

    PubMed

    Lamah, M; Chaudhry, H; Mortimer, P S; Dormandy, J A

    1996-01-01

    The reliability of intravital capillaroscopy for determining capillary density (CD) of skin has been questioned because it depends upon the variability of the measuring process and subjective interpretation of data as well as the intrinsic heterogeneity of capillary spacing. The aim of this study was to assess the repeatability of a standardised method for measuring CD of the skin of the dorsum of foot. In each of 30 subjects (10 controls and 20 patients with peripheral vascular disease), the foot was systematically mapped by examining 20 sites on the dorsum of foot and 2 sites on each toe, using white light (native) videomicroscopy at 40 x magnification. Off-line analysis of videoprints was then undertaken to determine CD at each site, by counting capillaries within areas of acceptable photographic quality only, having first defined the criteria for counting capillaries. The mean values were then calculated and taken to represent the CD of the foot or toes. Repeatability of the measuring equipment was first assessed by noting the presence or absence of each corresponding capillary in 2 prints, taken at intervals of hours or days (in 10 subjects) or months (in 2 patients), of an identical area of skin which was marked by a microtattoo on the first occasion. On average, 95% of corresponding capillaries were identified in both prints (from controls and patients), thus implying little intrinsic temporal variation of capillary anatomy as well as excellent repeatability of the measuring equipment. Repeatability of data analysis was assessed by the same observer reading the same 20 prints in a blinded manner on three separate occasions (intraobserver repeatability), and 2 observers reading the same 24 prints (interobserver repeatability). The mean coefficient of intraobserver variation of CD estimate was 5.6% and the interobserver correlation coefficient was 0.94. Finally, overall repeatability of the method was assessed by repeating the procedure on a subsequent

  17. Quantum cryptography with perfect multiphoton entanglement.

    PubMed

    Luo, Yuhui; Chan, Kam Tai

    2005-05-01

    Multiphoton entanglement in the same polarization has been shown theoretically to be obtainable by type-I spontaneous parametric downconversion (SPDC), which can generate bright pulses more easily than type-II SPDC. A new quantum cryptographic protocol utilizing polarization pairs with the detected type-I entangled multiphotons is proposed as quantum key distribution. We calculate the information capacity versus photon number corresponding to polarization after considering the transmission loss inside the optical fiber, the detector efficiency, and intercept-resend attacks at the level of channel error. The result compares favorably with all other schemes employing entanglement.

  18. Multiphoton polymerization using optical trap assisted nanopatterning

    NASA Astrophysics Data System (ADS)

    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.

  19. Studies on wide-field-of-view multiphoton imaging using the flexible clinical multiphoton tomograph MPTflex

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

    Multiphoton imaging systems are capable of high-resolution 3-D image acquisition of deep tissue. A first commercially available CE-certified biomedical system for subcelluar resolution of human skin has been launched by JenLab company with the DermaInspectR in 2002. The demand for more flexibility caused the development of the MPTflexR, which provides an increased flexibility and accessibility especially for clinical and cosmetic examinations. However the high resolution of clinical multiphoton tomographs are adherent with a small field-of-view (FOV) of about 360×360μm2. Especially time-consuming is the relocation of areas of interest (AOI) like lesions, sweat glands or hair shafts during a multiphoton examination. This limitation can be be overcome by macroscopic large-area (wide-field-ofview) multiphoton tomography, which is tested first within this work.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    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.

  2. Tumor Microvasculature and Microenvironment: Novel Insights Through Intravital Imaging in Pre-Clinical Models

    PubMed Central

    Fukumura, Dai; Duda, Dan G.; Munn, Lance L.; Jain, Rakesh K.

    2010-01-01

    Intravital imaging techniques have provided unprecedented insight into tumor microcirculation and microenvironment. For example, these techniques allowed quantitative evaluations of tumor blood vasculature to uncover its abnormal organization, structure and function (e.g., hyper-permeability, heterogeneous and compromised blood flow). Similarly, imaging of functional lymphatics has documented their absence inside tumors. These abnormalities result in elevated interstitial fluid pressure and hinder the delivery of therapeutic agents to tumors. In addition, they induce a hostile microenvironment characterized by hypoxia and acidosis, as documented by intravital imaging. The abnormal microenvironment further lowers the effectiveness of anti-tumor treatments such as radiation therapy and chemotherapy. In addition to these mechanistic insights, intravital imaging may also offer new opportunities to improve therapy. For example, tumor angiogenesis results in immature, dysfunctional vessels—primarily caused by an imbalance in production of pro- and anti-angiogenic factors by the tumors. Restoring the balance of pro- and anti-angiogenic signaling in tumors can “normalize” tumor vasculature and thus, improve its function, as demonstrated by intravital imaging studies in preclinical models and in cancer patients. Administration of cytotoxic therapy during periods of vascular normalization has the potential to enhance treatment efficacy. PMID:20374484

  3. Multiphoton Microscopy for Visualizing Lipids in Tissue.

    PubMed

    Lee, Martin; Serrels, Alan

    2016-01-01

    Visualizing the appearance of fat droplets and adipocytes in tissue can be realized using a label-free imaging method known as coherent anti-Stokes Raman spectroscopy (CARS). CARS is a nonlinear optical technique that allows label-free imaging of a material with contrast based on the same vibrational signatures of molecules found in Raman spectroscopy. CARS can be combined with other single and multiphoton imaging modes such as second harmonic generation and two-photon fluorescence to image a broad variety of biological structures.Here we describe the construction of a multiphoton microscope that will enable the study of both fluorescently labeled and unlabeled tissue. This has been used to monitor the contribution of Wt1 expressing cells towards the visceral fat depots during gestation. PMID:27417963

  4. Multiphoton tomography to detect chemo- and biohazards

    NASA Astrophysics Data System (ADS)

    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.

  5. Multiphoton tomography of intratissue tattoo nanoparticles

    NASA Astrophysics Data System (ADS)

    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.

  6. Assessing and benchmarking multiphoton microscopes for biologists.

    PubMed

    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.

  7. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling

    PubMed Central

    Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-01

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca2+). In the present study, we established conditions that allow the in vivo detection of Ca2+ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca2+ concentrations and, consequently, an increase in cell death in a p53-dependent pathway. PMID:25544762

  8. Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice.

    PubMed

    Herr, Nadine; Mauler, Maximilian; Bode, Christoph; Duerschmied, Daniel

    2015-01-01

    Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

  9. Elucidation of monocyte/macrophage dynamics and function by intravital imaging

    PubMed Central

    Rua, Rejane; McGavern, Dorian B.

    2015-01-01

    Monocytes and macrophages are a diverse population of innate immune cells that play a critical role in homeostasis and inflammation. These cells are surveillant by nature and closely monitor the vasculature and surrounding tissue during states of health and disease. Given their abundance and strategic positioning throughout the body, myeloid cells are among the first responders to any inflammatory challenge and are active participants in most immune-mediated diseases. Recent studies have shed new light on myeloid cell dynamics and function by use of an imaging technique referred to as intravital microscopy (IVM). This powerful approach allows researchers to gain real-time insights into monocytes and macrophages performing homeostatic and inflammatory tasks in living tissues. In this review, we will present a contemporary synopsis of how intravital microscopy has revolutionized our understanding of myeloid cell contributions to vascular maintenance, microbial defense, autoimmunity, tumorigenesis, and acute/chronic inflammatory diseases. PMID:26162402

  10. Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling.

    PubMed

    Giorgi, Carlotta; Bonora, Massimo; Missiroli, Sonia; Poletti, Federica; Ramirez, Fabian Galindo; Morciano, Giampaolo; Morganti, Claudia; Pandolfi, Pier Paolo; Mammano, Fabio; Pinton, Paolo

    2015-01-30

    One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca²⁺). In the present study, we established conditions that allow the in vivo detection of Ca²⁺ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca²⁺ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.

  11. Validation of a device for the active manipulation of the tumor microenvironment during intravital imaging

    PubMed Central

    Williams, James K.; Entenberg, David; Wang, Yarong; Avivar-Valderas, Alvaro; Padgen, Michael; Clark, Ashley; Aguirre-Ghiso, Julio A.; Castracane, James; Condeelis, John S.

    2016-01-01

    The tumor microenvironment is recognized as playing a significant role in the behavior of tumor cells and their progression to metastasis. However, tools to manipulate the tumor microenvironment directly, and image the consequences of this manipulation with single cell resolution in real time in vivo, are lacking. We describe here a method for the direct, local manipulation of microenvironmental parameters through the use of an implantable Induction Nano Intravital Device (iNANIVID) and simultaneous in vivo visualization of the results at single-cell resolution. As a proof of concept, we deliver both a sustained dose of EGF to tumor cells while intravital imaging their chemotactic response as well as locally induce hypoxia in defined microenvironments in solid tumors.

  12. First multiphoton tomography of brain in man

    NASA Astrophysics Data System (ADS)

    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. Studies of atmospheric molecules by multiphoton spectroscopy

    SciTech Connect

    Johnson, P.M.

    1991-10-01

    Carbon dioxide presents a great challenge to spectroscopy because of its propensity toward dissociation in all of its excited states. Multiphoton ionization spectroscopy is usually not applicable to the study of dissociating molecules because the dissociation competes effectively with ionization, resulting in no signal. We reasoned, however, that with high enough laser fluence, ionization could compete with dissociation in the longer lived states, exposing them for study from the continuous spectral background resulting from rapidly dissociating states. We describe the various spectroscopic and photophysical effects found through the multiphoton ionization and multiphoton photoelectron spectra. A recently developed variant of threshold ionization spectroscopy, usually called ZEKE, has shown a great deal of usefulness in providing the same information as traditional photoelectron spectroscopy but with higher resolution and much better signal-to-noise when using standard laboratory lasers. Threshold ionization techniques locate the states of an ion by scanning a light source across the ionization continuum of a neutral and somehow detecting when electrons are produced with no kinetic energy. We chose to develop our capabilities in threshold ionization spectroscopy using aromatic molecules because of their importance and because their electronic structure allows a pump-probe type of excitation scheme which avoids the use of vacuum ultraviolet laser beams. Among aromatics, the azines are noted for their small S{sub 1}-T{sub 1} energy gap which give them unique and interesting photophysical properties. We have continued our work on the multiphoton spectrum of metastable nitrogen produced by an electric discharge in supersonic beam. We have been able to assign more of the lines and simulated their rotational structure but many peaks remain unassigned.

  14. Quantitating therapeutic disruption of tumor blood flow with intravital video microscopy.

    PubMed

    Iga, Arthur M; Sarkar, Sandip; Sales, Kevin M; Winslet, Marc C; Seifalian, Alexander M

    2006-12-15

    Vascular-disrupting agents (VDA) kill tumor cells by selectively disrupting blood circulation in tumors. In vivo analysis of this intensely studied class of anticancer agents is invaluable for preclinical assessment of pharmacodynamic end points and effective therapeutic windows. In this review, we consider the role of intravital video microscopy in measuring tumor vascular response to VDAs, the potential of which lies in the opportunity to quantitate specific variables and to obtain real-time information on how VDAs affect tumor microcirculation.

  15. [Frontiers in Live Bone Imaging Researches. Novel drug discovery by means of intravital bone imaging technology].

    PubMed

    Ishii, Masaru

    2015-06-01

    Recent advances in intravital bone imaging technology has enabled us to grasp the real cellular behaviors and functions in vivo , revolutionizing the field of drug discovery for novel therapeutics against intractable bone diseases. In this chapter, I introduce various updated information on pharmacological actions of several antibone resorptive agents, which could only be derived from advanced imaging techniques, and also discuss the future perspectives of this new trend in drug discovery.

  16. Multiphoton harvesting metal–organic frameworks

    PubMed Central

    Quah, Hong Sheng; Chen, Weiqiang; Schreyer, Martin K.; Yang, Hui; Wong, Ming Wah; Ji, Wei; Vittal, Jagadese J.

    2015-01-01

    Multiphoton upconversion is a process where two or more photons are absorbed simultaneously to excite an electron to an excited state and, subsequently, the relaxation of electron gives rise to the emission of a photon with frequency greater than those of the absorbed photons. Materials possessing such property attracted attention due to applications in biological imaging, photodynamic therapy, three-dimensional optical data storage, frequency-upconverted lasing and optical power limiting. Here we report four-photon upconversion in metal–organic frameworks containing the ligand, trans, trans-9,10-bis(4-pyridylethenyl)anthracene. The ligand has a symmetrical acceptor–π–donor–π–acceptor structure and a singlet biradical electronic ground state, which boosted its multiphoton absorption cross-sections. We demonstrate that the upconversion efficiency can be enhanced by Förster resonance energy transfer within host–guest metal–organic frameworks consisting of encapsulated high quantum yielding guest molecules. Using these strategies, metal–organic framework materials, which can exhibit frequency-upconverted photoluminescence excited by simultaneous multiphoton absorption, can be rationally designed and synthesized. PMID:26245741

  17. Multiphoton harvesting metal-organic frameworks

    NASA Astrophysics Data System (ADS)

    Quah, Hong Sheng; Chen, Weiqiang; Schreyer, Martin K.; Yang, Hui; Wong, Ming Wah; Ji, Wei; Vittal, Jagadese J.

    2015-08-01

    Multiphoton upconversion is a process where two or more photons are absorbed simultaneously to excite an electron to an excited state and, subsequently, the relaxation of electron gives rise to the emission of a photon with frequency greater than those of the absorbed photons. Materials possessing such property attracted attention due to applications in biological imaging, photodynamic therapy, three-dimensional optical data storage, frequency-upconverted lasing and optical power limiting. Here we report four-photon upconversion in metal-organic frameworks containing the ligand, trans, trans-9,10-bis(4-pyridylethenyl)anthracene. The ligand has a symmetrical acceptor-π-donor-π-acceptor structure and a singlet biradical electronic ground state, which boosted its multiphoton absorption cross-sections. We demonstrate that the upconversion efficiency can be enhanced by Förster resonance energy transfer within host-guest metal-organic frameworks consisting of encapsulated high quantum yielding guest molecules. Using these strategies, metal-organic framework materials, which can exhibit frequency-upconverted photoluminescence excited by simultaneous multiphoton absorption, can be rationally designed and synthesized.

  18. Dynamic analysis of immune inflammation and bone destruction by intravital imaging.

    PubMed

    Kikuta, Junichi; Ishii, Masaru

    2016-01-01

      Rapid development of fluorescent imaging techniques enables us to understand cellular dynamics in vivo. We have originally established an advanced imaging system for visualizing living bone tissues with intravital two-photon microscopy. By means of the system, we have recently succeeded in visualization of the in vivo behavior of living mature osteoclasts on the bone surface, and identified different functional subsets of osteoclasts in terms of their motility and function, i.e., 'static - bone resorptive' and 'moving - non resorptive'. Pathological conditions changed the composition of these populations as well as the total number of mature osteoclasts. We also found that RANKL-bearing Th17 cells could control bone resorption of mature osteoclasts, demonstrating novel actions of Th17. Furthermore, we have also developed the imaging system to visualize bone destruction by osteoclasts in arthritic joints using intravital two-photon microscopy. In this review, we summarize the latest data of intravital imaging of osteoclast dynamics, and also discuss its further application. PMID:27212598

  19. Some simple mechanisms of multiphoton excitation in many - level systems

    NASA Astrophysics Data System (ADS)

    Donley, E. A.; Marquardt, R.; Quack, M.; Stohner, J.; Thanopulos, I.; Wallenborn, E.-U.

    Results are reported on coherent monochromatic multiphoton excitation in many-level systems, which are representative for some of the basic mechanisms for atomic and molecular multiphoton processes. Numerical solutions are discussed that use the Floquet and quasiresonant approximations in the framework of the URIMIR program package. The excitation schemes include direct three-photon excitation, two-photon excitation with diagonal coupling, Göppert-Mayer-type two-photon processes, multiphoton excitation with off-resonant intermediates, and practically irreversible coherent excitation into dense spectral structures. Several interesting phenomena are observed, such as nonlinear line shifts and broadenings of multiphoton resonances of relevance for multiphoton spectroscopy and almost constant intermediate population inversions, potentially useful for laser design. The accurate numerical results are compared with approximate solutions from perturbation theory, and with simple analytical solutions from Rabi-type formulae.

  20. Polymer microcantilevers fabricated via multiphoton absorption polymerization

    NASA Astrophysics Data System (ADS)

    Bayindir, Z.; Sun, Y.; Naughton, M. J.; LaFratta, C. N.; Baldacchini, T.; Fourkas, J. T.; Stewart, J.; Saleh, B. E. A.; Teich, M. C.

    2005-02-01

    We have used multiphoton absorption polymerization to fabricate a series of microscale polymer cantilevers. Atomic force microscopy has been used to characterize the mechanical properties of microcantilevers with spring constants that were found to span more than four decades. From these data, we extracted a Young's modulus of E =0.44GPa for these microscale cantilevers. The wide stiffness range and relatively low elastic modulus of the microstructures make them attractive candidates for a range of microcantilever applications, including measurements on soft matter.

  1. Characterization and prevention of phototoxic effects in intravital fluorescence microscopy in the hamster dorsal skinfold model.

    PubMed

    Steinbauer, M; Harris, A G; Abels, C; Messmer, K

    2000-07-01

    Intravital microscopy is widely used to study the microcirculation. However, the use of fluorescent dyes can induce phototoxic effects which may affect the measurements, particularly in tissue exposed to oxidative stress. The aim of the study was to determine the threshold light dose at which fluorescent microscopy is associated with phototoxic effects in the hamster dorsal skinfold chamber under normal and pathological conditions. The extent of phototoxicity in the microcirculation in the hamster skinfold chamber was investigated using intravital fluorescent microscopy during 60 min of illumination (1048 mW/cm2) applying two different concentrations of fluorescein isothiocyanate dextran under baseline conditions (groups A and B) and following 4 h of ischemia (groups C and D). In the second part of the study the microvasculature was analyzed regarding phototoxic effects during a standardized intravital microscopic examination after 4 h of pressure induced ischemia. Groups I and II (n=7) were studied using epiillumination after injection of fluorescein isothiocyanate dextran plus rhodamine 6G or rhodamine 6G only. In group III (n=7) only transillumination was used. Arteriolar vasospasm, microvascular perfusion failure, thrombus formation, and enhanced leukocyte endothelium interaction were observed as signs of a phototoxic effect in normal tissue. However, the light doses needed to induce these effects clearly exceeded those during standard examinations. The induction of a 4-h ischemia and reperfusion further enhanced these effects. Despite the predamage by ischemia/reperfusion the comparison of epiillumination and transillumination microscopy using a standard protocol showed no differences regarding the parameters analyzed at any time. This indicates that epiillumination and the fluorescent dyes per se did not affect the experimental results. These results show that ischemia/reperfusion studies in the dorsal skinfold chamber of the Syrian golden hamster can be

  2. The mouse dorsal skinfold chamber as a model for the study of thrombolysis by intravital microscopy.

    PubMed

    Boulaftali, Yacine; Lamrani, Lamia; Rouzaud, Marie-Catherine; Loyau, Stéphane; Jandrot-Perrus, Martine; Bouton, Marie-Christine; Ho-Tin-Noé, Benoît

    2012-05-01

    Although intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualised by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalisation. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalisation within 1 hour after treatment. Skin haemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1-/-) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi.

  3. Multiphoton ionization of large water clusters.

    PubMed

    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.

  4. Multiphoton ionization of large water clusters

    SciTech Connect

    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.

  5. Multimodal optoacoustic and multiphoton fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Sela, Gali; Razansky, Daniel; Shoham, Shy

    2013-03-01

    Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

  6. Multiphoton imaging with a nanosecond supercontinuum source

    NASA Astrophysics Data System (ADS)

    Lefort, Claire; O'Connor, Rodney P.; Blanquet, Véronique; Baraige, Fabienne; Tombelaine, Vincent; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

    2016-03-01

    Multiphoton microscopy is a well-established technique for biological imaging of several kinds of targets. It is classically based on multiphoton processes allowing two means of contrast simultaneously: two-photon fluorescence (TPF) and second harmonic generation (SHG). Today, the quasi exclusive laser technology used in that aim is femtosecond titanium sapphire (Ti: Sa) laser. We experimentally demonstrate that a nanosecond supercontinuum laser source (STM-250-VIS-IR-custom, Leukos, France; 1 ns, 600-2400 nm, 250 kHz, 1 W) allows to obtain the same kind of image quality in the case of both TPF and SHG, since it is properly filtered. The first set of images concerns the muscle of a mouse. It highlights the simultaneous detection of TPF and SHG. TPF is obtained thanks to the labelling of alpha-actinin with Alexa Fluor® 546 by immunochemistry. SHG is created from the non-centrosymmetric organization of myosin. As expected, discs of actin and myosin are superimposed alternatively. The resulting images are compared with those obtained from a standard femtosecond Ti: Sa source. The physical parameters of the supercontinuum are discussed. Finally, all the interest of using an ultra-broadband source is presented with images obtained in vivo on the brain of a mouse where tumor cells labeled with eGFP are grafted. Texas Red® conjugating Dextran is injected into the blood vessels network. Thus, two fluorophores having absorption wavelengths separated by 80 nm are imaged simultaneously with a single laser source.

  7. High-Resolution Molecular Imaging Via Intravital Microscopy: Illuminating Vascular Biology In Vivo

    PubMed Central

    Taqueti, Viviany R.; Jaffer, Farouc A.

    2012-01-01

    Complications of atherosclerosis and thrombosis are leading causes of death worldwide. While experimental investigations have yielded valuable insights into key molecular and cellular phenomena in these diseases of medium- and large-sized vessels, direct visualization of relevant in vivo biological processes has been limited. However, recent developments in molecular imaging technology, specifically fluorescence imaging agents coupled with high-resolution, high-speed intravital microscopy (IVM), are now enabling dynamic and longitudinal investigations into the mechanisms and progression of many vascular diseases. Here we review recent advances in IVM that have provided new in vivo biological insights into atherosclerosis and thrombosis. PMID:23135362

  8. Probing the role of the actin cytoskeleton during regulated exocytosis by intravital microscopy

    PubMed Central

    Milberg, Oleg; Tora, Muhibullah; Shitara, Akiko; Masedunskas, Andrius

    2015-01-01

    Summary The actin cytoskeleton plays a fundamental role in controlling several steps during regulated exocytosis. Here we describe a combination of procedures that are aimed at studying the dynamics and the mechanism of the actin cytoskeleton in the salivary glands of live rodents, a model for exocrine secretion. Our approach relies on intravital microscopy, an imaging technique that enables imaging biological events in live animals at a subcellular resolution, and it is complemented by the use of pharmacological agents and indirect immunofluorescence in the salivary tissue. PMID:24947398

  9. Intravital imaging technology reveals immune system dynamics in vivo.

    PubMed

    Ishii, Masaru

    2016-07-01

    Fluorescent 'intravital' imaging is a new research technique by which the interior of living tissues and organs (in living bodies, if possible) can be observed, revealing the kinetics of cell and molecular processes in real time. Recent technological innovations in optical equipment and fluorescence imaging techniques have enabled a variety of cellular phenomena in different tissues and organs to be characterized under completely native conditions. This shift from static to dynamic biology constitutes the beginning of a new era in biomedical sciences. PMID:27238377

  10. Intravital imaging of the effects of 5-fluorouracil on the murine liver microenvironment using 2-photon laser scanning microscopy

    PubMed Central

    OKIGAMI, MASATO; TANAKA, KOJI; INOUE, YASUHIRO; SAIGUSA, SUSUMU; OKUGAWA, YOSHINAGA; TOIYAMA, YUJI; MOHRI, YASUHIKO; KUSUNOKI, MASATO

    2016-01-01

    5-fluorouracil (5FU) is often used in the treatment of colorectal cancer. 5FU improves the median overall and disease-free survival rates and reduces recurrence rates in patients who have undergone curative surgical resection. However, in the adjuvant setting, whether 5FU eradicates clinically undetectable micrometastases in target organs such as the liver, or whether 5-FU inhibits the adhesion of circulating tumor cells has not yet been established. In the present study, 5FU was administered following the inoculation of red fluorescent protein-expressing HT29 cells into green fluorescent protein (GFP)-transgenic nude mice to examine its inhibitory effect. 2-photon laser scanning microscopy was performed at selected time points for time-series imaging of liver metastasis of GFP-transgenic mice. The cell number in vessels was quantified to evaluate the response of the tumor microenvironment to chemotherapy. HT29 cells were visualized in hepatic sinusoids at the single-cell level. A total of 2 hours after the injection (early stage), time-series imaging revealed that the number of caught tumor cells gradually reduced over time. In the 5FU treatment group, no significant difference was observed in the cell number in the early stage. One week after the injection (late stage), a difference in morphology was observed. The results of the present study indicated that 5FU eradicated clinically undetectable micrometastases in liver tissues by acting as a cytotoxic agent opposed to preventing adhesion. The present study indicated that time-series intravital 2-photon laser scanning microscopic imaging of metastatic tumor xenografts may facilitate the screening and evaluation of novel chemotherapeutic agents with less interindividual variability. PMID:27073493

  11. R-matrix Floquet theory of molecular multiphoton processes: II. Multiphoton ionization of H2

    NASA Astrophysics Data System (ADS)

    Colgan, J.; Glass, D. H.; Higgins, K.; Burke, P. G.

    2001-06-01

    Multiphoton ionization rates for H2 immersed in an intense linearly polarized laser field are calculated using the recently developed R-matrix Floquet theory of molecular multiphoton processes. We assume that the H2 molecule is aligned along the laser polarization direction and we adopt the fixed-nuclei approximation, in which the motion of the target electrons is calculated in the laser field and in the field of the nuclei, which are assumed to be fixed in space. An accurate multi-state wavefunction is employed to calculate one-, two- and four-photon ionization rates for H2 at several internuclear separations over a range of frequencies and intensities. Analysis of the ionization rates reveals the important role played both by resonances corresponding to Rydberg bound states converging to the H2+ ion ground state and by doubly excited states converging to the H2+ ion first excited state. These resonances give rise to resonant enhanced multiphoton ionization peaks in many of the ionization rates studied in this paper, and their possible role in controlling the vibrational population of the final H2+ ion is discussed.

  12. Assessment of microcirculatory effects of glycine by intravital microscopy in rats.

    PubMed

    Podoprigora, Guennady I; Blagosklonov, Oleg; Angoué, Orland; Boulahdour, Hatem; Nartsissov, Yaroslav R

    2012-01-01

    Experimental studies using laboratory animal models have shown a potential vasoactive effect of natural metabolites such as glycine. The present study used intravital microscopy in laboratory rat models to study the microcirculation in the brain pial and mesentery vessels. To investigate the pial microvasculature, a stereotaxis-like animal fixing device was used. The intravital microscopy unit consisted of a binocular microscope equipped with a digital photo-video camera, processor, monitor and printer. Using reflected light, a special contact lens with an amplified focus depth provided high-resolution images of nontransparent tissue objects that typically have insufficient light exposure. Glycine had a vasodilatory effect on microvessels in the rat brain and mesenterium. The diameter of pial arterioles increased after glycine application especially markedly (up to 250% of initial size). These changes were not observed when physiological saline was used. Even a very small amount of glycine (a drop on the needle) was sufficient to stop the early stages of histamine-induced blood stasis development in 3-5 s in mesenterial microvessels. The vasodilatory effect of glycine on the pial microcirculation correlates with its reported positive therapeutic effect in cerebral ischemic stroke. The ability of glycine to avoid or prevent histamine-induced microcirculatory alterations in mesenterial microvessels may have potential clinical applications. PMID:23366470

  13. Intravital lectin perfusion analysis of vascular permeability in human micro- and macro- blood vessels.

    PubMed

    Debbage, P L; Sölder, E; Seidl, S; Hutzler, P; Hugl, B; Ofner, D; Kreczy, A

    2001-10-01

    We previously applied intravital lectin perfusion in mouse models to elucidate mechanisms underlying vascular permeability. The present work transfers this technique to human models, analysing vascular permeability in macro- and microvessels. Human vascular endothelial surface carbohydrate biochemistry differs significantly from its murine counterpart, lacking alpha-galactosyl epitopes and expressing the L-fucose moiety in the glycocalyx; the poly-N-lactosamine glycan backbone is common to all mammals. We examined extensively lectin binding specificities in sections and in vivo, and then applied the poly-N-lactosamine-specific lectin LEA and the L-fucose-specific lectin UEA-I in human intravital perfusions. Transendothelial transport differed in macrovessels and microvessels. In microvessels of adult human fat tissue, rectal wall and rectal carcinomas, slow transendothelial transport by vesicles was followed by significant retention at the subendothelial basement membrane; paracellular passage was not observed. Passage time exceeded 1 h. Thus we found barrier mechanisms resembling those we described previously in murine tissues. In both adult and fetal macrovessels, the vena saphena magna and the umbilical vein, respectively, rapid passage across the endothelial lining was observed, the tracer localising completely in the subendothelial tissues within 15 min; vesicular transport was more rapid than in microvessels, and retention at the subendothelial basement membrane briefer.

  14. Fungal Infection in the Brain: What We Learned from Intravital Imaging.

    PubMed

    Shi, Meiqing; Mody, Christopher H

    2016-01-01

    Approximately 1.2 billion people suffer from fungal diseases worldwide. Arguably, the most serious manifestation occurs when pathogenic fungi infect the brain, often causing fatal meningoencephalitis. For most fungi, infection occurs via the vascular route. The organism must first be arrested in the brain microvasculature and transmigrate into the brain parenchyma across the blood-brain barrier. As a result, host immune cells are recruited into the brain to contain the fungi. However, it remains poorly understood how fungi traffic to, and migrate into the brain and how immune cells interact with invading fungi in the brain. A new era of intravital fluorescence microscopy has begun to provide insights. We are able to employ this powerful approach to study dynamic interactions of disseminating fungi with brain endothelial cells as well as resident and recruited immune cells during the brain infection. In this review, with a focus on Cryptococcus neoformans, we will provide an overview of the application of intravital imaging in fungal infections in the brain, discuss recent findings and speculate on possible future research directions. PMID:27532000

  15. Multicolor Fluorescent Intravital Live Microscopy (FILM) for Surgical Tumor Resection in a Mouse Xenograft Model

    PubMed Central

    Thurber, Greg M.; Figueiredo, Jose L.; Weissleder, Ralph

    2009-01-01

    Background Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM) where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time. Methodology/Principal Findings Theoretical simulations of imaging probe localization were carried out for three agents with specificity for cancer cells, stromal host response, or vascular perfusion. This transport analysis gave insight into the probe pharmacokinetics and tissue distribution, facilitating the experimental design and allowing predictions to be made about the localization of the probes in other animal models and in the clinic. The imaging probes were administered systemically at optimal time points based on the simulations, and the multicolor FILM images obtained in vivo were then compared to conventional pathological sections. Our data show the feasibility of real time in vivo pathology at cellular resolution and molecular specificity with excellent agreement between intravital and traditional in vitro immunohistochemistry. Conclusions/Significance Multicolor FILM is an accurate method for identifying malignant tissue and cells in vivo. The imaging probes distributed in a manner similar to predictions based on transport principles, and these models can be used to design future probes and experiments. FILM can provide critical real time feedback and should be a useful tool for more effective and complete cancer resection. PMID:19956597

  16. Intravital Imaging – Dynamic Insights into Natural Killer T Cell Biology

    PubMed Central

    Liew, Pei Xiong; Kubes, Paul

    2015-01-01

    Natural killer T (NKT) cells were first recognized more than two decades ago as a separate and distinct lymphocyte lineage that modulates an expansive range of immune responses. As innate immune cells, NKT cells are activated early during inflammation and infection, and can subsequently stimulate or suppress the ensuing immune response. As a result, researchers hope to harness the immunomodulatory properties of NKT cells to treat a variety of diseases. However, many questions still remain unanswered regarding the biology of NKT cells, including how these cells traffic from the thymus to peripheral organs and how they play such contrasting roles in different immune responses and diseases. In this new era of intravital fluorescence microscopy, we are now able to employ this powerful tool to provide quantitative and dynamic insights into NKT cell biology including cellular dynamics, patrolling, and immunoregulatory functions with exquisite resolution. This review will highlight and discuss recent studies that use intravital imaging to understand the spectrum of NKT cell behavior in a variety of animal models. PMID:26042123

  17. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Miller, Mark J.; Wei, Sindy H.; Cahalan, Michael D.; Parker, Ian

    2003-03-01

    The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naïve CD4+ T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of 11 μm·min1. T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 μm·min1. An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naïve T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.

  18. Intravital imaging of hair-cell development and regeneration in the zebrafish

    PubMed Central

    Pinto-Teixeira, Filipe; Muzzopappa, Mariana; Swoger, Jim; Mineo, Alessandro; Sharpe, James; López-Schier, Hernán

    2013-01-01

    Direct videomicroscopic visualization of organ formation and regeneration in toto is a powerful strategy to study cellular processes that often cannot be replicated in vitro. Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration. A general feature of this approach is its reliance on the optical isolation of defined cell types in the whole animals by transgenic expression of fluorescent markers. Here we describe a simple and robust method to analyze sensory hair-cell development and regeneration in the zebrafish lateral line by high-resolution intravital imaging using laser-scanning confocal microscopy (LSCM) and selective plane illumination microscopy (SPIM). The main advantage of studying hair-cell regeneration in the lateral line is that it occurs throughout the life of the animal, which allows its study in the most natural context. We detail protocols to achieve continuous videomicroscopy for up to 68 hours, enabling direct observation of cellular behavior, which can provide a sensitive assay for the quantitative classification of cellular phenotypes and cell-lineage reconstruction. Modifications to this protocol should facilitate pharmacogenetic assays to identify or validate otoprotective or reparative drugs for future clinical strategies aimed at preserving aural function in humans. PMID:24130521

  19. Fungal Infection in the Brain: What We Learned from Intravital Imaging

    PubMed Central

    Shi, Meiqing; Mody, Christopher H.

    2016-01-01

    Approximately 1.2 billion people suffer from fungal diseases worldwide. Arguably, the most serious manifestation occurs when pathogenic fungi infect the brain, often causing fatal meningoencephalitis. For most fungi, infection occurs via the vascular route. The organism must first be arrested in the brain microvasculature and transmigrate into the brain parenchyma across the blood–brain barrier. As a result, host immune cells are recruited into the brain to contain the fungi. However, it remains poorly understood how fungi traffic to, and migrate into the brain and how immune cells interact with invading fungi in the brain. A new era of intravital fluorescence microscopy has begun to provide insights. We are able to employ this powerful approach to study dynamic interactions of disseminating fungi with brain endothelial cells as well as resident and recruited immune cells during the brain infection. In this review, with a focus on Cryptococcus neoformans, we will provide an overview of the application of intravital imaging in fungal infections in the brain, discuss recent findings and speculate on possible future research directions. PMID:27532000

  20. Intravital microscopy: a novel tool to study cell biology in living animals

    PubMed Central

    Weigert, Roberto; Sramkova, Monika; Parente, Laura; Masedunskas, Andrius

    2011-01-01

    Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criteria. Indeed, first we will focus on those studies in which organs where imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures. PMID:20372919

  1. Imaging Circulating Tumor Cells in Freely Moving Awake Small Animals Using a Miniaturized Intravital Microscope

    PubMed Central

    Sasportas, Laura Sarah; Gambhir, Sanjiv Sam

    2014-01-01

    Metastasis, the cause for 90% of cancer mortality, is a complex and poorly understood process involving the invasion of circulating tumor cells (CTCs) into blood vessels. These cells have potential prognostic value as biomarkers for early metastatic risk. But their rarity and the lack of specificity and sensitivity in measuring them render their interrogation by current techniques very challenging. How and when these cells are circulating in the blood, on their way to potentially give rise to metastasis, is a question that remains largely unanswered. In order to provide an insight into this "black box" using non-invasive imaging, we developed a novel miniature intravital microscopy (mIVM) strategy capable of real-time long-term monitoring of CTCs in awake small animals. We established an experimental 4T1-GL mouse model of metastatic breast cancer, in which tumor cells express both fluorescent and bioluminescent reporter genes to enable both single cell and whole body tumor imaging. Using mIVM, we monitored blood vessels of different diameters in awake mice in an experimental model of metastasis. Using an in-house software algorithm we developed, we demonstrated in vivo CTC enumeration and computation of CTC trajectory and speed. These data represent the first reported use we know of for a miniature mountable intravital microscopy setup for in vivo imaging of CTCs in awake animals. PMID:24497977

  2. Multiphoton imaging of biological samples during freezing and heating

    NASA Astrophysics Data System (ADS)

    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.

  3. Widefield multiphoton microscopy with image-based adaptive optics

    NASA Astrophysics Data System (ADS)

    Chang, C.-Y.; Cheng, L.-C.; Su, H.-W.; Yen, W.-C.; Chen, S.-J.

    2012-10-01

    Unlike conventional multiphoton microscopy according to pixel by pixel point scanning, a widefield multiphoton microscope based on spatiotemporal focusing has been developed to provide fast optical sectioning images at a frame rate over 100 Hz. In order to overcome the aberrations of the widefield multiphoton microscope and the wavefront distortion from turbid biospecimens, an image-based adaptive optics system (AOS) was integrated. The feedback control signal of the AOS was acquired according to locally maximize image intensity, which were provided by the widefield multiphoton excited microscope, by using a hill climbing algorithm. Then, the control signal was utilized to drive a deformable mirror in such a way as to eliminate the aberration and distortion. A R6G-doped PMMA thin film is also increased by 3.7-fold. Furthermore, the TPEF image quality of 1 μm fluorescent beads sealed in agarose gel at different depths is improved.

  4. Single and Multiphoton Fluorescence Recovery after Photobleaching

    PubMed Central

    Sullivan, Kelley D.; Majewska, Ania K.

    2015-01-01

    Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules, and can be applied both in vitro and in vivo for two-and three-dimensional systems. This chapter discusses the three basic FRAP methods: traditional FRAP, multi-photon FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the appropriate mathematical analysis appropriate for situations in which the recovery kinetics are dictated by free diffusion. In some experiments, the recovery kinetics are dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Since the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section. PMID:25561627

  5. Multiphoton microscopy of cleared mouse organs

    NASA Astrophysics Data System (ADS)

    Parra, Sonia G.; Chia, Thomas H.; Zinter, Joseph P.; Levene, Michael J.

    2010-05-01

    Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 μm in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4096×4096 pixels.

  6. Multi-photon entanglement in high dimensions

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

    Forming the backbone of quantum technologies today, entanglement has been demonstrated in physical systems as diverse as photons, ions and superconducting circuits. Although steadily pushing the boundary of the number of particles entangled, these experiments have remained 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 dimensions are greater than two. Two photons in our state reside in a three-dimensional space, whereas the third lives in two dimensions. This asymmetric entanglement structure only appears in multiparticle entangled states with d > 2. Our method relies on combining two pairs of photons, high-dimensionally entangled in their orbital angular momentum. In addition, we show how this state enables a new type of ‘layered’ quantum communication protocol. Entangled states such as these serve as a manifestation of the complex dance of correlations that can exist within quantum mechanics.

  7. Multiphoton imaging with high peak power VECSELs

    NASA Astrophysics Data System (ADS)

    Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

    2016-03-01

    Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

  8. Point spread function engineering with multiphoton SPIFI

    NASA Astrophysics Data System (ADS)

    Wernsing, Keith A.; Field, Jeffrey J.; Domingue, Scott R.; Allende-Motz, Alyssa M.; DeLuca, Keith F.; Levi, Dean H.; DeLuca, Jennifer G.; Young, Michael D.; Squier, Jeff A.; Bartels, Randy A.

    2016-03-01

    MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes -- namely, second harmonic generation and two-photon fluorescence, respectively.1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.

  9. Rotational averaging of multiphoton absorption cross sections

    SciTech Connect

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

    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.

  10. Evaluation of Barrett Esophagus by Multiphoton Microscopy

    PubMed Central

    Chen, Jianxin; Wong, Serena; Nathanson, Michael H.; Jain, Dhanpat

    2014-01-01

    Context Multiphoton microscopy (MPM) based on 2-photon excitation fluorescence and second-harmonic generation allows simultaneous visualization of cellular details and extracellular matrix components of fresh, unfixed, and unstained tissue. Portable multiphoton microscopes, which could be placed in endoscopy suites, and multiphoton endomicroscopes are in development, but their clinical utility is unknown. Objectives To examine fresh, unfixed endoscopic biopsies obtained from the distal esophagus and gastroesophageal junction to (1) define the MPM characteristics of normal esophageal squamous mucosa and gastric columnar mucosa, and (2) evaluate whether diagnosis of intestinal metaplasia/Barrett esophagus (BE) could be made reliably with MPM. Design The study examined 35 untreated, fresh biopsy specimens from 25 patients who underwent routine upper endoscopy. A Zeiss LSM 710 Duo microscope (Carl Zeiss, Thornwood, New York) coupled to a Spectra-Physics (Mountain View, California) Tsunami Ti:sapphire laser was used to obtain a MPM image within 4 hours of fresh specimen collection. After obtaining MPM images, the biopsy specimens were placed in 10% buffered formalin and submitted for routine histopathologic examination. Then, the MPM images were compared with the findings in the hematoxylin-eosin–stained, formalin-fixed, paraffin-embedded sections. The MPM characteristics of the squamous, gastric-type columnar and intestinal-type columnar epithelium were analyzed. In biopsies with discrepancy between MPM imaging and hematoxylin-eosin–stained sections, the entire tissue block was serially sectioned and reevaluated. A diagnosis of BE was made when endoscopic and histologic criteria were satisfied. Results Based on effective 2-photon excitation fluorescence of cellular reduced pyridine nucleotides and flavin adenine dinucleotide and lack of 2-photon excitation fluorescence of mucin and cellular nuclei, MPM could readily identify and distinguish among squamous

  11. Multiphoton double ionization of the He atom

    NASA Astrophysics Data System (ADS)

    Li, Y.; Pindzola, M. S.

    2016-05-01

    Time-dependent close-coupling (TDCC) calculations are made for the multiphoton double ionization of the He atom under the influence of a fast pulse XUV laser. One set of TDCC calculations employs l1m1l2m2 coupling on a 2D (r1 ,r2) numerical lattice, a second set of TDCC calculations employs m1m2 coupling on a 4D (r1 ,θ1 ,r2 ,θ2) numerical lattice, and a third set of TDCC calculations employs m1m2 coupling on a 4D (ρ1 ,z1 ,ρ2 ,z2) numerical lattice. Studies are made to see which TDCC method is the most efficient at explaining measurements as the number of photons absorbed is increased. Work supported in part by Grants from NASA, NSF, and DOE.

  12. Generation of High-Order Squeezing in Multiphoton Micromaser

    NASA Technical Reports Server (NTRS)

    Li, Fu-Li; Huang, Qing

    1996-01-01

    The generation of steady state higher-order squeezing in the sense of Hong and Mandel and also of Hillery in a multiphoton micromaser is studied. The results show that the cotangent state which is generated by the coherent trapping scheme in a multiphoton micromaser can exhibit not only second-order squeezing but also fourth-order and squared field amplitude squeezings. The influence of the cavity loss on the squeezings is investigated.

  13. The Multiphoton Multiple Ionization of Molecules.

    NASA Astrophysics Data System (ADS)

    Hatherly, P. A.

    Available from UMI in association with The British Library. The multiphoton multiple ionization of a number of molecular systems has been studied using the picosecond laser facility at the Rutherford Appleton Laboratory. The laser produced 0.6ps pulses at 600nm, and 6ps at 248nm when used in conjunction with an excimer laser. The focused intensity in each case was >=q10 ^{15}W/cm^2 . A time of flight mass spectrometer designed and built at Reading University was capable of ion kinetic energy measurement, permitting the molecular dissociation dynamics to be investigated. One major question approached concerned the mode of multiphoton ionization of xenon. Specifically, does the ionization proceed in a sequential (single electron) or a collective (many electron) manner? To this end, experiments were performed with the isoelectronic molecule hydrogen iodide. The results, which were interpreted in terms of a Coulomb explosion mechanism, demonstrated the process to be sequential, rather than collective. Similar experiments on the isoelectronic pair, nitrogen and carbon monoxide tended to confirm this conclusion. These molecules were also studied at a number of laser wavelengths and pulse widths. Although the wavelength was found to have a minimal effect, the pulse width was of great importance. The results for hydrogen and deuterium contrasted with these results for other molecules, in that the energies could not be reconciled with a Coulomb explosion mechanism. Rather, dissociative autoionization or neutral dissociation followed by ionization of the atoms were considered to be the dominant processes. Finally, the existence of high energy protons (~eq100eV) from residual hydrocarbons in the vacuum chamber lead to a study of the alkanes from butane to dodecane. At 600nm, 0.6ps pulse width, the fragment energies were found to vary linearly with carbon chain length. At 248nm, 5ps though, only low energy protons were observed, independent of chain length.

  14. Studies of atmospheric molecules by multiphoton spectroscopy

    NASA Astrophysics Data System (ADS)

    Johnson, P. M.

    1990-12-01

    Resonance ionization processes can play an important role in understanding molecules important in combustion processes. They are a reflection of the dynamic as well as the static properties of atomic and molecular species. Due to the sequential or quasisequential nature of photon absorption in resonant multiphoton events, the lifetimes of the intermediate states play an essential role in the overall cross-sections if they are short enough to be competitive with subsequent photon interactions. In molecules, this is particularly important because there are many dissociative and other radiationless pathways which can contribute to a competitive channel. Under those conditions it should be possible to obtain information about the nature of the dynamics of the intermediate state from the multiphoton ionization process. This will involve looking at not only the ionization cross-section but also other observables such as the kinetic energy of the ejected electrons and possibly the distribution of fragment ions produced in the ionization event. Whether the ionization amplitude is affected or not, the time scales of the dynamic events which alter the ionization path can vary over a large range from the femtoseconds of dissociation to the microseconds of some radiationless transitions in large molecules. When the competing channel has a time scale shorter than the laser pulse length, the kinetics of the ionization are intimately tied into the precise nature of the laser pulse. For time scales longer than the laser pulse, pump-probe ionization schemes in which one laser prepares a state while another does the ionization provide a particularly simple method for investigating the dynamics of the intermediate state. Here the author discusses examples from each of these regimes. CO2 and pyrazine are examined.

  15. Combined application of dynamic light scattering imaging and fluorescence intravital microscopy in vascular biology

    NASA Astrophysics Data System (ADS)

    Kalchenko, V.; Ziv, K.; Addadi, Y.; Madar-Balakirski, N.; Meglinski, I.; Neeman, M.; Harmelin, A.

    2010-08-01

    The dynamic light scattering imaging (DLSI) system combined with the conventional fluorescence intravital microscope (FIM) has been applied for the examination of blood and lymph vessels in the mouse ear in vivo. While the CCD camera can be shared by both techniques the combined application of DLSI and FIM allows rapid switching between the modalities. In current study temporal speckles fluctuations are used for rendering blood vessels structure and monitoring blood perfusion with the higher spatial resolution, whereas FIM provides the images of lymphatic vessels. The results clearly demonstrate that combined application of DLSI and FIM approaches provides synchronic in vivo images of blood and lymph vessels with higher contrast and specificity. The use of this new dual-modal diagnostic system is particularly important and has a great potential to significantly expand the capabilities of vascular diagnostics providing synchronic in vivo images of blood and lymph vessels.

  16. Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

    PubMed Central

    Evans, Teresa A.; Barkauskas, Deborah S.; Myers, Jay T.; Huang, Alex Y.

    2014-01-01

    Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury. PMID:25489963

  17. Integrated intravital microscopy and mathematical modeling to optimize nanotherapeutics delivery to tumors

    NASA Astrophysics Data System (ADS)

    van de Ven, Anne L.; Wu, Min; Lowengrub, John; McDougall, Steven R.; Chaplain, Mark A. J.; Cristini, Vittorio; Ferrari, Mauro; Frieboes, Hermann B.

    2012-03-01

    Inefficient vascularization hinders the optimal transport of cell nutrients, oxygen, and drugs to cancer cells in solid tumors. Gradients of these substances maintain a heterogeneous cell-scale microenvironment through which drugs and their carriers must travel, significantly limiting optimal drug exposure. In this study, we integrate intravital microscopy with a mathematical model of cancer to evaluate the behavior of nanoparticle-based drug delivery systems designed to circumvent biophysical barriers. We simulate the effect of doxorubicin delivered via porous 1000 x 400 nm plateloid silicon particles to a solid tumor characterized by a realistic vasculature, and vary the parameters to determine how much drug per particle and how many particles need to be released within the vasculature in order to achieve remission of the tumor. We envision that this work will contribute to the development of quantitative measures of nanoparticle design and drug loading in order to optimize cancer treatment via nanotherapeutics.

  18. Intravital imaging of multicolor-labeled tumor immune microenvironment through skin-fold window chamber

    NASA Astrophysics Data System (ADS)

    Qi, Shuhong; Zhang, Zhihong

    2015-03-01

    Tumor immune microenvironment became very important for the tumor immunotherapy. There were several kinds of immune cells in tumor stromal, and they played very different roles in tumor growth. In order to observe the behaviors of multiple immune cells in tumor microenvironment and the interaction between immune cells and tumor cells at the same time, we generated a multicolor-labeled tumor immune microenvironment model. The tumor cells and immune cells were labeled by different fluorescent proteins. By using of skin-fold window chamber implanted into mice and intravital imaging technology, we could dynamically observe the different immune cells in tumor microenvironment. After data analysis from the video, we could know the behavior of TILs, DCs and Tregs in tumor immune microenvironment; furthermore, we could know these immune cells play different roles in the tumor microenvironment.

  19. Impact of rapamycin on phenotype and tolerogenic function of dendritic cells via intravital optical imaging

    NASA Astrophysics Data System (ADS)

    Luo, Meijie; Zhang, Zhihong

    2014-03-01

    Rapamycin (RAPA) as a unique tolerance-promoting therapeutic drug is crucial to successful clinical organ transplantation. DC (Dendritic cells) play a critical role in antigen presentation to T cells to initiate immune responses involved in tissue rejection. Although the influence of RAPA on DC differentiation and maturation had been reported by some research groups, it is still controversial and unclear right now. In addition, it is also lack of study on investigating the role of DC in DTH reaction via intravital optical imaging. Herein, we investigated the effect of rapamycin on phenotype and function of bone marrow monocyte-derived DC both in vitro and in vivo. In vitro experiments by flow cytometry (FACS) showed that DC displayed decreased cell size and lower expression levels of surface molecule CD80 induced by RAPA; Furthermore, the phagocytic ability to OVA of DC was inhibited by RAPA started from 1 h to 2 h post co-incubation, but recovered after 4 h; In addition, the capacity of DC to activate naïve OT-II T cell proliferation was also inhibited at 3 day post co-incubation, but had no effect at 5 day, the data indicated this effect was reversible when removing the drug. More importantly, the DC-T interaction was monitored both in vitro and in intravital lymph node explant, and showed that RAPA-DC had a significant lower proportion of long-lived (>15min) contacts. Thus, RAPA displayed immunosuppressive to phenotypic and functional maturation of DC, and this phenomenon induced by RAPA may favorable in the clinical organ transplantation in future.

  20. X-ray intravital microscopy for functional imaging in rat hearts using synchrotron radiation coronary microangiography

    SciTech Connect

    Umetani, K.; Fukushima, K.

    2013-03-15

    An X-ray intravital microscopy technique was developed to enable in vivo visualization of the coronary, cerebral, and pulmonary arteries in rats without exposure of organs and with spatial resolution in the micrometer range and temporal resolution in the millisecond range. We have refined the system continually in terms of the spatial resolution and exposure time. X-rays transmitted through an object are detected by an X-ray direct-conversion type detector, which incorporates an X-ray SATICON pickup tube. The spatial resolution has been improved to 6 {mu}m, yielding sharp images of small arteries. The exposure time has been shortened to around 2 ms using a new rotating-disk X-ray shutter, enabling imaging of beating rat hearts. Quantitative evaluations of the X-ray intravital microscopy technique were extracted from measurements of the smallest-detectable vessel size and detection of the vessel function. The smallest-diameter vessel viewed for measurements is determined primarily by the concentration of iodinated contrast material. The iodine concentration depends on the injection technique. We used ex vivo rat hearts under Langendorff perfusion for accurate evaluation. After the contrast agent is injected into the origin of the aorta in an isolated perfused rat heart, the contrast agent is delivered directly into the coronary arteries with minimum dilution. The vascular internal diameter response of coronary arterial circulation is analyzed to evaluate the vessel function. Small blood vessels of more than about 50 {mu}m diameters were visualized clearly at heart rates of around 300 beats/min. Vasodilation compared to the control was observed quantitatively using drug manipulation. Furthermore, the apparent increase in the number of small vessels with diameters of less than about 50 {mu}m was observed after the vasoactive agents increased the diameters of invisible small blood vessels to visible sizes. This technique is expected to offer the potential for direct

  1. Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations

    PubMed Central

    Lohela, Marja; Casbon, Amy-Jo; Olow, Aleksandra; Bonham, Lynn; Branstetter, Daniel; Weng, Ning; Smith, Jeffrey; Werb, Zena

    2014-01-01

    Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R–dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti–CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1+ neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment. PMID:25385645

  2. In vivo evaluation of venular glycocalyx during hemorrhagic shock in rats using intravital microscopy.

    PubMed

    Torres Filho, Ivo; Torres, Luciana N; Sondeen, Jill L; Polykratis, I Amy; Dubick, Michael A

    2013-01-01

    Hemorrhage is responsible for a large percentage of trauma-related deaths but the mechanisms underlying tissue ischemia are complex and not well understood. Despite the evidence linking glycocalyx degradation and hemorrhagic shock, there is no direct data obtained in vivo showing glycocalyx thickness reduction in skeletal muscle venules after hemorrhage. We hypothesize that damage to the endothelial glycocalyx is a key element in hemorrhage pathophysiology and tested the hypothesis that hemorrhage causes glycocalyx degradation in cremaster muscle microvessels. We utilized intravital microscopy to estimate glycocalyx thickness in 48 microvessels while other microvascular parameters were measured using non-invasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. We studied 27 post-capillary venules (<16 μm diameter) of 8 anesthetized rats subjected to hemorrhage (40% of total blood volume). Six control rats were equally instrumented but not bled. Dextrans of different molecular weights labeled with FITC or Texas Red were injected. Glycocalyx thickness was estimated from the widths of the fluorescence columns and from anatomical diameter. While control rats did not show remarkable responses, a statistically significant decrease of about 59% in glycocalyx thickness was measured in venules after hemorrhagic shock. Venular glycocalyx thickness and local blood flow changes were correlated: venules with the greatest flow reductions showed the largest decreases in glycocalyx. These changes may have a significant impact in shock pathophysiology. Intravital microscopy and integrated systems such as the one described here may be important tools to identify mechanisms by which resuscitation fluids may improve tissue recovery and outcome following hemorrhage.

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

    PubMed

    Soulet, Denis; Paré, Alexandre; Coste, Julien; Lacroix, Steve

    2013-01-01

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

  4. X-ray intravital microscopy for functional imaging in rat hearts using synchrotron radiation coronary microangiography

    NASA Astrophysics Data System (ADS)

    Umetani, K.; Fukushima, K.

    2013-03-01

    An X-ray intravital microscopy technique was developed to enable in vivo visualization of the coronary, cerebral, and pulmonary arteries in rats without exposure of organs and with spatial resolution in the micrometer range and temporal resolution in the millisecond range. We have refined the system continually in terms of the spatial resolution and exposure time. X-rays transmitted through an object are detected by an X-ray direct-conversion type detector, which incorporates an X-ray SATICON pickup tube. The spatial resolution has been improved to 6 μm, yielding sharp images of small arteries. The exposure time has been shortened to around 2 ms using a new rotating-disk X-ray shutter, enabling imaging of beating rat hearts. Quantitative evaluations of the X-ray intravital microscopy technique were extracted from measurements of the smallest-detectable vessel size and detection of the vessel function. The smallest-diameter vessel viewed for measurements is determined primarily by the concentration of iodinated contrast material. The iodine concentration depends on the injection technique. We used ex vivo rat hearts under Langendorff perfusion for accurate evaluation. After the contrast agent is injected into the origin of the aorta in an isolated perfused rat heart, the contrast agent is delivered directly into the coronary arteries with minimum dilution. The vascular internal diameter response of coronary arterial circulation is analyzed to evaluate the vessel function. Small blood vessels of more than about 50 μm diameters were visualized clearly at heart rates of around 300 beats/min. Vasodilation compared to the control was observed quantitatively using drug manipulation. Furthermore, the apparent increase in the number of small vessels with diameters of less than about 50 μm was observed after the vasoactive agents increased the diameters of invisible small blood vessels to visible sizes. This technique is expected to offer the potential for direct

  5. High-resolution multimodal clinical multiphoton tomography of skin

    NASA Astrophysics Data System (ADS)

    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.

  6. The multiphoton ionization of uranium hexafluoride

    SciTech Connect

    Armstrong, D.P. . UEO Enrichment Technical Operations Div.)

    1992-05-01

    Multiphoton ionization (MPI) time-of-flight mass spectroscopy and photoelectron spectroscopy studies of UF{sub 6} have been conducted using focused light from the Nd:YAG laser fundamental ({lambda}=1064 nm) and its harmonics ({lambda}=532, 355, or 266 nm), as well as other wavelengths provided by a tunable dye laser. The MPI mass spectra are dominated by the singly and multiply charged uranium ions rather than by the UF{sub x}{sup +} fragment ions even at the lowest laser power densities at which signal could be detected. The laser power dependence of U{sup n+} ions signals indicates that saturation can occur for many of the steps required for their ionization. In general, the doubly-charged uranium ion (U{sup 2+}) intensity is much greater than that of the singly-charged uranium ion (U{sup +}). For the case of the tunable dye laser experiments, the U{sup n+} (n = 1- 4) wavelength dependence is relatively unstructured and does not show observable resonance enhancement at known atomic uranium excitation wavelengths. The dominance of the U{sup 2+} ion and the absence or very small intensities of UF{sub x}{sup +} fragments, along with the unsaturated wavelength dependence, indicate that mechanisms may exist other than ionization of bare U atoms after the stepwise photodissociation of F atoms from the parent molecule.

  7. Unconditionally secure key distillation from multiphotons

    SciTech Connect

    Tamaki, Kiyoshi; Lo, Hoi-Kwong

    2006-01-15

    In this paper, we prove that the unconditionally secure key can be surprisingly extracted from multiphoton emission part in the photon polarization-based quantum key distribution. One example is shown by explicitly proving that one can indeed generate an unconditionally secure key from Alice's two-photon emission part proposed by Scarani [et al. Phys. Rev. Lett. 92, 057901 (2004)]. Which is called the Scarani-Acin-Ribordy-Gisin (SARG04) protocol. This protocol uses the same four states as in Bennett-Brassard 1984 (BB84) and differs only in the classical postprocessing protocol. It is, thus, interesting to see how the classical postprocessing of quantum key distribution might qualitatively change its security. We also show that one can generate an unconditionally secure key from the single to the four-photon part in a generalized SARG04 protocol that uses six states. Finally, we also compare the bit error rate threshold of these protocols with the one in the BB84 protocol and the original six-state protocol assuming a depolarizing channel.

  8. Infrared Multiphoton Dissociation for Quantitative Shotgun Proteomics

    PubMed Central

    Ledvina, Aaron R.; Lee, M. Violet; McAlister, Graeme C.; Westphall, Michael S.; Coon, Joshua J.

    2012-01-01

    We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low pressure trap of a dual-cell quadrupole linear ion trap (dual cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly outperforms resonant excitation CAD for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT RF amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass-to-charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides. PMID:22480380

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

    PubMed

    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.

  10. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

    NASA Astrophysics Data System (ADS)

    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.

  11. High-intensity laser heating in liquids: Multiphoton absorption

    SciTech Connect

    Longtin, J.P.; Tien, C.L.

    1995-12-31

    At high laser intensities, otherwise transparent liquids can absorb strongly by the mechanism of multiphoton absorption, resulting in absorption and heating several orders of magnitude greater than classical, low-intensity mechanisms. The use of multiphoton absorption provides a new mechanism for strong, controlled energy deposition in liquids without bulk plasma formation, shock waves, liquid ejection, etc., which is of interest for many laser-liquid applications, including laser desorption of liquid films, laser particle removal, and laser water removal from microdevices. This work develops a microscopically based model of the heating during multiphoton absorption in liquids. The dependence on pulse duration, intensity, wavelength, repetition rate, and liquid properties is discussed. Pure water exposed to 266 nm laser radiation is investigated, and a novel heating mechanism for water is proposed that uses multiple-wavelength laser pulses.

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

    PubMed

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

    2016-01-01

    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. PMID:27599635

  13. Post conductive keratoplasty visualization of rabbit cornea by multiphoton microscopy

    NASA Astrophysics Data System (ADS)

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

    2007-07-01

    Conductive keratoplasty (CK) is a new refractive surgery for presbyopia and hyperopia patients. By applying radio frequency current at the peripheral regions of cornea, collagen, the most abundant composition of corneal stroma, shrinks due to the heat generated. The shrinkage at the periphery alters the corneal architecture and achieves clearer focus for near vision. In this work we use multiphoton microscopy to observe the post surgery structure variation at both submicron resolution and over a large region within the tissue. Since collagen can be induced to generate strong second harmonic generation (SHG) signal, multiphoton excitation provide direct visualization of collagen orientation within corneal stroma. In addition, since the SHG intensity of collagen tissue deteriorates with increasing thermal damage [1-3], our methodology can be used to characterize the extent of corneal stroma damage from the CK procedure. Finally, the influence of CK on the morphology and distribution of keratocytes can also be investigated by detecting multiphoton excited autofluorescence from the cells.

  14. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

    PubMed Central

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

    2016-01-01

    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. PMID:27599635

  15. Evaluation of multiphoton effects in down-conversion

    SciTech Connect

    Yoshimi, Kazuyoshi; Koshino, Kazuki

    2010-04-15

    Multiphoton effects in down-conversion are investigated based on the full-quantum multimode formalism by considering a three-level system as a prototype nonlinear system. We analytically derive the three-photon output wave function for two input photons, where one of the two input photons is down-converted and the other one is not. Using this output wave function, we calculate the down-conversion probability, the purity, and the fidelity to evaluate the entanglement between a down-converted photon pair and a non-down-converted photon. It is shown that the saturation effect occurs by multiphoton input and that it affects both the down-conversion probability and the quantum correlation between the down-converted photon pair and the non-down-converted photon. We also reveal the necessary conditions for multiphoton effects to be strong.

  16. Multiphoton absorption is probably not the primary threshold damage mechanism for femtosecond laser pulse exposures in the retinal pigment epithelium

    NASA Astrophysics Data System (ADS)

    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.

  17. Fibre-coupled multiphoton microscope with adaptive motion compensation.

    PubMed

    Sherlock, Ben; Warren, Sean; Stone, James; Neil, Mark; Paterson, Carl; Knight, Jonathan; French, Paul; Dunsby, Chris

    2015-05-01

    To address the challenge of sample motion during in vivo imaging, we present a fibre-coupled multiphoton microscope with active axial motion compensation. The position of the sample surface is measured using optical coherence tomography and fed back to a piezo actuator that adjusts the axial location of the objective to compensate for sample motion. We characterise the system's performance and demonstrate that it can compensate for axial sample velocities up to 700 µm/s. Finally we illustrate the impact of motion compensation when imaging multiphoton excited autofluorescence in ex vivo mouse skin.

  18. Effect of multiphoton ionization on performance of crystalline lens.

    PubMed

    Gupta, Pradeep Kumar; Singh, Ram Kishor; Strickland, D; Campbell, M C W; Sharma, R P

    2014-12-15

    This Letter presents a model for propagation of a laser pulse in a human crystalline lens. The model contains a transverse beam diffraction effect, laser-induced optical breakdown for the creation of plasma via a multiphoton ionization process, and the gradient index (GRIN) structure. Plasma introduces the nonlinearity in the crystalline lens which affects the propagation of the beam. The multiphoton ionization process generates plasma that changes the refractive index and hence leads to the defocusing of the laser beam. The Letter also points out the relevance of the present investigation to cavitation bubble formation for restoring the elasticity of the eyes.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Fibre-coupled multiphoton microscope with adaptive motion compensation

    PubMed Central

    Sherlock, Ben; Warren, Sean; Stone, James; Neil, Mark; Paterson, Carl; Knight, Jonathan; French, Paul; Dunsby, Chris

    2015-01-01

    To address the challenge of sample motion during in vivo imaging, we present a fibre-coupled multiphoton microscope with active axial motion compensation. The position of the sample surface is measured using optical coherence tomography and fed back to a piezo actuator that adjusts the axial location of the objective to compensate for sample motion. We characterise the system’s performance and demonstrate that it can compensate for axial sample velocities up to 700 µm/s. Finally we illustrate the impact of motion compensation when imaging multiphoton excited autofluorescence in ex vivo mouse skin. PMID:26137387

  1. Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

    PubMed Central

    Vučinić, Dejan; Bartol, Thomas M.; Sejnowski, Terrence J.

    2010-01-01

    Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task. PMID:16880851

  2. Effect of multiphoton ionization on performance of crystalline lens.

    PubMed

    Gupta, Pradeep Kumar; Singh, Ram Kishor; Strickland, D; Campbell, M C W; Sharma, R P

    2014-12-15

    This Letter presents a model for propagation of a laser pulse in a human crystalline lens. The model contains a transverse beam diffraction effect, laser-induced optical breakdown for the creation of plasma via a multiphoton ionization process, and the gradient index (GRIN) structure. Plasma introduces the nonlinearity in the crystalline lens which affects the propagation of the beam. The multiphoton ionization process generates plasma that changes the refractive index and hence leads to the defocusing of the laser beam. The Letter also points out the relevance of the present investigation to cavitation bubble formation for restoring the elasticity of the eyes. PMID:25502994

  3. Optical clearing and multiphoton imaging of paraffin-embedded specimens

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    New labeling, imaging, or analysis tools could provide new retrospective insights when applied to archived, paraffin-embedded samples. Deep-tissue multiphoton microscopy of paraffin-embedded specimens is achieved using optical clearing with mineral oil. We tested a variety of murine tissue specimens including skin, lung, spleen, kidney, and heart, acquiring multiphoton autofluorescence and second-harmonic generation, and pump-probe images This technique introduces the capability for non-destructive 3-dimensional microscopic imaging of existing archived pathology specimens, enabling retrospective studies.

  4. Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging.

    PubMed

    Ding, Dan; Goh, Chi Ching; Feng, Guangxue; Zhao, Zujin; Liu, Jie; Liu, Rongrong; Tomczak, Nikodem; Geng, Junlong; Tang, Ben Zhong; Ng, Lai Guan; Liu, Bin

    2013-11-13

    Ultrabright organic dots with aggregation-induced emission characteristics (AIE dots) are prepared and shown to exhibit a high quantum yield, a, large two-photon absorption cross-section, and low in vivo toxicity. Real-time two-photon intravital blood vascular imaging in various tissues substantiates that the AIE dots are effective probes for in vivo vasculature imaging in a deep and high-contrast manner.

  5. Localization of near-infrared contrast agents in tumors by intravital microscopy

    NASA Astrophysics Data System (ADS)

    Becker, Andreas; Schneider, Guenther; Riefke, Bjoern; Licha, Kai; Semmler, Wolfhard

    1999-01-01

    In this contribution we use intravital microscopy to study the dynamics of extravasation into normal and tumor tissue of several hydrophilic cyanine dyes used as near-infrared (NIR) contrast agents. The technique provides information about the angiographic properties of the dyes and about their interaction with tumor tissue under dynamic conditions in vivo. In our previous work we demonstrated that several NIR- absorbing fluorescent dyes enable in vivo fluorescence detection of tumors in mice and rats. However, the mechanism leading to dye accumulation and enhanced fluorescence in tumors is not fully understood. Increased extravasation of dyes into tumor tissue due to pathologically altered tumor vessels may be an important factor in this process. Indocyanine green (ICG) displayed predominantly intravascular distribution and rapid elimination resulting in enhanced fluorescence signal of vessels during the first 15 min after administration only. No elevated extravasation into tumor tissue was observed with ICG. A hydrophilic indotricarbocyanine derivative with a high molecular weight displayed prolonged intravascular distribution and increased fluorescence signal of the vasculature compared to surrounding tissue for up to five hours. Rapid extravasation and accumulation in tumor areas, yielding elevated contrast of tumors up to 15 min after administration, was observed with hydrophilic, low molecular weight indotricarbocyanine derivatives.

  6. Intravital fluorescence microscopic study of the behavior of long-circulating liposomes during microvascular thrombosis

    NASA Astrophysics Data System (ADS)

    Dvoisselle, Jean-Marie; Begu, Sylvie; Tourne-Peteilh, Corine; Buys, Bruno; Mordon, Serge R.

    2002-06-01

    Treatment of thrombosis depends on the selectivity of thrombolytic agents to the clot. It has been already demonstrated that liposomes can provide a better selectivity of such agents to the clot site. We have recently shown that intravital fluorescence microscopy is a powerful tool to image in situ and in real time the labeling of leukocytes by long circulating liposomes. The aim of this study was to monitor the in vivo behavior of such liposomes in a clot site. Carboxyfluorescein-loaded long circulating liposomes were prepared and characterized in term of size and permeability. The liposomes suspension was injected intravenously to golden hamsters. The skin microcirculation was observed using a dorsal skin-fold chamber by fluorescence microscopy. Thrombosis were obtained as the consequence of the inflammatory response due to the surgery. Using this model, fluorescent dots were observed at the site of the clot. Liposomes accumulate at the clot site whatever the mechanism (passive deposition or uptake). There is a period of latency and 30 seconds after the blood flow stop, fluorescence increases very rapidly and a bright fluorescent spot is observed at the site of the clot. Further studies are needed to determine the exact localization of liposomes in the clot and the mechanism of interaction.

  7. Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation

    PubMed Central

    van Ham, Tjakko J.; Brady, Colleen A.; Kalicharan, Ruby D.; Oosterhof, Nynke; Kuipers, Jeroen; Veenstra-Algra, Anneke; Sjollema, Klaas A.; Peterson, Randall T.; Kampinga, Harm H.; Giepmans, Ben N. G.

    2014-01-01

    Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage. PMID:24973753

  8. Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.

    PubMed

    Kataoka, Hanae; Ushiyama, Akira; Kawakami, Hayato; Akimoto, Yoshihiro; Matsubara, Sachie; Iijima, Takehiko

    2016-01-01

    Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy.

  9. Dorsal Skinfold Chamber Preparation in Mice: Studying Angiogenesis by Intravital Microscopy.

    PubMed

    Sckell, Axel; Leunig, Michael

    2016-01-01

    Intravital microscopy represents an internationally accepted and sophisticated experimental method to study angiogenesis, microcirculation, and many other parameters in a wide variety of neoplastic and nonneoplastic tissues. Since 1924, when the first transparent chamber model in animals was introduced, many other chamber models have been described in the literature for studying angiogenesis and microcirculation. Because angiogenesis is an active and dynamic process, one of the major strengths of chamber models is the possibility of monitoring angiogenesis in vivo continuously for up to several weeks with high spatial and temporal resolution. In addition, after the termination of experiments, tissue samples can be excised easily and further examined by various ex vivo methods such as histology, immunohistochemistry, and molecular biology. This chapter describes the protocol for the surgical preparation of a dorsal skinfold chamber in mice as well as the method to implant tumors in this chamber for further investigations of angiogenesis and other microcirculatory parameters. However, the application of the dorsal skinfold chamber model is not limited to the investigation of neoplastic tissues. To this end, the investigation of angiogenesis and other microcirculatory parameters of nonneoplastic tissues such as tendons, osteochondral grafts, or pancreatic islets has been an object of interest.

  10. Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

    PubMed Central

    Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M.; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

    2013-01-01

    A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging. PMID:23401517

  11. Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy.

    PubMed

    Hirata, Eishu; Kiyokawa, Etsuko

    2016-09-20

    Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.

  12. Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy.

    PubMed

    Hirata, Eishu; Kiyokawa, Etsuko

    2016-09-20

    Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena. PMID:27475975

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

    NASA Astrophysics Data System (ADS)

    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

  14. Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie; Tora, Muhibullah; Milberg, Oleg; Weigert, Roberto

    2013-01-01

    Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton. PMID:24022089

  15. Intravital Imaging Reveals Angiotensin II-Induced Transcytosis of Albumin by Podocytes.

    PubMed

    Schießl, Ina Maria; Hammer, Anna; Kattler, Veronika; Gess, Bernhard; Theilig, Franziska; Witzgall, Ralph; Castrop, Hayo

    2016-03-01

    Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (P<0.001), and 239.4±34.6 µm(3) (P<0.001) albumin-containing vesicles per glomerulus, respectively, compared with none at baseline or 10 ng/kg per minute Ang II. Immunostaining of Ang II-infused kidneys confirmed the presence of albumin-containing vesicles, which colocalized with megalin, in podocin-positive cells. Furthermore, podocyte endocytosis of albumin was markedly reduced in the presence of gentamicin, a competitive inhibitor of megalin-dependent endocytosis. Ang II infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and gentamicin further increased this concentration. Some endocytic vesicles were acidified and colocalized with LysoTracker. Most vesicles migrated from the capillary to the apical aspect of the podocyte and were eventually released into the urinary space. This transcytosis accounted for approximately 10% of total albumin filtration. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. Ang II enhances the endocytosis and transcytosis of plasma albumin by podocytes, which may eventually impair podocyte function.

  16. Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow

    PubMed Central

    Shi, Xiaorui; Zhang, Fei; Urdang, Zachary; Dai, Min; Neng, Lingling; Zhang, Jinhui; Chen, Songlin; Ramamoorthy, Sripriya; Nuttall, Alfred L.

    2014-01-01

    Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions. PMID:24780131

  17. Multi-photon UV photolysis of gaseous polycyclic aromatic hydrocarbons: extinction spectra and dynamics.

    PubMed

    Walsh, A J; Ruth, A A; Gash, E W; Mansfield, M W D

    2013-08-01

    The extinction spectra of static naphthalene and static biphenylene vapor, each buffered with a noble gas at room temperature, were measured as a function of time in the region between 390 and 850 nm after UV multi-photon laser photolysis at 308 nm. Employing incoherent broadband cavity enhanced absorption spectroscopy (IBBCEAS), the spectra were found to be unstructured with a general lack of isolated features suggesting that the extinction was not solely based on absorption but was in fact dominated by scattering from particles formed in the photolysis of the respective polycyclic aromatic hydrocarbon. Following UV multi-photon photolysis, the extinction dynamics of the static (unstirred) closed gas-phase system exhibits extraordinary quasi-periodic and complex oscillations with periods ranging from seconds to many minutes, persisting for up to several hours. Depending on buffer gas type and pressure, several types of dynamical responses could be generated (classified as types I, II, and III). They were studied as a function of temperature and chamber volume for different experimental conditions and possible explanations for the oscillations are discussed. A conclusive model for the observed phenomena has not been established. However, a number of key hypotheses have made based on the measurements in this publication: (a) Following the multi-photon UV photolysis of naphthalene (or biphenylene), particles are formed on a timescale not observable using IBBCEAS. (b) The observed temporal behavior cannot be described on basis of a chemical reaction scheme alone. (c) The pressure dependence of the system's responses is due to transport phenomena of particles in the chamber. (d) The size distribution and the refractive indices of particles are time dependent and evolve on a timescale of minutes to hours. The rate of particle coagulation, involving coalescent growth and particle agglomeration, affects the observed oscillations. (e) The walls of the chamber act as a sink

  18. Multi-photon UV photolysis of gaseous polycyclic aromatic hydrocarbons: Extinction spectra and dynamics

    SciTech Connect

    Walsh, A. J.; Gash, E. W.; Mansfield, M. W. D.; Ruth, A. A.

    2013-08-07

    The extinction spectra of static naphthalene and static biphenylene vapor, each buffered with a noble gas at room temperature, were measured as a function of time in the region between 390 and 850 nm after UV multi-photon laser photolysis at 308 nm. Employing incoherent broadband cavity enhanced absorption spectroscopy (IBBCEAS), the spectra were found to be unstructured with a general lack of isolated features suggesting that the extinction was not solely based on absorption but was in fact dominated by scattering from particles formed in the photolysis of the respective polycyclic aromatic hydrocarbon. Following UV multi-photon photolysis, the extinction dynamics of the static (unstirred) closed gas-phase system exhibits extraordinary quasi-periodic and complex oscillations with periods ranging from seconds to many minutes, persisting for up to several hours. Depending on buffer gas type and pressure, several types of dynamical responses could be generated (classified as types I, II, and III). They were studied as a function of temperature and chamber volume for different experimental conditions and possible explanations for the oscillations are discussed. A conclusive model for the observed phenomena has not been established. However, a number of key hypotheses have made based on the measurements in this publication: (a) Following the multi-photon UV photolysis of naphthalene (or biphenylene), particles are formed on a timescale not observable using IBBCEAS. (b) The observed temporal behavior cannot be described on basis of a chemical reaction scheme alone. (c) The pressure dependence of the system's responses is due to transport phenomena of particles in the chamber. (d) The size distribution and the refractive indices of particles are time dependent and evolve on a timescale of minutes to hours. The rate of particle coagulation, involving coalescent growth and particle agglomeration, affects the observed oscillations. (e) The walls of the chamber act as a sink

  19. A simple model of multiphoton micromachining in silk hydrogels

    NASA Astrophysics Data System (ADS)

    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. Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

    PubMed Central

    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

    2016-01-01

    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. PMID:27283889

  1. Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

    NASA Astrophysics Data System (ADS)

    Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

    2015-07-01

    Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

  2. Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

    NASA Astrophysics Data System (ADS)

    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.

  3. Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    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.

  4. Experimental Resonance Enhanced Multiphoton Ionization (REMPI) studies of small molecules

    NASA Technical Reports Server (NTRS)

    Dehmer, J. L.; Dehmer, P. M.; Pratt, S. T.; Ohalloran, M. A.; Tomkins, F. S.

    1987-01-01

    Resonance enhanced multiphoton ionization (REMPI) utilizes tunable dye lasers to ionize an atom or molecule by first preparing an excited state by multiphoton absorption and then ionizing that state before it can decay. This process is highly selective with respect to both the initial and resonant intermediate states of the target, and it can be extremely sensitive. In addition, the products of the REMPI process can be detected as needed by analyzing the resulting electrons, ions, fluorescence, or by additional REMPI. This points to a number of exciting opportunities for both basic and applied science. On the applied side, REMPI has great potential as an ultrasensitive, highly selective detector for trace, reactive, or transient species. On the basic side, REMPI affords an unprecedented means of exploring excited state physics and chemistry at the quantum-state-specific level. An overview of current studies of excited molecular states is given to illustrate the principles and prospects of REMPI.

  5. Differentiation of normal and cancerous lung tissues by multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Wu, Ruei-Jhih; Hovhannisyan, Vladimir A.; Lin, Wei-Chou; Lin, Sung-Jan; So, Peter T. C.; Dong, Chen-Yuan

    2009-07-01

    We utilize multiphoton microscopy for the label-free diagnosis of noncancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from humans. Our results show that the combination of second-harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from noncancerous lung tissues. Specifically, noncancerous lung tissues are largely fibrotic in structure, while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55+/-0.23 and 0.87+/-0.15, respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50+/-0.12 and 0.72+/-0.13, respectively. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from noncancerous tissues.

  6. In vivo multiphoton imaging of obstructive cholestasis in mice

    NASA Astrophysics Data System (ADS)

    Li, Feng-Chieh; Lee, Yu Yang; Chiou, Ling-Ling; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2010-02-01

    Combining multiphoton microscopy with a newly designed hepatic imaging window, we acquired in vivo images of mice obstructive cholestasis. We observed that in mice with bile duct ligation, bile canaliculi failed to appear during the whole observation period over 100 minutes following carboxyfluorescein diacetate injection, whereas the fluorescence was retained much longer within sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in hepatocytes during the course.

  7. Relaxation channels of multi-photon excited xenon clusters

    SciTech Connect

    Serdobintsev, P. Yu.; Melnikov, A. S.; Rakcheeva, L. P. Murashov, S. V.; Khodorkovskii, M. A.; Lyubchik, S.; Timofeev, N. A.; Pastor, A. A.

    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.

  8. Intravital Computer Morphometry on Protozoa: A Method for Monitoring of the Morphofunctional Disorders in Cells Exposed in the Cell Phone Communication Electromagnetic Field.

    PubMed

    Uskalova, D V; Igolkina, Yu V; Sarapultseva, E I

    2016-08-01

    Morphofunctional disorders in unicellular aquatic protozoa - Spirostomum ambiguum infusorians after 30-, 60-, and 360-min exposure in electromagnetic field at a radiation frequency of 1 GHz and energy flow density of 50 μW/cm(2) were analyzed by intravital computer morphometry. Significant disorders in morphometric values correlated with low mobility of the protozoa. The results suggested the use of intravital computer morphometry on the protozoa for early diagnosis of radiation-induced effects of the mobile communication electromagnetic field, for example, low mobility of spermatozoa.

  9. Intravital Computer Morphometry on Protozoa: A Method for Monitoring of the Morphofunctional Disorders in Cells Exposed in the Cell Phone Communication Electromagnetic Field.

    PubMed

    Uskalova, D V; Igolkina, Yu V; Sarapultseva, E I

    2016-08-01

    Morphofunctional disorders in unicellular aquatic protozoa - Spirostomum ambiguum infusorians after 30-, 60-, and 360-min exposure in electromagnetic field at a radiation frequency of 1 GHz and energy flow density of 50 μW/cm(2) were analyzed by intravital computer morphometry. Significant disorders in morphometric values correlated with low mobility of the protozoa. The results suggested the use of intravital computer morphometry on the protozoa for early diagnosis of radiation-induced effects of the mobile communication electromagnetic field, for example, low mobility of spermatozoa. PMID:27591872

  10. Development of a spectrally resolved multifocal multiphoton microscope

    NASA Astrophysics Data System (ADS)

    Liu, Lixin; Shao, Yonghong; Qu, Junle; Li, Heng; Guo, Baoping; Liu, Wenqing; Niu, Hanben

    2008-12-01

    Multifocal Multiphoton Microscopy (MMM) can acquire three-dimensional (3D) fluorescence microscopic images of samples by multiphoton excitation with the advantages of high speed, reduced photobleaching, enhanced penetration depth and high signal-to-noise ratio. As fluorescence spectrum can provide information about the components of the sample, it is becoming increasingly popular in biomedicine to combine fluorescence spectrum measurement with multidimensional fluorescence microscopy. In this paper, we present the development of a spectrally resolved multifocal multiphoton microscope (SR-MMM). A microlens array is employed in the MMM system to produce 2D excitation foci on the sample for simultaneous two-photon excitation and a pair of galvo mirrors is used to scan the excitation foci across the sample. A liquid crystal tunable filter (LCTF) is mounted in front of a high-speed cooled CCD camera and is used to change the detection wavelength of the MMM system. Depth-resolved and spectrum-resolved two-photon excitation fluorescence images of a few samples are obtained with the SR-MMM system.

  11. Controlled Damage in Thick Specimens by Multiphoton ExcitationV⃞

    PubMed Central

    Galbraith, James A.; Terasaki, Mark

    2003-01-01

    Controlled damage by light energy has been a valuable tool in studies of cell function. Here, we show that the Ti:Sapphire laser in a multiphoton microscope can be used to cause localized damage within unlabeled cells or tissues at greater depths than previously possible. We show that the damage is due to a multiphoton process and made wounds as small as 1 μm in diameter 20 μm from the surface. A characteristic fluorescent scar allows monitoring of the damage and identifies the wound site in later observations. We were able to lesion a single axon within a bundle of nerves, locally interrupt organelle transport within one axon, cut dendrites in a zebrafish embryo, ablate a mitotic pole in a sea urchin egg, and wound the plasma membrane and nuclear envelope in starfish oocytes. The starfish nucleus collapsed ∼1 h after wounding, indicating that loss of compartmentation barrier makes the structure unstable; surprisingly, the oocyte still completed meiotic divisions when exposed to maturation hormone, indicating that the compartmentalization and translocation of cdk1 and its regulators is not required for this process. Multiphoton excitation provides a new means for producing controlled damage deep within tissues or living organisms. PMID:12802057

  12. Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source.

    PubMed

    Lefort, Claire; O'Connor, Rodney P; Blanquet, Véronique; Magnol, Laetitia; Kano, Hideaki; Tombelaine, Vincent; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

    2016-07-01

    Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red(®) are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three-photon ones. PMID:26872004

  13. Differentiation of normal and cancerous lung tissues by multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Wu, Ruei-Jr; Hovhannisyan, Vladimir A.; Lin, Wei-Chou; Lin, Sung-Jan; So, Peter T. C.; Dong, Chen-Yuan

    2010-02-01

    In this work, we utilized multiphoton microscopy for the label-free diagnosis of non-cancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from human. Our results show that the combination of second harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from non-cancerous lung tissues. Specifically, non-cancerous lung tissues are largely fibrotic in structure while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI or SAAID) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55 +/-0.23 and 0.87+/-0.15 respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50+/-0.12 and 0.72+/-0.13 respectively. Intrinsic fluorescence ratio (FAD/NADH) of SCC and non-cancerous tissues are 0.40+/-0.05 and 0.53+/-0.05 respectively, the redox ratio of SCC diminishes significantly, indicating that increased cellular metabolic activity. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from non-cancerous tissues. With additional development, multiphoton microscopy may be used for the clinical diagnosis of lung cancers.

  14. Multiphoton imaging of excised normal skin and keloid scar: preliminary investigations

    NASA Astrophysics Data System (ADS)

    Brewer, Michael B.; Yeh, Alvin T.; Torkian, Behrooz; Sun, Chung-Ho; Tromberg, Bruce J.; Wong, Brian J.

    2004-07-01

    Wound healing is a physiologic process that acts to repair disruptions in the continuity of tissue caused by injury or surgical incision. Keloids and hypertrophic scars are forms of aberrant wound healing, which are characterized by the overproduction of collagen, resulting in an excessive amount of scar tissue. Keloid tumors, by definition, grow outside the boundary of the original tissue damage. Multiphoton microscopy (MPM) is an imaging technique which allows imaging of living specimens, without the use of fixation or stains. Images of collagen fibers are produced by the second harmonic signal intensity generated by endogenous fluorescence through excitation by infrared laser light. A postauricular keloid tumor was excised from a patient. The tissue was dissected, and a portion was imaged using MPM. Normal skin tissue was isolated from a patient undergoing a facelift. A portion of this tissue was also dissected and imaged using MPM. MPM images were taken using a 63X water immersion objective lens on a two-photon microscope and a titanium-sapphire laser. Images were taken beginning at the surface of the tissue and moving in at intervals of 200 nm to a final depth of 30 μm. The two-photon images were used to reconstruct three-dimensional representations of the collagen matrix within the tissues, which are readily contrasted. Density of the collagen within each tissue was also ascertained using depth dependant decay of the image intensity. Multiphoton imaging was successfully used to image the collagen matrix of normal skin and a keloid scar, demonstrating differences in their microstructures.

  15. Direct observation of liposome uptake by leukocytes in vivo in skin blood vessels using intravital fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Devoisselle, Jean-Marie; Mordon, Serge R.; Begu, Sylvie; Desmettre, Thomas

    2000-04-01

    This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5,6-CF-encapsulated PEGylated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using these model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 hours. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up liposomes. This observation is in accordance with previous in vitro studies.

  16. Automatic detection of motion blur in intravital video microscopy image sequences via directional statistics of log-Gabor energy maps.

    PubMed

    Ferrari, Ricardo J; Pinto, Carlos H Villa; da Silva, Bruno C Gregório; Bernardes, Danielle; Carvalho-Tavares, Juliana

    2015-02-01

    Intravital microscopy is an important experimental tool for the study of cellular and molecular mechanisms of the leukocyte-endothelial interactions in the microcirculation of various tissues and in different inflammatory conditions of in vivo specimens. However, due to the limited control over the conditions of the image acquisition, motion blur and artifacts, resulting mainly from the heartbeat and respiratory movements of the in vivo specimen, will very often be present. This problem can significantly undermine the results of either visual or computerized analysis of the acquired video images. Since only a fraction of the total number of images are usually corrupted by severe motion blur, it is necessary to have a procedure to automatically identify such images in the video for either further restoration or removal. This paper proposes a new technique for the detection of motion blur in intravital video microscopy based on directional statistics of local energy maps computed using a bank of 2D log-Gabor filters. Quantitative assessment using both artificially corrupted images and real microscopy data were conducted to test the effectiveness of the proposed method. Results showed an area under the receiver operating characteristic curve (AUC) of 0.95 (AUC = 0.95; 95 % CI 0.93-0.97) when tested on 329 video images visually ranked by four observers.

  17. Three-Dimensional Analysis of Cell Division Orientation in Epidermal Basal Layer Using Intravital Two-Photon Microscopy

    PubMed Central

    Nemoto, Tomomi

    2016-01-01

    Epidermal structures are different among body sites, and proliferative keratinocytes in the epidermis play an important role in the maintenance of the epidermal structures. In recent years, intravital skin imaging has been used in mammalian skin research for the investigation of cell behaviors, but most of these experiments were performed with rodent ears. Here, we established a non-invasive intravital imaging approach for dorsal, ear, hind paw, or tail skin using R26H2BEGFP hairless mice. Using four-dimensional (x, y, z, and time) imaging, we successfully visualized mitotic cell division in epidermal basal cells. A comparison of cell division orientation relative to the basement membrane in each body site revealed that most divisions in dorsal and ear epidermis occurred in parallel, whereas the cell divisions in hind paw and tail epidermis occurred both in parallel and oblique orientations. Based on the quantitative analysis of the four-dimensional images, we showed that the epidermal thickness correlated with the basal cell density and the rate of the oblique divisions. PMID:27657513

  18. Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice.

    PubMed

    Yao, Yi; Liu, Yun; Takashima, Akira

    2016-01-01

    Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal - used as a measure of priming 2) in vivo neutrophil labeling - achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models. PMID:27403648

  19. Evaluation of pulsatile and nonpulsatile flow in capillaries of goat skeletal muscle using intravital microscopy.

    PubMed

    Lee, J J; Tyml, K; Menkis, A H; Novick, R J; Mckenzie, F N

    1994-11-01

    It is commonly believed that pulsatile flow generated by the pumping action of the heart is dampened out by the time it reaches the microcirculation. In clinical practice, most of the cardiopulmonary bypass pumps and ventricular assist devices are nonpulsatile. To test the hypothesis that pulsatile flow generated by the heart does exist at the microvascular level, intravital microscopy of a large animal model (goat) was developed to visualize and to videorecord the surface microcirculation of the flexor carpi ulnaris muscle from the right forelimb. Density of perfused capillaries and red blood cell velocity in capillaries were measured in five goats during pulsatile perfusion provided by the heart and during a subsequent 3-hr period of nonpulsatile perfusion provided by a centrifugal ventricular assist device (Centrimed, Sarns 3M) that bypassed the heart. Throughout the experiment, the heart rate, innominate artery mean blood pressure, and flow remained unchanged. During the pulsatile regimen, velocities showed regular fluctuations that coincided with the period of the cardiac cycle (range of periods: 0.5-0.8 sec). The peak velocity amplitudes (range: 0.25-0.55 mm/sec) correlated directly with the amplitude of the pulse pressure. During the nonpulsatile regimen, no such correlations were seen. During pulsatile flow and during the 3-hr nonpulsatile period, capillary density remained stable at 24 capillaries/mm of test line but there were significant increases in red cell velocity, from 0.8 to 1.2 mm/sec (P < 0.05), and in coefficient of variation of velocity (used as an index of flow heterogeneity), from 19 to 34% (P < 0.05). We conclude that (1) pulsatility exists in the capillary bed and that it directly correlates with the pumping action of the heart and (2) nonpulsatile flow produced by the ventricular assist device does not cause an acute deterioration in microvascular perfusion. We interpret the increase in heterogeneity of flow as an early sign of

  20. Spatiotemporal Analyses of Osteogenesis and Angiogenesis via Intravital Imaging in Cranial Bone Defect Repair

    PubMed Central

    Huang, Chunlan; Ness, Vincent P.; Yang, Xiaochuan; Chen, Hongli; Luo, Jiebo; Brown, Edward B; Zhang, Xinping

    2015-01-01

    Osteogenesis and angiogenesis are two integrated components in bone repair and regeneration. A deeper understanding of osteogenesis and angiogenesis has been hampered by technical difficulties of analyzing bone and neovasculature simultaneously in spatiotemporal scales and in three-dimensional formats. To overcome these barriers, a cranial defect window chamber model was established that enabled high-resolution, longitudinal, and real-time tracking of angiogenesis and bone defect healing via Multiphoton Laser Scanning Microscopy (MPLSM). By simultaneously probing new bone matrix via second harmonic generation (SHG), neovascular networks via intravenous perfusion of fluorophore, and osteoblast differentiation via 2.3kb collagen type I promoter driven GFP (Col2.3GFP), we examined the morphogenetic sequence of cranial bone defect healing and further established the spatiotemporal analyses of osteogenesis and angiogenesis coupling in repair and regeneration. We demonstrated that bone defect closure was initiated in the residual bone around the edge of the defect. The expansion and migration of osteoprogenitors into the bone defect occurred during the first 3 weeks of healing, coupled with vigorous microvessel angiogenesis at the leading edge of the defect. Subsequent bone repair was marked by matrix deposition and active vascular network remodeling within new bone. Implantation of bone marrow stromal cells (BMSCs) isolated from Col2.3GFP mice further showed that donor-dependent bone formation occurred rapidly within the first 3 weeks of implantation, in concert with early angiogenesis. The subsequent bone wound closure was largely host-dependent, associated with localized modest induction of angiogenesis. The establishment of a live imaging platform via cranial window provides a unique tool to understand osteogenesis and angiogenesis in repair and regeneration, enabling further elucidation of the spatiotemporal regulatory mechanisms of osteoprogenitor cell interactions

  1. Multiphoton ionization of hydrogen by a strong multimode field

    SciTech Connect

    Basile, S.; Trombetta, F.; Ferrante, G.; Burlon, R.; Leone, C.

    1988-02-01

    Multiphoton ionization of hydrogen atoms by a strong multimode field when several final continuum states are populated is treated theoretically within a model based on the S-matrix formalism. The field is taken in dipole approximation, with zero bandwidth. Field-dressed Coulomb wave functions are used for the electron final states. Ionization rates are calculated for different numbers of photons absorbed in excess of the minimum number required to go into the continuum. In general, the calculated quantities are in good qualitative agreement with the corresponding experimental observations.

  2. Robust distant entanglement generation using coherent multiphoton scattering.

    PubMed

    Chan, Ching-Kit; Sham, L J

    2013-02-15

    We describe a protocol to entangle two qubits at a distance by using resonance fluorescence. The scheme makes use of the postselection of large and distinguishable fluorescence signals corresponding to entangled and unentangled qubit states and has the merits of both high success probability and high entanglement fidelity owing to the multiphoton nature. Our result shows that the entanglement generation is robust against photon fluctuations in the fluorescence signals for a wide range of driving fields. We also demonstrate that this new protocol has an average entanglement duration within the decoherence time of corresponding qubit systems, based on current experimental photon efficiency.

  3. Anomalous multiphoton photoelectric effect in ultrashort time scales.

    PubMed

    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.

  4. The nature of multiphoton fluorescence from red blood cells

    NASA Astrophysics Data System (ADS)

    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.

  5. Microstructure imaging of human rectal mucosa using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liu, N. R.; Chen, G.; Chen, J. X.; Yan, J.; Zhuo, S. M.; Zheng, L. Q.; Jiang, X. S.

    2011-01-01

    Multiphoton microscopy (MPM) has high resolution and sensitivity. In this study, MPM was used to image microstructure of human rectal mucosa. The morphology and distribution of the main components in mucosa layer, absorptive cells and goblet cells in the epithelium, abundant intestinal glands in the lamina propria and smooth muscle fibers in the muscularis mucosa were clearly monitored. The variations of these components were tightly relevant to the pathology in gastrointestine system, especially early rectal cancer. The obtained images will be helpful for the diagnosis of early colorectal cancer.

  6. Quantum radiation reaction effects in multiphoton Compton scattering.

    PubMed

    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.

  7. Multiphoton ionization of ions, neutrals, and clusters. Final report

    SciTech Connect

    Wessel, J.

    1995-12-28

    A multiyear research program investigating molecular detection methods based on multiphoton spectroscopy has been completed under DOE sponsorship. A number of new laser-based spectroscopic methods were developed and applied to a variety of aromatic hydrocarbons, including monomer and cluster species. The objectives of sensitivities approaching single molecule detection combined with high selectivity were achieved. This report references the status of the field at the beginning of this work and summarizes the significant progress during the period from 1987 onward. Detailed scientific findings from the studies are presented in the published literature referenced throughout this report.

  8. Multiphoton ionization of ions, neutrals, and clusters. Progress report

    SciTech Connect

    Wessel, J.

    1991-06-28

    Scientific results are summarized from a three year research program on multiphoton ionization in aromatic molecules, clusters, and their ions. As originally proposed, the studies elucidated a new cluster ionization mechanism, characterized properties of long range intermolecular interactions, and investigated electronic transitions of aromatic cations cooled in a supersonic beam. The studies indicate that the new cluster ionization mechanism is highly efficient and dominates conventional 1 + 1 resonant ionization. In the case of the dimer of the large aromatic molecule fluorene, the results suggest that excimer formation competes with a direct ionization process. Highly selective excitonic spectra have been identified for several cluster species.

  9. Integrated spectrometer design with application to multiphoton microscopy.

    PubMed

    Chandler, Eric V; Durfee, Charles G; Squier, Jeffrey A

    2011-01-01

    We present a prism-based spectrometer integrated into a multifocal, multiphoton microscope. The multifocal configuration facilitates interrogation of samples under different excitation conditions. Notably, the image plane of the microscope and the image plane of the spectrometer are coincident eliminating the need for an intermediate image plane containing an entrance slit. An EM-CCD detector provides sufficient gain for spectral interrogation of single-emitters. We employ this spectrometer to observe spectral shifts in the two-photon excitation fluorescence emission of single CdSe nanodots as a function of excitation polarization. PMID:21263548

  10. Quantum Radiation Reaction Effects in Multiphoton Compton Scattering

    SciTech Connect

    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.

  11. Inherent contradictions in the tunneling-multiphoton dichotomy

    SciTech Connect

    Reiss, H. R.

    2007-03-15

    Strong-field phenomena are currently described as being multiphoton or tunneling, based on whether the Keldysh parameter {gamma} is greater than or less than unity. There are disqualifying features in this categorization. It is specific to the length gauge, dependent on only one intensity parameter, and backward in the sense that ionization with {gamma}>>1 can occur only by tunneling, and ionization with {gamma}<<1 must be over the barrier, and hence is not achieved by tunneling. As {gamma} becomes smaller, ionization becomes increasingly distant from tunneling, and eventually reaches conditions where the dipole approximation fails and there is no meaningful scalar-potential diagram at all.

  12. Exploration of multiphoton entangled states by using weak nonlinearities

    PubMed Central

    He, Ying-Qiu; Ding, Dong; Yan, Feng-Li; Gao, Ting

    2016-01-01

    We propose a fruitful scheme for exploring multiphoton entangled states based on linear optics and weak nonlinearities. Compared with the previous schemes the present method is more feasible because there are only small phase shifts instead of a series of related functions of photon numbers in the process of interaction with Kerr nonlinearities. In the absence of decoherence we analyze the error probabilities induced by homodyne measurement and show that the maximal error probability can be made small enough even when the number of photons is large. This implies that the present scheme is quite tractable and it is possible to produce entangled states involving a large number of photons. PMID:26751044

  13. Multifocal multiphoton microscopy based on a spatial light modulator

    PubMed Central

    Shao, Y.; Qin, W.; Liu, H.; Peng, X.; Niu, H.

    2013-01-01

    We present a new multifocal multiphoton microscope that employs a programmable spatial light modulator to generate dynamic multifocus arrays which can be rapidly scanned by changing the incident angle of the laser beam using a pair of galvo scanners. Using this microscope, we can rapidly select the number and the spatial density of focal points in a multifocus array, as well as the locations and shapes of arrays according to the features of the areas of interest in the field of view without any change to the hardware. PMID:23894222

  14. Long- and short-term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury

    PubMed Central

    Fenrich, Keith K; Weber, Pascal; Rougon, Geneviève; Debarbieux, Franck

    2013-01-01

    After spinal cord injury (SCI), resident and peripheral myelomonocytic cells are recruited to the injury site and play a role in injury progression. These cells are important for clearing cellular debris, and can modulate the retraction and growth of axons in vitro. However, their precise spatiotemporal recruitment dynamics is unknown, and their respective roles after SCI remain heavily debated. Using chronic, quantitative intravital two-photon microscopy of adult mice with SCI, here we show that infiltrating lysozyme M (LysM(+)) and resident CD11c(+) myelomonocytic cells have distinct spatiotemporal recruitment profiles, and exhibit changes in morphology, motility, phagocytic activity and axon interaction patterns over time. This study provides the first in vivo description of the influx of inflammatory and resident myelomonocytic cells into the injured spinal cord and their interactions with cut axons, and underscores the importance of precise timing and targeting of specific cell populations in developing therapies for SCI. PMID:23918770

  15. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice.

    PubMed

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  16. Simultaneous three-dimensional optical coherence tomography and intravital microscopy for imaging subpleural pulmonary alveoli in isolated rabbit lungs

    NASA Astrophysics Data System (ADS)

    Meissner, Sven; Knels, Lilla; Krueger, Alexander; Koch, Thea; Koch, Edmund

    2009-09-01

    There is a growing interest in analyzing lung mechanics at the level of the alveoli in order to understand stress-related pathogenesis and possibly avoid ventilator associated lung injury. Emerging quantitative models to simulate fluid mechanics and the associated stresses and strains on delicate alveolar walls require realistic quantitative input on alveolar geometry and its dynamics during ventilation. Here, three-dimensional optical coherence tomography (OCT) and conventional intravital microscopy are joined in one setup to investigate the geometric changes of subpleural alveoli during stepwise pressure increase and release in an isolated and perfused rabbit lung model. We describe good qualitative agreement and quantitative correlation between the OCT data and video micrographs. Our main finding is the inflation and deflation of individual alveoli with noticeable hysteresis. Importantly, this three-dimensional geometry data can be extracted and converted into input data for numerical simulations.

  17. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

    2016-09-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  18. Intravital fluorescence imaging of mouse brain using implantable semiconductor devices and epi-illumination of biological tissue

    PubMed Central

    Takehara, Hiroaki; Ohta, Yasumi; Motoyama, Mayumi; Haruta, Makito; Nagasaki, Mizuki; Takehara, Hironari; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Ohta, Jun

    2015-01-01

    The application of the fluorescence imaging method to living animals, together with the use of genetically engineered animals and synthesized photo-responsive compounds, is a powerful method for investigating brain functions. Here, we report a fluorescence imaging method for the brain surface and deep brain tissue that uses compact and mass-producible semiconductor imaging devices based on complementary metal-oxide semiconductor (CMOS) technology. An image sensor chip was designed to be inserted into brain tissue, and its size was 1500 × 450 μm. Sample illumination is also a key issue for intravital fluorescence imaging. Hence, for the uniform illumination of the imaging area, we propose a new method involving the epi-illumination of living biological tissues, and we performed investigations using optical simulations and experimental evaluation. PMID:26137364

  19. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  20. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice.

    PubMed

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  1. Ultrafast multiphoton transient absorption of β-carotene

    NASA Astrophysics Data System (ADS)

    Buckup, Tiago; Weigel, Alexander; Hauer, Jürgen; Motzkus, Marcus

    2010-07-01

    Multiphoton spectroscopy is able to directly excite electronic states, which are one-photon forbidden. Under single photon conditions, such one-photon forbidden states are exclusively populated via internal relaxation. Hence, transient absorption with two-photon excitation has the potential of clarifying complex relaxation networks by using aimed excitation. In this work we exploited ultrafast two-photon spectroscopy to investigate the excitation of dark states of β-carotene in solution. After direct excitation of the vibronic manifold of S2Ag- from S 0 via two-photon transition, the characteristic internal conversion via hot-S 1 → S 1 → S 0 was observed in the respective spectral region. Additional slow dynamics in the blue-wing of excited-state absorption (ESA) and in the NIR were detected, which is not directly observable with one-photon excitation transient absorption. These features are associated here to resonant multiphoton processes, which lead simultaneously to ultrafast intersystem crossing between singlet and triplet systems as well as to excitation of doublet states. Furthermore, we identify a 340-400 fs relaxation component in the near-infrared region after two-photon resonant excitation and discuss the role of additional dark states ( 3Ag- and 1Bu-) in this process.

  2. Scaling up multiphoton neural scanning: the SSA algorithm.

    PubMed

    Schuck, Renaud; Annecchino, Luca A; Schultz, Simon R

    2014-01-01

    In order to reverse-engineer the information processing capabilities of the cortical circuit, we need to densely sample neural circuit; it may be necessary to sample the activity of thousands of neurons simultaneously. Frame scanning techniques do not scale well in this regard, due to the time "wasted" scanning extracellular space. For scanners in which inertia can be neglected, path length minimization strategies enable large populations to be imaged at relatively high sampling rates. However, in a standard multiphoton microscope, the scanners responsible for beam deflection are inertial, indicating that an optimal solution should take rotor and mirror momentum into account. We therefore characterized the galvanometric scanners of a commercial multiphoton microscope, in order to develop and validate a MATLAB model of microscope scanning dynamics. We tested the model by simulating scan paths across pseudo-randomly positioned neuronal populations of differing neuronal density and field of view. This model motivated the development of a novel scanning algorithm, Adaptive Spiral Scanning (SSA), in which the radius of a circular trajectory is constantly updated such that it follows a spiral trajectory scanning all the cells. Due to the kinematic efficiency of near-circular trajectories, this algorithm achieves higher sampling rates than shortest path approaches, while retaining a relatively efficient coverage fraction in comparison to raster or resonance based frame-scanning approaches. PMID:25570582

  3. Rigid and high NA multiphoton fluorescence GRIN-endoscopes

    NASA Astrophysics Data System (ADS)

    Schenkl, Selma; Ehlers, Alexander; Le Harzic, Ronan; Stark, Martin; Riemann, Iris; Messerschmidt, Bernhard; Kaatz, Martin; König, Karsten

    2007-07-01

    Multiphoton autofluorescence imaging offers minimal-invasive examination of cells without the need of staining and complicated confocal detection systems. Therefore, it is especially interesting for non-invasive clinical diagnostics. To extend this sophisticated technique from superficial regions to deep lying cell layers, internal body parts and specimens difficult of access, the bulky optics need to be reduced in diameter. This is done by tiny GRIN-optics, based on a radial gradient in the reflective index. Of especial interest for multi-photon applications is the newly developed GRIN-lens assembly with increased numerical aperture. High resolution images of plant tissue, hair and cells show the improved image quality,compared to classical GRIN-lenses. The rigid GRIN-endoscopes are already applied in wound healing studies. Here, the GRIN-lenses with diameters smaller than 3 mm enter small skin depressions. They reproduce the focus of a conventional laser scanning tomograph tens of mm apart in the specimen under study. We present first clinical measurements of elastin and SHG of collagen of in-vivo human skin of venous ulcers (ulcer curis).

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Multiphoton imaging: a view to understanding sulfur mustard lesions

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J. S.; Madren-Whalley, Janna S.

    2003-07-01

    It is well known that topical exposure to sulfur mustard (SM) produces persistent, incapacitating blisters of the skin. However, the primary lesions effecting epidermal-dermal separation and disabling of mechanisms for cutaneous repair remain uncertain. Immunofluorescent staining plus multiphoton imaging of human epidermal tissues and keratinocytes exposed to SM (400 μM x 5 min)have revealed that SM disrupts adhesion-complex molecules which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin-14 showed early, progressive, postexposure collapse of the K5/K14 cytoskeleton that resulted in ventral displacement of the nuclei beneath its collapsing filaments. This effectively corrupted the dynamic filament assemblies that link basal-cell nuclei to the extracellular matrix via α6β4-integrin and laminin-5. At 1 h postexposure, there was disruption in the surface organization of α6β4 integrins, associated displacement of laminin-5 anchoring sites and a concomitant loss of functional asymmetry. Accordingly, our multiphoton images are providing compelling evidence that SM induces prevesicating lesions that disrupt the receptor-ligand organization and cytoskeletal systems required for maintaining dermal-epidermal attachment, signal transduction, and polarized mobility.

  6. Multifunctional gold nanorod theragnostics probed by multi-photon imaging.

    PubMed

    Book Newell, Brittany; Wang, Yuling; Irudayaraj, Joseph

    2012-02-01

    This study exhibits the fabrication of target-specific Gold nanorods (GNRs) coupled with an anti-tumorigenic apoptotic drug and provides tracking of the labeled particles as they migrate through cells and release their drug-load to targeted cancer cells. We utilize the photoluminescence property of GNRs and their ability to be conjugated with multiple agents to transform facile rods to a targeted drug delivery vehicle. GNRs of aspect ratio 2.8 were conjugated with a targeting ligand, folic acid and an anthracycline drug, Doxorubicin. The multifunctional nanorods were then used to target folate receptor expressing cancers cells for the delivery of a concentration dependent dosage of Doxorubicin (DOX). By utilizing the photoluminescence of GNRs and the innate fluorescence of DOX, multi-photon fluorescence lifetime imaging was utilized to monitor the uptake of functionalized nanorods, the release of the drug and its localization in living cells. We show that these nano-vehicles successfully targeted cancer cells over expressing folate receptors and showed low toxicity to control cell lines. Release of DOX was observed in the cytoplasmic region and after 16 h was found to be redistributed in the nucleus resulting in cell death. Our theragnostic approach demonstrates the fabrication of multifunctional GNRs for targeted drug delivery and monitoring of the drug and the vehicle by multi-photon microscopy using fluorescence intensity and lifetime imaging.

  7. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.

    PubMed

    Larson, Daniel R; Zipfel, Warren R; Williams, Rebecca M; Clark, Stephen W; Bruchez, Marcel P; Wise, Frank W; Webb, Watt W

    2003-05-30

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

  8. Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo

    NASA Astrophysics Data System (ADS)

    Larson, Daniel R.; Zipfel, Warren R.; Williams, Rebecca M.; Clark, Stephen W.; Bruchez, Marcel P.; Wise, Frank W.; Webb, Watt W.

    2003-05-01

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

  9. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.

    PubMed

    Larson, Daniel R; Zipfel, Warren R; Williams, Rebecca M; Clark, Stephen W; Bruchez, Marcel P; Wise, Frank W; Webb, Watt W

    2003-05-30

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales. PMID:12775841

  10. Fringe-free, Background-free, Collinear Third Harmonic Generation FROG Measurements for Multiphoton Microscopy

    SciTech Connect

    Chadwick, R; Spahr, E; Squier, J A; Durfee, C G; Walker, B C; Fittinghoff, D N

    2006-07-21

    Collinear pulse measurement tools useful at the full numerical aperture (NA) of multiphoton microscope objectives are a necessity for a quantitative characterization of the femtosecond pulses focused by these systems. In this letter, we demonstrate a simple new technique, for characterizing the pulse at the focus in a multiphoton microscope. This technique, a background-free, fringe-free, form of frequency-resolved optical gating, uses the third harmonic signal generated from a glass coverslip. Here it is used to characterize 100 fs pulses (typical values for a multiphoton microscope) at the focus of a 0.65 NA objective.

  11. Lehmann-Symanzik-Zimmermann reduction approach to multiphoton scattering in coupled-resonator arrays

    NASA Astrophysics Data System (ADS)

    Shi, T.; Sun, C. P.

    2009-05-01

    We present a quantum field theoretical approach based on the Lehmann-Symanzik-Zimmermann reduction for the multiphoton scattering process in a nanoarchitecture consisting of the coupled-resonator arrays (CRA), which are also coupled to some artificial atoms as a controlling quantum node. By making use of this approach, we find the bound states of a single photon for an elementary unit, the T -type CRA, and explicitly obtain its multiphoton scattering S matrix in various situations. We also use this method to calculate the multiphoton S matrices for the more complex quantum network constructed with main T -type CRAs, such as a H -type CRA waveguide.

  12. Aqueous multiphoton lithography with multifunctional silk-centred bio-resists

    NASA Astrophysics Data System (ADS)

    Sun, Yun-Lu; Li, Qi; Sun, Si-Ming; Huang, Jing-Chun; Zheng, Bo-Yuan; Chen, Qi-Dai; Shao, Zheng-Zhong; Sun, Hong-Bo

    2015-10-01

    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.

  13. Aqueous multiphoton lithography with multifunctional silk-centred bio-resists

    PubMed Central

    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

  14. Aqueous multiphoton lithography with multifunctional silk-centred bio-resists.

    PubMed

    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 10(4)-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.

  15. Aqueous multiphoton lithography with multifunctional silk-centred bio-resists.

    PubMed

    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 10(4)-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

  16. Resonance Enhanced Multi-photon Spectroscopy of DNA

    NASA Astrophysics Data System (ADS)

    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

  17. Nonperturbative methods in the problem of multiphoton excitation of atom by squeezed light

    NASA Technical Reports Server (NTRS)

    Belousov, A. V.; Kovarsky, V. A.

    1993-01-01

    Multiphoton detectors for the strong squeezed light vacuum are considered. The result is compared with the perturbation theory. It is shown that as the degree of squeezing is increased the statistical factor decreases.

  18. Multiphoton FLIM: a reliable FRET detection tool in cell biological applications

    NASA Astrophysics Data System (ADS)

    Krishnan, Ramanujan V.; Biener, Eva; Centonze, Victoria E.; Gertler, Arieh; Herman, Brian A.

    2004-06-01

    Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.

  19. High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues

    PubMed Central

    Lin, Chun-Yu; Li, Pei-Kao; Cheng, Li-Chung; Li, Yi-Cheng; Chang, Chia-Yuan; Chiang, Ann-Shyn; Dong, Chen Yuan; Chen, Shean-Jen

    2015-01-01

    In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 106 μm3/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen. PMID:25780739

  20. Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

    PubMed Central

    Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

    2009-01-01

    We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

  1. High-Resolution Multiphoton Imaging of Tumors In Vivo

    PubMed Central

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

    2014-01-01

    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. PMID:21969629

  2. Multi-photon absorption limits to heralded single photon sources

    PubMed Central

    Husko, Chad A.; Clark, Alex S.; Collins, Matthew J.; De Rossi, Alfredo; Combrié, Sylvain; Lehoucq, Gaëlle; Rey, Isabella H.; Krauss, Thomas F.; Xiong, Chunle; Eggleton, Benjamin J.

    2013-01-01

    Single photons are of paramount importance to future quantum technologies, including quantum communication and computation. Nonlinear photonic devices using parametric processes offer a straightforward route to generating photons, however additional nonlinear processes may come into play and interfere with these sources. Here we analyse spontaneous four-wave mixing (SFWM) sources in the presence of multi-photon processes. We conduct experiments in silicon and gallium indium phosphide photonic crystal waveguides which display inherently different nonlinear absorption processes, namely two-photon (TPA) and three-photon absorption (ThPA), respectively. We develop a novel model capturing these diverse effects which is in excellent quantitative agreement with measurements of brightness, coincidence-to-accidental ratio (CAR) and second-order correlation function g(2)(0), showing that TPA imposes an intrinsic limit on heralded single photon sources. We build on these observations to devise a new metric, the quantum utility (QMU), enabling further optimisation of single photon sources. PMID:24186400

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Monitoring wound healing by multiphoton tomography/endoscopy

    NASA Astrophysics Data System (ADS)

    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.

  5. Multiphoton Microscopy and Interaction of Intense Light Pulses with Polymers

    NASA Astrophysics Data System (ADS)

    Guay, Jean-Michel

    2011-07-01

    The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

  6. Performance evaluation of a sensorless adaptive optics multiphoton microscope.

    PubMed

    Skorsetz, Martin; Artal, Pablo; Bueno, Juan M

    2016-03-01

    A wavefront sensorless adaptive optics technique was combined with a custom-made multiphoton microscope to correct for specimen-induced aberrations. A liquid-crystal-on-silicon (LCoS) modulator was used to systematically generate Zernike modes during image recording. The performance of the instrument was evaluated in samples providing different nonlinear signals and the benefit of correcting higher order aberrations was always noticeable (in both contrast and resolution). The optimum aberration pattern was stable in time for the samples here involved. For a particular depth location within the sample, the wavefront to be precompensated was independent on the size of the imaged area (up to ∼ 360 × 360 μm(2)). The mode combination optimizing the recorded image depended on the Zernike correction control sequence; however, the final images hardly differed. At deeper locations, a noticeable dominance of spherical aberration was found. The influence of other aberration terms was also compared to the effect of the spherical aberration.

  7. Molecule-specific darkfield and multiphoton imaging using gold nanocages

    NASA Astrophysics Data System (ADS)

    Powless, Amy J.; Jenkins, Samir V.; McKay, Mary Lee; Chen, Jingyi; Muldoon, Timothy J.

    2015-03-01

    Due to their robust optical properties, biological inertness, and readily adjustable surface chemistry, gold nanostructures have been demonstrated as contrast agents in a variety of biomedical imaging applications. One application is dynamic imaging of live cells using bioconjugated gold nanoparticles to monitor molecule trafficking mechanisms within cells; for instance, the regulatory pathway of epidermal growth factor receptor (EGFR) undergoing endocytosis. In this paper, we have demonstrated a method to track endocytosis of EGFR in MDA-MB-468 breast adenocarcinoma cells using bioconjugated gold nanocages (AuNCs) and multiphoton microscopy. Dynamic imaging was performed using a time series capture of 4 images every minute for one hour. Specific binding and internalization of the bioconjugated AuNCs was observed while the two control groups showed non-specific binding at fewer surface sites, leading to fewer bound AuNCs and no internalization.

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

    PubMed

    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.

  9. Multi-photon absorption limits to heralded single photon sources

    NASA Astrophysics Data System (ADS)

    Husko, Chad A.; Clark, Alex S.; Collins, Matthew J.; de Rossi, Alfredo; Combrié, Sylvain; Lehoucq, Gaëlle; Rey, Isabella H.; Krauss, Thomas F.; Xiong, Chunle; Eggleton, Benjamin J.

    2013-11-01

    Single photons are of paramount importance to future quantum technologies, including quantum communication and computation. Nonlinear photonic devices using parametric processes offer a straightforward route to generating photons, however additional nonlinear processes may come into play and interfere with these sources. Here we analyse spontaneous four-wave mixing (SFWM) sources in the presence of multi-photon processes. We conduct experiments in silicon and gallium indium phosphide photonic crystal waveguides which display inherently different nonlinear absorption processes, namely two-photon (TPA) and three-photon absorption (ThPA), respectively. We develop a novel model capturing these diverse effects which is in excellent quantitative agreement with measurements of brightness, coincidence-to-accidental ratio (CAR) and second-order correlation function g(2)(0), showing that TPA imposes an intrinsic limit on heralded single photon sources. We build on these observations to devise a new metric, the quantum utility (QMU), enabling further optimisation of single photon sources.

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

    PubMed

    Antonello, Jacopo; van Werkhoven, Tim; Verhaegen, Michel; Truong, Hoa H; Keller, Christoph U; Gerritsen, Hans C

    2014-06-01

    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 aberration correction is achieved by maximizing a suitable image quality metric. We implement a model-based aberration correction algorithm in a second-harmonic microscope. The tip, tilt, and defocus aberrations are removed from the basis functions used for the control of the DM, as these aberrations induce distortions in the acquired images. We compute the parameters of a quadratic polynomial that is used to model the image quality metric directly from experimental input-output measurements. Finally, we apply the aberration correction by maximizing the image quality metric using the least-squares estimate of the unknown aberration.

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

    PubMed

    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.

  12. Watching stem cells at work with a flexible multiphoton tomograph

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada; Hoffmann, Robert; Weinigel, Martin; König, Karsten

    2012-03-01

    There is a high demand for non-invasive imaging techniques that allow observation of stem cells in their native environment without significant input on cell metabolism, reproduction, and behavior. Easy accessible hair follicle pluripotent stem cells in the bulge area and dermal papilla are potential sources for stem cell based therapy. It has been shown that these cells are able to generate hair, non-follicle skin cells, nerves, vessels, smooth muscles etc. and may participate in wound healing processes. We report on the finding of nestin-GFP expressing stem cells in their native niche in the bulge of the hair follicle of living mice by using high-resolution in-vivo multiphoton tomography. The 3D imaging with submicron resolution was based on two-photon induced fluorescence and second harmonic generation (SHG) of collagen. Migrating stem cells from the bulge to their microenvironment have been detected inside the skin during optical deep tissue sectioning.

  13. Design of a fiber-optic multiphoton microscopy handheld probe

    PubMed Central

    Zhao, Yuan; Sheng, Mingyu; Huang, Lin; Tang, Shuo

    2016-01-01

    We have developed a fiber-optic multiphoton microscopy (MPM) system with handheld probe using femtosecond fiber laser. Here we present the detailed optical design and analysis of the handheld probe. The optical systems using Lightpath 352140 and 352150 as objective lens were analyzed. A custom objective module that includes Lightpath 355392 and two customized corrective lenses was designed. Their performances were compared by wavefront error, field curvature, astigmatism, F-θ error, and tolerance in Zemax simulation. Tolerance analysis predicted the focal spot size to be 1.13, 1.19 and 0.83 µm, respectively. Lightpath 352140 and 352150 were implemented in experiment and the measured lateral resolution was 1.22 and 1.3 µm, respectively, which matched with the prediction. MPM imaging by the handheld probe were conducted on leaf, fish scale and rat tail tendon. The MPM resolution can potentially be improved by the custom objective module.

  14. Design of a fiber-optic multiphoton microscopy handheld probe

    PubMed Central

    Zhao, Yuan; Sheng, Mingyu; Huang, Lin; Tang, Shuo

    2016-01-01

    We have developed a fiber-optic multiphoton microscopy (MPM) system with handheld probe using femtosecond fiber laser. Here we present the detailed optical design and analysis of the handheld probe. The optical systems using Lightpath 352140 and 352150 as objective lens were analyzed. A custom objective module that includes Lightpath 355392 and two customized corrective lenses was designed. Their performances were compared by wavefront error, field curvature, astigmatism, F-θ error, and tolerance in Zemax simulation. Tolerance analysis predicted the focal spot size to be 1.13, 1.19 and 0.83 µm, respectively. Lightpath 352140 and 352150 were implemented in experiment and the measured lateral resolution was 1.22 and 1.3 µm, respectively, which matched with the prediction. MPM imaging by the handheld probe were conducted on leaf, fish scale and rat tail tendon. The MPM resolution can potentially be improved by the custom objective module. PMID:27699109

  15. Plasma induced by resonance enhanced multiphoton ionization in inert gas

    SciTech Connect

    Shneider, Mikhail N.; Zhang Zhili; Miles, Richard B.

    2007-12-15

    We present a detailed model for the evolution of resonance enhanced multiphoton ionization (REMPI) produced plasma during and after the ionizing laser pulse in inert gas (argon, as an example) at arbitrary pressures. Our theory includes the complete process of the REMPI plasma generation and losses, together with the changing gas thermodynamic parameters. The model shows that the plasma expansion follows a classical ambipolar diffusion and that gas heating results in a weak shock or acoustic wave. The gas becomes involved in the motion not only from the pressure gradient due to the heating, but also from the momentum transfer from the charged particles to gas atoms. The time dependence of the total number of electrons computed in theory matches closely with the results of coherent microwave scattering experiments.

  16. Multiphoton ionization and third-harmonic generation in atoms and molecules

    SciTech Connect

    Compton, R.N.

    1982-01-01

    Resonantly enhanced multiphoton ionization (REMPI) provides a powerful new method for investigating atomic and molecular energy levels. The method is particularly useful in discovering and characterizing certain optically forbidden transitions. The method is particularly well suited for studying Rydberg transitions in molecules and is experimentally easier than the traditional use of far ultraviolet radiation in conventional spectroscopy. Research on multiphoton ionization and third-harmonic generation is reviewed. (WHK)

  17. High (1 GHz) repetition rate compact femtosecond laser: A powerful multiphoton tool for nanomedicine and nanobiotechnology

    NASA Astrophysics Data System (ADS)

    Ehlers, A.; Riemann, I.; Martin, S.; Le Harzic, R.; Bartels, A.; Janke, C.; König, K.

    2007-07-01

    Multiphoton tomography of human skin and nanosurgery of human chromosomes have been performed with a 1GHz repetition rate laser by the use of the commercially available femtosecond multiphoton laser tomograph DermaInspect as well as a compact galvoscanning microscope. We performed the autofluorescence tomography up to 100μm in the depth of human skin. Submicron cutting lines and hole drillings have been conducted on labeled human chromosomes.

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

    NASA Astrophysics Data System (ADS)

    Gard, Bryan; Cross, Robert; Anisimov, Petr; Lee, Hwang; Dowling, Jonathan

    2012-06-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Separation of deuterium by IR multiphoton decomposition of chlorodifluoromethane. IR multiphoton absorption by and decomposition of a CF 2DCl/CF 2HCl mixture

    NASA Astrophysics Data System (ADS)

    Kutschke, K. O.; Gauthier, M.; Hackett, P. A.

    1983-08-01

    IR multiphoton absorption by various pressures of CF 2HCl or of 1??? CF 2DCl in CF 2HCl was studied at several 9P and 10R lines of the CO 2 laser using temporally unmodified pulses. Values of < n> for the individual molecules, and thus the optical selectivities, obtained from these data indicate that the molecule is not a promising candidate for deuterium separation in these frequency ranges. A measurement of D-isotope depletion during the multiphoton decomposition of the same mixture at 9P42 (1025.2 cm -1) gave a minimum estimate of the isotopic selectivity of the dissociation process of 35.

  2. Intravital microscopy of the capillary perfusion in the corium limbi of the third toe of the minipig.

    PubMed

    Hiebl, B; Mrowietz, C; Braune, S; Franke, R P; Plendl, J; Jung, F

    2009-01-01

    Several methods are available today for the investigation of microcirculation in animal models, but they can be invasive and time-consuming depending on the area investigated. In particular, non-invasive methods that can be conducted rapidly and without dye or tracer injections are in demand. The cutaneous microcirculation can be easily studied in the dorsal corium limbi of the third toe of the porcine forelimb using intravital microscopy - analogous to nail fold capillary microscopy in humans. The capillary microscopy system consists of a reflected-light microscope with a cold light source, green and infrared filters and a video camera. The video sequences were recorded using the image capture system Framegrabber (Imagenation PXC-200) and a PC (with an Intel Core 2 Duo processor, 1024 MB RAM, 160 GB hard disk, Windows XP Pro), and stored via a DVD recorder (Panasonic LQ-MD800). Quantification of capillary erythrocyte flow velocities was performed using the computer-assisted image analysis system Cap Image Version 8.5 which includes a movie tool as a video sequence storage medium. The method allows estimation of capillary density and tortuosity as well as capillary circulation in the anesthetized pig within a few minutes. First measurements were made after anesthesia induction followed by further measurements during anesthesia maintenance (3 minutes each). No differences in capillary circulation were found. The present method is thus very well suited for long-term microcirculation measurements in pigs, e.g., to evaluate therapeutic interventions in the ischemic limb model. PMID:19713612

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

    PubMed

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

    2013-06-01

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

  4. Intravital Two-Photon Imaging of Lymphocytes Crossing High Endothelial Venules and Cortical Lymphatics in the Inguinal Lymph Node.

    PubMed

    Park, Chung; Hwang, Il-Young; Kehrl, John H

    2016-01-01

    Lymphocyte recirculation through lymph nodes (LNs) requires their crossing of endothelial barriers present in blood vessels and lymphatics by means of chemoattractant-triggered cell migration. The chemoattractant-chemoattractant receptor axes that predominately govern the trafficking of lymphocytes into, and out of, LNs are CCL19/CCR7 and sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1PR1), respectively. Blood-borne lymphocytes downregulate S1PR1 and use CCR7 signaling to adhere to high endothelial venules (HEVs) for transmigration. During their LN residency, recirculating lymphocytes reacquire S1PR1 and attenuate their sensitivity to chemokines. Eventually lymphocytes exit the LN by entering the cortical or medullary lymphatics, a process that depends upon S1PR1 signaling. Upon entering into the lymph, lymphocytes lose their polarity, downregulate their sensitivity to S1P due to the high concentration of S1P, and upregulate their sensitivity to chemokines. However, many of the details of lymphocyte transmigration across endothelial barriers remain poorly understood. Intravital two-photon imaging with advanced microscope technologies not only allows the real-time observation of immune cells in intact LN of a live mouse, but also provides a means to monitor the interactions between circulating lymphocytes and stromal barriers. Here, we describe procedures to visualize lymphocytes engaging and crossing HEVs, and approaching and crossing the cortical lymphatic endothelium to enter the efferent lymph in live mice. PMID:27271904

  5. REAL-TIME INTRAVITAL IMAGING ESTABLISHES TUMOUR-ASSOCIATED MACROPHAGES AS THE EXTRASKELETAL TARGET OF BISPHOSPHONATE ACTION IN CANCER

    PubMed Central

    Junankar, Simon; Shay, Gemma; Jurczyluk, Julie; Ali, Naveid; Down, Jenny; Pocock, Nicholas; Parker, Andrew; Nguyen, Akira; Sun, Shuting; Kashemirov, Boris; McKenna, Charles E.; Croucher, Peter I.; Swarbrick, Alexander; Weilbaecher, Katherine; Phan, Tri Giang; Rogers, Michael J.

    2014-01-01

    Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their anti-resorptive effects on the skeleton. However, since bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their anti-tumour action, particularly on cells outside of bone, remain unknown. Here we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumours, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumour-associated macrophages (TAMs), but not tumour cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumour of a patient and showed the resected tumour to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumours outside the skeleton and that their anti-tumour activity is likely to be mediated via TAMs. PMID:25312016

  6. 4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis.

    PubMed

    Sullivan, David P; Watson, Richard L; Muller, William A

    2016-09-01

    Leukocyte transendothelial migration (TEM) is an essential component of the inflammatory response. In vitro studies with human cells have demonstrated that platelet/endothelial cell adhesion molecule (PECAM) functions upstream of CD99 during TEM; however, results in vivo with mice have been apparently contradictory. In this study we use four-dimensional (4D) intravital microscopy to demonstrate that the site and order of function of PECAM and CD99 in vivo are dependent on the strain of mice. In FVB/n mice, PECAM functions upstream of CD99, as in human cells in vitro, and blocking antibodies against either molecule arrest neutrophils before they traverse the endothelium. However, in C57BL/6 mice, PECAM and CD99 appear to function at a different step, as the same antibodies arrest leukocyte migration through the endothelial basement membrane. These results are the first direct comparison of PECAM and CD99 function in different murine strains as well as the first demonstration of the sequential function of PECAM and CD99 in vivo. PMID:27422987

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

    NASA Astrophysics Data System (ADS)

    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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    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.

  10. Application of Multiphoton Microscopy in Dermatological Studies: a Mini-Review

    PubMed Central

    Yew, Elijah; Rowlands, Christopher

    2014-01-01

    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. PMID:25075226

  11. The multiphoton ionization of uranium hexafluoride. Revision 1

    SciTech Connect

    Armstrong, D.P.

    1992-05-01

    Multiphoton ionization (MPI) time-of-flight mass spectroscopy and photoelectron spectroscopy studies of UF{sub 6} have been conducted using focused light from the Nd:YAG laser fundamental ({lambda}=1064 nm) and its harmonics ({lambda}=532, 355, or 266 nm), as well as other wavelengths provided by a tunable dye laser. The MPI mass spectra are dominated by the singly and multiply charged uranium ions rather than by the UF{sub x}{sup +} fragment ions even at the lowest laser power densities at which signal could be detected. The laser power dependence of U{sup n+} ions signals indicates that saturation can occur for many of the steps required for their ionization. In general, the doubly-charged uranium ion (U{sup 2+}) intensity is much greater than that of the singly-charged uranium ion (U{sup +}). For the case of the tunable dye laser experiments, the U{sup n+} (n = 1- 4) wavelength dependence is relatively unstructured and does not show observable resonance enhancement at known atomic uranium excitation wavelengths. The dominance of the U{sup 2+} ion and the absence or very small intensities of UF{sub x}{sup +} fragments, along with the unsaturated wavelength dependence, indicate that mechanisms may exist other than ionization of bare U atoms after the stepwise photodissociation of F atoms from the parent molecule.

  12. Superresolved multiphoton microscopy with spatial frequency-modulated imaging.

    PubMed

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

    2016-06-14

    Superresolved 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 superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved 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 2η below the diffraction limit, where η is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media. PMID:27231219

  13. Security of quantum key distribution with multiphoton components.

    PubMed

    Yin, Hua-Lei; Fu, Yao; Mao, Yingqiu; Chen, Zeng-Bing

    2016-01-01

    Most qubit-based quantum key distribution (QKD) protocols extract the secure key merely from single-photon component of the attenuated lasers. However, with the Scarani-Acin-Ribordy-Gisin 2004 (SARG04) QKD protocol, the unconditionally secure key can be extracted from the two-photon component by modifying the classical post-processing procedure in the BB84 protocol. Employing the merits of SARG04 QKD protocol and six-state preparation, one can extract secure key from the components of single photon up to four photons. In this paper, we provide the exact relations between the secure key rate and the bit error rate in a six-state SARG04 protocol with single-photon, two-photon, three-photon, and four-photon sources. By restricting the mutual information between the phase error and bit error, we obtain a higher secure bit error rate threshold of the multiphoton components than previous works. Besides, we compare the performances of the six-state SARG04 with other prepare-and-measure QKD protocols using decoy states. PMID:27383014

  14. Security of quantum key distribution with multiphoton components.

    PubMed

    Yin, Hua-Lei; Fu, Yao; Mao, Yingqiu; Chen, Zeng-Bing

    2016-07-07

    Most qubit-based quantum key distribution (QKD) protocols extract the secure key merely from single-photon component of the attenuated lasers. However, with the Scarani-Acin-Ribordy-Gisin 2004 (SARG04) QKD protocol, the unconditionally secure key can be extracted from the two-photon component by modifying the classical post-processing procedure in the BB84 protocol. Employing the merits of SARG04 QKD protocol and six-state preparation, one can extract secure key from the components of single photon up to four photons. In this paper, we provide the exact relations between the secure key rate and the bit error rate in a six-state SARG04 protocol with single-photon, two-photon, three-photon, and four-photon sources. By restricting the mutual information between the phase error and bit error, we obtain a higher secure bit error rate threshold of the multiphoton components than previous works. Besides, we compare the performances of the six-state SARG04 with other prepare-and-measure QKD protocols using decoy states.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Security of quantum key distribution with multiphoton components

    PubMed Central

    Yin, Hua-Lei; Fu, Yao; Mao, Yingqiu; Chen, Zeng-Bing

    2016-01-01

    Most qubit-based quantum key distribution (QKD) protocols extract the secure key merely from single-photon component of the attenuated lasers. However, with the Scarani-Acin-Ribordy-Gisin 2004 (SARG04) QKD protocol, the unconditionally secure key can be extracted from the two-photon component by modifying the classical post-processing procedure in the BB84 protocol. Employing the merits of SARG04 QKD protocol and six-state preparation, one can extract secure key from the components of single photon up to four photons. In this paper, we provide the exact relations between the secure key rate and the bit error rate in a six-state SARG04 protocol with single-photon, two-photon, three-photon, and four-photon sources. By restricting the mutual information between the phase error and bit error, we obtain a higher secure bit error rate threshold of the multiphoton components than previous works. Besides, we compare the performances of the six-state SARG04 with other prepare-and-measure QKD protocols using decoy states. PMID:27383014

  17. Multiphoton imaging the disruptive nature of sulfur mustard lesions

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J.; Braue, Catherine R.; Dillman, James F.

    2005-03-01

    Sulfur mustard [bis-2-chloroethyl sulfide] is a vesicating agent first used as a weapon of war in WWI. It causes debilitating blisters at the epidermal-dermal junction and involves molecules that are also disrupted by junctional epidermolysis bullosa (JEB) and other blistering skin diseases. Despite its recurring use in global conflicts, there is still no completely effective treatment. We have shown by imaging human keratinocytes in cell culture and in intact epidermal tissues that the basal cells of skin contain well-organized molecules (keratins K5/K14, α6β4 integrin, laminin 5 and α3β1 integrin) that are early targets of sulfur mustard. Disruption and collapse of these molecules is coincident with nuclear displacement, loss of functional asymmetry, and loss of polarized mobility. The progression of this pathology precedes basal cell detachment by 8-24 h, a time equivalent to the "clinical latent phase" that defines the extant period between agent exposure and vesication. Our images indicate that disruption of adhesion-complex molecules also impairs cytoskeletal proteins and the integration of structures required for signal transduction and tissue repair. We have recently developed an optical system to test this hypothesis, i.e., to determine whether and how the early disruption of target molecules alters signal transduction. This environmentally controlled on-line system provides a nexus for real-time correlation of imaged lesions with DNA microarray analysis, and for using multiphoton microscopy to facilitate development of more effective treatment strategies.

  18. Dynamics of cavityless lasing generated by ultrafast multiphoton excitation

    NASA Astrophysics Data System (ADS)

    Kimberg, Victor; Polyutov, Sergey; Gel'Mukhanov, Faris; Ågren, Hans; Baev, Alexander; Zheng, Qingdong; He, Guang S.

    2006-09-01

    A dynamical theory is developed with the purpose of explaining recent experimental results on multiphoton-excited amplified stimulated emission (ASE). Several conspicuous features of this experiment are analyzed, like the threshold dependence of the spectral profile on the pump intensity, and spectral shifts of the ASE pulses co- and counterpropagating relative to the pump pulse. Two models are proposed and evaluated, one based on the isolated molecule and another which involves solvent interaction. The spectral shift between the forward and backward ASE pulses arises in the first model through the competition between the ASE transitions from the pumped vibrational levels and from the bottom of the excited-state well, while in the solvent-related model the dynamical solute-solvent interaction leads to a relaxed excited state, producing an additional ASE channel. In the latter model the additional redshifted ASE channel makes the dynamics of ASE essentially different from that in the molecular model because the formation of the relaxed state takes a longer time. The variation of the pump intensity influences strongly the relative intensities of the different ASE channels and, hence, the spectral shape of ASE in both models. The regime of ASE changes character when the pump intensity crosses a threshold value. Such a phase transition occurs when the ASE rate approaches the rate of vibrational relaxation or the rate of solute-solvent relaxation in the first excited state.

  19. Multi-photon processes in alkali metal vapors

    NASA Astrophysics Data System (ADS)

    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.

  20. Performance evaluation of a sensorless adaptive optics multiphoton microscope.

    PubMed

    Skorsetz, Martin; Artal, Pablo; Bueno, Juan M

    2016-03-01

    A wavefront sensorless adaptive optics technique was combined with a custom-made multiphoton microscope to correct for specimen-induced aberrations. A liquid-crystal-on-silicon (LCoS) modulator was used to systematically generate Zernike modes during image recording. The performance of the instrument was evaluated in samples providing different nonlinear signals and the benefit of correcting higher order aberrations was always noticeable (in both contrast and resolution). The optimum aberration pattern was stable in time for the samples here involved. For a particular depth location within the sample, the wavefront to be precompensated was independent on the size of the imaged area (up to ∼ 360 × 360 μm(2)). The mode combination optimizing the recorded image depended on the Zernike correction control sequence; however, the final images hardly differed. At deeper locations, a noticeable dominance of spherical aberration was found. The influence of other aberration terms was also compared to the effect of the spherical aberration. PMID:26469361

  1. Security of quantum key distribution with multiphoton components

    NASA Astrophysics Data System (ADS)

    Yin, Hua-Lei; Fu, Yao; Mao, Yingqiu; Chen, Zeng-Bing

    2016-07-01

    Most qubit-based quantum key distribution (QKD) protocols extract the secure key merely from single-photon component of the attenuated lasers. However, with the Scarani-Acin-Ribordy-Gisin 2004 (SARG04) QKD protocol, the unconditionally secure key can be extracted from the two-photon component by modifying the classical post-processing procedure in the BB84 protocol. Employing the merits of SARG04 QKD protocol and six-state preparation, one can extract secure key from the components of single photon up to four photons. In this paper, we provide the exact relations between the secure key rate and the bit error rate in a six-state SARG04 protocol with single-photon, two-photon, three-photon, and four-photon sources. By restricting the mutual information between the phase error and bit error, we obtain a higher secure bit error rate threshold of the multiphoton components than previous works. Besides, we compare the performances of the six-state SARG04 with other prepare-and-measure QKD protocols using decoy states.

  2. Laser multiphoton ionization of tetrakis(dimethylamino)ethylene.

    PubMed

    Smith, Byron H; Compton, Robert N

    2014-09-01

    The tetrakis(dimethylamino)ethylene (TDAE) molecule possesses the lowest known molecular ionization potential (<5.4 eV) and exhibits an intense Rydberg series between the first and second ionization limit (∼14 eV). The ionization of TDAE using multiphoton ionization photoelectron spectroscopy was carried out using laser light at a variety of wavelengths with a hemispherical energy analyzer. Interestingly, photoelectron signal due to direct two-photon ionization was not seen, rather ionization from a fluorescent charge-transfer state located ∼2.5 eV below the ionization limit was evident and in general agreement with a previous study. In addition, a second intense peak exists corresponding to thermal energy electrons. Measurements of the angular distribution for the electrons due to photoionization from the intermediate state are peaked along the electric field vector of the laser and the thermal electrons direction is independent of this angle. From this, we propose that the thermal peak is most likely due to thermionic emission initiated through excitation of a known long-lived Rydberg state at ∼6.5 eV. Alternately, we speculate that excitation leading to thermionic emission could result from a "collective" excitation mechanism.

  3. Compact fixed wavelength femtosecond oscillators for multi-photon imaging

    NASA Astrophysics Data System (ADS)

    Hakulinen, T.; Klein, J.; Zadoyan, R.; Baldacchini, T.; Franke, T.

    2015-03-01

    In recent years two-photon microscopy with fixed-wavelength has raised increasing interest in life-sciences: Two-photon (2P) absorption spectra of common dyes are broader than single-photon ones. Therefore, excitation of several dyes simultaneously with a single IR laser wavelength is feasible and could be seen as an advantage in 2P microscopy. We used pulsed fixed-wavelength infrared lasers with center wavelength at 1040 nm, for two-photon microscopy in a variety of biologically relevant samples, among these a mouse brain sample, a mouse artery (within the animal, acute preparation), and a preparation of mouse bladder. The 1040 nm laser proved to be efficient not only in exciting fluorescence from yellow fluorescent protein (YFP) and red fluorescent dyes, but also for second harmonic generation (SHG) signals from muscle tissue and collagen. With this work we demonstrate that economical, small-footprint fixedwavelength lasers can present an interesting alternative to tunable lasers that are commonly used in multiphoton microscopy.

  4. Dynamics of cavityless lasing generated by ultrafast multiphoton excitation

    SciTech Connect

    Kimberg, Victor; Polyutov, Sergey; Gel'mukhanov, Faris; A ring gren, Hans; Baev, Alexander; Zheng Qingdong; He, Guang S.

    2006-09-15

    A dynamical theory is developed with the purpose of explaining recent experimental results on multiphoton-excited amplified stimulated emission (ASE). Several conspicuous features of this experiment are analyzed, like the threshold dependence of the spectral profile on the pump intensity, and spectral shifts of the ASE pulses co- and counterpropagating relative to the pump pulse. Two models are proposed and evaluated, one based on the isolated molecule and another which involves solvent interaction. The spectral shift between the forward and backward ASE pulses arises in the first model through the competition between the ASE transitions from the pumped vibrational levels and from the bottom of the excited-state well, while in the solvent-related model the dynamical solute-solvent interaction leads to a relaxed excited state, producing an additional ASE channel. In the latter model the additional redshifted ASE channel makes the dynamics of ASE essentially different from that in the molecular model because the formation of the relaxed state takes a longer time. The variation of the pump intensity influences strongly the relative intensities of the different ASE channels and, hence, the spectral shape of ASE in both models. The regime of ASE changes character when the pump intensity crosses a threshold value. Such a phase transition occurs when the ASE rate approaches the rate of vibrational relaxation or the rate of solute-solvent relaxation in the first excited state.

  5. Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Mulholland, William J.; Kendall, Mark A. F.; White, Nick; Bellhouse, Brian J.

    2004-11-01

    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.

  6. Multiphoton and photothermal imaging of molecular events in cancer

    NASA Astrophysics Data System (ADS)

    Skala, Melissa

    2010-10-01

    Optical techniques are attractive for monitoring disease processes in living tissues because they are relatively cheap, non-invasive and provide a wealth of functional information. Multiphoton microscopy (MPM) and Optical Coherence Tomography (OCT) are two types of three-dimensional optical imaging modalities that have demonstrated great utility in pre-clinical models of disease. These techniques are particularly useful for identifying metabolic and molecular biomarkers in cancer. These biomarkers can be used to identify the mechanisms of tumor growth, and to predict the response of a particular tumor to treatment. Specifically, MPM of the co-enzymes NADH and FAD was used to quantify metabolic changes associated with developing cancers in vivo. This imaging technique exploits intrinsic sources of tissue contrast and thus does not require contrast agents. Ongoing work combines this metabolic imaging technique with vascular imaging to provide a comprehensive picture of oxygen supply and demand with tumor therapy. Molecular signaling represents a third critical component in tumor physiology. To this end we have recently developed photothermal OCT, which combines coherent detection with laser-heated gold nanoparticles to achieve high-resolution molecular contrast at deeper depths than MPM. This multi-functional imaging platform will provide unprecedented insight into oxygen supply and demand, and molecular signaling in response to tumor growth and targeted cancer therapies in pre-clinical models.

  7. The analysis of aging skin based on multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Achieving molecular selectivity in imaging using multiphoton Raman spectroscopy techniques

    SciTech Connect

    Holtom, Gary R. ); Thrall, Brian D. ); Chin, Beek Yoke ); Wiley, H Steven ); Colson, Steven D. )

    2000-12-01

    In the case of most imaging methods, contrast is generated either by physical properties of the sample (Differential Image Contrast, Phase Contrast), or by fluorescent labels that are localized to a particular protein or organelle. Standard Raman and infrared methods for obtaining images are based upon the intrinsic vibrational properties of molecules, and thus obviate the need for attached flurophores. Unfortunately, they have significant limitations for live-cell imaging. However, an active Raman method, called Coherent Anti-Stokes Raman Scattering (CARS), is well suited for microscopy, and provides a new means for imaging specific molecules. Vibrational imaging techniques, such as CARS, avoid problems associated with photobleaching and photo-induced toxicity often associated with the use of fluorescent labels with live cells. Because the laser configuration needed to implement CARS technology is similar to that used in other multiphoton microscopy methods, such as two -photon fluorescence and harmonic generation, it is possible to combine imaging modalities, thus generating simultaneous CARS and fluorescence images. A particularly powerful aspect of CARS microscopy is its ability to selectively image deuterated compounds, thus allowing the visualization of molecules, such as lipids, that are chemically indistinguishable from the native species.

  10. Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

    The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis.

    PubMed

    Vanheule, Eline; Geerts, Anja M; Van Huysse, Jacques; Schelfhout, Daphné; Praet, Marleen; Van Vlierberghe, Hans; De Vos, Martine; Colle, Isabelle

    2008-12-01

    This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl(4)) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (alpha-SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl(4) and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl(4) mice, CV were enlarged, with marked sinusoidal-free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and alpha-SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl(4) and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.

  13. Possibility of efficient generation of multiphoton entangled states using a one-dimensional nonlinear photonic crystal

    SciTech Connect

    Dong Yunxia; Zhang Xiangdong

    2010-03-15

    A rigorous quantum theory for the generation of multiphoton entangled states based on two consecutive three-frequency interactions of waves in a one-dimensional nonlinear photonic crystal is developed using the field expansion and differentiation methods. The three-photon correlation coefficient and the average photon numbers generated in the structure are calculated. All order expansion terms are included in the calculation. The generation conditions for multiphoton entangled states in such a structure are also analyzed. It is shown that the created photons in the present structures obey the super-Poisson statistics at the interacting frequencies and are in a multiparticle entangled state. This means the nonlinear photonic crystal can be applied as a highly efficient source of an entangled multiphoton for highly integrated all-optical circuits.

  14. Label-free multiphoton imaging and photoablation of preinvasive cancer cells

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Wu, Guizhu; Zhu, Xiaoqin; Jiang, Xingshan; Xie, Shusen

    2012-01-01

    Detection and treatment of early lesions in epithelial tissue offer several possibilities for curing cancer, but it is challenging. Here, we present an optical technique, the combination of multiphoton imaging and absorption, to label-freely detect and ablate preinvasive cancer cells in epithelial tissue. We find that multiphoton imaging can label-freely visualize the principal features of nuclear atypia associated with epithelial precancerous lesions, and the spatial localization of multiphoton absorption can perform targeted ablation of preinvasive cancer cells with micrometer-sized volume precision. These results indicate that this optical technique has the capability to label-freely visualize and remove preinvasive cancer cells in epithelial tissue. This study highlights the potential of this technique as a "seek-and-treat" tool for early lesions in epithelial tissue.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging.

    PubMed

    Kumar, S; Dunsby, C; De Beule, P A A; Owen, D M; Anand, U; Lanigan, P M P; Benninger, R K P; Davis, D M; Neil, M A A; Anand, P; Benham, C; Naylor, A; French, P M W

    2007-10-01

    We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor. PMID:19550524

  17. Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two-photon microscopy.

    PubMed

    Ricard, Clément; Lamasse, Lisa; Jaouen, Alexandre; Rougon, Geneviève; Debarbieux, Franck

    2016-06-01

    Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord.

  18. What ticks do under your skin: two-photon intravital imaging of Ixodes scapularis feeding in the presence of the lyme disease spirochete.

    PubMed

    Bockenstedt, Linda K; Gonzalez, David; Mao, Jialing; Li, Ming; Belperron, Alexia A; Haberman, Ann

    2014-03-01

    Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.

  19. What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete

    PubMed Central

    Bockenstedt, Linda K.; Gonzalez, David; Mao, Jialing; Li, Ming; Belperron, Alexia A.; Haberman, Ann

    2014-01-01

    Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding. PMID:24600332

  20. Real-time optical diagnosis of gastric cancer with serosal invasion using multiphoton imaging

    PubMed Central

    Yan, Jun; Zheng, Yu; Zheng, Xiaoling; Liu, Zhangyuanzhu; Liu, Wenju; Chen, Dexin; Dong, Xiaoyu; Li, Kai; Liu, Xiumin; Chen, Gang; Lu, Jianping; Chen, Jianxin; Zhuo, Shuangmu; Li, Guoxin

    2016-01-01

    A real-time optical biopsy, which could determine tissue histopathology, would be of extraordinary benefit to staging laparoscopy for gastric cancer with serosal invasion (T4) that requires downstage treatment. We investigated the feasibility of using multiphoton imaging to perform a real-time optical diagnosis of gastric cancer with or without serosal invasion. First, a pilot study was performed to establish the optical diagnostic features of gastric cancer with or without serosal invasion using multiphoton imaging compared with hematoxylin-eosin staining and Masson’s trichrome staining. Second, a blinded study was performed to compare the diagnostic sensitivity, specificity, and accuracy of multiphoton imaging and endoscopic ultrasonography (EUS) for T4 gastric cancer. In the pilot study, multiphoton imaging revealed collagen loss and degradation and cellular and nuclear pleomorphism in gastric cancer with serosal invasion. The collagen content in gastric cancer with or without serosal invasion was 0.36 ± 0.18 and 0.79 ± 0.16 (p < 0.001), respectively. In the blinded study, the sensitivity, specificity, and accuracy of EUS and multiphoton imaging for T4 gastric cancer were 70% and 90% (p = 0.029), 66.67% and 96.67% (p = 0.003), and 68.33% and 93.33% (p = 0.001), respectively. It is feasible to use multiphoton imaging to make a real-time optical diagnosis of gastric cancer with or without serosal invasion. PMID:27499365

  1. Electric field effects in the infrared multiphoton dissociation of CF2HCl

    NASA Astrophysics Data System (ADS)

    Gozel, P.; van den Bergh, H.

    1981-02-01

    The infrared multiphoton dissociation of chlorodifluoromethane is studied in a dc electric field. The reaction yield is measured as a function of the laser energy fluence, the CF2HCl pressure, and the electric field strength in the range 0⩽E⩽5.4 kV cm-1. At low fluence (F<1J cm-2) and intensity, under essentially collision free conditions, the multiphoton dissociation is strongly enhanced by the electric field. This is tentatively explained by a breakdown in the angular momentum selection rules caused by the applied field.

  2. Enhancement of molecular ions in mass spectrometry using an ultrashort optical pulse in multiphoton ionization.

    PubMed

    Shimizu, Takashi; Watanabe-Ezoe, Yuka; Yamaguchi, Satoshi; Tsukatani, Hiroko; Imasaka, Tomoko; Zaitsu, Shin-Ichi; Uchimura, Tomohiro; Imasaka, Totaro

    2010-05-01

    The spectral domain of an ultraviolet femtosecond laser was expanded by stimulated Raman scattering/four-wave Raman mixing, and the resulting laser pulse was compressed using a pair of gratings. The pulse width was then measured using an autocorrelator comprised of a Michelson interferometer equipped with a multiphoton ionization/mass spectrometer which was used as a two-photon detector. A gas chromatograph/mass spectrometer was employed to analyze triacetone triperoxide (TATP), and the molecular ion induced by multiphoton ionization was substantially enhanced by decreasing the laser pulse width. PMID:20364824

  3. Diagrammatic analysis of multiphoton processes in a ladder-type three-level atomic system

    SciTech Connect

    Noh, Heung-Ryoul; Moon, Han Seb

    2011-11-15

    We present a diagrammatic method for complete characterization of multiphoton processes in three-level atomic systems. By considering the interaction routes of the coupling and probe photons for a ladder-type, three-level, noncycling (or cycling) atomic system, we are able to completely discriminate between the pure one-photon and the pure two-photon resonance effects, and the effect of their combination in electromagnetically induced transparency (EIT) using our diagrammatic method. We show that the proposed diagrammatic method is very useful for the analysis of multiphoton processes in ladder-type EIT.

  4. Large field of view multiphoton microscopy of human skin

    NASA Astrophysics Data System (ADS)

    Balu, Mihaela; Mikami, Hideharu; Hou, Jue; Potma, Eric O.; Tromberg, Bruce J.

    2016-03-01

    Clinical examination crucially relies on the ability to quickly examine large tissue areas and rapidly zoom in to regions of interest. Skin lesions often show irregularity in color and appearance in general, especially when they start to progress towards malignancy. Large field of view (FOV) and automatic translation of the imaging area are critical in the assessment of the entire lesion. Imaging of limited FOVs of the lesion can easily result in false negative diagnosis. We present a multiphoton microscope based on two-photon excited fluorescence and second-harmonic generation that images FOVs of about 0.8 mm2 (without stitching adjacent FOVs) at speeds of 10 frames/second (800 x 800 pixels) with lateral and axial resolutions of 0.5 μm and 2.5 μm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, relay optics, a beam expander and a high NA objective lens. We optimized the system based on the Olympus 25x, 1.05NA water immersion lens, that features a long working distance of 1 mm. Proper tailoring of the beam expander, which consists of the scan and tube lens elements, enables scaling of the FOV. The design criteria include a flat wavefront of the beam, minimum field curvature, and suppressed spherical aberrations. All aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide FOVs in normal human skin.

  5. Multiphoton microscopy in the evaluation of human bladder biopsies

    PubMed Central

    Jain, Manu; Robinson, Brian D.; Scherr, Douglas S.; Sterling, Joshua; Lee, Ming-Ming; Wysock, James; Rubin, Mark A.; Maxfield, Frederick R.; Zipfel, Warren R.; Webb, Watt W.; Mukherjee, Sushmita

    2013-01-01

    Context Multiphoton microscopy (MPM) is a nonlinear imaging approach, providing cellular and subcellular details from fresh (unprocessed) tissue by exciting intrinsic tissue emissions. With miniaturization and substantially decreased cost on the horizon, MPM is an emerging in vivo imaging technique with many potential clinical applications. Objective To assess the imaging ability and diagnostic accuracy of MPM for human bladder biopsies. Design Seventy-seven fresh bladder biopsies were imaged by MPM and subsequently submitted for routine surgical pathology diagnosis. Twelve cases were excluded due to extensive cautery artifact that prohibited definitive diagnosis. Comparison was made between MPM imaging and gold standard hematoxylin and eosin (H&E) stained sections of each specimen. Results In 57/65 cases (88%), accurate MPM diagnoses (benign or neoplastic) were given based upon the architecture and/or cytologic grade. The sensitivity and specificity of MPM in our study were 90.4% and 76.9%, respectively. A positive (neoplastic) diagnosis on MPM had a high predictive value (94%), and negative (benign) diagnoses were sustained on histopathology in two-thirds of cases. Architecture (papillary vs. flat) was correctly determined in 56/65 cases (86%), and cytologic grade (benign/low vs. high grade) was assigned correctly in 38/56 cases (68%). Conclusions MPM images alone provide sufficient detail to classify most lesions as either benign or neoplastic using the same basic diagnostic criteria as histopathology (architecture and cytologic grade). Future developments in MPM technology may provide urologists and pathologists with additional screening and diagnostic tools for early detection of bladder cancer. Additional applications of such emerging technologies warrant exploration. PMID:22540300

  6. In Vivo Multiphoton Microscopy of Basal Cell Carcinoma

    PubMed Central

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

    2015-01-01

    Importance Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment. Objectives To demonstrate the capability of MPM to image in vivo BCC lesions in human skin, and to evaluate if histopathologic criteria can be identified in MPM images. Design, Setting, and Participants Imaging in patients with BCC was performed at the University of California–Irvine Health Beckman Laser Institute & Medical Clinic, Irvine, between September 2012 and April 2014, with a clinical MPM-based tomograph. Ten BCC lesions were imaged in vivo in 9 patients prior to biopsy. The MPM images were compared with histopathologic findings. Main Outcomes and Measures MPM imaging identified in vivo and noninvasively the main histopathologic feature of BCC lesions: nests of basaloid cells showing palisading in the peripheral cell layer at the dermoepidermal junction and/or in the dermis. Results The main MPM feature associated with the BCC lesions involved nests of basaloid cells present in the papillary and reticular dermis. This feature correlated well with histopathologic examination. Other MPM features included elongated tumor cells in the epidermis aligned in 1 direction and parallel collagen and elastin bundles surrounding the tumors. Conclusions and Relevance This study demonstrates, in a limited patient population, that noninvasive in vivo MPM imaging can provide label-free contrast that reveals several characteristic features of BCC lesions. Future studies are needed to validate the technique and correlate MPM performance with histopathologic examination. PMID:25909650

  7. Energetics from Slow Infrared Multiphoton Dissociation of Biomolecules

    PubMed Central

    Jockusch, Rebecca A.; Paech, Kolja

    2005-01-01

    Photodissociation kinetics of the protonated pentapeptide leucine enkephalin measured using a cw CO2 laser and a Fourier-transform mass spectrometer are reported. A short induction period, corresponding to the time required to raise the internal energy of the ion population to a (dissociating) steady state, is observed. After this induction period, the dissociation data are accurately fit by first-order kinetics. A plot of the log of the unimolecular dissociation rate constant, kuni, as a function of the log of laser power is linear at low laser powers (<9 W, kuni <0.05 s−1), but tapers off at high laser power (9–33 W, kuni = 0.05–7 s−1). The entire measured dissociation curve can be accurately fit by an exponential function plus a constant. The experiment is simulated using a master equation formalism. In the model, the laser radiation is described as an energetically flat-topped distribution which is spatially uniform. This description is consistent with experimental results which indicate that ion motion within the cell averages out spatial inhomogeneities in the laser light. The model has several adjustable parameters. The effect of varying these parameters on the calculated kinetics and power dependence curves is discussed. A procedure for determining a limited range of threshold dissociation energy, Eo, which fits both the measured induction period and power dependence curves, is presented. Using this procedure, Eo of leucine enkephalin is determined to be 1.12–1.46 eV. This result is consistent with, although less precise than, values measured previously using blackbody infrared radiative dissociation. Although the blackbody dissociation results were used as a starting point to search for fits of the master equation model to experiment, these results demonstrate that it is, in principle, possible to determine a limited range of Eo from slow infrared multiphoton dissociation data alone. PMID:16467893

  8. Semiclassical analysis of long-wavelength multiphoton processes: The Rydberg atom

    NASA Astrophysics Data System (ADS)

    Vela-Arevalo, Luz V.; Fox, Ronald F.

    2004-06-01

    We study the problem of multiphoton processes for intense, long-wavelength irradiation of atomic and molecular electrons. An exact, nonperturbative approach is applied to the standard vector potential coupling Hamiltonian for a three-dimensional hydrogenlike atom in a microwave field treated semiclassically. Multiphoton probability exchange is calculated in both the velocity and the length gauges, by applying the Goeppert-Mayer gauge transformation. The expansion of the time-dependent solution in terms of Floquet states delineates the mechanism of multiphoton transitions. A detailed analysis of the Floquet states and quasienergies as functions of the field parameters allows us to describe the relation between avoided quasienergy crossings and multiphoton probability exchange. We formulate analytical expressions for the variation of quasienergies and Floquet states with respect to the field parameters, and demonstrate that avoided quasienergy crossings are accompanied by dramatic changes in the Floquet states. Analysis of the Floquet states, for small values of the field strength, yields selection rules for the avoided quasienergy crossings. In the case of strong fields, the simultaneous choice of frequency and strength of the field producing an avoided crossing results in improved ionization probability.

  9. Multi-photon transitions and Rabi resonance in continuous wave EPR.

    PubMed

    Saiko, Alexander P; Fedaruk, Ryhor; Markevich, Siarhei A

    2015-10-01

    The study of microwave-radiofrequency multi-photon transitions in continuous wave (CW) EPR spectroscopy is extended to a Rabi resonance condition, when the radio frequency of the magnetic-field modulation matches the Rabi frequency of a spin system in the microwave field. Using the non-secular perturbation theory based on the Bogoliubov averaging method, the analytical description of the response of the spin system is derived for all modulation frequency harmonics. When the modulation frequency exceeds the EPR linewidth, multi-photon transitions result in sidebands in absorption EPR spectra measured with phase-sensitive detection at any harmonic. The saturation of different-order multi-photon transitions is shown to be significantly different and to be sensitive to the Rabi resonance. The noticeable frequency shifts of sidebands are found to be the signatures of this resonance. The inversion of two-photon lines in some spectral intervals of the out-of-phase first-harmonic signal is predicted under passage through the Rabi resonance. The inversion indicates the transition from absorption to stimulated emission or vice versa, depending on the sideband. The manifestation of the primary and secondary Rabi resonance is also demonstrated in the time evolution of steady-state EPR signals formed by all harmonics of the modulation frequency. Our results provide a theoretical framework for future developments in multi-photon CW EPR spectroscopy, which can be useful for samples with long spin relaxation times and extremely narrow EPR lines. PMID:26295168

  10. Multiphoton ionization studies of WF{sub 6} and UF{sub 6}

    SciTech Connect

    Harkins, D.A.; Armstrong, D.P.; Compton, R.N.; Ding, Dajun

    1993-07-01

    Multiphoton ionization of UF{sub 6} and WF{sub 6} has been studied from low ({approximately} 10{sup 5}) to high ({approximately}10{sup 16} W/cm{sup 2}) power density. The role of ``collective states`` in MPI is considered.

  11. Electron-nuclear energy sharing in above-threshold multiphoton dissociative ionization of H2.

    PubMed

    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.

  12. The Multiphoton Interaction of Lambda Model Atom and Two-Mode Fields

    NASA Technical Reports Server (NTRS)

    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.

  13. Clinical combination of multiphoton tomography and high frequency ultrasound imaging for evaluation of skin diseases

    NASA Astrophysics Data System (ADS)

    König, K.; Speicher, M.; Koehler, M. J.; Scharenberg, R.; Elsner, P.; Kaatz, M.

    2010-02-01

    For the first time, high frequency ultrasound imaging, multiphoton tomography, and dermoscopy were combined in a clinical study. 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 DermaInspectTM and (iii) dermoscopes. Dermoscopy provides two-dimensional color imaging of the skin surface with a magnification up to 70x. Ultrasound images are generated from reflections of the emitted ultrasound signal, based on inhomogeneities of the tissue. These echoes are converted to electrical signals. Depending on the ultrasound frequency the penetration depth varies from about 1 mm to 16 mm in dermatological application. The 100-MHz-ultrasound system provided an axial resolution down to 16 μm and a lateral resolution down to 32 μm. In contrast to the wide-field ultrasound images, multiphoton tomography provided horizontal optical sections of 0.36×0.36 mm2 down to 200 μm tissue depth with submicron resolution. The autofluorescence of mitochondrial coenzymes, melanin, and elastin as well as the secondharmonic- generation signal of the collagen network were imaged. The combination of ultrasound and multiphoton tomography provides a novel opportunity for diagnostics of skin disorders.

  14. Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

    PubMed

    Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

    2014-01-01

    Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

  15. Simultaneous resonant enhanced multiphoton ionization and electron avalanche ionization in gas mixtures

    SciTech Connect

    Shneider, Mikhail N.; Zhang Zhili; Miles, Richard B.

    2008-07-15

    Resonant enhanced multiphoton ionization (REMPI) and electron avalanche ionization (EAI) are measured simultaneously in Ar:Xe mixtures at different partial pressures of mixture components. A simple theory for combined REMPI+EAI in gas mixture is developed. It is shown that the REMPI electrons seed the avalanche process, and thus the avalanche process amplifies the REMPI signal. Possible applications are discussed.

  16. Electron energy spectrum and maximum disruption angle under multi-photon beamstrahlung

    SciTech Connect

    Yokoya, Kaoru; Chen, Pisin

    1989-03-01

    The final electron energy spectrum under multi-photon beamstrahlung process is derived analytically in the classical and the intermediate regimes. The maximum disruption angle from the low energy tail of the spectrum is also estimated. The results are then applied to the TLC and the CLIC parameters. 6 refs., 1 fig., 1 tab.

  17. Fluorescence excitation and multiphoton ionization spectroscopy of 3-methylindole in a supersonic jet

    NASA Astrophysics Data System (ADS)

    Hays, T. R.; Henke, W. E.; Selzle, H. L.; Schlag, E. W.

    1983-05-01

    The fluorescence excitation and multiphoton ionization spectroscopy of 3-methylindole (skatole) is reported. One electronic origin ( 1L b) is assigned at 34875 cm -1, the second ( 1L a) suspected at 35483 cm -1. Some 1L b vibrational assignments are also made. Complex formation between skatole and some small molecules is indicated but not directly observed.

  18. A multiphoton ionization technique for the determination of the ionization threshold of molecules in fluid media

    SciTech Connect

    Faidas, H.; Christophorou, L.G.

    1987-01-01

    A new laser-induced multiphoton conductivity technique for the determination of the ionization threshold of molecules in fluids (liquids and dense gases) is described. Results are presented and discussed on aromatic molecules in nonpolar fluids. 2 refs., 1 fig., 1 tab.

  19. Experimental measurements of multiphoton enhanced air breakdown by a subthreshold intensity excimer laser

    SciTech Connect

    Way, Jesse; Hummelt, Jason; Scharer, John

    2009-10-15

    This work presents density, spectroscopic temperature, and shockwave measurements of laser induced breakdown plasma in atmospheric air by subthreshold intensity (5.5x10{sup 9} W/cm{sup 2}) 193 nm laser radiation. Using molecular spectroscopy and two-wavelength interferometry, it is shown that substantial ionization (>10{sup 16} cm{sup -3}) occurs that is not predicted by collisional cascade (CC) breakdown theory. While the focused laser irradiance is three orders of magnitude below the theoretical collisional breakdown threshold, the substantial photon energy at 193 nm (6.42 eV/photon) compared with the ionization potential of air (15.6 eV) significantly increases the probability of multiphoton ionization effects. By spectroscopically monitoring the intensity of the N{sub 2}{sup +} first negative system (B {sup 2}SIGMA{sub u}{sup +}-X {sup 2}SIGMA{sub g}{sup +}) vibrational bandhead (v{sup '}=0,v{sup ''}=0) at low pressure (20 Torr) where multiphoton effects are dominant, it is shown that two photon excitation, resonant enhanced multiphoton ionization is the primary mechanism for quantized ionization of N{sub 2} to the N{sub 2}{sup +}(B {sup 2}SIGMA{sub u}{sup +}) state. This multiphoton effect then serves to amplify the collisional breakdown process at higher pressures by electron seeding, thereby reducing the threshold intensity from that required via CC processes for breakdown and producing high density laser formed plasmas.

  20. Nanoparticle-assisted-multiphoton microscopy for in vivo brain imaging of mice

    NASA Astrophysics Data System (ADS)

    Qian, Jun

    2015-03-01

    Neuro/brain study has attracted much attention during past few years, and many optical methods have been utilized in order to obtain accurate and complete neural information inside the brain. Relying on simultaneous absorption of two or more near-infrared photons by a fluorophore, multiphoton microscopy can achieve deep tissue penetration and efficient light detection noninvasively, which makes it very suitable for thick-tissue and in vivo bioimaging. Nanoparticles possess many unique optical and chemical properties, such as anti-photobleaching, large multiphoton absorption cross-section, and high stability in biological environment, which facilitates their applications in long-term multiphoton microscopy as contrast agents. In this paper, we will introduce several typical nanoparticles (e.g. organic dye doped polymer nanoparticles and gold nanorods) with high multiphoton fluorescence efficiency. We further applied them in two- and three-photon in vivo functional brain imaging of mice, such as brain-microglia imaging, 3D architecture reconstruction of brain blood vessel, and blood velocity measurement.

  1. Ex vivo applications of multiphoton microscopy in urology

    NASA Astrophysics Data System (ADS)

    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

  2. Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two-photon microscopy

    PubMed Central

    Ricard, Clément; Lamasse, Lisa; Jaouen, Alexandre; Rougon, Geneviève; Debarbieux, Franck

    2016-01-01

    Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord. PMID:27375951

  3. Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

    PubMed

    Pang, Yonggang; Tsigkou, Olga; Spencer, Joel A; Lin, Charles P; Neville, Craig; Grottkau, Brian

    2015-10-01

    Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad

  4. High-fidelity spatially resolved multiphoton counting for quantum imaging applications.

    PubMed

    Chrapkiewicz, Radosław; Wasilewski, Wojciech; Banaszek, Konrad

    2014-09-01

    We present a method for spatially resolved multiphoton counting based on an intensified camera with the retrieval of multimode photon statistics fully accounting for nonlinearities 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 the Mandel parameter and the noise reduction factor in contrast to raw statistics of camera photo-events. Detector operation is reliable for illumination levels up to the average of one detected photon per an event area-substantially higher than in previous approaches to characterize quantum statistical properties of light with spatial resolution. PMID:25166081

  5. Semiclassical analysis of long-wavelength multiphoton processes: The periodically driven harmonic oscillator

    NASA Astrophysics Data System (ADS)

    Fox, Ronald F.; Vela-Arevalo, Luz V.

    2002-11-01

    The problem of multiphoton processes for intense, long-wavelength irradiation of atomic and molecular electrons is presented. The recently developed method of quasiadiabatic time evolution is used to obtain a nonperturbative analysis. When applied to the standard vector potential coupling, an exact auxiliary equation is obtained that is in the electric dipole coupling form. This is achieved through application of the Goeppert-Mayer gauge. While the analysis to this point is general and aimed at microwave irradiation of Rydberg atoms, a Floquet analysis of the auxiliary equation is presented for the special case of the periodically driven harmonic oscillator. Closed form expressions for a complete set of Floquet states are obtained. These are used to demonstrate that for the oscillator case there are no multiphoton resonances.

  6. Nonperturbative treatments of nonresonant multiphoton ionization of the hydrogen atom: weak field limit

    SciTech Connect

    Trombetta, F.; Basile, S.; Ferrante, G.

    1989-04-01

    A nonperturbative treatment of the multiphoton ionization of the hydrogen atom based on the S matrix and devised for nonresonant strong-field situations is analyzed in the weak-field limit. Comparisons are presented with other S matrices as well as other nonperturbative approaches. Our treatment is found to perform generally better than similar S-matrix treatments. The usual perturbative results are recovered provided that the photon wavelengths are sufficiently short and are off resonance with the atomic transitions. Important indications are obtained as to the role of the atomic structure, the relevance of the gauge consistency, and the reliability and improvement of the present nonperturbative treatment. The results represent a significant step toward an assessment of the S-matrix-based treatments of multiphoton ionization.

  7. Longer wavelengths require lower intensity in multiphoton detachment of negative ions

    NASA Astrophysics Data System (ADS)

    Davidson, M. D.; Schumacher, D. W.; Bucksbaum, P. H.; Muller, H. G.; van Linden van den Heuvell, H. B.

    1992-12-01

    Negative chlorine ions, held in a Penning ion trap, are irradiated by intense (up to 2×1016 W/m2) Raman-shifted light of a Nd:YAlG laser with a wavelength of 1098 nm and a pulse duration of 30 ps. Electron-energy spectra are obtained showing up to 11-photon absorption. In the case of circularly polarized light suppression of the low-energy channels is observed. A saturation intensity of (8.5+/-2.5)×1015 W/m2 is found for multiphoton detachment of Cl- by 1908-nm light. This result is counterintuitive because it is lower than the saturation intensity for multiphoton detachment at 1064 nm.

  8. Multiphoton microscopy with clearing for three dimensional histology of kidney biopsies

    PubMed Central

    Olson, Eben; Levene, Michael J.; Torres, Richard

    2016-01-01

    We present a multiphoton microscopy approach with clearing optimized for pathology evaluation producing image quality comparable to traditional histology. Use of benzyl alcohol/benzyl benzoate with 4',6-diamidino-2-phenylindole and eosin in an optimized imaging setup results in optical sections nearly indistinguishable from traditionally-cut sections. Application to human renal tissue demonstrates diagnostic-level image quality can be maintained through 1 millimeter of tissue. Three dimensional perspectives reveal changes of glomerular capsule cells not evident on single sections. Collagen-derived second harmonic generation can be visualized through entire biopsies. Multiphoton microscopy with clearing has potential for increasing the yield of histologic evaluation of biopsy specimens. PMID:27570700

  9. Effect of Size-Dependent Photodestructive Efficacy by Gold Nanomaterials with Multiphoton Laser.

    PubMed

    Chang, Wen-Tsan; Chen, Shean-Jen; Chang, Chia-Yuan; Liu, Yi-Hsien; Chen, Chang-Hsin; Yang, Chen-Han; Chou, Lawrence Chao-Shan; Chang, Jui-Cheng; Cheng, Li-Chung; Kuo, Wen-Shuo; Wang, Jiu-Yao

    2015-08-12

    The photostability, photodestructive efficacy, two-photon excitation cross section, and two-photon fluorescence of gold nanoparticles conjugated with a hydrophilic photosensitizer, indocyanine green, via multiphoton laser exhibited an increased size effect in methicillin-resistant Staphylococcus aureus and A549 cancer cells that was dependent on the size of multifunctional gold nanomaterials, but the effect only occurred when nanomaterials within 100 nm in diameter were used. Besides, the enhanced effectiveness of photodestruction, photostability, and contrast probe indicated an additive effect in the therapeutic and imaging efficiency of multifunctional gold nanomaterials. Consequently, the preparation of the multifunctional gold nanomaterials and their use in biomedical applications via multiphoton laser is an alternative and potential therapeutic approach for killing bacteria and for ablating cancer cells.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    Monaghan, Michael G.; Kroll, Sebastian; Brucker, Sara Y.

    2016-01-01

    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. PMID:27018844

  12. Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas

    PubMed Central

    Gualda, Emilio J.; Vázquez de Aldana, Javier R.; Martínez-García, M. Carmen; Moreno, Pablo; Hernández-Toro, Juan; Roso, Luis; Artal, Pablo; Bueno, Juan M.

    2011-01-01

    The performance of femtosecond (fs) laser intrastromal ablation was evaluated with backscattering-mode adaptive-optics multiphoton microscopy in ex vivo chicken corneas. The pulse energy of the fs source used for ablation was set to generate two different ablation patterns within the corneal stroma at a certain depth. Intrastromal patterns were imaged with a custom adaptive-optics multiphoton microscope to determine the accuracy of the procedure and verify the outcomes. This study demonstrates the potential of using fs pulses as surgical and monitoring techniques to systematically investigate intratissue ablation. Further refinement of the experimental system by combining both functions into a single fs laser system would be the basis to establish new techniques capable of monitoring corneal surgery without labeling in real-time. Since the backscattering configuration has also been optimized, future in vivo implementations would also be of interest in clinical environments involving corneal ablation procedures. PMID:22076258

  13. Identification of normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections

    NASA Astrophysics Data System (ADS)

    Zhou, Yi; Chen, Zhifen; Kang, Deyong; li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Guan, Guoxian; Chen, Jianxin

    2016-01-01

    Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a potential diagnostic tool is attractive. MPM can effectively provide information about morphological and biochemical changes in biological tissues at the molecular level. In this paper, we attempt to identify normal and cancerous human colorectal muscularis propria by multiphoton microscopy in different sections (both in transverse and longitudinal sections). The results show that MPM can display different microstructure changes in the transverse and longitudinal sections of colorectal muscularis propria. MPM also can quantitatively describe the alteration of collagen content between normal and cancerous muscle layers. These are important pathological findings that MPM images can bring more detailed complementary information about tissue architecture and cell morphology through observing the transverse and longitudinal sections of colorectal muscularis propria. This work demonstrates that MPM can be better for identifying the microstructural characteristics of normal and cancerous human colorectal muscularis propria in different sections.

  14. Label-free identification of intestinal metaplasia in the stomach using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Wu, G.; Wei, J.; Zheng, Z.; Ye, J.; Zeng, S.

    2014-06-01

    The early diagnosis of intestinal metaplasia (IM) in the stomach together with effective therapeutic interventions is crucial to reducing the mortality-rates of the patients associated with gastric cancer. However, it is challenging during conventional white-light endoscopy, and histological analysis remains the ‘gold standard’ for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), for the identification of IM in the stomach. It was found that multiphoton imaging provides cellular and subcellular details to the identification of IM from normal gastric tissues. In particular, there is significant difference in the population density of goblet cells between normal and IM gastric tissues, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early gastric lesions. To our knowledge, this is the first demonstration of the potential of MPM for the identification of IM.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. ACT: Acting Out Central Theme.

    ERIC Educational Resources Information Center

    Kise, Joan Duff

    1982-01-01

    The author describes ACT (Acting Out Central Theme), a method for dealing with psychomotor, cognitive, and affective domains in slow readers. The ACT approach involves three sessions which focus on discussion of a theme such as friendship, presentaton of the theme as a skit, and assignment of topics to individual students. (SW)

  17. Twofold symmetric angular distributions in multiphoton ionization with elliptically polarized light

    SciTech Connect

    Basile, S.; Trombetta, F.; Ferrante, G.

    1988-11-21

    The angular distributions of electrons in multiphoton multichannel ionization of hydrogen for the case of elliptically polarized laser light are calculated within a nonperturbative theoretical model taking into account the Coulomb interaction in the final state. It is found that the ellipticity of the radiation not only modifies the shape but also lowers the fourfold rotational symmetry occurring in linear polarization to a twofold one.

  18. Influence of the ac Stark effect on multiphoton transitions in molecules

    NASA Astrophysics Data System (ADS)

    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

  19. Multiphoton Process and Anomalous Potential of Cell Membrane by Laser Radiation

    NASA Technical Reports Server (NTRS)

    Zhang, Kaixi; Zhao, Qingxun; Cui, Zhiyun; Zhar, Ping; Dong, Lifang

    1996-01-01

    In this paper, by the use of quantum biology and quantum optics, the laser induced potential variation of cell membrane has been studied. Theoretically, we have found a method of calculating the monophoton and multiphoton processes in the formation of the anomalous potential of cell membrane. In contrast with the experimental results, our numerical result is in the same order. Therefore, we have found the possibility of cancer caused by the laser induced anomalous cell potential.

  20. Multiphoton lasing in atomic potassium: Steady-state and dynamic behavior

    SciTech Connect

    Font, J. L.; Fernandez-Soler, J. J.; Vilaseca, R.; Gauthier, Daniel J.

    2005-12-15

    We show theoretically that it is possible to generate laser light based on two-photon and other high-order multiphoton processes when an atomic beam of optically driven potassium atoms crosses a high-finesse optical cavity. We use a rigorous model that takes into account all the atomic substates involved in the optical interactions and is valid for any drive and lasing field intensities. The polarizations of the drive and lasing fields are assumed to be fixed. Stable and unstable laser emission branches are obtained, which are represented as a function of cavity detuning and are analyzed in terms of the fundamental quantum processes yielding them. Closed-curve laser-emission profiles are obtained for multiphoton lasing based on processes involving more than one lasing photon. Two-photon laser emission branches show relatively long segments of stationary emission, combined in general with some segments of nonstationary emission, or with segments of mixture with three-photon emission processes. Rayleigh and hyper-Rayleigh processes can become simultaneously resonant, entailing in such case a large and fast transfer of population from the atomic initial ground sublevel to other ground sublevels with different z components of the total angular momentum. They could be useful in generating multiphoton correlated field states. In all cases the largest laser emission intensities are obtained from the highest-order processes, rather than the lowest. These results open the way to the understanding of experiments performed in the past years and suggest possibilities for more efficient and varied types of multiphoton laser operation.

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

    PubMed Central

    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

  2. Studies of atmospheric molecules by multiphoton spectroscopy. Progress report, July 15, 1989--October, 1991

    SciTech Connect

    Johnson, P.M.

    1991-10-01

    Carbon dioxide presents a great challenge to spectroscopy because of its propensity toward dissociation in all of its excited states. Multiphoton ionization spectroscopy is usually not applicable to the study of dissociating molecules because the dissociation competes effectively with ionization, resulting in no signal. We reasoned, however, that with high enough laser fluence, ionization could compete with dissociation in the longer lived states, exposing them for study from the continuous spectral background resulting from rapidly dissociating states. We describe the various spectroscopic and photophysical effects found through the multiphoton ionization and multiphoton photoelectron spectra. A recently developed variant of threshold ionization spectroscopy, usually called ZEKE, has shown a great deal of usefulness in providing the same information as traditional photoelectron spectroscopy but with higher resolution and much better signal-to-noise when using standard laboratory lasers. Threshold ionization techniques locate the states of an ion by scanning a light source across the ionization continuum of a neutral and somehow detecting when electrons are produced with no kinetic energy. We chose to develop our capabilities in threshold ionization spectroscopy using aromatic molecules because of their importance and because their electronic structure allows a pump-probe type of excitation scheme which avoids the use of vacuum ultraviolet laser beams. Among aromatics, the azines are noted for their small S{sub 1}-T{sub 1} energy gap which give them unique and interesting photophysical properties. We have continued our work on the multiphoton spectrum of metastable nitrogen produced by an electric discharge in supersonic beam. We have been able to assign more of the lines and simulated their rotational structure but many peaks remain unassigned.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Multiphoton imaging of upconverting lanthanide nanoparticles in three dimensional models of cancer

    NASA Astrophysics Data System (ADS)

    Gainer, Christian F.; Romanowski, Marek

    2013-02-01

    While upconverting lanthanide nanoparticles have numerous advantages over other exogenous contrast agents used in scanned multiphoton imaging, their long luminescence lifetimes cause images collected with non-descanned detection to be greatly blurred. We demonstrate herein the use of Richardson-Lucy deconvolution to deblur luminescence images obtained via multiphoton scanning microscopy. Images were taken of three dimensional models of colon and ovarian cancer following incubation with NaYF4:Yb,Er nanoparticles functionalized with an antibody for EGFR and folic acid respectively. Following deconvolution, images had a lateral resolution on par with the optimal performance of the imaging system used, ~1.2 μm, and an axial resolution of ~5 μm. Due to the relatively high multiphoton excitation efficiency of these nanoparticles, it is possible to follow binding of individual particles in tissue. In addition, their extreme photostability allows for prolonged imaging without significant loss in luminescence signal. With these advantageous properties in mind, we also discuss the potential application of upconverting lanthanide nanoparticles for tracking of specific, cancer relevant receptors in tissue.

  5. Intrinsic Indicator of Photodamage during Label-Free Multiphoton Microscopy of Cells and Tissues

    PubMed Central

    Andresen, Elisabeth F.; Geiger, Kathrin D.; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    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. PMID:25343251

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

    PubMed Central

    Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada; Rebane, Aleksander; Lindquist, Jake R.; Santos, Sofia; Kats, Ilia; Davidson, Michael W.; Mazitschek, Ralph; Hughes, Thomas E.; Drobizhev, Mikhail; Knop, Michael; Shah, Jagesh V.

    2015-01-01

    We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. PMID:25877871

  9. In vitro characterization of corneal wound healing using multiphoton autofluorescence and second harmonic generation (SHG) microscopy

    NASA Astrophysics Data System (ADS)

    Sun, Yen; Lo, Wen; Chen, Wei-Liang; Teng, Shu-Wen; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this investigation is to characterize corneal wound healing under in vitro conditions. Multiphoton autofluorescence and second harmonic generation (SHG) microscopy will be used to visualize cells and collagen fibers associated with corneal wound healing. 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. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. To acquire both high resolution and tissue-level information of the specimen, a sample positioning stage is used in conjunction with the beam scanning system. Finally, the organ level image can be assembled from individual area scans. The in vitro samples we used are cornea buttons acquired from porcine eyes. Localized wounds will be induced by #11 blade and imaged using multiphoton microscopy. Based on these results, we envision the in vitro imaging chamber to be able to follow the wound healing process without damaging histological procedures. We envision this approach will enable us to further understand wound healing process associated with corneal scar and can lead to in vivo methodology for diagnosing cornea damage.

  10. Diffusion of Nerve Growth Factor in Rat Striatum as Determined by Multiphoton Microscopy

    PubMed Central

    Stroh, Mark; Zipfel, Warren R.; Williams, Rebecca M.; Webb, Watt W.; Saltzman, W. Mark

    2003-01-01

    Neurotrophins such as nerve growth factor (NGF) may be useful for treating diseases in the central nervous system; our ability to harness the potential therapeutic benefit of NGF is directly related to our understanding of the fate of exogenously supplied factors in brain tissue. We utilized multiphoton microscopy to quantify the dynamic behavior of NGF in coronal, 400-μm thick, fresh rat brain tissue slices. We administered a solution containing bioactive rhodamine nerve growth factor conjugate via pressure injection and monitored the dispersion in the striatal region of the coronal slices. Multiphoton microscopy facilitated repeated imaging deep (∼200 μm) into tissue slices with minimal photodamage of tissue and photobleaching of label. The pressure injection paradigm approximated diffusion from a point source, and we therefore used the corresponding solution to the diffusion equation to estimate an apparent diffusion coefficient in brain tissue (Db(34°C)) of 2.75 ± 0.24 × 10−7 cm2/s (average ± SE). In contrast, we determined a corresponding free diffusion coefficient in buffered solution (Df(34°C)) of 12.6 ± 0.9 × 10−7 cm2/s using multiphoton fluorescence photobleaching recovery. The tortuosity, defined as the square root of the ratio of Df to Db, was 2.14 and moderate in magnitude. PMID:12829512

  11. Polarization control of intermediate state absorption in resonance-mediated multi-photon absorption process

    NASA Astrophysics Data System (ADS)

    Xu, Shuwu; Huang, Yunxia; Yao, Yunhua; Jia, Tianqing; Ding, Jingxin; Zhang, Shian; Sun, Zhenrong

    2015-07-01

    We theoretically and experimentally demonstrate the control of the intermediate state absorption in an (n + m) resonance-mediated multi-photon absorption process by the polarization-modulated femtosecond laser pulse. An analytical solution of the intermediate state absorption in a resonance-mediated multi-photon absorption process is obtained based on the time-dependent perturbation theory. Our theoretical results show that the control efficiency of the intermediate state absorption by the polarization modulation is independent of the laser intensity when the transition from the intermediate state to the final state is coupled by the single-photon absorption, but will be affected by the laser intensity when this transition is coupled by the non-resonant multi-photon absorption. These theoretical results are experimentally confirmed via a two-photon fluorescence control in (2 + 1) resonance-mediated three-photon absorption of Coumarin 480 dye and a single-photon fluorescence control in (1 + 2) resonance-mediated three-photon absorption of IR 125 dye.

  12. Arbitrary two-dimensional multiphoton excitation patterns with temporally focused digital holograms

    NASA Astrophysics Data System (ADS)

    Oron, Dan; Papagiakoumou, Eirini; de-Sars, Vincent; Emiliani, Valentina

    2009-02-01

    Multiphoton excitation has recently found application in the fields of bioimaging, uncaging and lithography. In order to fully exploit the advantages of nonlinear excitation, in particular the axial resolution due to nonlinearity, most systems to date operate with point or multipoint excitation, while scanning either the laser beam or the sample to generate the illumination pattern. Here we combine the recently introduced technique of scanningless multiphoton excitation by temporal focusing with recent advances in digital holography to generate arbitrarily shaped, depth resolved, two-dimensional excitation patterns completely without scanning. This is of particular importance in applications requiring uniform excitation of large areas over short time scales, such as neuronal activation by multiphoton uncaging of neurotransmitters. We present an experimental and theoretical analysis of the effect of spatial patterning on the depth resolution achieved in temporal focusing microscopy. It is shown that the depth resolution for holographic excitation is somewhat worse than that achieved for uniform illumination. This is also accompanied by the appearance of a speckle pattern at the temporal focal plane. The origin of the two effects, as well as means to overcome them, are discussed.

  13. Multiphoton and tunneling ionization probability of atoms and molecules in an intense laser field

    NASA Astrophysics Data System (ADS)

    Zhao, Song-Feng; Liu, Lu; Zhou, Xiao-Xin

    2014-02-01

    We theoretically studied ionization of atoms exposed to an intense laser field by using three different methods, i.e., the numerical solution of the single-active-electron approximation based time-dependent Schrödinger equation (SAE-TDSE), the Perelomov-Popov-Terent'ev (PPT) model, and the Ammosov-Delone-Krainov (ADK) model. The ionization of several linear molecules in a strong laser field is also investigated with the molecular ADK (MO-ADK) and the molecular PPT (MO-PPT) model. We show that the ionization probability from the PPT and the MO-PPT model agrees well with the corresponding SAE-TDSE result in both the multiphoton and tunneling ionization regimes. By considering the volume effect of the laser field, the ionization signal obtained from the PPT and the MO-PPT model fits well the experimental data in the whole range of the multiphoton and tunneling ionization regimes. However, both the ADK and MO-ADK models seriously underestimate the ionization probabilities (or signals) in the multiphoton regime.

  14. Coupling CARS with multiphoton fluorescence and high harmonic generation imaging modalities using a femtosecond laser source

    NASA Astrophysics Data System (ADS)

    Chen, Hongtao; Slipchenko, Mikhail N.; Zhu, Jiabin; Buhman, Kimberly K.; Cheng, Ji-Xin

    2009-02-01

    Multimodal nonlinear optical imaging has opened new opportunities and becomes a powerful tool for imaging complex tissue samples with inherent 3D spatial resolution.. We present a robust and easy-to-operate approach to add the coherent anti-stokes Raman scattering (CARS) imaging modality to a widely used multiphoton microscope. The laser source composed of a Mai Tai femtosecond laser and an optical parametric oscillator (OPO) offers one-beam, two-beam and three-beam modalities. The Mai Tai output at 790 nm is split into two beams, with 80% of the power being used to pump the OPO. The idler output at 2036 nm from OPO is doubled using a periodically poled lithium niobate (PPLN) crystal. This frequency-doubled idler beam at 1018 nm is sent through a delay line and collinearly combined with the other Mai Tai beam for CARS imaging on a laser-scanning microscope. This Mai Tai beam is also used for multiphoton fluorescence and second harmonic generation (SHG) imaging. The signal output at 1290 nm from OPO is used for SHG and third-harmonic generation (THG) imaging. External detectors are installed for both forward and backward detection, whereas two internal lamda-scan detectors are employed for microspectroscopy analysis. This new system allows vibrationally resonant CARS imaging of lipid bodies, SHG imaging of collagen fibers, and multiphoton fluorescence analysis in fresh tissues. As a preliminary application, the effect of diacylglycerol acyltransferase 1 (DGAT1) deficiency on liver lipid metabolism in mice was investigated.

  15. Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

    PubMed Central

    Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

    2012-01-01

    Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes. PMID:22876358

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Role of quantum trajectory in high-order harmonic generation in the Keldysh multiphoton regime

    NASA Astrophysics Data System (ADS)

    Li, Peng-Cheng; Chu, Shih-I.

    2016-05-01

    We present a systematic study of quantum-trajectory analysis 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. We perform the time-frequency transform to explore the spectral characteristics of the HHG. We find that the time-frequency spectra exhibit a broken distribution at above-threshold HHG due to the competition associated with the short- and long-trajectories when the ionization process is pushed from the multiphoton regime into the tunneling regime, it implies that the harmonic emission in the broken regions of time-frequency spectra are suppressed. In addition, we present a time-dependent density-functional theory approach for an ab initio study of the effect of correlated multielectron responses on the harmonic emission of Ar atom associated with the quantum trajectories in the multiphoton regime. This work is partially supported by DOE.

  18. In vivo multiphoton imaging of collagen remodeling after microablative fractional rejuvenation

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Kapsokalyvas, Dimitrios; Troiano, Michela; Campolmi, Piero; Morini, Cristiano; Cosci, Alessandro; Massi, Daniela; Lotti, Torello; Pavone, Francesco S.

    2011-03-01

    The potential of multiphoton microscopy in providing in-vivo early diagnosis of skin lesions has already been demonstrated, while its capability in therapy follow-up has not been deeply explored so far. Two-photon excited fluorescence and second-harmonic generation microscopy were used in combination to follow-up collagen remodeling after laser micro-ablative rejuvenation. Treated regions of volunteers were imaged with multiphoton microscopy before and after treatment, and we found a strong age-dependence of the treatment effectiveness. In particular, the photorejuvenating effect was negligible in young subjects (< 30 years), whereas a significant production of new collagen was observed in aged subjects (> 70 years). Quantification of the amount of newly produced collagen and its organization were performed by means of visual examination of two-photon images. The obtained results demonstrate the performance of laser fractional micro-ablative rejuvenation without the need of an invasive biopsy as well as the wide applicability range of applications for multiphoton microscopy in clinical dermatology.

  19. Design, synthesis, characterization and applications of multi-photon absorbing chromophores

    NASA Astrophysics Data System (ADS)

    Zheng, Qingdong

    Recent development in multi-photon based applications including optical power limiting, frequency up-conversion lasing, three-dimensional data storage, two-photon fluorescence microscopy and two-photon photodynamic therapy has benefited a lot from a number of chromophores with large multi-photon absorption. This thesis was focused on the development of novel two- and three-photon active chromophores and their applications. Chapter 1 describes a theoretical background of multi-photon absorption, and recent development of multi-photon based applications. Some molecular design strategies were proposed after a literature review of chromophores with large two-photon absorption. In Chapter 2, a series of stilbazolium salts with varying electron donors and anions were synthesized and characterized. The two-photon absorption and two-photon pumped cavity lasing properties for these dyes were studied by using 1064 nm nano-second laser beam. By using tunable femto-second laser, three-photon pumped cavity-less lasing properties of these dyes have also been comprehensively studied. Four-photon pumped stimulated emission was achieved in some of these stilbazolium dyes. Unsymmetrical emission behaviors under 3- and 4-photon pump conditions for all these stilbazolium dyes were observed, explained and verified. In Chapter 3, DNA was successfully used as a matrix for one-, two-, and three-photon pumped stimulated emission or lasing by intercalating a multi-photon active chromophore. In Chapter 4, it is experimentally shown that both two- and three-photon absorption in a highly concentrated chromophore system can be more efficiently utilized to accomplish optical power limiting and stabilization at laser wavelengths of 1.064 mum and ˜1.3 mum, respectively. In Chapter 5, three novel 1,10-phenanthroline containing pi-conjugated chromophores with varied electron donors were synthesized and characterized together with their corresponding nickel(II) chelated complexes. Large two

  20. Spontaneous Raman and Coherent Anti-Stokes Raman Spectroscopy of Infrared Multiphoton-Excited Molecules.

    NASA Astrophysics Data System (ADS)

    Chen, Kuei-Hsien

    This thesis is a study of infrared multiphoton excitation using spontaneous and coherent anti-Stokes Raman spectroscopy. The spontaneous Raman measurements provide information on the intramolecular vibrational energy distribution over the different modes. This information is complemented by the CARS measurements which make it possible to perform state-specific studies of the vibrational and rotational distribution. For SF_6, the time-resolved spontaneous Raman measurements show complete equilibrium of energy from the pump mode to other vibrational modes. In contrast, for smaller molecules such as CF_2 Cl_2, a nonthermal energy distribution is observed after excitation. These measurements therefore disprove the general belief that the intramolecular energy distribution in infrared multiphoton molecules is always in equilibrium. The CARS measurements on bulk OCS provide values for the anharmonicities and for the energy transfer rates between modes. In addition the spectra show a very fast relaxation of the vibrational energy within the nu_2 mode. For SO_2 , the CARS measurements show that it is the nu_1 symmetric stretching mode and not the overtone excitation of the nu_2 bending mode that is pumped by the CO_2 laser. Moreover, it is shown that the hot bands of SO_2 have been incorrectly assigned up to now. Corrected values for the anharmonicities are given. In the second half of the thesis, a pulsed supersonic molecular beam is added to the infrared multiphoton excitation study. Combined with the state-specific CARS technique, the collisionless and internally cooled molecules in the beam open the door to a more detailed study of the excitation process. Pure rotational CARS is used to study the change in rotational distribution of ethylene due to infrared excitation in the beam. The appearance of rotational holes reveal which rotational states are pumped by the CO _2 laser. For OCS the evolution of the overtone population into a thermal distribution is studied

  1. Juggling Act

    ERIC Educational Resources Information Center

    Rudalevige, Andrew

    2009-01-01

    Two education bills from George W. Bush's first term are long overdue for reauthorization. One, of course, is the No Child Left Behind Act (NCLB), passed in late 2001. The other is the Education Sciences Reform Act (ESRA), which in November 2002 replaced the Office of Educational Research and Improvement (OERI) with a new Institute of Education…

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

    NASA Astrophysics Data System (ADS)

    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.

  3. ACT Test

    MedlinePlus

    ... this page helpful? Also known as: ACT; Activated Coagulation Time Formal name: Activated Clotting Time Related tests: ... in the blood called platelets and proteins called coagulation factors are activated in a sequence of steps ...

  4. Acting Atoms.

    ERIC Educational Resources Information Center

    Farin, Susan Archie

    1997-01-01

    Describes a fun game in which students act as electrons, protons, and neutrons. This activity is designed to help students develop a concrete understanding of the abstract concept of atomic structure. (DKM)

  5. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy

    PubMed Central

    Rupprecht, Peter; Prendergast, Andrew; Wyart, Claire; Friedrich, Rainer W

    2016-01-01

    There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio. PMID:27231612

  6. Fast volumetric imaging with patterned illumination via digital micro-mirror device-based temporal focusing multiphoton microscopy

    PubMed Central

    Chang, Chia-Yuan; Hu, Yvonne Yuling; Lin, Chun-Yu; Lin, Cheng-Han; Chang, Hsin-Yu; Tsai, Sheng-Feng; Lin, Tzu-Wei; Chen, Shean-Jen

    2016-01-01

    Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination. PMID:27231617

  7. Fast volumetric imaging with patterned illumination via digital micro-mirror device-based temporal focusing multiphoton microscopy.

    PubMed

    Chang, Chia-Yuan; Hu, Yvonne Yuling; Lin, Chun-Yu; Lin, Cheng-Han; Chang, Hsin-Yu; Tsai, Sheng-Feng; Lin, Tzu-Wei; Chen, Shean-Jen

    2016-05-01

    Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination. PMID:27231617

  8. Fast volumetric imaging with patterned illumination via digital micro-mirror device-based temporal focusing multiphoton microscopy.

    PubMed

    Chang, Chia-Yuan; Hu, Yvonne Yuling; Lin, Chun-Yu; Lin, Cheng-Han; Chang, Hsin-Yu; Tsai, Sheng-Feng; Lin, Tzu-Wei; Chen, Shean-Jen

    2016-05-01

    Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination.

  9. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy.

    PubMed

    Rupprecht, Peter; Prendergast, Andrew; Wyart, Claire; Friedrich, Rainer W

    2016-05-01

    There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio. PMID:27231612

  10. Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities.

    PubMed

    Heskes, A M; Lincoln, C N; Goodger, J Q D; Woodrow, I E; Smith, T A

    2012-07-01

    Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8-cineole, together with an immiscible, non-volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 μm, revealing a unique spatial organization of cavity metabolites whereby the non-volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non-volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil.

  11. Multiphoton imaging of quantum dot bioconjugates in cultured cells following Nd:YLF laser excitation

    NASA Astrophysics Data System (ADS)

    Serrano, Elba E.; Knight, V. B.

    2005-04-01

    Quantum dot bioconjugates offer unprecedented opportunities for monitoring biological processes and molecular interactions in cells, tissues, and organs. We are interested in developing applications that permit investigation of physiological processes and cytoskeletal organization in live cells, and allow imaging of complex organs, such as the auditory and vestibular sensory structures of the inner ear. Multiphoton microscopy is a powerful technique for acquiring images from deep within a sample while reducing phototoxic effects of laser light exposure on cells. Previous studies have established that a solid-state Nd:YLF laser can be used to acquire two-photon and three-photon images from live cells while minimizing phototoxic side effects (Wokosin et al., 1996, Bioimaging, 4:208-214; Squirrell et al., 1999, Nature Biotechnology, 8:763-767). We present here the results of experiments using an all-solid-state Nd:YLF 1047 nm femtosecond laser (Microlase DPM1000) source to excite quantum dot bioconjugates. Cells were labeled with Qdot (Quantum Dot Corporation) bioconjugates or with Alexa Fluor (Molecular Probes) bioconjugates and then imaged with a BioRad 1024 confocal microscope configured for multiphoton imaging using internal or external (non-descanned) detectors. Results demonstrate that the Nd:YLF laser can be used to stimulate fluorescence emission of quantum dots and Alexa Fluor bioconjugates in cultured amphibian (Xenopus) and mammalian (rat, chinese hamster) cells. We conclude that the Nd:YLF laser is a viable excitation source that extends the applicability of quantum dots for investigation of biological processes using multiphoton microscopy.

  12. Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways

    PubMed Central

    Kretschmer, Sarah; Pieper, Mario; Hüttmann, Gereon; Bölke, Torsten; Wollenberg, Barbara; Marsh, Leigh M; Garn, Holger; König, Peter

    2016-01-01

    The basic understanding of inflammatory airway diseases greatly benefits from imaging the cellular dynamics of immune cells. Current imaging approaches focus on labeling specific cells to follow their dynamics but fail to visualize the surrounding tissue. To overcome this problem, we evaluated autofluorescence multiphoton microscopy for following the motion and interaction of cells in the airways in the context of tissue morphology. Freshly isolated murine tracheae from healthy mice and mice with experimental allergic airway inflammation were examined by autofluorescence multiphoton microscopy. In addition, fluorescently labeled ovalbumin and fluorophore-labeled antibodies were applied to visualize antigen uptake and to identify specific cell populations, respectively. The trachea in living mice was imaged to verify that the ex vivo preparation reflects the in vivo situation. Autofluorescence multiphoton microscopy was also tested to examine human tissue from patients in short-term tissue culture. Using autofluorescence, the epithelium, underlying cells, and fibers of the connective tissue, as well as blood vessels, were identified in isolated tracheae. Similar structures were visualized in living mice and in the human airway tissue. In explanted murine airways, mobile cells were localized within the tissue and we could follow their migration, interactions between individual cells, and their phagocytic activity. During allergic airway inflammation, increased number of eosinophil and neutrophil granulocytes were detected that moved within the connective tissue and immediately below the epithelium without damaging the epithelial cells or connective tissues. Contacts between granulocytes were transient lasting 3 min on average. Unexpectedly, prolonged interactions between granulocytes and antigen-uptaking cells were observed lasting for an average of 13 min. Our results indicate that autofluorescence-based imaging can detect previously unknown immune cell

  13. Fast three-dimensional random access multi-photon microscopy for functional recording of neuronal activity

    NASA Astrophysics Data System (ADS)

    Reddy, Duemani; Saggau, Peter

    2007-07-01

    The dendritic processes of neurons have been shown to possess active and dynamic properties that give them the ability to modulate synaptic integration and shape individual synaptic responses. Effectively studying these properties at multiple locations on a live neuron in highly light scattering brain tissue requires an imaging/recording mechanism with high spatio-temporal resolution as well as optical sectioning and random access site selection capabilities. Our lab has made significant steps in developing such a system by combining the spatial resolution and optical sectioning ability of advanced imaging techniques such as confocal and multi-photon microscopy with the temporal resolution and random access capability provided by acousto-optic laser scanning. However, all systems that have been developed to date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which many neurons can be studied since they represent complex three-dimensional (3D) structures. We have previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of acoustooptic deflectors and does not require axial movements of the objective lens. We have also shown how, when used with the ultra-fast laser pulses needed in multi-photon microscopy, this scheme inherently compensates for the spatial dispersion which would otherwise significantly reduce the resolution of acousto-optic based multi-photon microscopy. We have now coupled this scanning scheme to a modified commercial research microscope and use the combined system to effectively image user-defined sites of interest on fluorescent 3D structures with positioning times that are in the low microsecond (μs) range. The resulting random-access scanning mechanism allows for functional imaging of complex 3D structures such as neuronal dendrites at several thousand volumes per second.

  14. Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.

    PubMed

    Kretschmer, Sarah; Pieper, Mario; Hüttmann, Gereon; Bölke, Torsten; Wollenberg, Barbara; Marsh, Leigh M; Garn, Holger; König, Peter

    2016-08-01

    The basic understanding of inflammatory airway diseases greatly benefits from imaging the cellular dynamics of immune cells. Current imaging approaches focus on labeling specific cells to follow their dynamics but fail to visualize the surrounding tissue. To overcome this problem, we evaluated autofluorescence multiphoton microscopy for following the motion and interaction of cells in the airways in the context of tissue morphology. Freshly isolated murine tracheae from healthy mice and mice with experimental allergic airway inflammation were examined by autofluorescence multiphoton microscopy. In addition, fluorescently labeled ovalbumin and fluorophore-labeled antibodies were applied to visualize antigen uptake and to identify specific cell populations, respectively. The trachea in living mice was imaged to verify that the ex vivo preparation reflects the in vivo situation. Autofluorescence multiphoton microscopy was also tested to examine human tissue from patients in short-term tissue culture. Using autofluorescence, the epithelium, underlying cells, and fibers of the connective tissue, as well as blood vessels, were identified in isolated tracheae. Similar structures were visualized in living mice and in the human airway tissue. In explanted murine airways, mobile cells were localized within the tissue and we could follow their migration, interactions between individual cells, and their phagocytic activity. During allergic airway inflammation, increased number of eosinophil and neutrophil granulocytes were detected that moved within the connective tissue and immediately below the epithelium without damaging the epithelial cells or connective tissues. Contacts between granulocytes were transient lasting 3 min on average. Unexpectedly, prolonged interactions between granulocytes and antigen-uptaking cells were observed lasting for an average of 13 min. Our results indicate that autofluorescence-based imaging can detect previously unknown immune cell

  15. Effects of multi-photon interferences from internally generated fields in strongly resonant systems

    NASA Astrophysics Data System (ADS)

    Deng, Lu; Payne, Marvin G.; Garrett, William R.

    2006-06-01

    In studies of various nonlinear optical phenomena, strong resonant features in the atomic or molecular response to multi-photon driven processes have been used to greatly enhance the visibility of otherwise weak higher-order processes. However, there are well defined circumstances where a multi-photon-resonant response of a target system leads to the generation of one or more new electromagnetic fields that can drastically change the overall system response from what would be expected from the imposed laser fields alone. New effects can occur and dominate some aspects of the nonlinear optical response because of the constructive or destructive interference between transition amplitudes along multiple excitation pathways between a given set of optically coupled states, where one of the pathways involve internally generated field(s). Under destructive interference some resonant enhancements can become completely canceled (suppressed). This review focuses on the class of optical interference effects associated with internally generated fields, that have been found to be capable of influencing a very significant number of basic physical phenomena in gas or vapor phase systems. It provides a historical overview of experimental and theoretical developments and a modern understanding of the underlying physics and its various manifestations that include: suppression of multi-photon excitation processes, suppression of stimulated emissions (Raman, hyper-Raman, and optically pumped stimulated emissions), saturation of parametric wave-mixing, pressure and beam-geometry dependent shifting of multi-photon-resonant absorption lines, and the suppression of Autler-Townes splitting and ac-stark shifts. Additionally, optical interference effects in some modern contexts, such as achieving multi-photon induced transparency, establishing single-photon self-interference based induced transparency, and generating entangled single photon states, are reviewed.

  16. Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

    PubMed

    Moalli, Federica; Proulx, Steven T; Schwendener, Reto; Detmar, Michael; Schlapbach, Christoph; Stein, Jens V

    2015-01-01

    Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy. PMID:25821451

  17. Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser

    PubMed Central

    Maruyama, Atsushi; Oshima, Yusuke; Kajiura-Kobayashi, Hiroko; Nonaka, Shigenori; Imamura, Takeshi; Naruse, Kiyoshi

    2014-01-01

    Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon–excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon–excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high–pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low–numerical aperture (NA) objective lens and high–peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec. PMID:25360352

  18. Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue

    PubMed Central

    Erami, Zahra; Herrmann, David; Warren, Sean C.; Nobis, Max; McGhee, Ewan J.; Lucas, Morghan C.; Leung, Wilfred; Reischmann, Nadine; Mrowinska, Agata; Schwarz, Juliane P.; Kadir, Shereen; Conway, James R.W.; Vennin, Claire; Karim, Saadia A.; Campbell, Andrew D.; Gallego-Ortega, David; Magenau, Astrid; Murphy, Kendelle J.; Ridgway, Rachel A.; Law, Andrew M.; Walters, Stacey N.; Grey, Shane T.; Croucher, David R.; Zhang, Lei; Herzog, Herbert; Hardeman, Edna C.; Gunning, Peter W.; Ormandy, Christopher J.; Evans, T.R. Jeffry; Strathdee, Douglas; Sansom, Owen J.; Morton, Jennifer P.; Anderson, Kurt I.; Timpson, Paul

    2015-01-01

    Summary E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments. PMID:26725115

  19. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

    PubMed Central

    Grandjean, Capucine L.; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A.; Bousso, Philippe

    2016-01-01

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. PMID:27698437

  20. Intravital and Whole-Organ Imaging Reveals Capture of Melanoma-Derived Antigen by Lymph Node Subcapsular Macrophages Leading to Widespread Deposition on Follicular Dendritic Cells

    PubMed Central

    Moalli, Federica; Proulx, Steven T.; Schwendener, Reto; Detmar, Michael; Schlapbach, Christoph; Stein, Jens V.

    2015-01-01

    Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy. PMID:25821451

  1. In situ quantitative monitoring of polyplexes and polyplex micelles in the blood circulation using intravital real-time confocal laser scanning microscopy.

    PubMed

    Nomoto, Takahiro; Matsumoto, Yu; Miyata, Kanjiro; Oba, Makoto; Fukushima, Shigeto; Nishiyama, Nobuhiro; Yamasoba, Tatsuya; Kataoka, Kazunori

    2011-04-30

    Surface modification using poly(ethylene glycol) (PEG) is a widely used strategy to improve the biocompatibility of cationic polymer-based nonviral gene vectors (polyplexes). A novel method based on intravital real-time confocal laser scanning microscopy (IVRTCLSM) was applied to quantify the dynamic states of polyplexes in the bloodstream, thereby demonstrating the efficacy of PEGylation to prevent their agglomeration. Blood flow in the earlobe blood vessels of experimental animals was monitored in a noninvasive manner to directly observe polyplexes in the circulation. Polyplexes formed distinct aggregates immediately after intravenous injection, followed by interaction with platelets. To quantify aggregate formation and platelet interaction, the coefficient of variation and Pearson's correlation coefficient were adopted. In contrast, polyplex micelles prepared through self-assembly of plasmid DNA with PEG-based block catiomers had dense PEG palisades, revealing no formation of aggregates without visible interaction with platelets during circulation. This is the first report of in situ monitoring and quantification of the availability of PEGylation to prevent polyplexes from agglomeration over time in the blood circulation. This shows the high utility of IVRTCLSM in drug and gene delivery research.

  2. ATOMIC AND MOLECULAR PHYSICS: Multiphoton ionization of the hydrogen atom exposed to circularly or linearly polarized laser pulses

    NASA Astrophysics Data System (ADS)

    Wang, Pei-Jie; He, Feng

    2009-12-01

    This paper studies the multiphoton ionization of the hydrogen atom exposed to the linearly or circularly polarized laser pulses by solving the time-dependent Schrödinger equation. It finds that the ratio of the ionization probabilities by linearly and circularly polarized laser pulses varies with the numbers of absorbing photons. With the same laser intensity, the circularly polarized laser pulse favors to ionize the atom with more ease than the linearly polarized laser pulse if only two or three photons are necessary to be absorbed. For the higher order multiphoton ionization, the linearly polarized laser pulse has the advantage over circularly polarized laser pulse to ionize the atom.

  3. Enhanced high-order-harmonic generation and wave mixing via two-color multiphoton excitation of atoms and molecules

    NASA Astrophysics Data System (ADS)

    Avetissian, H. K.; Avchyan, B. R.; Mkrtchian, G. F.

    2016-07-01

    We consider harmonics generation and wave mixing by two-color multiphoton resonant excitation of three-level atoms and 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 a generalized rotating wave approximation are in a good agreement with numerical calculations. The results applied to the hydrogen atoms and homonuclear diatomic molecular ions show that one can achieve efficient generation of moderately high multicolor harmonics via multiphoton resonant excitation by appropriate laser pulses.

  4. Multiphoton microscopy of transdermal quantum dot delivery using two photon polymerization-fabricated polymer microneedles

    PubMed Central

    Gittard, Shaun D; Miller, Philip R; Boehm, Ryan D; Ovsianikov, Aleksandr; Chichkov, Boris N; Heiser, Jeremy; Gordon, John; Monteiro-Riviere, Nancy A; Narayan, Roger J

    2010-01-01

    Due to their ability to serve as fluorophores and drug delivery vehicles, quantum dots are a powerful tool for theranostics-based clinical applications. In this study, microneedle devices for transdermal drug delivery were fabricated by means of two-photon polymerization of an acrylate-based polymer. We examined proliferation of cells on this polymer using neonatal human epidermal keratinocytes and human dermal fibroblasts. The microneedle device was used to inject quantum dots into porcine skin; imaging of the quantum dots was performed using multiphoton microscopy. PMID:21413181

  5. Multiphoton Absorption by Helium, Magnesium and H2 at High Frequencies and Intensities

    NASA Astrophysics Data System (ADS)

    Taylor, K. T.

    We review principally some recent work carried out in Belfast and Heraklion which handles the few-electron dynamics of atomic and molecular systems exposed to high frequency, high intensity laser fields. The design and application of the quantitatively accurate computational methods is discussed. The Belfast work is illustrated by results for double ionization of helium and the hydrogen molecule where in each case the two electrons have been handled in full-dimensionality. The first results for multiphoton, double ionization of a complex atom, namely magnesium demonstrate an important application of the Heraklion approach.

  6. Strong-field high-frequency approximation to the multiphoton ionization of hydrogen

    SciTech Connect

    Trombetta, F. ); Basile, S.; Ferrante, G. )

    1990-04-01

    The strong-field multiphoton ionization of atoms is considered and a theoretical approach dealing nonperturbatively with the radiation field formulated. The general computational scheme is the conventional perturbation theory, but the intermediate states are dressed by the field. We present in detail a method to dress the continuum states and to study the dipole transitions within the continuum. In the high-frequency domain, the proposed procedure rapidly converges over a wide range of field intensity and offers an interesting framework for calculating ionization rates for arbitrary numbers of absorbed (above-threshold) photons and field polarization.

  7. Multiphoton resonances for all-optical quantum logic with multiple cavities

    NASA Astrophysics Data System (ADS)

    Everitt, Mark S.; Garraway, Barry M.

    2014-07-01

    We develop a theory for the interaction of multilevel atoms with multimode cavities yielding cavity-enhanced multiphoton resonances. The locations of the resonances are predicted from the use of effective two- and three-level Hamiltonians. As an application we show that quantum gates can be realized when photonic qubits are encoded on the cavity modes in arrangements where ancilla atoms transit the cavity. The fidelity of operations is increased by conditional measurements on the atom and by the use of a selected, dual-rail, Hilbert space. A universal set of gates is proposed, including the Fredkin gate and iswap operation; the system seems promising for scalability.

  8. Histology in vivo: chemical contrast combined with clinical multimodal multiphoton tomography

    NASA Astrophysics Data System (ADS)

    Weinigel, Martin; Breunig, Hans Georg; Koenig, Karsten

    2015-03-01

    Label-free multiphoton tomography based on two-photon autofluorescence, fluorescence lifetime, and second harmonic generation imaging can be supplemented by coherent anti-Stokes Raman scattering. We present a compact, mobile and flexible clinical tomograph equipped with a novel detector design with multiple miniaturized detectors for individual acquisition of all four contrast mechanisms. Imaging of endogenous fluorophores, SHG-active collagen as well as nonfluorescent lipids in human skin in vivo is possible with this clinical tomograph paving the way towards in vivo histology.

  9. Measurement of the absolute cross section for multiphoton ionization of atomic hydrogen at 248 nm

    SciTech Connect

    Kyrala, G.A.; Nichols, T.D.

    1990-01-01

    We present measurements of the absolute rates for multiphoton ionization of the ground state from atomic hydrogen by a linearly polarized, subpicosecond KrF laser pulse at a wavelength of 248 nm. A laser crossed atomic beam technique is used. The irradiance was varied from 3{times}10{sup 12} w/cm{sup 2} to 2{times}10{sup 14} w/cm{sup 2} and three above threshold ionization peaks were observed. The measured rate for total electron production is less than predicted by the numerical and perturbation calculations, but significantly higher than calculated by the Reiss and Keldysh methods. 21 refs., 7 figs.

  10. Waveguide-integrated single- and multi-photon detection at telecom wavelengths using superconducting nanowires

    SciTech Connect

    Ferrari, Simone; Kahl, Oliver; Kovalyuk, Vadim; Goltsman, Gregory N.; Korneev, Alexander; Pernice, Wolfram H. P.

    2015-04-13

    We investigate single- and multi-photon detection regimes of superconducting nanowire detectors embedded in silicon nitride nanophotonic circuits. At near-infrared wavelengths, simultaneous detection of up to three photons is observed for 120 nm wide nanowires biased far from the critical current, while narrow nanowires below 100 nm provide efficient single photon detection. A theoretical model is proposed to determine the different detection regimes and to calculate the corresponding internal quantum efficiency. The predicted saturation of the internal quantum efficiency in the single photon regime agrees well with plateau behavior observed at high bias currents.

  11. Generation of multiphoton entangled quantum states by means of integrated frequency combs.

    PubMed

    Reimer, Christian; Kues, Michael; Roztocki, Piotr; Wetzel, Benjamin; Grazioso, Fabio; Little, Brent E; Chu, Sai T; Johnston, Tudor; Bromberg, Yaron; Caspani, Lucia; Moss, David J; Morandotti, Roberto

    2016-03-11

    Complex optical photon states with entanglement shared among several modes are critical to improving our fundamental understanding of quantum mechanics and have applications for quantum information processing, imaging, and microscopy. We demonstrate that optical integrated Kerr frequency combs can be used to generate several bi- and multiphoton entangled qubits, with direct applications for quantum communication and computation. Our method is compatible with contemporary fiber and quantum memory infrastructures and with chip-scale semiconductor technology, enabling compact, low-cost, and scalable implementations. The exploitation of integrated Kerr frequency combs, with their ability to generate multiple, customizable, and complex quantum states, can provide a scalable, practical, and compact platform for quantum technologies.

  12. In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

    2014-03-01

    The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

  13. Imaging NO elimination in the infrared multiphoton dissociation of nitroalkanes and alkyl nitrites

    NASA Astrophysics Data System (ADS)

    Fernando, Ravin; Ariyasingha, Nuwandi M.; Suits, Arthur G.

    2016-02-01

    We present a DC slice imaging study of the decomposition of C2, C3 and C4 nitroalkanes and alkyl nitrites, focusing on the NO elimination channel, possibly a minor pathway. Infrared multiphoton dissociation (IRMPD) is used to induce dissociation on the ground electronic state under collisionless conditions. The channels that produced NO as a product were studied and compared among the target molecules to gain a better understanding of the isomerization of the nitroalkanes prior to dissociation. Trends in the total translational energy and NO rotational temperatures obtained from the images are discussed.

  14. Blind frequency-resolved optical-gating pulse characterization for quantitative differential multiphoton microscopy.

    PubMed

    Field, Jeffrey J; Durfee, Charles G; Squier, Jeff A

    2010-10-15

    We use a unique multifocal multiphoton microscope to directly characterize the pulse in the focal plane of a high-NA objective using second-harmonic generation frequency-resolved optical gating (FROG). Because of the nature of the optical setup, femtosecond laser pulses of orthogonal polarization states are generated in the focal plane, each acquiring a different spectral dispersion. By applying an additional constraint on the phase extraction algorithm, we simultaneously extract both the gate and probe pulses from a single spectrogram with a FROG error of 0.016. PMID:20967069

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Electroluminescence and multiphoton effects in a resonator driven by a tunnel junction

    NASA Astrophysics Data System (ADS)

    Jin, Jinshuang; Marthaler, Michael; Schön, Gerd

    2015-02-01

    We study a transmission line resonator which is driven by electrons tunneling through a voltage-biased tunnel junction. Using the Born-Markovian quantum master equation in the polaron basis we investigate the nonequilibrium photon state and emission spectrum of the resonator as well as properties of the transport current across the tunnel junction and its noise spectrum. The electroluminescence is optimized, with maximum peak height and narrow linewidth, when the back-action of the tunnel junction on the resonator and the damping of the resonator are similar in strength. For strong coupling between the resonator and tunnel junction, multiphoton effects create signatures in the transport current and current noise spectrum.

  17. Coherent control of radiation patterns of nonlinear multiphoton processes in nanoparticles.

    PubMed

    Papoff, Francesco; McArthur, Duncan; Hourahine, Ben

    2015-07-09

    We propose 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. We derive conditions on the 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. The control introduces narrow features in the response of the particles that result in high sensitivity to small variations in the local environment, including subwavelength spatial shifts.

  18. Characterization of multiphoton laser scanning device optical parameters for image restoration

    NASA Astrophysics Data System (ADS)

    Fischer, Frank; Konig, Karsten; Puschmann, Stefan; Wepf, Roger; Riemann, Iris; Ulrich, Volker; Fischer, Peter

    2004-09-01

    Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 μm tissue depth, was improved.

  19. Experimental quantum teleportation and multiphoton entanglement via interfering narrowband photon sources

    SciTech Connect

    Yang Jian; Zhang Han; Peng Chengzhi; Chen Zengbing; Bao Xiaohui; Chen Shuai; Pan Jianwei

    2009-10-15

    In this paper, we report a realization of synchronization-free quantum teleportation and narrowband three-photon entanglement through interfering narrowband photon sources. Since both the single-photon and the entangled photon pair utilized are completely autonomous, it removes the requirement of high-demanding synchronization techniques in long-distance quantum communication with pulsed spontaneous parametric down-conversion sources. The frequency linewidth of the three-photon entanglement realized is on the order of several MHz, which matches the requirement of atomic ensemble based quantum memories. Such a narrowband multiphoton source will have applications in some advanced quantum communication protocols and linear optical quantum computation.

  20. Characterization of multiphoton photoacoustic spectroscopy for subsurface brain tissue diagnosis and imaging

    NASA Astrophysics Data System (ADS)

    Dahal, Sudhir; Cullum, Brian M.

    2016-04-01

    The development and demonstration of a multiphoton photoacoustic imaging technique capable of providing high spatial resolution chemical images of subsurface tissue components as deep as 1.4 cm below the tissue surface is described. By combining multiphoton excitation in the diagnostic window (650 to 1100 nm), with ultrasonic detection of nonradiative relaxation events, it is possible to rapidly reconstruct three-dimensional, chemical specific, images of samples underneath overlying structures as well as chemical species of the same material. Demonstration of this technique for subsurface tissue differentiation is shown, with the ability to distinguish between grade III astrocytoma tissue and adjacent healthy tissue in blind studies. By employing photoacoustic signal detection, the high nonradiative relaxation rates of most biological tissue components (>90%) and the minimal signal attenuation of the resulting ultrasound compensate for excitation efficiency losses associated with two-photon absorption. Furthermore, the two-photon absorption process results in a highly localized excitation volume (ca., 60 μm). Characterization of the probing depth, spatial resolution, and ability to image through overlying structures is also demonstrated in this paper using tissue phantoms with well-characterized optical scattering properties, mimicking those of tissues.

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

    PubMed

    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.

  2. Multiphoton absorption and decomposition of fluoroform-d: Laser isotope separation of deuterium

    SciTech Connect

    Evans, D.K.; McAlpine, R.D.; Adams, H.M.

    1982-10-01

    Multiphoton absorption (MPA) studies of fluoroform-d, a molecule of interest for potential laser based hydrogen isotope separation processes, are reported for CDF/sub 3/ pressures 0.2--1.3 kPa, and for a variety of 10 ..mu..m CO/sub 2/ laser lines with pulse widths of 2 or 6 ns and fluences within the range 10/sup -3/--70 J/cm/sup 2/. Unlike SF/sub 6/, no red shift of the MPA spectrum relative to the small signal spectrum was observed at high fluence. Selective multiphoton decomposition (MPD) experiments using the 10R(26) line, 6 ns pulse to excite the CDF/sub 3/ component in natural-abundance CHF/sub 3/ (approx. 150 ppm D/H) at a pressure of 13.3 kPa resulted in the recovery of water enriched up to 30% in deuterium: a measured isotope enrichment of > or =2000 fold. This demonstrates that a product, highly enriched in deuterium, can be recovered from the selective MPD of fluoroform.

  3. Multiphoton Ionization of Atoms and Molecules with Soft and Hard X-rays

    NASA Astrophysics Data System (ADS)

    Rolles, Daniel

    2015-05-01

    We have recently extended our previous investigations of the multiphoton ionization of heavy atoms, such as Kr and Xe, and of high-Z atom containing molecules from the soft into the hard X-ray range as well as into the XUV regime. Using the 100-nm focus environment at LCLS, we were able to reach peak intensities up to 1019W/cm2 at photon energies between 5 to 9 keV. This allows studying atomic and molecular ionization processes under unprecedented X-ray intensities and, in particular, under the identical conditions where typical coherent diffractive imaging experiments are performed. Our results are thus important benchmarks for calculating radiation damage effects in FEL-based X-ray imaging experiments. Using new micro-focusing capabilities at FLASH, we also extended our studies into the XUV range between 70 and 200 eV photon energy and observed significantly higher charge states than previously reported. I will present the results from our recent measurements at LCLS and FLASH and discuss the different multiphoton ionization mechanisms that play a role in the XUV, soft, and hard X-ray range.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Hybrid Multiphoton Volumetric Functional Imaging of Large Scale Bioengineered Neuronal Networks

    PubMed Central

    Paluch, Shir; Dvorkin, Roman; Brosh, Inbar; Shoham, Shy

    2014-01-01

    Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bio-engineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes/sec of structures with mm-scale dimensions containing a network of over 1000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances. PMID:24898000

  8. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin.

    PubMed Central

    Masters, B R; So, P T; Gratton, E

    1997-01-01

    Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 PMID:9168018

  9. Imaging sulfur mustard lesions in human epidermal tissues and keratinocytes by confocal and multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert; Madren-Whalley, Janna S.

    2002-06-01

    Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.

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

    PubMed

    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.

  11. Role of quantum trajectory in high-order harmonic generation in the Keldysh multiphoton regime.

    PubMed

    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. PMID:27410589

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

    SciTech Connect

    Borglin, Johan; Guldbrand, Stina; Evenbratt, Hanne; Kirejev, Vladimir; Ericson, Marica B.; Grönbeck, Henrik

    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.

  13. Characterization of multiphoton photoacoustic spectroscopy for subsurface brain tissue diagnosis and imaging

    NASA Astrophysics Data System (ADS)

    Dahal, Sudhir; Cullum, Brian M.

    2016-04-01

    The development and demonstration of a multiphoton photoacoustic imaging technique capable of providing high spatial resolution chemical images of subsurface tissue components as deep as 1.4 cm below the tissue surface is described. By combining multiphoton excitation in the diagnostic window (650 to 1100 nm), with ultrasonic detection of nonradiative relaxation events, it is possible to rapidly reconstruct three-dimensional, chemical specific, images of samples underneath overlying structures as well as chemical species of the same material. Demonstration of this technique for subsurface tissue differentiation is shown, with the ability to distinguish between grade III astrocytoma tissue and adjacent healthy tissue in blind studies. By employing photoacoustic signal detection, the high nonradiative relaxation rates of most biological tissue components (>90%) and the minimal signal attenuation of the resulting ultrasound compensate for excitation efficiency losses associated with two-photon absorption. Furthermore, the two-photon absorption process results in a highly localized excitation volume (ca., 60 μm). Characterization of the probing depth, spatial resolution, and ability to image through overlying structures is also demonstrated in this paper using tissue phantoms with well-characterized optical scattering properties, mimicking those of tissues.

  14. Minimum Copies of Schrödinger's Cat State in the Multi-Photon System.

    PubMed

    Lu, Yiping; Zhao, Qing

    2016-01-01

    Multi-photon entanglement has been successfully studied by many theoretical and experimental groups. However, as the number of entangled photons increases, some problems are encountered, such as the exponential increase of time necessary to prepare the same number of copies of entangled states in experiment. In this paper, a new scheme is proposed based on the Lagrange multiplier and Feedback, which cuts down the required number of copies of Schrödinger's Cat state in multi-photon experiment, which is realized with some noise in actual measurements, and still keeps the standard deviation in the error of fidelity unchanged. It reduces about five percent of the measuring time of eight-photon Schrödinger's Cat state compared with the scheme used in the usual planning of actual measurements, and moreover it guarantees the same low error in fidelity. In addition, we also applied the same approach to the simulation of ten-photon entanglement, and we found that it reduces in priciple about twenty two percent of the required copies of Schrödinger's Cat state compared with the conventionally used scheme of the uniform distribution; yet the distribution of optimized copies of the ten-photon Schrödinger's Cat state gives better fidelity estimation than the uniform distribution for the same number of copies of the ten-photon Schrödinger's Cat state. PMID:27576585

  15. Photon-momentum transfer in multiphoton ionization and in time-resolved holography with photoelectrons

    NASA Astrophysics Data System (ADS)

    Chelkowski, Szczepan; Bandrauk, André D.; Corkum, Paul B.

    2015-11-01

    In most models and theoretical calculations describing multiphoton ionization by infrared light, the dipole approximation is used. This is equivalent to setting the very small photon momentum to zero. Using numerical solutions of the two-dimensional (2-D) time-dependent Schrödinger equation for one electron (H-like) systems, we show that, for linear polarization, the radiation pressure on photoelectrons is very sensitive to the details of the ionization mechanism. The directly ionized photoelectrons, those that never recollide with the parent ion, are driven in the direction of the laser photon momentum, whereas a fraction of slower photoelectrons are pushed in the opposite direction, leading to the counterintuitive shifts observed in recent experiments [Phys. Rev. Lett. 113, 243001 (2014), 10.1103/PhysRevLett.113.243001]. This complex response is due to the interplay between the Lorentz force and the Coulomb attraction from the ion. On average, however, the photoelectron momentum is in the direction of the photon momentum as in the case of circular polarization. The influence of the photon momentum is shown to be discernible in the holographic patterns of time-resolved atomic and molecular holography with photoelectrons, thus suggesting a new research subject in multiphoton ionization.

  16. Development of a portable multiphoton photo-acoustic spectroscopy system for tumor diagnostics

    NASA Astrophysics Data System (ADS)

    Chandrasekharan, Nirmala; Kiser, John B.; Cullum, Brian M.

    2004-12-01

    In this paper we describe the development of a novel fiber optic probe for subsurface tumor diagnostics, based on non-resonant multiphoton photoacoustic spectroscopy (NMPPAS). In this technique, endogenous biomarkers present in tissues are irradiated in the near infrared, using a tunable high-power laser. The resulting multiphoton excitation events are detected as an acoustic (i.e. ultrasonic) signal, using an ultrasonic piezoelectric transducer. The signal from the piezoelectric transducer is then corrected for laser power fluctuations by normalizing the NMPPAS signal at each wavelength with the laser intensity recorded, from an optical diode. By scanning the laser excitation over the appropriate wavelength range for the tissue of interest, absorption differences between normal and tumor tissues can be measured and analyzed. The fiber optic probe was characterized and optimized for transmission efficiency as well as its time dependent response to high power laser pulses. The focusing optics were optimized and a piezoelectric transducer film detector chosen based on its sensitivity in the ultrasonic frequency range of interest. Using this probe system NMPPAS measurements were performed on several common fluorescent dyes including rhodamine 6G as well as well-characterized biomarkers like tryptophan. Furthermore, the technique was further successfully applied to the differentiation of tumorous and healthy human brain tissues.

  17. Femtosecond Laser-Induced Upconversion Luminescence in Rare-Earth Ions by Nonresonant Multiphoton Absorption.

    PubMed

    Yao, Yunhua; Xu, Cheng; Zheng, Ye; Yang, Chengshuai; Liu, Pei; Jia, Tianqing; Qiu, Jianrong; Sun, Zhenrong; Zhang, Shian

    2016-07-21

    The upconversion luminescence of rare-earth ions has attracted considerable interest because of its important applications in photoelectric conversion, color display, laser device, multiplexed biolabeling, and security printing. Previous studies mainly explored the upconversion luminescence generation through excited state absorption, energy transfer upconversion, and photon avalanche under the continuous wave laser excitation. Here, we focus on the upconversion luminescence generation through a nonresonant multiphoton absorption by using the intense femtosecond pulsed laser excitation and study the upconversion luminescence intensity control by varying the femtosecond laser phase and polarization. We show that the upconversion luminescence of rare-earth ions under the intense femtosecond laser field excitation is easy to be obtained due to the nonresonant multiphoton absorption through the nonlinear interaction between light and matter, which is not available by the continuous wave laser excitation in previous works. We also show that the upconversion luminescence intensity can be effectively controlled by varying the femtosecond pulsed laser phase and polarization, which can open a new technological opportunity to generate and control the upconversion luminescence of rare-earth ions and also can be further extended to the relevant application areas. PMID:27367751

  18. Multiphoton crosslinking for biocompatible 3D printing of type I collagen.

    PubMed

    Bell, Alex; Kofron, Matthew; Nistor, Vasile

    2015-09-01

    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.

  19. A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

    PubMed Central

    Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

    2015-01-01

    We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

  20. Minimum Copies of Schrödinger’s Cat State in the Multi-Photon System

    PubMed Central

    Lu, Yiping; Zhao, Qing

    2016-01-01

    Multi-photon entanglement has been successfully studied by many theoretical and experimental groups. However, as the number of entangled photons increases, some problems are encountered, such as the exponential increase of time necessary to prepare the same number of copies of entangled states in experiment. In this paper, a new scheme is proposed based on the Lagrange multiplier and Feedback, which cuts down the required number of copies of Schrödinger’s Cat state in multi-photon experiment, which is realized with some noise in actual measurements, and still keeps the standard deviation in the error of fidelity unchanged. It reduces about five percent of the measuring time of eight-photon Schrödinger’s Cat state compared with the scheme used in the usual planning of actual measurements, and moreover it guarantees the same low error in fidelity. In addition, we also applied the same approach to the simulation of ten-photon entanglement, and we found that it reduces in priciple about twenty two percent of the required copies of Schrödinger’s Cat state compared with the conventionally used scheme of the uniform distribution; yet the distribution of optimized copies of the ten-photon Schrödinger’s Cat state gives better fidelity estimation than the uniform distribution for the same number of copies of the ten-photon Schrödinger’s Cat state. PMID:27576585

  1. Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection.

    PubMed

    Warren, Sean C; Kim, Youngchan; Stone, James M; Mitchell, Claire; Knight, Jonathan C; Neil, Mark A A; Paterson, Carl; French, Paul M W; Dunsby, Chris

    2016-09-19

    This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging. PMID:27661887

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Design and implementation of fiber-based multiphoton endoscopy with microelectromechanical systems scanning

    PubMed Central

    Tang, Shuo; Jung, Woonggyu; McCormick, Daniel; Xie, Tuqiang; Su, Jiangping; Ahn, Yeh-Chan; Tromberg, Bruce J.; Chen, Zhongping

    2010-01-01

    A multiphoton endoscopy system has been developed using a two-axis microelectromechanical systems (MEMS) mirror and double-cladding photonic crystal fiber (DCPCF). The MEMS mirror has a 2-mm-diam, 20-deg optical scanning angle, and 1.26-kHz and 780-Hz resonance frequencies on the x and y axes. The maximum number of resolvable focal spots of the MEMS scanner is 720×720 on the x and y axes, which indicates that the MEMS scanner can potentially support high-resolution multiphoton imaging. The DCPCF is compared with standard single-mode fiber and hollow-core photonic bandgap fiber on the basis of dispersion, attenuation, and coupling efficiency properties. The DCPCF has high collection efficiency, and its dispersion can be compensated by grating pairs. Three configurations of probe design are investigated, and their imaging quality and field of view are compared. A two-lens configuration with a collimation and a focusing lens provides the optimum imaging performance and packaging flexibility. The endoscope is applied to image fluorescent microspheres and bovine knee joint cartilage. PMID:19566298

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

    NASA Astrophysics Data System (ADS)

    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.

  6. DNA Multiphoton Absorption Generates Localized Damage for Studying Repair Dynamics in Live Cells

    PubMed Central

    Daddysman, Matthew K.; Fecko, Christopher J.

    2011-01-01

    Investigations into the spatiotemporal dynamics of DNA repair using live-cell imaging are aided by the ability to generate well defined regions of ultravioletlike photolesions in an optical microscope. We demonstrate that multiphoton excitation of DNA in live cells with visible femtosecond pulses produces thymine cyclopyrimidine dimers (CPDs), the primary ultraviolet DNA photoproduct. The CPDs are produced with a cubic to supercubic power dependence using pulses in the wavelength range from at least 400 to 525 nm. We show that the CPDs are confined in all three spatial dimensions, making multiphoton excitation of DNA with visible light an ideal technique for generating localized DNA photolesions in a wide variety of samples, from cultured cells to thicker tissues. We demonstrate the utility of this method by applying it to investigate the spatiotemporal recruitment of GFP-tagged topoisomerase I (TopI) to sites of localized DNA damage in polytene chromosomes within live cells of optically thick Drosophila salivary glands. PMID:22067170

  7. Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities

    PubMed Central

    Wokosin, David L.; Squirrell, Jayne M.; Eliceiri, Kevin W.; White, John G.

    2008-01-01

    Experimental laser microbeam techniques have become established tools for studying living specimens. A steerable, focused laser beam may be used for a variety of experimental manipulations such as laser microsurgery, optical trapping, localized photolysis of caged bioactive probes, and patterned photobleaching. Typically, purpose-designed experimental systems have been constructed for each of these applications. In order to assess the consequences of such experimental optical interventions, long-term, microscopic observation of the specimen is often required. Multiphoton excitation, because of its ability to obtain high-contrast images from deep within a specimen with minimal phototoxic effects, is a preferred technique for in vivo imaging. An optical workstation is described that combines the functionality of an experimental optical microbeam apparatus with a sensitive multiphoton imaging system designed for use with living specimens. Design considerations are discussed and examples of ongoing biological applications are presented. The integrated optical workstation concept offers advantages in terms of flexibility and versatility relative to systems implemented with separate imaging and experimental components. PMID:18607511

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Characterizing liver capsule microstructure via in situ bulge test coupled with multiphoton imaging.

    PubMed

    Jayyosi, C; Coret, M; Bruyère-Garnier, K

    2016-02-01

    The characterization of biological tissue at the microscopic scale is the starting point of many applications in tissue engineering and especially in the development of structurally based constitutive models. In the present study, focus is made on the liver capsule, the membrane encompassing hepatic parenchyma, which takes a huge part in liver mechanical properties. An in situ bulge test experiment under a multiphoton microscope has been developed to assess the microstructure changes that arise with biaxial loading. Multiphoton microscopy allows to observe the elastin and collagen fiber networks simultaneously. Thus a description of the microstructure organization of the capsule is given, characterizing the shapes, geometry and arrangement of fibers. The orientation of fibers is calculated and orientation distribution evolution with loading is given, in the case of an equibiaxial and two non equibiaxial loadings, thanks to a circular and elliptic set up of the bulge test. The local strain fields have also been computed, by the mean of a photobleaching grid, to get an idea of what the liver capsule might experience when subjected to internal pressure. Results show that strain fields present some heterogeneity due to anisotropy. Reorientation occurs in non equibiaxial loadings and involves fibers layers from the inner to the outer surface as expected. Although there is a fiber network rearrangement to accommodate with loading in the case of equibiaxial loading, there is no significant reorientation of the main fibers direction of the different layers.

  10. Minimum Copies of Schrödinger's Cat State in the Multi-Photon System.

    PubMed

    Lu, Yiping; Zhao, Qing

    2016-08-31

    Multi-photon entanglement has been successfully studied by many theoretical and experimental groups. However, as the number of entangled photons increases, some problems are encountered, such as the exponential increase of time necessary to prepare the same number of copies of entangled states in experiment. In this paper, a new scheme is proposed based on the Lagrange multiplier and Feedback, which cuts down the required number of copies of Schrödinger's Cat state in multi-photon experiment, which is realized with some noise in actual measurements, and still keeps the standard deviation in the error of fidelity unchanged. It reduces about five percent of the measuring time of eight-photon Schrödinger's Cat state compared with the scheme used in the usual planning of actual measurements, and moreover it guarantees the same low error in fidelity. In addition, we also applied the same approach to the simulation of ten-photon entanglement, and we found that it reduces in priciple about twenty two percent of the required copies of Schrödinger's Cat state compared with the conventionally used scheme of the uniform distribution; yet the distribution of optimized copies of the ten-photon Schrödinger's Cat state gives better fidelity estimation than the uniform distribution for the same number of copies of the ten-photon Schrödinger's Cat state.

  11. Minimum Copies of Schrödinger’s Cat State in the Multi-Photon System

    NASA Astrophysics Data System (ADS)

    Lu, Yiping; Zhao, Qing

    2016-08-01

    Multi-photon entanglement has been successfully studied by many theoretical and experimental groups. However, as the number of entangled photons increases, some problems are encountered, such as the exponential increase of time necessary to prepare the same number of copies of entangled states in experiment. In this paper, a new scheme is proposed based on the Lagrange multiplier and Feedback, which cuts down the required number of copies of Schrödinger’s Cat state in multi-photon experiment, which is realized with some noise in actual measurements, and still keeps the standard deviation in the error of fidelity unchanged. It reduces about five percent of the measuring time of eight-photon Schrödinger’s Cat state compared with the scheme used in the usual planning of actual measurements, and moreover it guarantees the same low error in fidelity. In addition, we also applied the same approach to the simulation of ten-photon entanglement, and we found that it reduces in priciple about twenty two percent of the required copies of Schrödinger’s Cat state compared with the conventionally used scheme of the uniform distribution; yet the distribution of optimized copies of the ten-photon Schrödinger’s Cat state gives better fidelity estimation than the uniform distribution for the same number of copies of the ten-photon Schrödinger’s Cat state.

  12. Chronic imaging of amyloid plaques in the live mouse brain using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Bacskai, Brian J.; Kajdasz, Stephen T.; Christie, R. H.; Zipfel, Warren R.; Williams, Rebecca M.; Kasischke, Karl A.; Webb, Watt W.; Hyman, B. T.

    2001-04-01

    Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.

  13. Secure satellite communication using multi-photon tolerant quantum communication protocol

    NASA Astrophysics Data System (ADS)

    Darunkar, Bhagyashri; Punekar, Nikhil; Verma, Pramode K.

    2015-09-01

    This paper proposes and analyzes the potential of a multi-photon tolerant quantum communication protocol to secure satellite communication. For securing satellite communication, quantum cryptography is the only known unconditionally secure method. A number of recent experiments have shown feasibility of satellite-aided global quantum key distribution (QKD) using different methods such as: Use of entangled photon pairs, decoy state methods, and entanglement swapping. The use of single photon in these methods restricts the distance and speed over which quantum cryptography can be applied. Contemporary quantum cryptography protocols like the BB84 and its variants suffer from the limitation of reaching the distances of only Low Earth Orbit (LEO) at the data rates of few kilobits per second. This makes it impossible to develop a general satellite-based secure global communication network using the existing protocols. The method proposed in this paper allows secure communication at the heights of the Medium Earth Orbit (MEO) and Geosynchronous Earth Orbit (GEO) satellites. The benefits of the proposed method are two-fold: First it enables the realization of a secure global communication network based on satellites and second it provides unconditional security for satellite networks at GEO heights. The multi-photon approach discussed in this paper ameliorates the distance and speed issues associated with quantum cryptography through the use of contemporary laser communication (lasercom) devices. This approach can be seen as a step ahead towards global quantum communication.

  14. Development and characterization of non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) for brain tumor margining

    NASA Astrophysics Data System (ADS)

    Dahal, Sudhir

    During tumor removal surgery, due to the problems associated with obtaining high-resolution, real-time chemical images of where exactly the tumor ends and healthy tissue begins (tumor margining), it is often necessary to remove a much larger volume of tissue than the tumor itself. In the case of brain tumor surgery, however, it is extremely unsafe to remove excess tissue. Therefore, without an accurate image of the tumor margins, some of the tumor's finger-like projections are inevitably left behind in the surrounding parenchyma to grow again. For this reason, the development of techniques capable of providing high-resolution real-time images of tumor margins up to centimeters below the surface of a tissue is ideal for the diagnosis and treatment of tumors, as well as surgical guidance during brain tumor excision. A novel spectroscopic technique, non-resonant multiphoton photoacoustic spectroscopy (NMPPAS), is being developed with the capabilities of obtaining high-resolution subsurface chemical-based images of underlying tumors. This novel technique combines the strengths of multiphoton tissue spectroscopy and photoacoustic spectroscopy into a diagnostic methodology that will, ultimately, provide unparalleled chemical information and images to provide the state of sub-surface tissues. The NMPPAS technique employs near-infrared light (in the diagnostic window) to excite ultraviolet and/or visible light absorbing species deep below the tissue's surface. Once a multiphoton absorption event occurs, non-radiative relaxation processes generates a localized thermal expansion and subsequent acoustic wave that can be detected using a piezoelectric transducer. Since NMPPAS employs an acoustic detection modality, much deeper diagnoses can be performed than that is possible using current state of the art high-resolution chemical imaging techniques such as multiphoton fluorescence spectroscopy. NMPPAS was employed to differentiate between excised brain tumors (astrocytoma III

  15. Statistical analysis on activation and photo-bleaching of step-wise multi-photon activation fluorescence of melanin

    NASA Astrophysics Data System (ADS)

    Gu, Zetong; Lai, Zhenhua; Zhang, Xi; Yin, Jihao; DiMarzio, Charles A.

    2015-03-01

    Melanin is regarded as the most enigmatic pigments/biopolymers found in most organisms. We have shown previously that melanin goes through a step-wise multi-photon absorption process after the fluorescence has been activated with high laser intensity. No melanin step-wise multi-photon activation fluorescence (SMPAF) can be obtained without the activation process. The step-wise multi-photon activation fluorescence has been observed to require less laser power than what would be expected from a non-linear optical process. In this paper, we examined the power dependence of the activation process of melanin SMPAF at 830nm and 920nm wavelengths. We have conducted research using varying the laser power to activate the melanin in a point-scanning mode for multi-photon microscopy. We recorded the fluorescence signals and position. A sequence of experiments indicates the relationship of activation to power, energy and time so that we can optimize the power level. Also we explored regional analysis of melanin to study the spatial relationship in SMPAF and define three types of regions which exhibit differences in the activation process.

  16. Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats

    NASA Astrophysics Data System (ADS)

    Thorling, Camilla A.; Liu, Xin; Burczynski, Frank J.; Fletcher, Linda M.; Gobe, Glenda C.; Roberts, Michael S.

    2011-11-01

    Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery. In orthotopic transplantation, the donor liver is exposed to periods of ischemia and when oxygenated blood is reintroduced to the liver, oxidative stress may develop and lead to graft failure. The aim of this project was to investigate whether noninvasive multiphoton and fluorescence lifetime imaging microscopy, without external markers, were useful in detecting early liver damage caused by I/R injury. Localized hepatic ischemia was induced in rats for 1 h followed by 4 h reperfusion. Multiphoton and fluorescence lifetime imaging microscopy was conducted prior to ischemia and up to 4 h of reperfusion and compared to morphological and biochemical assessment of liver damage. Liver function was significantly impaired at 2 and 4 h of reperfusion. Multiphoton microscopy detected liver damage at 1 h of reperfusion, manifested by vacuolated cells and heterogeneous spread of damage over the liver. The damage was mainly localized in the midzonal region of the liver acinus. In addition, fluorescence lifetime imaging showed a decrease in cellular metabolic activity. Multiphoton and fluorescence lifetime imaging microscopy detected evidence of early I/R injury both structurally and functionally. This provides a simple noninvasive technique useful for following progressive liver injury without external markers.

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

    EPA Science Inventory

    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...

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Verification Results of Jet Resonance-enhanced Multiphoton Ionization as a Real-time PCDD/F Emission Monitor

    EPA Science Inventory

    The Jet REMPI (Resonance Enhanced Multiphoton Ionization) monitor was tested on a hazardous waste firing boiler for its ability to determine concentrations of polychlorinated dibenzodioxins and dibenzofurans (PCDDs/Fs). Jet REMPI is a real time instrument capable of highly selec...

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

    EPA Science Inventory

    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...

  1. Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy.

    PubMed

    Labouta, Hagar I; Hampel, Martina; Thude, Sibylle; Reutlinger, Katharina; Kostka, Karl-Heinz; Schneider, Marc

    2012-01-01

    Multiphoton microscopy has become popular in studying dermal nanoparticle penetration. This necessitates studying the imaging parameters of multiphoton microscopy in skin as an imaging medium, in terms of achievable detection depths and the resolution limit. This would simulate real-case scenarios rather than depending on theoretical values determined under ideal conditions. This study has focused on depth profiling of sub-resolution gold nanoparticles (AuNP) in reconstructed (fixed and unfixed) and human skin using multiphoton microscopy. Point spread functions (PSF) were determined for the used water-immersion objective of 63×/NA = 1.2. Factors such as skin-tissue compactness and the presence of wrinkles were found to deteriorate the accuracy of depth profiling. A broad range of AuNP detectable depths (20-100 μm) in reconstructed skin was observed. AuNP could only be detected up to ∼14 μm depth in human skin. Lateral (0.5 ± 0.1 μm) and axial (1.0 ± 0.3 μm) PSF in reconstructed and human specimens were determined. Skin cells and intercellular components didn't degrade the PSF with depth. In summary, the imaging parameters of multiphoton microscopy in skin and practical limitations encountered in tracking nanoparticle penetration using this approach were investigated.

  2. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

    PubMed Central

    Kumar, Devender; Ristow, Laura C.; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A.; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

    2015-01-01

    Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration. PMID:26684456

  3. Periodicity in tumor vasculature targeting kinetics of ligand-functionalized nanoparticles studied by dynamic contrast enhanced magnetic resonance imaging and intravital microscopy

    PubMed Central

    Cebulla, Jana; Huuse, Else Marie; Davies, Catharina de L.; Mulder, Willem J. M.; Larsson, Henrik B.W.; Haraldseth, Olav

    2014-01-01

    In the past two decades advances in the development of targeted nanoparticles have facilitated their application as molecular imaging agents and targeted drug delivery vehicles. Nanoparticle-enhanced molecular imaging of the angiogenic tumor vasculature has been of particular interest. Not only because angiogenesis plays an important role in various pathologies, but also since endothelial cell surface receptors are directly accessible for relatively large circulating nanoparticles. Typically, nanoparticle targeting towards these receptors is studied by analyzing the contrast distribution on tumor images acquired before and at set time points after administration. Although several exciting proof-of-concept studies demonstrated qualitative assessment of relative target concentration and distribution, these studies did not provide quantitative information on the nanoparticle targeting kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of αvβ3-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging, and using compartment modeling we were able to quantify nanoparticle targeting rates. As such, this approach can facilitate optimization of targeted nanoparticle design and it holds promise for providing more quantitative information on in vivo receptor levels. Interestingly, we also observed a periodicity in the accumulation kinetics of αvβ3-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof useful for vascular targeting in general. PMID:23982332

  4. Imaging photoelectron circular dichroism of chiral molecules by femtosecond multiphoton coincidence detection

    NASA Astrophysics Data System (ADS)

    Lehmann, C. Stefan; Ram, N. Bhargava; Powis, Ivan; Janssen, Maurice H. M.

    2013-12-01

    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

  5. Imaging photoelectron circular dichroism of chiral molecules by femtosecond multiphoton coincidence detection

    SciTech Connect

    Lehmann, C. Stefan; Ram, N. Bhargava; Janssen, Maurice H. M.; Powis, Ivan

    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

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

    NASA Astrophysics Data System (ADS)

    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

  7. Effect of cage charges on multiphoton absorptions: first-principles study on metallofullerenes Sc(2)C(2)@C(68) and Sc(3)N@C(68).

    PubMed

    Cheng, W-D; Hu, H; Wu, D-S; Wang, J-Y; Huang, S-P; Xe, Z; Zhang, H

    2009-05-21

    A combined method of the time-dependent density functional theory (TDDFT) and sum-overstate (SOS) formula was implemented to model multiphoton absorption spectra, including two-photon absorption (2PA) and three-photon absorption (3PA), of Sc(2)C(2)@C(68) and Sc(3)N@C(68) endohedral metallofullerenes (EMFs). This method has been proved to be effective by comparisons between the calculated and experimental results of trans-4,4'-bis[diphenylamino]stilbene. It was found that the multiphoton absorption cross sections were larger for Sc(2)C(2)@C(68) than that of Sc(3)N@C(68). The electronic origin of multiphoton absorption has been identified with respect to the molecular orbitals involved in charge transfer process. It shows that the increase of pi-charges on the cage of C(68) results in a large multiphoton absorption cross section in EMFs.

  8. Engineering entangled microwave photon states through multiphoton interactions between two cavity fields and a superconducting qubit

    PubMed Central

    Zhao, Yan-Jun; Wang, Changqing; Zhu, Xiaobo; Liu, Yu-xi

    2016-01-01

    It has been shown that there are not only transverse but also longitudinal couplings between microwave fields and a superconducting qubit with broken inversion symmetry of the potential energy. Using multiphoton processes induced by longitudinal coupling fields and frequency matching conditions, we design a universal algorithm to produce arbitrary superpositions of two-mode photon states of microwave fields in two separated transmission line resonators, which are coupled to a superconducting qubit. Based on our algorithm, we analyze the generation of evenly-populated states and NOON states. Compared to other proposals with only single-photon process, we provide an efficient way to produce entangled microwave photon states when the interactions between superconducting qubits and microwave fields are in the strong and ultrastrong regime. PMID:27033558

  9. Multimodal In Vivo Skin Imaging with Integrated Optical Coherence and Multiphoton Microscopy

    PubMed Central

    Graf, Benedikt W.; Boppart, Stephen A.

    2014-01-01

    In this paper, we demonstrate high-resolution, multimodal in vivo imaging of human skin using optical coherence (OCM) and multiphoton microscopy (MPM). These two modalities are integrated into a single instrument to enable simultaneous acquisition and coregistration. The system design and the OCM image processing architecture enable sufficient performance of both modalities for in vivo imaging of human skin. Examples of multimodal in vivo imaging are presented as well as time lapse imaging of blood flow in single capillary loops. By making use of multiple intrinsic contrast mechanisms this integrated technique improves the ability to noninvasively visualize living tissue. Integrated OCM and MPM has potential applications for in vivo diagnosis of various pathological skin conditions, such as skin cancer, as well as potential pharmaceutical and cosmetic research applications. PMID:25673966

  10. Multiphoton fluorescence spectra and lifetimes of biliverdins and their protein-associated complex

    NASA Astrophysics Data System (ADS)

    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 (<100ps) was much shorter than those of porphyrins (~10ns), which is another commonly present metabolites in living cells. Further mixing biliverdins with proteins like fetal bovine serum (FBS), biliverdins reductase A (BVRA), or heme oxygenase-1 (HO-1), the yields of red autofluorescences didn't change a lot, but the corresponding lifetimes with HO-1 and BSA were lengthened to 200~300ps. This indicates that biliverdin can have an association with these proteins and change its lifetime. These spectral and temporal characteristics of fluorescence make biliverdin a potential marker fluorophore for hyperspectral diagnosis on the heme catabolism in human cells or tissues.

  11. The stepwise multi-photon activation fluorescence guided ablation of melanin

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Gu, Zetong; DiMarzio, Charles

    2015-02-01

    Previous research has shown that the stepwise multi-photon activation fluorescence (SMPAF) of melanin, activated and excited by a continuous-wave (CW) mode near infrared (NIR) laser, is a low-cost and reliable method for detecting melanin. We have developed a device utilizing the melanin SMPAF to guide the ablation of melanin with a 975 nm CW laser. This method provides the ability of targeting individual melanin particles with micrometer resolution, and enables localized melanin ablation to be performed without collateral damage. Compared to the traditional selective photothermolysis, which uses pulsed lasers for melanin ablation, this method demonstrates higher precision and lower cost. Therefore, the SMPAF guided selective ablation of melanin is a promising tool of melanin ablation for both medical and cosmetic purposes.

  12. Spatiotemporal control of degenerate multiphoton fluorescence microscopy with delay-tunable femtosecond pulse pairs

    NASA Astrophysics Data System (ADS)

    Das, Dhiman; Bhattacharyya, Indrajit; Goswami, Debabrata

    2016-07-01

    Selective excitation of a particular fluorophore in an ensemble of different fluorophores with overlapping fluorescence spectra is shown to be dependent on the time delay of femtosecond pulse pairs in multiphoton fluorescence microscopy. In particular, the two-photon fluorescence behavior of the Texas Red and DAPI dye pair inside Bovine Pulmonary Artery Endothelial (BPAE) cells depends strongly on the center wavelength of the laser, as well as the delay between two identical laser pulses in one-color femtosecond pulse-pair excitation scheme. Thus, we present a novel design concept using pairs of femtosecond pulses at different central wavelengths and tunable pulse separations for controlling the image contrast between two spatially and spectrally overlapping fluorophores. This femtosecond pulse-pair technique is unique in utilizing the variation of dye dynamics inside biological cells as a contrast mode in microscopy of different fluorophores.

  13. The Effect of the Argon Carrier Gas in the Multiphoton Dissociation-Ionization of Tetracene

    PubMed Central

    Poveda, Juan Carlos; Román, Alejandro San; Guerrero, Alfonso; Álvarez, Ignacio; Cisneros, Carmen

    2008-01-01

    The multiphoton dissociation-ionization of tetracene at 355 nm using 6.5 nanosecond laser pulses, with and without argon as a carrier gas (CG), has been studied and compared. Ion fragments were analyzed in a time-of-flight mass spectrometer and separated according to their mass-to-charge ratio (m/z). The results show that the dynamic of photodissociation at ∼1010 W cm−2 intensities is strongly influenced by the CG. The suppression of fragmentation channels primarily those relating to the formation of the CHm+ (m = 2, 4), C2H4+ and C5H4+2 ions. CH5+ and CH6+ were observed which have not been reported before in photodissociation tetracene experiments. PMID:19325732

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

    PubMed

    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. PMID:18059779

  15. Vibrationally resolved electron-nuclear energy sharing in above-threshold multiphoton dissociation of CO

    NASA Astrophysics Data System (ADS)

    Sun, Xufei; Li, Min; Shao, Yun; Liu, Ming-Ming; Xie, Xiguo; Deng, Yongkai; Wu, Chengyin; Gong, Qihuang; Liu, Yunquan

    2016-07-01

    We study the photon energy sharing between the photoelectron and the nuclei in the process of above-threshold multiphoton dissociative ionization of CO molecules by measuring the joint energy spectra. The experimental observation shows that the electron-nuclear energy sharing strongly depends on the vibrational state. The experimental observation shows that both the energy deposited to the nuclei of C O+ and the emitted photoelectron decrease with increasing the vibrational level. Through studying the vibrationally resolved nuclear kinetic energy release and photoelectron energy spectra at different laser intensities, for each vibrational level of C O+ , the nuclei always tend to take the same amount of energy in every vibrational level regardless of the laser intensity, while the energy deposited to the photoelectron varies with respect to the laser intensity because of the ponderomotive shifted energy and the distinct dissociative ionization mechanisms.

  16. 3D super-resolved in vitro multiphoton microscopy by saturation of excitation.

    PubMed

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

    2015-08-24

    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. PMID:26368235

  17. Multiphoton ionization and fragmentation of iodine-containing molecules by femtosecond ultraintense hard X-rays

    NASA Astrophysics Data System (ADS)

    Robatjazi, S. J.; Li, X.; Rolles, D.; Rudenko, A.; Erk, B.; Boll, R.; Bomme, C.; Savelyev, E.; Rudek, B.; Foucar, L.; Bostedt, Ch.; Southworth, S.; Lehmann, C. S.; Kraessig, B.; Young, L.; Marchenko, T.; Simon, M.; Ueda, K.; Ferguson, K. R.; Bucher, M.; Gorkhover, T.; Carron, S.; Alonso-Mori, R.; Williams, G.; Boutet, S.

    2016-05-01

    We present ion charge state distributions and kinetic energy spectra resulting from the breakup of CH3 I and C6 H5 I molecules induced by femtosecond X-ray pulses from the Linac Coherent Light Source (LCLS) at 8.3 keV photon energy. Using a few-hundred nm focus of the LCLS CXI beamline, we reach peak intensities of up to 1020 W/ cm2, resulting in stripping of more than 50 electrons per molecule within few tens of fs. We find that in this regime the interplay between multiphoton absorption and subsequent charge rearrangement considerably differs from earlier observations for soft X-rays or for weaker hard X-rays. We discuss the pulse duration dependence of the data, and compare the results for seeded and unseeded LCLS pulses. Supported by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U. S. DOE.

  18. Resonance-Enhanced Multiphoton Ionization Mass Spectrometry (REMPI-MS): Applications for Process Analysis

    NASA Astrophysics Data System (ADS)

    Streibel, Thorsten; Zimmermann, Ralf

    2014-06-01

    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.

  19. Marginal characteristics of skin scarred dermis quantitatively extracted from multiphoton microscopic imaging

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

    Multiphoton microscopy based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was applied to examine the marginal regions at dermis of normal, atrophic and keloid scars. High-contrast, high-resolution image showed an obvious boundary at scar margin and different morphological patterns of collagen or elastin on the two sides. Since the degree of the morphological alteration between the two sides of boundary at scar margin was varied among different types of scars, alteration degree of SHG-to-TPEF index was defined as a quantitative indicator for discrimination. It will help to determine the most appropriate clinical treatment strategy for different types of scars and potentially monitor therapy in vivo.

  20. Graphene oxide-based micropatterns via high-throughput multiphoton-induced reduction and ablation.

    PubMed

    Li, Yi-Cheng; Yeh, Te-Fu; Huang, Hsin-Chieh; Chang, Hsin-Yu; Lin, Chun-Yu; Cheng, Li-Chung; Chang, Chia-Yuan; Teng, Hsisheng; Chen, Shean-Jen

    2014-08-11

    In this study, a developed temporal focusing-based femtosecond laser system provides high-throughput multiphoton-induced reduction and ablation of graphene oxide (GO) films. Integrated with a digital micromirror device to locally control the laser pulse numbers, GO-based micropatterns can be quickly achieved instantly. Furthermore, the degree of reduction and ablation can be precisely adjusted via controlling the laser wavelength, power, and pulse number. Compared to point-by-point scanning laser direct writing, this approach offers a high-throughput and multiple-function approach to accomplish a large area of micro-scale patterns on GO films. The high-throughput micropatterning of GO via the temporal focusing-based femtosecond laser system fulfills the requirement of mass production for GO-based applications in microelectronic devices. PMID:25321055

  1. 2010 MULTIPHOTON PROCESSES GORDON RESEARCH CONFERENCE, JUNE 6-11, 2010, TILTON, NH

    SciTech Connect

    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.

  2. 2008 Multiphoton Processes Gordon Research Conferences - June 8-13, 2008

    SciTech Connect

    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.

  3. Mesenchymal stem cell interactions with 3D ECM modules fabricated via multiphoton excited photochemistry.

    PubMed

    Su, Ping-Jung; Tran, Quyen A; Fong, Jimmy J; Eliceiri, Kevin W; Ogle, Brenda M; Campagnola, Paul J

    2012-09-10

    To understand complex micro/nanoscale ECM stem cell interactions, reproducible in vitro models are needed that can strictly recapitulate the relative content and spatial arrangement of native tissue. Additionally, whole ECM proteins are required to most accurately reflect native binding dynamics. To address this need, we use multiphoton excited photochemistry to create 3D whole protein constructs or "modules" to study how the ECM governs stem cell migration. The constructs were created from mixtures of BSA/laminin (LN) and BSA alone, whose comparison afforded studying how the migration dynamics are governed from the combination of morphological and ECM cues. We found that mesenchymal stem cells interacted for significantly longer durations with the BSA/LN constructs than pure BSA, pointing to the importance of binding cues of the LN. Critical to this work was the development of an automated system with feedback based on fluorescence imaging to provide quality control when synthesizing multiple identical constructs.

  4. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation

    NASA Astrophysics Data System (ADS)

    Zipfel, Warren R.; Williams, Rebecca M.; Christie, Richard; Nikitin, Alexander Yu; Hyman, Bradley T.; Webb, Watt W.

    2003-06-01

    Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.

  5. Robert Feulgen Prize Lecture. Laser tweezers and multiphoton microscopes in life sciences.

    PubMed

    König, K

    2000-08-01

    Near infrared (NIR) laser microscopy enables optical micromanipulation, piconewton force determination, and sensitive fluorescence studies by laser tweezers. Otherwise, fluorescence images with high spatial and temporal resolution of living cells and tissues can be obtained via non-resonant fluorophore excitation with multiphoton NIR laser scanning microscopes. Furthermore, NIR femtosecond laser pulses at TW/cm2 intensities can be used to realize non-invasive contact-free surgery of nanometer-sized structures within living cells and tissues. Applications of these novel versatile NIR laser-based tools for the determination of motility forces, coenzyme and chlorophyll imaging, three-dimensional multigene detection, non-invasive optical sectioning of tissues ("optical biopsy"), functional protein imaging, and nanosurgery of chromosomes are described.

  6. Specific local induction of DNA strand breaks by infrared multi-photon absorption

    PubMed Central

    Träutlein, D.; Deibler, M.; Leitenstorfer, A.; Ferrando-May, E.

    2010-01-01

    Highly confined DNA damage by femtosecond laser irradiation currently arises as a powerful tool to understand DNA repair in live cells as a function of space and time. However, the specificity with respect to damage type is limited. Here, we present an irradiation procedure based on a widely tunable Er/Yb : fiber femtosecond laser source that favors the formation of DNA strand breaks over that of UV photoproducts by more than one order of magnitude. We explain this selectivity with the different power dependence of the reactions generating strand breaks, mainly involving reactive radical intermediates, and the direct photochemical process leading to UV-photoproducts. Thus, localized multi-photon excitation with a wavelength longer than 1 µm allows for the selective production of DNA strand breaks at sub-micrometer spatial resolution in the absence of photosensitizers. PMID:19906733

  7. Feasible Study for Multi-photon Stereolithography Method of Electro Conductive Polymer Actuator with Complex Shape

    NASA Astrophysics Data System (ADS)

    Sone, Junji; Asami, Akihisa; Yamada, Katsumi; Chen, Jun

    Recently, a soft actuator was developed using an electro-conducting polymer and an ionic conducting polymer. Moreover, stereolithography that uses a femtosecond laser was researched as a method of multiphoton-sensitized polymerization. In this study, we tried a more stable and more rapid stereolithography method for fabricating an electro-conducting polymer using a protein material. From the results of this study, we found that the method was 10 times faster when an aqueous solid that included an electro-conducting polymer, a catalyst, and gelatine was used. In addition, it was stable in that the temperature of the aqueous solid was controlled at 10 degree. We built a 3D shape using the newly developed method, and we will apply this method to a complex actuator.

  8. Engineering entangled microwave photon states through multiphoton interactions between two cavity fields and a superconducting qubit

    NASA Astrophysics Data System (ADS)

    Zhao, Yan-Jun; Wang, Changqing; Zhu, Xiaobo; Liu, Yu-Xi

    2016-04-01

    It has been shown that there are not only transverse but also longitudinal couplings between microwave fields and a superconducting qubit with broken inversion symmetry of the potential energy. Using multiphoton processes induced by longitudinal coupling fields and frequency matching conditions, we design a universal algorithm to produce arbitrary superpositions of two-mode photon states of microwave fields in two separated transmission line resonators, which are coupled to a superconducting qubit. Based on our algorithm, we analyze the generation of evenly-populated states and NOON states. Compared to other proposals with only single-photon process, we provide an efficient way to produce entangled microwave photon states when the interactions between superconducting qubits and microwave fields are in the strong and ultrastrong regime.

  9. Role of multiphoton bunching in high-order ghost imaging with thermal light sources

    SciTech Connect

    Liu Qian; Chen Xihao; Luo Kaihong; Wu Lingan; Wu Wei

    2009-05-15

    The intrinsic higher-order correlation of intensities which gives a measure of 'pure' correlations among photons (corresponding to multiphoton bunching) is investigated with regard to ghost imaging with thermal light. The synchronous detection of the same light field by all reference detectors, which is a necessary condition for achieving an Nth-order ghost image based on N-photon bunching, is discussed. Furthermore, it is found that the enhanced high visibility of Nth-order ghost imaging is a consequence of the contribution of N-photon bunching, which is not a small value but is equal to the sum of all contributions from (N-1)-photon bunching. These results differ from those obtained by certain other groups.

  10. Multiphoton Ionization Time-of-Flight Mass Spectrometry for the Detection of Bioactive Lignan.

    PubMed

    Uchimura, Tomohiro; Tokumoto, Goro; Batnyam, Onon; Chou, Chih-Wei; Fujita, Satoshi

    2016-01-01

    Multiphoton ionization time-of-flight mass spectrometry (MPI-TOFMS) combined with a pulsed laser for sample vaporization was developed for the detection of a low-volatile compound in a solution. A solution containing Taiwanin A ((3E,4E)-3,4-bis(1,3-benzodioxol-5-ylmethylene)dihydro-2(3H)-furanone), which is a lignan that has an anticancer effect, was employed in the present study. Consequently, Taiwanin A could be detected by irradiating a laser pulse for vaporization to an inlet nozzle, rather than by heating. Therefore, the present method could be effective for detecting compounds with lower volatilities in a liquid sample. PMID:26860576

  11. Influence of multi-photon excitation on the atomic above-threshold ionization

    NASA Astrophysics Data System (ADS)

    Tian, Yuan-Ye; Wang, Chun-Cheng; Li, Su-Yu; Guo, Fu-Ming; Ding, Da-Jun; Wim-G, Roeterdink; Chen, Ji-Gen; Zeng, Si-Liang; Liu, Xue-Shen; Yang, Yu-Jun

    2015-04-01

    Using the time-dependent pseudo-spectral scheme, we solve the time-dependent Schrödinger equation of a hydrogen-like atom in a strong laser field in momentum space. The intensity-resolved photoelectron energy spectrum in above-threshold ionization is obtained and further analyzed. We find that with the increase of the laser intensity, the above-threshold ionization emission spectrum exhibits periodic resonance structure. By analyzing the population of atomic bound states, we find that it is the multi-photon excitation of bound state that leads to the occurrence of this phenomenon, which is in fairly good agreement with the experimental results. Project supported by the National Basic Research Program of China (Grant No. 2013CB922200), the National Natural Science Foundation of China (Grants Nos. 11274141, 11034003, 11304116, 11274001, and 11247024), and the Jilin Provincial Research Foundation for Basic Research, China (Grant No. 20140101168JC).

  12. Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging.

    PubMed

    Ulrich, Volker; Fischer, Peter; Riemann, Iris; Königt, Karsten

    2004-01-01

    An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution. PMID:15536977

  13. High-resolution distributed temperature sensing with the multiphoton-timing technique

    NASA Astrophysics Data System (ADS)

    Höbel, M.; Ricka, J.; Wüthrich, M.; Binkert, Th.

    1995-06-01

    We report on a multiphoton-timing distributed temperature sensor (DTS) based on the concept of distributed anti-Stokes Raman thermometry. The sensor combines the advantage of very high spatial resolution (40 cm) with moderate measurement times. In 5 min it is possible to determine the temperature of as many as 4000 points along an optical fiber with an accuracy Delta T less than 2 deg C. The new feature of the DTS system is the combination of a fast single-photon avalanche diode with specially designed real-time signal-processing electronics. We discuss various parameters that affect the operation of analog and photon-timing DTS systems. Particular emphasis is put on the consequences of the nonideal behavior of sensor components and the corresponding correction procedures.

  14. Resonant and nonresonant multiphoton ionization processes in the mass spectrometry of explosives.

    PubMed

    Hamachi, Akifumi; Okuno, Tomoya; Imasaka, Tomoko; Kida, Yuichiro; Imasaka, Totaro

    2015-03-01

    Multiphoton ionization processes were studied for three types of explosives using a line-tunable ultraviolet femtosecond laser. When peroxides such as triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD) were ionized through a nonresonant two-photon process, a molecular ion was dominantly observed by reducing the excess energy remaining in the ion. However, an aromatic nitro compound such as 2,4,6-trinitrotoluene (TNT) produced large signals arising from molecular and fragment ions by resonant two-photon ionization. In addition, only fragment ions were produced from a nonaromatic nitro compound such as 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), even when a resonant two-photon ionization process was employed, suggesting that a further reduction in excess energy would be necessary if a molecular ion were to be observed. PMID:25622138

  15. In-vivo imaging of psoriatic lesions with polarization multispectral dermoscopy and multiphoton microscopy

    PubMed Central

    Kapsokalyvas, Dimitrios; Cicchi, Riccardo; Bruscino, Nicola; Alfieri, Domenico; Prignano, Francesca; Massi, Daniela; Lotti, Torello; Pavone, Francesco S.

    2014-01-01

    Psoriasis is a skin autoimmune disease characterized by hyperkeratosis, hyperproliferation of the epidermis and dilatation of dermal papillary blood vessels. Healthy skin (5 volunteers) and psoriatic lesions (3 patients) were visualized in vivo, with high contrast and resolution, with a Polarization Multispectral Dermoscope and a Multiphoton Microscope. Psoriatic features were identified and quantified. The effective diameter of the superficial blood vessels was measured at 35.2 ± 7.2 μm and the elongated dermal papillae had an effective diameter of 64.2 ± 22.6 μm. The methodologies developed could be employed for quantitative diagnostic purposes and furthermore serve as a monitoring method of the effect of personalized treatments. PMID:25071974

  16. Multiphoton electron emission from Cu and W: An angle-resolved study

    SciTech Connect

    Damascelli, A.; Gabetta, G.; Lumachi, A.; Fini, L.; Parmigiani, F.

    1996-09-01

    The experimental results of multiphoton electron emission from Cu and W induced by 2-eV 100-fs laser pulses with {ital s} and {ital p} polarizations at incidence angles between 0{degree} and 85{degree} and different intensities are reported. The data show a third-order nonlinear photoemission process for Cu and a fourth-order behavior for W. For both metals the electron emission is higher for the polarization in the incidence plane, with a maximum value at the pseudo-Brewster angle, while the electron yield as a function of the incidence angle exhibits an unambiguous dependence on the bulk absorption coefficient and it can be accounted for on the basis of the Fresnel equations. {copyright} {ital 1996 The American Physical Society.}

  17. Selective IR multiphoton dissociation of CF{sub 3}I in a nonequilibrium pressure shock

    SciTech Connect

    Makarov, Grigorii N; Mochalov, S A; Petin, A N

    2001-03-31

    The isotopically selective IR multiphoton dissociation of CF{sub 3}I is studied in a nonequilibrium pressure shock formed in the interaction of a pulsed gas-dynamically cooled molecular flow with a solid surface. The yield of a C{sub 2}F{sub 6} product and its enrichment factor by the {sup 13}C isotope were measured experimentally. The time-of-flight spectra of CF{sub 3}I were obtained in the flow interacting with the surface and in an unperturbed flow. The spectral dependences of the yield of a C{sub 2}F{sub 6} product in an unperturbed flow and in a pressure shock were studied along with the process selectivity. It was shown that the efficiency of isotopically selective molecule dissociation can be significantly increased due to the formation of a pressure shock. The origin of the results observed are discussed. (laser applications and other topics in quantum electronics)

  18. Characterization of fibrillar collagen types using multi-dimensional multiphoton laser scanning microscopy.

    PubMed

    Lutz, V; Sattler, M; Gallinat, S; Wenck, H; Poertner, R; Fischer, F

    2012-04-01

    In dermal photodamage the ratio of the collagen types III to I changes. This makes the investigation of the fibrillar collagen type characteristics interesting for skin research. In this study collagen types were characterized using 5-dimensional multiphoton laser scanning microscopy (5D-IVT) that can be applied in vivo. Second harmonic generation (SHG) signals and fluorescence lifetimes of the collagen autofluorescence were analysed. Collagen type I generates a higher SHG intensity and a longer fluorescence lifetime compared to collagen type III. Thus, the SHG intensity decrease found in photodamaged skin might be explained by the increase in collagen type III. Calculating the in vivo relevant increase of collagen type III gives a negligible difference in fluorescence lifetime not qualifying this method for the determination of collagen type changes in dermal photodamage in vivo in human skin. However, for pathologies that exhibit higher differences in collagen types 5D-IVT analysis might be a suitable method.

  19. Multiphoton fluorescence microscopy: behavior of biological specimens under high-intensity illumination

    NASA Astrophysics Data System (ADS)

    Cheng, Ping C.; Lin, Bai-Ling; Kao, Fu-Jen; Sun, Chi-Kuang

    2000-07-01

    Recent development in multi-photon fluorescence microscopy, second and third harmonic generation microscopy (SHG and THG) and CARS open new dimensions in biological studies. Not only the technologies allow probing the biological specimen both functionally and structurally with increasing spatial and temporal resolution, but also raise the interest in how biological specimens respond to high intensity illumination commonly used in these types of microscopy. We have used maize leaf protoplast as a model system to evaluate the photo-induced response of living sample under high intensity illumination. It was found that cells can be seriously damaged by high intensity NIR irradiation even the linear absorption coefficient in low in these wavelengths. Micro-spectroscopy of single chloroplast also allows us to gain insight on the possible photo-damage mechanism. In addition to fluorescence emission, second harmonic generation was observed in the maize protoplasts.

  20. Label-free multi-photon imaging of dysplasia in Barrett’s esophagus

    PubMed Central

    Mehravar, Soroush; Banerjee, Bhaskar; Chatrath, Hemant; Amirsolaimani, Babak; Patel, Krunal; Patel, Charmi; Norwood, Robert A; Peyghambarian, Nasser; Kieu, Khanh

    2015-01-01

    Barrett’s esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barrett’s. PMID:26819824