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Sample records for act intravital multiphoton

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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

  6. Characterization of multiphoton microscopy in the bone marrow following intravital laser osteotomy.

    PubMed

    Turcotte, Raphaël; Alt, Clemens; Mortensen, Luke J; Lin, Charles P

    2014-10-01

    The bone marrow is an important site where all blood cells are formed from hematopoietic stem cells and where hematologic malignancies such as leukemia emerge. It is also a frequent site for metastasis of solid tumors such as breast cancer and prostate cancer. Intravital microscopy is a powerful tool for studying the bone marrow with single cell and sub-cellular resolution. To improve optical access to this rich biological environment, plasma-mediated laser ablation with sub-microjoule femtosecond pulses was used to thin cortical bone. By locally removing a superficial layer of bone (local laser osteotomy), significant improvements in multiphoton imaging were observed in individual bone marrow compartments in vivo. This work demonstrates the utility of scanning laser ablation of hard tissue with sub-microjoule pulses as a preparatory step to imaging. PMID:25360374

  7. From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin.

    PubMed

    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

  8. Deep-tissue multiphoton fluorescence lifetime microscopy for intravital imaging of protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Fruhwirth, G. O.; Matthews, D. R.; Brock, A.; Keppler, M.; Vojnovic, B.; Ng, T.; Ameer-Beg, S.

    2009-02-01

    Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo. For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we show the application of this method for intravital imaging of direct protein-protein interactions.

  9. In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

    PubMed Central

    He, Wei; Wang, Haifeng; Hartmann, Lynn C.; Cheng, Ji-Xin; Low, Philip S.

    2007-01-01

    Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at ≈2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease. PMID:17601776

  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. 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

  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. 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.

  14. 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. PMID:25504510

  15. Intravital microscopy

    PubMed Central

    Masedunskas, Andrius; Milberg, Oleg; Porat-Shliom, Natalie; Sramkova, Monika; Wigand, Tim; Amornphimoltham, Panomwat; Weigert, Roberto

    2012-01-01

    Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs. PMID:22992750

  16. 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

  17. 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.

  18. 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.

  19. Microbial pathogenesis revealed by intravital microscopy: pros, cons and cautions.

    PubMed

    Stolp, Bettina; Melican, Keira

    2016-07-01

    Intravital multiphoton imaging allows visualization of infections and pathogenic mechanisms within intact organs in their physiological context. Today, most organs of mice and rats are applicable to in vivo or ex vivo imaging, opening completely new avenues for many researchers. Advances in fluorescent labeling of pathogens and infected cells, as well as improved small animal models for human pathogens, led to the increased application of in vivo imaging in infectious diseases research in recent years. Here, we review the latest literature on intravital or ex vivo imaging of viral and bacterial infections and critically discuss requirements, benefits and drawbacks of applied animal models, labeling strategies, and imaged organs. PMID:26938770

  20. Video-rate resonant scanning multiphoton microscopy

    PubMed Central

    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.

    2013-01-01

    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

  1. 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

  2. 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

  3. 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.

  4. Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

    PubMed Central

    Andresen, Volker; Sporbert, Anje

    2014-01-01

    Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells – on the level of a few protein molecules in germinal centers. PMID:24748007

  5. 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.

  6. 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

  7. 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

  8. 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

  9. High-Resolution Intravital Microscopy

    PubMed Central

    Andresen, Volker; Pollok, Karolin; Rinnenthal, Jan-Leo; Oehme, Laura; Günther, Robert; Spiecker, Heinrich; Radbruch, Helena; Gerhard, Jenny; Sporbert, Anje; Cseresnyes, Zoltan; Hauser, Anja E.; Niesner, Raluca

    2012-01-01

    Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology

  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. Understanding liver immunology using intravital microscopy.

    PubMed

    Marques, Pedro Elias; Oliveira, André Gustavo; Chang, Lynne; Paula-Neto, Heitor Affonso; Menezes, Gustavo Batista

    2015-09-01

    The liver has come a long way since it was considered only a metabolic organ attached to the gastrointestinal tract. The simultaneous ascension of immunology and intravital microscopy evidenced the liver as a central axis in the immune system, controlling immune responses to local and systemic agents as well as disease tolerance. The multiple hepatic cell populations are organized in a vascular environment that promotes intimate cellular interactions, including initiation of innate and adaptive immune responses, rapid leukocyte recruitment, pathogen clearance and production of a variety of immune mediators. In this review, we focus on the advances in liver immunology supported by intravital microscopy in diseases such as isquemia/reperfusion, acute liver injury and infections. PMID:26055800

  13. [Analysis of bone tissues by intravital imaging].

    PubMed

    Mizuno, Hiroki; Yamashita, Erika; Ishii, Masaru

    2016-05-01

    In recent years,"the fluorescent imaging techniques"has made rapid advances, it has become possible to observe the dynamics of living cells in individuals or tissues. It has been considered that it is extremely difficult to observe the living bone marrow directly because bone marrow is surrounded by a hard calcareous. But now, we established a method for observing the cells constituting the bone marrow of living mice in real time by the use of the intravital two-photon imaging system. In this article, we show the latest data and the reports about the hematopoietic stem cells and the leukemia cells by using the intravital imaging techniques, and also discuss its further application. PMID:27117619

  14. 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

  15. 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.

  16. 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

  17. 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

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

    PubMed

    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. PMID:25710308

  19. Regulated exocytosis: novel insights from intravital microscopy

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie

    2012-01-01

    Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electro-physiology, and electron microscopy. However, recent developments in time-lapse light microscopy have made possible imaging individual exocytic events hence advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy a light microscopy-based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. Intravital microscopy has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings, and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for intravital microscopy being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions. PMID:22243493

  20. 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

  1. Topiramate inhibits trigeminovascular activation: an intravital microscopy study

    PubMed Central

    Akerman, Simon; Goadsby, Peter J

    2005-01-01

    Activation, or the altered perception of activation, of trigeminal nerves that innervate the cranial vasculature is considered to be a pivotal component of the pathophysiology of acute migraine. Calcitonin gene-related peptide (CGRP) levels are increased during migraine and after trigeminal nerve stimulation in the cat. Both CGRP and nitric oxide (NO) infusion causes headache and delayed migraine in migraineurs. Neurogenic stimulation of a cranial window, CGRP and NO injection all cause meningeal artery dilation in the rat when viewed using intravital microscopy. Topiramate is an antiepileptic drug with established efficacy as a migraine preventive, and has recently been shown to inhibit neurons of the trigeminocervical complex after superior sagittal sinus stimulation. In this study, we used intravital microscopy with neurogenic dural vasodilation, and CGRP- and NO-induced dilation to examine whether intravenous topiramate has effects on the trigeminovascular system. Topiramate was able to attentuate neurogenic dural vasodilation maximally after 15 min by 52% at 30 mg kg−1 (t5=6.78, n=6); there was no significant inhibition at 10 mg kg−1. There was also significant attenuation of the NO-induced dilation maximally after 15 min, at both 10 and 30 mg kg−1 by 21% (t6=6.09, n=7) and 41% (t6=5.3, n=7), respectively. CGRP-induced dilation was not inhibited at either dose of topiramate. The study demonstrates that topiramate is likely to inhibit neurogenic dural vasodilation by inhibiting the release of CGRP from prejunctional trigeminal neurons, thus attenuating the dural vasodilation. Topiramate is not able to act postsynaptically at the blood vessels themselves as the CGRP-induced dilation was not attenuated. The data are consistent with an effect of topiramate on trigeminovascular activation which may form part of its preventive antimigraine mechanisms of action. PMID:15980877

  2. Intravital Microscopic Interrogation of Peripheral Taste Sensation

    PubMed Central

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

    2015-01-01

    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. PMID:25726964

  3. 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.

  4. 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.

  5. Multiphoton microscopy in neuroscience

    NASA Astrophysics Data System (ADS)

    Denk, Winfried

    2002-06-01

    The study of the nervous system requires to an exceptional extent observation of and experimentation on intact tissue. There, in particular, high-resolution optical microscopy benefits from the inherent advantages of multi-photon fluorescence excitation. Several cases will be presented from a number of different tissues and organisms, where multi-photon excited laser scanning fluorescence microscopy has been an essential experimental tool. Those examples include the discovery of biochemical coincidence detection in synaptic spines and the clarification of the underlying mechanism; the observation of sensory evoked dendritic signaling in intact animals and the observation of light induced calcium signals in the intact retina. Recently a fiber coupled two-photon microscopy has been developed that allows the imaging in moving animal.

  6. 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.

  7. 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.

  8. 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

  9. Real-time intravital microscopy of individual nanoparticle dynamics in liver and tumors of live mice

    PubMed Central

    van de Ven, Anne L; Kim, Pilhan; Ferrari, Mauro; Yun, Seok Hyun

    2013-01-01

    Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laser-scanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response. PMID:25383179

  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. Calculation of multiphoton ionization processes

    NASA Technical Reports Server (NTRS)

    Chang, T. N.; Poe, R. T.

    1976-01-01

    We propose an accurate and efficient procedure in the calculation of multiphoton ionization processes. In addition to the calculational advantage, this procedure also enables us to study the relative contributions of the resonant and nonresonant intermediate states.

  12. 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.

  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. 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.

  15. 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

  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

    2016-01-01

    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

  17. Intravital models of infection lay the foundation for tissue microbiology.

    PubMed

    Choong, Ferdinand X; Regberg, Jakob; Udekwu, Klas I; Richter-Dahlfors, Agneta

    2012-04-01

    In complex environments, such as those found in the human host, pathogenic bacteria constantly battle the unfavorable conditions imposed by the host response to their presence. During Escherichia coli-induced pyelonephritis, a cascade of events are shown in an intravital animal model to occur in a timely and sequential manner, representing the dynamic interplay between host and pathogen. Today, intravital techniques allow for observing infection in the living host. At resolutions almost on the single-cell level, improved detection methods offer a movie-like description of infection dynamics. Tissue microbiology involves monitoring host-pathogen interaction within the dynamic microecology of infectious sites in the live host. This new field holds great promise for insightful research into microbial disease intervention strategies. PMID:22439728

  18. Intravital FRET: Probing Cellular and Tissue Function in Vivo.

    PubMed

    Radbruch, Helena; Bremer, Daniel; Mothes, Ronja; Günther, Robert; Rinnenthal, Jan Leo; Pohlan, Julian; Ulbricht, Carolin; Hauser, Anja E; Niesner, Raluca

    2015-01-01

    The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo-ratiometrically and time-resolved by fluorescence lifetime imaging-and show their concrete application in the context of neuroinflammation in adult mice. PMID:26006244

  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 Effects in Rutile.

    NASA Astrophysics Data System (ADS)

    Royce, Gerald A.

    Multiphoton effects are investigated in crystalline rutile TiO(,2) using Nd:YAG laser photons. The 1.06 micron laser is operated in Q-switched mode with intensities up to 1.4 x 10('6) watts/cm('2) on the rutile crystal. Photoconductivity measurements provide data indicating a mixture of modes for electrons to be photoionized. Assuming aluminum impurity as the contributing sites, the first order photionization cross section is found to be 1.5 x 10('-26) cm('2) and second order cross section is found to be 7.7 x 10('-51) cm('4)-s. No appreciable change in cross section is observed for circular versus linear polarization of the laser. Observations of the photo-emission of the laser illuminated crystal provide radiative relaxation times on the order of 100 nanoseconds with emission peaks at 4500 and 5000 angstroms plus a near infrared continuum out to 1 micron. The thermoluminescence of rutile shows a number of trapping levels between 0.4 and 0.8 eV below the conduction band. These are attributed to an aluminum impurity.

  1. 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.

  2. 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.

  3. Advances in multiphoton microscopy technology

    PubMed Central

    Hoover, Erich E.; Squier, Jeff A.

    2013-01-01

    Multiphoton microscopy has enabled unprecedented dynamic exploration in living organisms. A significant challenge in biological research is the dynamic imaging of features deep within living organisms, which permits the real-time analysis of cellular structure and function. To make progress in our understanding of biological machinery, optical microscopes must be capable of rapid, targeted access deep within samples at high resolution. In this Review, we discuss the basic architecture of a multiphoton microscope capable of such analysis and summarize the state-of-the-art technologies for the quantitative imaging of biological phenomena. PMID:24307915

  4. 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.

  5. 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

  6. 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

  7. Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

    NASA Astrophysics Data System (ADS)

    Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

    2010-03-01

    In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

  8. Compact non-contact total emission detection for in vivo multiphoton excitation microscopy.

    PubMed

    Combs, C A; Smirnov, A; Glancy, B; Karamzadeh, N S; Gandjbakhche, A H; Redford, G; Kilborn, K; Knutson, J R; Balaban, R S

    2014-02-01

    We describe a compact, non-contact design for a total emission detection (c-TED) system for intra-vital multiphoton 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), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold 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 multiphoton imaging methods is discussed. PMID:24251437

  9. 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

  10. 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

  11. 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.

  12. 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

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

    PubMed

    Vennin, Claire; Herrmann, David; 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

  14. Intravital microscopy of subpleural alveoli via transthoracic endoscopy

    NASA Astrophysics Data System (ADS)

    Schwenninger, David; Runck, Hanna; Schumann, Stefan; Haberstroh, Jörg; Meissner, Sven; Koch, Edmund; Guttmann, Josef

    2011-04-01

    Transfer of too high mechanical energy from the ventilator to the lung's alveolar tissue is the main cause for ventilator-induced lung injury (VILI). To investigate the effects of cyclic energy transfer to the alveoli, we introduce a new method of transthoracic endoscopy that provides morphological as well as functional information about alveolar geometry and mechanics. We evaluate the new endoscopic method to continuously record images of focused subpleural alveoli. The method is evaluated by using finite element modeling techniques and by direct observation of subpleural alveoli both in isolated rat lungs as well as in intact animals (rats). The results confirm the overall low invasiveness of the endoscopic method insofar as the mechanical influences on the recorded alveoli are only marginal. It is, hence, a suited method for intravital microscopy in the rat model as well as in larger animals.

  15. 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

  16. 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.

  17. 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

  18. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

    PubMed Central

    Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

    2016-01-01

    Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU. PMID:26901051

  19. Intravital Microscopic Research of Microembolization with Degradable Starch Microspheres

    PubMed Central

    Ebert, Juergen; Berger, Gerd

    2013-01-01

    Treatment efficacy in cancer patients using systemically applied cytostatic drugs is decreased by cytotoxic side effects, which limits the use of efficient dosages. Degradable starch microspheres (DSM) are used to apply drugs into blood vessels which supply the target organ leading to drug accumulation in the target organ by reduction of the blood flow. The present investigations show that DSM is a very effective embolization material leading to effective and enhanced accumulation of 5-FU within the liver tumor tissue of experimental induced liver cancer in rats. By using intravital microscopy, a rapid deceleration of the blood flow into the target organ is observed immediately after application of DSM. The microspheres are stepwise degraded in the direction of the systemic blood flow and are totally dissolved after 25 minutes. These stepwise processes leave the degraded material during the degradation process within the vessels leading to temporally reciprocal blood flow via some of the side-arms of the major blood vessels. By using DMS in transarterial chemoembolization (TACE), severe adverse side effects like postembolization syndrome are rarely observed when compared to other embolization materials. The complete degradation of DSM causes only a short-lasting temporary vascular occlusion, which allows a repeat application of DSM in TACE. PMID:24324891

  20. 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

  1. 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

  2. The multiphoton AC Stark effect

    NASA Astrophysics Data System (ADS)

    Rudolph, T. G.; Ficek, Z.; Freedhoff, H. S.

    1998-02-01

    We study the interaction of a two-level atom with two intense lasers: a strong laser of Rabi frequency 2Ω on resonance with the atomic transition, and a weaker laser detuned by 2Ω/n, i.e. by a subharmonic of the Rabi frequency of the first. The second laser "dresses" the dressed states created by the first in an n-photon process. We calculate the energy levels and eigenstates of this "doubly-dressed" atom, and find a new phenomenon: the splitting of the energy levels due to an n-photon coupling between them, resulting in a multiphoton AC Stark effect. We illustrate this effect in the fluorescence spectrum, and show that the spectrum contains triplets at the subharmonic as well as harmonic resonance frequencies with a clear dependence on the order n of the resonance and the ratio α of the Rabi frequencies of the lasers

  3. Multiphoton-Excited Serotonin Photochemistry

    PubMed Central

    Gostkowski, Michael L.; Allen, Richard; Plenert, Matthew L.; Okerberg, Eric; Gordon, Mary Jane; Shear, Jason B.

    2004-01-01

    We report photochemical and photophysical studies of a multiphoton-excited reaction of serotonin that previously has been shown to generate a photoproduct capable of emitting broadly in the visible spectral region. The current studies demonstrate that absorption of near-infrared light by an intermediate state prepared via three-photon absorption enhances the photoproduct formation yield, with the largest action cross sections (∼10−19 cm2) observed at the short-wavelength limit of the titanium:sapphire excitation source. The intermediate state is shown to persist for at least tens of nanoseconds and likely to be different from a previously reported oxygen-sensitive intermediate. In addition, the two-photon fluorescence action spectrum for the fluorescent photoproduct was determined and found to have a maximum at ∼780 nm (3.2 eV). A general mechanism for this photochemical process is proposed. PMID:15111435

  4. 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

  5. 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

  6. Multiphoton ionization of uranium hexafluoride

    NASA Astrophysics Data System (ADS)

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

    1994-01-01

    Multiphoton ionization (MPI) time-of-flight mass spectroscopy (TOFMS) and photoelectron spectroscopy (PES) studies of UF6 are reported using focused light from the Nd:YAG laser fundamental (λ=1064 nm) and its harmonics (λ=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+x fragment ions, even at the lowest laser power densities at which signal could be detected. In general, the doubly charged uranium ion (U2+) intensity is much greater than that of the singly charged uranium ion (U+). For the case of the tunable dye laser experiments, the Un+ (n=1-4) wavelength dependence is relatively unstructured and does not show observable resonance enhancement at known atomic uranium excitation wavelengths. The MPI-PES studies reveal only very slow electrons (≤0.5 eV) for all wavelengths investigated. The dominance of the U2+ ion, the absence or very small intensities of UF+x (x=1-3) fragments, the unstructured wavelength dependence, and the preponderance of slow electrons all indicate that mechanisms may exist other than ionization of bare U atoms following the stepwise photodissociation of F atoms from the parent molecule. The data also argue against stepwise photodissociation of UF+x (x=5,6) ions. Neither of the traditional MPI mechanisms (``neutral ladder'' or the ``ionic ladder'') are believed to adequately describe the ionization phenomena observed. We propose that the multiphoton excitation of UF6 under these experimental conditions results in a highly excited molecule, superexcited UF6**. The excitation of highly excited UF6** is proposed to be facilitated by the well known ``giant resonance,'' whose energy level lies in the range of 12-14 eV above that of ground state UF6. The highly excited molecule then primarily dissociates, via multiple channels, into Un+, UF+x, fluorine atoms, and ``slow'' electrons, although dissociation

  7. [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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Polarization phenomena in multiphoton ionization of atoms

    NASA Technical Reports Server (NTRS)

    Jacobs, V. L.

    1973-01-01

    The theory of multiphoton ionization for an atomic system of arbitrary complexity is developed using a density matrix formalism. An expression is obtained which determines the differential N-photon ionization cross section as a function of the polarization states of the target atom and the incident radiation. The parameters which characterize the photoelectron angular distribution are related to the general reduced matrix elements for the N-photon transition. Two-photon ionization of unpolarized atoms is treated as an illustration of the use of the theory. The dependence of the multiphoton ionization cross section on the polarization state of the incident radiation, which has been observed in two- and three-photon ionization of Cs, is accounted for by the theory. Finally, the photoelectron spin polarization produced by the multiphoton ionization of unpolarized atoms, like the analogous polarization resulting from single-photon ionization, is found to depend on the circular polarization of the incident radiation.

  13. Multiphoton absorption in amyloid protein fibres

    NASA Astrophysics Data System (ADS)

    Hanczyc, Piotr; Samoc, Marek; Norden, Bengt

    2013-12-01

    Fibrillization of peptides leads to the formation of amyloid fibres, which, when in large aggregates, are responsible for diseases such as Alzheimer's and Parkinson's. Here, we show that amyloids have strong nonlinear optical absorption, which is not present in native non-fibrillized protein. Z-scan and pump-probe experiments indicate that insulin and lysozyme β-amyloids, as well as α-synuclein fibres, exhibit either two-photon, three-photon or higher multiphoton absorption processes, depending on the wavelength of light. We propose that the enhanced multiphoton absorption is due to a cooperative mechanism involving through-space dipolar coupling between excited states of aromatic amino acids densely packed in the fibrous structures. This finding will provide the opportunity to develop nonlinear optical techniques to detect and study amyloid structures and also suggests that new protein-based materials with sizable multiphoton absorption could be designed for specific applications in nanotechnology, photonics and optoelectronics.

  14. 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.

  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. Multiphoton coherent control in complex systems

    PubMed Central

    Goswami, Debabrata

    2005-01-01

    Control of multiphoton transitions is demonstrated for a multilevel system by generalizing the instantaneous phase of any chirped pulse as individual terms of a Taylor series expansion. In the case of a simple two-level system, all odd terms in the series lead to population inversion while the even terms lead to self-induced transparency. The results hold for multiphoton transitions that do not have any lower-order photon resonance or any intermediate virtual state dynamics within the laser pulse width. PMID:17396157

  17. 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.

  18. 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.

  19. 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.

  20. Real-time intravital imaging of pH variation associated with osteoclast activity.

    PubMed

    Maeda, Hiroki; Kowada, Toshiyuki; Kikuta, Junichi; Furuya, Masayuki; Shirazaki, Mai; Mizukami, Shin; Ishii, Masaru; Kikuchi, Kazuya

    2016-08-01

    Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption. PMID:27272564

  1. 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

  2. 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. PMID:26325284

  3. Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

    PubMed Central

    Masedunskas, Andrius; Sramkova, Monika; Parente, Laura; Sales, Katiuchia Uzzun; Amornphimoltham, Panomwat; Bugge, Thomas H.; Weigert, Roberto

    2011-01-01

    The regulation and the dynamics of membrane trafficking events have been studied primarily in in vitro models that often do not fully reflect the functional complexity found in a living multicellular organism. Here we used intravital microscopy in the salivary glands of live rodents to investigate regulated exocytosis, a fundamental process in all of the secretory organs. We found that β-adrenergic stimulation elicits exocytosis of large secretory granules, which gradually collapse with the apical plasma membrane without any evidence of compound exocytosis, as was previously described. Furthermore, we show that the driving force required to complete the collapse of the granules is provided by the recruitment of F-actin and nonmuscle myosin II on the granule membranes that is triggered upon fusion with the plasma membrane. Our results provide information on the machinery controlling regulated secretion and show that intravital microscopy provides unique opportunities to address fundamental questions in cell biology under physiological conditions. PMID:21808006

  4. 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

  5. Intravital microscopy in tumor biology - current status and future perspectives (review).

    PubMed

    Leunig, M; Messmer, K

    1995-02-01

    To date, most progress in biomedical research is reported from cellular and/or molecular studies identifying important disease mechanisms and suggesting novel strategies in cancer therapy. Although these findings are fundamental for the understanding and treatment of neoplastic disease they often fail to be demonstrable in vivo. Many tumors resist complete eradication by anti-cancer agents indicating that caution has to be exercised in the extrapolation of in vitro observations to the clinical situation. This review emphasizes intravital microscopy as a quantitative method to analyze in vivo mechanisms of neoplastic disease and to test the in vivo function of novel strategies in cancer therapy derived from in vitro observations. Intravital microscopy facilitates a comprehensive analysis of the tumor microcirculation and microenvironment which should aid to improve the current understanding of tumor biology. PMID:21556553

  6. Formalism for multiphoton plasmon excitation in jellium clusters

    NASA Astrophysics Data System (ADS)

    Connerade, Jean-Patrick; Solov'yov, Andrey V.

    2002-07-01

    We present a formalism for the description of multiphoton plasmon excitation processes in jellium clusters. By using our method, we demonstrate that, in addition to dipole plasmon excitations, the multipole plasmons (quadrupole, octupole, etc.) can be excited in a cluster by multiphoton absorption processes, which results in a significant difference between plasmon resonance profiles in the cross sections for multiphoton as compared to single-photon absorption. We calculate the cross sections for multiphoton absorption and analyze the balance between the surface and volume plasmon contributions to multipole plasmons.

  7. 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

  8. Clinical multiphoton endoscopy with FLIM capability

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    Multiphoton endoscopy can be applied for intra-corporeal imaging as well as to examine otherwise hard-to-access tissue areas like chronic wounds. Using high-NA (NA = 0.8) gradient-index (GRIN) lens-based endoscopes with a diameter of 1.4 mm and effective lengths of 7 mm and 20 mm, respectively, two-photon excitation of endogenous fluorophores and second-harmonic generation (SHG) is used for multimodal in vivo imaging of human skin. A further imaging modality is fluorescence lifetime imaging (FLIM) which allows functional imaging to investigate the healing mechanism of chronic wounds and the corresponding cell metabolism. We performed first in vivo measurements using FLIM endoscopy with the medically-certified multiphoton tomograph MPTflex® in combination with a computer-controlled motorized scan head and a GRIN-lens endoscope.

  9. In vivo multiphoton tomography of skin cancer

    NASA Astrophysics Data System (ADS)

    König, Karsten; Riemann, Iris; Ehlers, Alexander; Buckle, Rainer; Dimitrow, Enrico; Kaatz, Martin; Fluhr, Joachim; Elsner, Peter

    2006-02-01

    The multiphoton tomograph DermaInspect was used to perform first clinical studies on the early non-invasive detection of skin cancer based on non-invasive optical sectioning of skin by two-photon autofluorescence and second harmonic generation. In particular, deep-tissue pigmented lesions -nevi- have been imaged with intracellular resolution using near infrared (NIR) femtosecond laser radiation. So far, more than 250 patients have been investigated. Cancerous tissues showed significant morphological differences compared to normal skin layers. In the case of malignant melanoma, the occurrence of luminescent melanocytes has been detected. Multiphoton tomography will become a novel non-invasive method to obtain high-resolution 3D optical biopsies for early cancer detection, treatment control, and in situ drug screening.

  10. 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.

  11. 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.

  12. Multiphoton Microwave Ionization of Rydberg Atoms

    NASA Astrophysics Data System (ADS)

    Gurian, Joshua Houston

    This thesis describes a series of multiphoton microwave experiments on Rydberg atoms when the microwave frequency is much greater than the classical Kepler frequency of the excited atoms. A new kHz pulse repetition frequency dye laser system was constructed for Rydberg lithium excitation with a linewidth as narrow as 3 GHz. This new laser system is used for first experiments of multiphoton microwave ionization of Rydberg lithium approaching the photoionization limit using 17 and 36 GHz microwave pulses. A multi-channel quantum defect model is presented that well describes the experimental results, indicating that these results are due to the coherent coupling of many atomic levels both above and below the classical ionization limit. Finally, preliminary results of measuring the final-state distributions of high lying Rydberg states after 17 GHz microwave pulses are presented.

  13. Multiphoton microscopy with near infrared contrast agents

    NASA Astrophysics Data System (ADS)

    Yazdanfar, Siavash; Joo, Chulmin; Zhan, Chun; Berezin, Mikhail Y.; Akers, Walter J.; Achilefu, Samuel

    2010-05-01

    While multiphoton microscopy (MPM) has been performed with a wide range of excitation wavelengths, fluorescence emission has been limited to the visible spectrum. We introduce a paradigm for MPM of near-infrared (NIR) fluorescent molecular probes via nonlinear excitation at 1550 nm. This all-NIR system expands the range of available MPM fluorophores, virtually eliminates background autofluorescence, and allows for use of fiber-based, turnkey ultrafast lasers developed for telecommunications.

  14. 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.

  15. Medium-induced multi-photon radiation

    NASA Astrophysics Data System (ADS)

    Ma, Hao; Salgado, Carlos A.; Tywoniuk, Konrad

    2011-01-01

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

  16. Fundamental studies of molecular multiphoton ionization

    SciTech Connect

    Miller, J.C.; Compton, R.N.

    1984-04-01

    For several years the authors have performed fundamental studies of molecular multiphoton ionization (MPI). We will present a potpourri of techniques and results chosen to illustrate the interesting complexities of molecular MPI. Techniques used include time-of-flight mass spectroscopy, photoelectron spectroscopy, supersonic expansion cooling of molecular beams, harmonic generation, two-color laser MPI, and polarization spectroscopy. Whenever possible the relevance of these results to resonance ionization spectroscopy schemes will be delineated. 23 references, 10 figures.

  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. 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

  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. Multi-photon excited luminescence of magnetic FePt core-shell nanoparticles

    PubMed Central

    Seemann, K.M.; Kuhn, B.

    2014-01-01

    We present magnetic FePt nanoparticles with a hydrophilic, inert, and biocompatible silico-tungsten oxide shell. The particles can be functionalized, optically detected, and optically manipulated. To show the functionalization the fluorescent dye NOPS was bound to the FePt core-shell nanoparticles with propyl-triethoxy-silane linkers and fluorescence of the labeled particles were observed in ethanol (EtOH). In aqueous dispersion the NOPS fluorescence is quenched making them invisible using 1-photon excitation. However, we observe bright luminescence of labeled and even unlabeled magnetic core-shell nanoparticles with multi-photon excitation. Luminescence can be detected in the near ultraviolet and the full visible spectral range by near infrared multi-photon excitation. For optical manipulation, we were able to drag clusters of particles, and maybe also single particles, by a focused laser beam that acts as optical tweezers by inducing an electric dipole in the insulated metal nanoparticles. In a first application, we show that the luminescence of the core-shell nanoparticles is bright enough for in vivo multi-photon imaging in the mouse neocortex down to cortical layer 5. PMID:25071977

  1. Pulse front adaptive optics in multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Sun, B.; Salter, P. S.; Booth, M. J.

    2016-03-01

    The accurate focusing of ultrashort laser pulses is extremely important in multiphoton microscopy. Using adaptive optics to manipulate the incident ultrafast beam in either the spectral or spatial domain can introduce significant benefits when imaging. Here we introduce pulse front adaptive optics: manipulating an ultrashort pulse in both the spatial and temporal domains. A deformable mirror and a spatial light modulator are operated in concert to modify contours of constant intensity in space and time within an ultrashort pulse. Through adaptive control of the pulse front, we demonstrate an enhancement in the measured fluorescence from a two photon microscope.

  2. 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.

  3. 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

  4. 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

  5. Widefield multiphoton excited fluorescence microscopy for animal study in vivo

    NASA Astrophysics Data System (ADS)

    Cheng, L.-C.; Chang, C.-Y.; Lin, C.-H.; Su, Y.-D.; Huang, T.-Y.; Chen, S.-J.

    2010-08-01

    Unlike conventional multiphoton excited microscopy according to pixel-by-pixel point scanning, a widefield multiphoton excited microscopy based on spatiotemporal focusing has been developed to construct three-dimensional (3D) multiphoton fluorescence images only with the need of an axial scanning. By implementing a 4.0 W 10 kHz femtosecond laser amplifier with an instant strong peak power and a fast TE-cooled EMCCD camera with an ultra-sensitive fluorescence detection, the multiphoton excited fluorescence images with the excitation area over 100 μm x 100 μm can be achieved at a frame rate up to 80 Hz. A mechanical shutter is utilized to control the exposure time of 1 ms, i.e. average ten laser pulses reach the fluorescent specimen, and hence an uniform enough multiphoton excited fluorescence image can be attained with less photobleaching. The Brownian motion of microbeads and 3D neuron cells of a rat cerebellum have been observed with a lateral spatial resolution of 0.24 μm and an axial resolution of 2.5 μm. Therefore, the developed widefield multiphoton microscopy can provide fast and high-resolution multiphoton excited fluorescence images for animal study in vivo.

  6. Multifocal multiphoton microscopy with adaptive optical correction

    NASA Astrophysics Data System (ADS)

    Coelho, Simao; Poland, Simon; Krstajic, Nikola; Li, David; Monypenny, James; Walker, Richard; Tyndall, David; Ng, Tony; Henderson, Robert; Ameer-Beg, Simon

    2013-02-01

    Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg-Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.

  7. 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.

  8. The infrared multiphoton dissociation of three nitrolkanes

    NASA Astrophysics Data System (ADS)

    Wodtke, A. M.; Hintsa, E. J.; Lee, Y. T.

    1986-01-01

    Infrared multiphoton dissociation in a molecular beam has been studied in order to elucidate the collision free, 'thermal' chemistry and dynamics of nitromethane, nitroethane and 2-nitropropane. The isomerization of CH3NO2 to CH3ONO was observed by detecting the CH3O and NO products from the dissociation of the very internally hot, isomerized nitromethane. A novel application of RRKM theory was used to estimate the barrier height to isomerization at 55.5 kcal/mol. The barrier height determination method was tested and found to give excellent results by applying it to the determintaion of the barrier height to HONO elimination from nitroethane, a value which is well known from activation energy measurements. The method was then applied to the case of HONO elimination from 2-nitropropane and it appears that there is good to believe that the barrier height is 3-5 kcal/mol lower in 2-nitropropane than in nitroethane. The success of this method for determining barrier heights shows how a microscopic molecular beam experiment, using infrared multiphoton dissociation where the concept of temperature has no place, can be quantitatively related to pyrolysis experiments which are conducted under collisional, thermal conditions and measure phenomenological quantities such as activation energies.

  9. 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.

  10. Controllable infrared continuum source for multiphoton imaging

    NASA Astrophysics Data System (ADS)

    de Mauro, C.; Alfieri, D.; Arrigoni, M.; Armstrong, D.; Pavone, F. S.

    2010-02-01

    We report on multiphoton imaging of biological samples performed with continuum infrared source generated in photonic crystal fibers (PCFs). We studied the spectra generated in PCFs with dispersion profiles designed to maximize the power density in the 700-1000 nm region, where the two-photon absorption cross sections of the most common dyes lie. Pumping in normal dispersion region, with <140 femtosecond pulses delivered by a tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), results in a limitation of nonlinear broadening up to a mean power density above 2 mW/nm. Axial and lateral resolution obtained with a scanning multiphoton system has been measureed to be near the theoretical limit. The possibility of simultaneous two-photon excitation of different dyes in the same sample and high image resolution are demonstrated at tens of microns in depth. Signal-to-noise ratio and general performances are found to be comparable with those of a single wavelength system, used for comparison.

  11. Development of an applicator for multiphoton PDT

    NASA Astrophysics Data System (ADS)

    Graschew, Georgi; Bastian, Matthias; Rakowsky, Stefan; Roelofs, Theo A.; Balanos, Evangelos; Schlag, Peter M.; Steinmeyer, Gunter; Elsaesser, Thomas

    2004-09-01

    Multiphoton excitation of photosensitizers for laser induced fluorescence diagnosis (LIFD) and photodynamic therapy (PDT) of tumors has the advantage of greater tissue penetration due to the longer wavelength of irradiation. However, multiphoton LIFD and PDT are presently not clinically applicable as there are no applicators available for the delivery of the pulsed laser radiation to the operating room. As an approach, in this contribution the beam delivery through photonic crystal fibers has been investigated. Pulses of a Ti:sapphire laser of 100 fs pulse duration and an average power of 150 mW have been transported through such a fiber of 25 m length and the resulting pulses show the absence of nonlinear contributions but still a broadening of the pulse to 2 ps due to the dispersion of the fiber. It is planned to compensate this broadening by a grating in front of the fiber. Alternatively, the transport of laser radiation of 150 fs and 100 mW through a mirror-joint-arm used for conventional CO2 lasers has been tested showing no broadening of the laser pulses. Two-photon photodynamic activity of mTHPC-CMPEG4 shall serve as a test of the laser light transport system.

  12. 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.

  13. Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex

    PubMed Central

    Dorand, R Dixon; Barkauskas, Deborah S; Evans, Teresa A; Petrosiute, Agne; Huang, Alex Y

    2014-01-01

    Fluorescent imaging coupled with high-resolution femto-second pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intra-vital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation. PMID:25568834

  14. 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

  15. Self-organized pattern formation in laser-induced multiphoton ionization

    NASA Astrophysics Data System (ADS)

    Buschlinger, Robert; Nolte, Stefan; Peschel, Ulf

    2014-05-01

    We use finite-difference time-domain modeling to investigate plasma generation induced by multiphoton absorption of intense laser light in dielectrics with tiny inhomogeneities. Plasma generation is found to be strongly amplified around nanometer-sized inhomogeneities as present in glasses. Each inhomogeneity acts as the seed of a plasma structure growing against the direction of light propagation. Plasma structures originating from randomly distributed inhomogeneities are found to interact strongly and to organize in regularly spaced planes oriented perpendicularly to the laser polarization. We discuss similarities between our results and nanogratings in fused silica written by laser beams with spatially homogeneous as well as radial and azimuthal polarizations.

  16. Coherent Scattering of a Multiphoton Quantum Superposition by a Mirror BEC

    SciTech Connect

    De Martini, Francesco; Sciarrino, Fabio; Vitelli, Chiara; Cataliotti, Francesco S.

    2010-02-05

    We present the proposition of an experiment in which the multiphoton quantum superposition consisting of Napprox =10{sup 5} particles generated by a quantum-injected optical parametric amplifier, seeded by a single-photon belonging to an Einstein-Podolsky-Rosen entangled pair, is made to interact with a mirror-Bose-Einstein condensate (BEC) shaped as a Bragg interference structure. The overall process will realize a macroscopic quantum superposition involving a microscopic single-photon state of polarization entangled with the coherent macroscopic transfer of momentum to the BEC structure, acting in spacelike separated distant places.

  17. 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

  18. 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

  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. High resolution intravital imaging of subcellular structures of mouse abdominal organs using a microstage device.

    PubMed

    Cao, Liqin; Kobayakawa, Satoru; Yoshiki, Atsushi; Abe, Kuniya

    2012-01-01

    Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox. PMID:22479464

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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. PMID:11702193

  6. 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.

  7. In vivo multiphoton tomography of inflammatory tissue and melanoma

    NASA Astrophysics Data System (ADS)

    Riemann, Iris; Dimitrow, Enrico; Kaatz, Martin; Fluhr, Joachim; Elsner, Peter; Kobow, Jens; Konig, Karsten

    2005-04-01

    Multiphoton optical tomography provides the capability of non-invasive optical sectioning of skin with high spatial and intracellular resolution as well as high NIR (near infrared) light penetration into pigmented skin areas. The imaging system DermaInspect based on femtosecond laser pulses was used to perform multiphoton optical tomography in clinical studies. Patients with abnormal pigmented tissues were imaged in vivo. After the multiphoton imaging procedure, biopsies were taken, imaged again and further processed with standard histological methods. We report on preliminary results. The visualization of pigmented cell clusters based on non-linear luminescence using the novel multiphoton device was possible. These clusters could be clearly distinguished from non-pigmented cells. Cancerous tissues showed significant differences in the cell structure of the epidermal layers. The system DermaInspect might become a high resolution diagnostic tool for melanoma diagnostics.

  8. 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.

  9. REVIEW ARTICLE Multiphoton polymerization of hybrid materials

    NASA Astrophysics Data System (ADS)

    Farsari, Maria; Vamvakaki, Maria; Chichkov, Boris N.

    2010-12-01

    Multiphoton polymerization has been developed as a direct laser writing technique for the preparation of complex 3D structures with resolution beyond the diffraction limit of light. The combination of two or more hybrid materials with different functionalities in the same system has allowed the preparation of structures with advanced properties and functions. Furthermore, the surface functionalization of the 3D structures opens new avenues for their applications in a variety of nanobiotechnological fields. This paper describes the principles of 2PP and the experimental set-up used for 3D structure fabrication. It also gives an overview of the materials that have been employed in 2PP so far and depicts the perspectives of this technique in the development of new active components.

  10. 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.

  11. Enhancing Multiphoton Rates with Quantum Memories

    NASA Astrophysics Data System (ADS)

    Nunn, J.; Langford, N. K.; Kolthammer, W. S.; Champion, T. F. M.; Sprague, M. R.; Michelberger, P. S.; Jin, X.-M.; England, D. G.; Walmsley, I. A.

    2013-03-01

    Single photons are a vital resource for optical quantum information processing. Efficient and deterministic single photon sources do not yet exist, however. To date, experimental demonstrations of quantum processing primitives have been implemented using nondeterministic sources combined with heralding and/or postselection. Unfortunately, even for eight photons, the data rates are already so low as to make most experiments impracticable. It is well known that quantum memories, capable of storing photons until they are needed, are a potential solution to this “scaling catastrophe.” Here, we analyze in detail the benefits of quantum memories for producing multiphoton states, showing how the production rates can be enhanced by many orders of magnitude. We identify the quantity ηB as the most important figure of merit in this connection, where η and B are the efficiency and time-bandwidth product of the memories, respectively.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Multiphoton nanosurgery in cells and tissues

    NASA Astrophysics Data System (ADS)

    Riemann, Iris; Anhut, Tiemo; Stracke, Frank; Le Harzic, Ronan; Koenig, Karsten

    2005-04-01

    Multiphoton Microscopy with a femtosecond pulsed Ti:sapphire laser in the near infrared (NIR) enables the user not only to image cells and tissues with a subcellular resolution but also to perform highly precise nanosurgery. Intratissue compartments, single cells and even cell organelles like mitochondria, membranes or chromosomes can be manipulated and optically knocked out. Working at transient TW/cm2 laser intensities, single cells of tumor-sphaeroids were eliminated efficiently inside the sphaeroid without damaging the neighbour cells. Also single organelles of cells inside tissues could be optically knocked out with the nanoscalpel without collateral damage. Tissue structures inside a human tooth have been ablated with sizes below 1 μm. This method may become a useful instrument for nano-manipulating and surgery in several fields of science, including targeted transfection.

  17. Nonlinear magic: multiphoton microscopy in the biosciences.

    PubMed

    Zipfel, Warren R; Williams, Rebecca M; Webb, Watt W

    2003-11-01

    Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes. PMID:14595365

  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. 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.

  20. 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

  1. Automated Identification and Localization of Hematopoietic Stem Cells in 3D Intravital Microscopy Data

    PubMed Central

    Khorshed, Reema A.; Hawkins, Edwin D.; Duarte, Delfim; Scott, Mark K.; Akinduro, Olufolake A.; Rashidi, Narges M.; Spitaler, Martin; Lo Celso, Cristina

    2015-01-01

    Summary Measuring three-dimensional (3D) localization of hematopoietic stem cells (HSCs) within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components. PMID:26120058

  2. Intravital Microscopy Reveals Differences in the Kinetics of Endocytic Pathways between Cell Cultures and Live Animals

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie; Rechache, Kamil; Aye, Myo-Pale’; Weigert, Roberto

    2012-01-01

    Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events. PMID:24710546

  3. 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

  4. A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats

    PubMed Central

    Mizuno, Makoto; Tomizawa, Atsuyuki; Ohno, Kousaku; Jakubowski, Joseph A.; Sugidachi, Atsuhiro

    2016-01-01

    Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 μM ADP and 10 μg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl3, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl3 caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl3, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, P = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents. PMID:27128503

  5. 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.

  6. 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

  7. A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats.

    PubMed

    Mizuno, Makoto; Tomizawa, Atsuyuki; Ohno, Kousaku; Jakubowski, Joseph A; Sugidachi, Atsuhiro

    2016-01-01

    Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 μM ADP and 10 μg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl3, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl3 caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl3, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, P = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents. PMID:27128503

  8. 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.

  9. 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.

  10. Multiphoton excitation of fluorescent DNA base analogs

    NASA Astrophysics Data System (ADS)

    Katilius, Evaldas; Woodbury, Neal W.

    2006-07-01

    Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5×10-50 cm4s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  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. Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

    PubMed Central

    Nunes-Silva, Albená; Bernardes, Priscila T. T.; Rezende, Bárbara M.; Lopes, Fernando; Gomes, Elisa C.; Marques, Pedro E.; Lima, Paulo M. A.; Coimbra, Cândido C.; Menezes, Gustavo B.; Teixeira, Mauro M.; Pinho, Vanessa

    2014-01-01

    Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process. PMID:24798414

  18. Convergent perturbation analysis of intense coherent multiphoton interactions

    NASA Technical Reports Server (NTRS)

    Gower, M. C.; Yee, T. K.; Gustafson, T. K.; Fan, B.

    1979-01-01

    Use has been made of flow graphs to deduce Feenberg perturbation expansions for radiative interactions. It is demonstrated that these expansions can in certain cases be summed to provide closed form expressions for the molecular response. In particular, it is shown that the coherent state response can be obtained by the summation of a continued fraction perturbation expansion for the harmonic oscillator. Anharmonicity in the lower levels is treated and its shown to introduce Rabi flopping identifiable with multiphoton transitions among isolated tightly coupled subsystems of levels. Relevance to laser induced multiphoton excitation and energy level shift calculations in the presence of a strong field are also discussed.

  19. Nonlinear optical imaging characteristics of colonic adenocarcinoma using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Nenrong; Chen, Rong; Li, Hongsheng; Chen, Jianxin

    2012-12-01

    Multiphoton microscopy (MPM), a noninvasive optical method with high resolution and high sensitivity, can obtain detailed microstructures of biotissues at submolecular level. In this study, MPM is used to image microstructure varieties of human colonic mucosa and submucosa with adenocarcinoma. Some parameters, such as gland configuration, SHG/TPEF intensity ratio, and collagen orientation and so on, should serve the indicators of early colorectal cancer. The exploratory results show that it's potential for the development of multiphoton mini-endoscopy in real-time early diagnosis of colorectal cancer.

  20. 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.

  1. 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

  2. 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

  3. Photonic near-field imaging in multiphoton photoemission electron microscopy

    NASA Astrophysics Data System (ADS)

    Fitzgerald, J. P. S.; Word, R. C.; Saliba, S. D.; Könenkamp, R.

    2013-05-01

    We report the observation of optical near fields in a photonic waveguide of conductive indium tin oxide (ITO) using multiphoton photoemission electron microscopy (PEEM). Nonlinear two-photon photoelectron emission is enhanced at field maxima created by interference between incident 410-nm and coherently excited guided photonic waves, providing strong phase contrast. Guided modes are observed under both transverse magnetic field (TM) and transverse electric field (TE) polarized illuminations and are consistent with classical electromagnetic theory. Implications on the role of multiphoton PEEM in optical near-field imaging are discussed.

  4. Unambiguous atomic Bell measurement assisted by multiphoton states

    NASA Astrophysics Data System (ADS)

    Torres, Juan Mauricio; Bernád, József Zsolt; Alber, Gernot

    2016-05-01

    We propose and theoretically investigate an unambiguous Bell measurement of atomic qubits assisted by multiphoton states. The atoms interact resonantly with the electromagnetic field inside two spatially separated optical cavities in a Ramsey-type interaction sequence. The qubit states are postselected by measuring the photonic states inside the resonators. We show that if one is able to project the photonic field onto two coherent states on opposite sites of phase space, an unambiguous Bell measurement can be implemented. Thus, our proposal may provide a core element for future components of quantum information technology such as a quantum repeater based on coherent multiphoton states, atomic qubits and matter-field interaction.

  5. Multiphoton interband excitations of quantum gases in driven optical lattices

    NASA Astrophysics Data System (ADS)

    Weinberg, M.; Ölschläger, C.; Sträter, C.; Prelle, S.; Eckardt, A.; Sengstock, K.; Simonet, J.

    2015-10-01

    We report on the observation of multiphoton interband absorption processes for quantum gases in shaken light crystals. Periodic inertial forcing, induced by a spatial motion of the lattice potential, drives multiphoton interband excitations of up to the ninth order. The occurrence of such excitation features is systematically investigated with respect to the potential depth and the driving amplitude. Ab initio calculations of resonance positions as well as numerical evaluation of their strengths exhibit good agreement with experimental data. In addition our findings could make it possible to reach novel phases of quantum matter by tailoring appropriate driving schemes.

  6. Multiphoton Coherent Manipulation in Large Spin Qubits

    NASA Astrophysics Data System (ADS)

    Chiorescu, Irinel

    2009-03-01

    Manipulation of quantum information allows certain algorithms to be performed at unparalleled speeds. Photons are an ideal choice to manipulate qubits as they interact with quantum systems in predictable ways. They are a versatile tool for manipulating, reading/coupling qubits and for encoding/transferring quantum information over long distances. Spin-based qubits have well known behavior under photon driving and can be potentially operated up to room temperature. When diluted enough to avoid uncontrolled spin-spin interactions, a variety of spin qubits show long coherence times, e.g. the nitrogen vacancies in pure diamonds (1,2), nitrogen atoms trapped in a C60 cage (3), Ho3+ and Cr5+ ions (4,5) and molecular magnets (6,7). We have used large spin Mn2+ ions (S=5/2) to realize a six level system that can be operated by means of single as well as multi-photon coherent Rabi oscillations (8). This spin system has a very small anisotropy whose effect can be tuned in-situ to turn the system into a multi-level harmonic system. This offer new ways of manipulating, reading and resetting a spin qubit. Decoherence effects are strongly reduced by the quasi-isotropic electron interaction with the crystal field and with the 55Mn nuclear spins. [0pt] 1. R. Hanson et al., Science 320, 352 (2008). [0pt] 2. M.V. Gurudev Dutt et al., Science 316, 1312 (2007). [0pt] 3. G.W. Morley et al., Phys. Rev. Lett. 98, 220501 (2007). [0pt] 4. S. Bertaina et al., Nat. Nanotech. 2, 39 (2007). [0pt] 5. S. Nellutla et al., Phys. Rev. Lett. 99, 137601 (2007). [0pt] 6. A. Ardavan et al., Phys. Rev. Lett. 98, 057201 (2007). [0pt] 7. S. Bertaina et al., Nature 453, 203,(2008). [0pt] 8. S. Bertaina et al., submitted.

  7. Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

    PubMed Central

    Radbruch, Helena; Andresen, Volker; Mossakowski, Agata; Siffrin, Volker; Seelemann, Thomas; Spiecker, Heinrich; Moll, Ingrid; Herz, Josephine; Hauser, Anja E.; Zipp, Frauke; Behne, Martin J.; Niesner, Raluca

    2013-01-01

    Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm2) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm2) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal

  8. Toward a comprehensive interpretation of intravital microscopy images in studies of lung tissue dynamics

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Schirrmann, Kerstin; Schnabel, Christian; Meissner, Sven; Kertzscher, Ulrich; Kirsten, Lars; Koch, Edmund

    2015-06-01

    Intravital microscopy (IVM) is a well-established imaging technique for real-time monitoring of microscale lung tissue dynamics. Although accepted as a gold standard in respiratory research, its characteristic image features are scarcely understood, especially when trying to determine the actual position of alveolar walls. To allow correct interpretation of these images with respect to the true geometry of the lung parenchyma, we analyzed IVM data of alveoli in a mouse model in comparison with simultaneously acquired optical coherence tomography images. Several IVM characteristics, such as double ring structures or disappearing alveoli in regions of liquid filling, could be identified and related to the position of alveoli relative to each other. Utilizing a ray tracing approach based on an idealized geometry of the mouse lung parenchyma, two major reflection processes could be attributed to the IVM image formation: partial reflection and total internal reflection between adjacent alveoli. Considering the origin of the reflexes, a model was developed to determine the true position of alveolar walls within IVM images. These results allow thorough understanding of IVM data and may serve as a basis for the correction of alveolar sizes for more accurate quantitative analysis within future studies of lung tissue dynamics.

  9. Intra-vital microscopy of lung tissue: A simulation based analysis of the image formation

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Schirrmann, Kerstin; Schnabel, Christian; Meissner, Sven; Kertzscher, Ulrich; Kirsten, Lars; Koch, Edmund

    2013-06-01

    In the course of pulmonary research, understanding alveolar tissue dynamics plays a critical role in the treatment of patients suffering from acute lung diseases. As a gold standard technique for monitoring micro scale changes of lung tissue, real-time intra-vital microscopy (IVM) has been established to evaluate the behavior of the alveolar tissue. To allow profound qualitative and quantitative conclusions, characteristic features of the obtained images have to be thoroughly understood. These factors are strongly influenced by the imaging setup and physiological condition of the lung. To circumvent misinterpretations, a ray-tracing approach has been applied in this study using an idealized geometry of the mouse lung parenchyma deduced from optical coherence tomography (OCT) as a complementary imaging technique. Basic features of IVM images are double ring structures and disappearing of alveoli related to liquid infiltration. Ray propagation analysis reveals the formation of these features by two major reflection processes: partial reflection and total internal reflection. The results give rise to quantification errors of the alveolar area related to reflexes misinterpreted as alveolar borders and should further be used to yield a correction factor for future IVM lung tissue studies.

  10. Visualizing leukocyte trafficking in the living brain with 2-photon intravital microscopy

    PubMed Central

    Pai, Saparna; Danne, Karyn J.; Qin, Jim; Cavanagh, Lois L.; Smith, Adrian; Hickey, Michael J.; Weninger, Wolfgang

    2012-01-01

    Intravital imaging of the superficial brain tissue in mice represents a powerful tool for the dissection of the cellular and molecular cues underlying inflammatory and infectious central nervous system (CNS) diseases. We present here a step-by-step protocol that will enable a non-specialist to set up a two-photon brain-imaging model. The protocol offers a two-part approach that is specifically optimized for imaging leukocytes but can be easily adapted to answer varied CNS-related biological questions. The protocol enables simultaneous visualization of fluorescently labeled immune cells, the pial microvasculature and extracellular structures such as collagen fibers at high spatial and temporal resolution. Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF). Experiments typically require 1–2 h of preparation, which is followed by variable periods of immune cell tracking. Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis. We exemplify the utility of this protocol by tracking leukocytes in transgenic mice in the pial vessels under steady-state conditions. PMID:23316136

  11. Intravital analysis of vascular permeability in mice using two-photon microscopy.

    PubMed

    Egawa, Gyohei; Nakamizo, Satoshi; Natsuaki, Yohei; Doi, Hiromi; Miyachi, Yoshiki; Kabashima, Kenji

    2013-01-01

    Blood vessel endothelium forms a semi-permeable barrier and its permeability controls the traffics of plasma contents. Here we report an intravital evaluation system for vascular permeability in mice using two-photon microscopy. We used various sizes of fluorescein-conjugated dextran as a tracer and its efflux was quantified by measuring the changes of fluorescent intensity both on the blood vessel area and the interstitial space. Using this system, we demonstrated that skin blood vessels limited the passage of dextran larger than 70 kDa under homeostatic conditions. We evaluated the kinetics of vascular permeability in histamine- or IgE-induced type I allergic models and a hapten-induced type IV allergic model. In such inflammatory conditions, the hyperpermeability was selectively induced in the postcapillary venules and dextran as large as 2000-kDa leaked from the bloods. Taken together, our study provides a convenient method to characterize the skin blood vessels as a traffic barrier in physiological conditions. PMID:23732999

  12. Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration and Phagocytosis of Plasmodium-Infected Erythrocytes

    PubMed Central

    de Moraes, Luciana Vieira; Tadokoro, Carlos Eduardo; Gómez-Conde, Iván; Olivieri, David N.; Penha-Gonçalves, Carlos

    2013-01-01

    Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE. PMID:23382682

  13. 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.

  14. Intravital fluorescent microscopic evaluation of bacterial cellulose as scaffold for vascular grafts.

    PubMed

    Esguerra, Maricris; Fink, Helen; Laschke, Matthias W; Jeppsson, Anders; Delbro, Dick; Gatenholm, Paul; Menger, Michael D; Risberg, Bo

    2010-04-01

    Although commonly used synthetic vascular grafts perform satisfactorily in large caliber blood vessels, they are prone to thrombosis in small diameter vessels. Therefore, small vessels might benefit from tissue engineered vascular grafts. This study evaluated bacterial cellulose (BC) as a potential biomaterial for biosynthetic blood vessels. We implanted the dorsal skinfold chambers in three groups of Syrian golden hamsters with BC (experimental group), polyglycolic acid, or expanded polytetrafluorethylene (control groups). Following implantation, we used intravital fluorescence microscopy, histology, and immunohistochemistry to analyze the biocompatibility, neovascularization, and incorporation of each material over a time period of 2 weeks. Biocompatibility was good in all groups, as indicated by the absence of leukocyte activation upon implantation. All groups displayed angiogenic response in the host tissue, but that response was highest in the polyglycolic acid group. Histology revealed vascularized granulation tissue surrounding all three biomaterials, with many proliferating cells and a lack of apoptotic cell death 2 weeks after implantation. In conclusion, BC offers good biocompatibility and material incorporation compared with commonly used materials in vascular surgery. Thus, BC represents a promising new biomaterial for tissue engineering of vascular grafts. PMID:19536832

  15. Expanding Applications of the Nano Intravital Device as a Platform for Exploring Tumor Microenvironments

    NASA Astrophysics Data System (ADS)

    Padgen, Michael R.

    The tumor microenvironment has been demonstrated to be a key determinant in the progression of cancer. Unfortunately, the mechanisms behind the different microenvironments (cytokine gradients, hypoxia, hypoglycemia, etc) have not been fully elucidated. Identifying these mechanisms can lead to targeted, individualized therapy to prevent metastasis. The Nano Intravital Device (NANIVID) is a microfabricated, implantable device designed to initiate specific microenvironments in vivo so that the time course of the effects can be observed. With both spatial and temporal control over the induced environments, the affected regions of the tumor can be compared to the rest of the tumor. The NANIVID was first used to establish cytokine gradients to monitor the migration of invasive cancer cells. The three projects that comprise this work expand the applications of the NANIVID to establish the device as a robust platform for investigating tumor microenvironment interactions. The first project released chemical mimics from the device to induce the cellular hypoxic response in tumors to determine how hypoxia affects the fate of disseminated tumor cells. The second project used the NANIVID in combination with an atomic force microscope to investigate the altered mechanics of migrating invasive cancer cells. The final project was to develop a cell counter to monitor the isolation of the invasive subpopulation of cells that were drawn into the device using a chemoattractant. These three projects demonstrate the potential of the NANIVID as a platform for investigating the tumor microenvironment.

  16. Intravital Fluorescence Facilitates Measurement of Multiple Physiologic Functions and Gene Expression in Tumors of Live Animals

    PubMed Central

    Dewhirst, Mark W.; Shan, S.; Cao, Yiting; Moeller, Benjamin; Yuan, Fan; Li, Chuan-Yuan

    2002-01-01

    The purpose of this report is to present an overview of the use of fluorescence imaging in vivo, with particular emphasis on oncology. It is important to note, however, that many of the methods described herein have been applied to the study of non-malignant tissues as well. Modern medicine and biology research has benefited greatly from an ever-expanding assortment of fluorescent markers and labels. These markers and labels have allowed investigators to observe the behavior and properties of cell and molecular entities of interest in the context of complicated biological systems such as a mammalian cell or a whole mouse. Methods developed to image fluorescence in whole mice have been valuable in studying patterns of tumor growth and metastases. Alternatively, more detailed information and a wide variety of endpoints can be obtained using “intravital” preparations. This review focuses on use of fluorescence imaging for intravital preparations. For detail on fluorescence imaging of whole animals, refer to reviews on this subject [1,2]. For oncologic applications, studies have focused primarily on window chamber preparations that allow for real-time visualization of tumor growth, vascularity, vascular responses to stimulation, vascular permeability, vascular orientation, flow instability, and the like. These endpoints have been used to show that there are functional differences between tumor and normal tissues with respect to these functions under baseline conditions and after therapeutic manipulation. Examples of some of these differences are provided in this review as a means to illustrate how they can be used. PMID:14646042

  17. Contribution of selectins to leucocyte sequestration in pulmonary microvessels by intravital microscopy in rabbits.

    PubMed Central

    Kuebler, W M; Kuhnle, G E; Groh, J; Goetz, A E

    1997-01-01

    1. Sequestration of leucocytes in the lung is the net result of leucocyte rolling and sticking in pulmonary arterioles and venules and their retention in alveolar capillaries. 2. In order to investigate whether adhesion molecules of the selectin family contribute to these phenomena the effects of fucoidin (an inhibitor of L- and P-selectin) on microhaemodynamics and leucocyte kinetic were studied in pulmonary arterioles, capillaries and venules by means of intravital fluorescence microscopy in a rabbit model. 3. Fucoidin reduced leucocyte rolling in pulmonary arterioles and venules by 75 and 83%, respectively, without affecting leucocyte sticking. In alveolar capillaries, fucoidin reduced leucocyte retention and accelerated leucocyte passage, thus reducing the alveolar transit time of leucocytes by 62%. 4. It is concluded that rolling of leucocytes in pulmonary microvessels is mediated by selectins, whereas sticking relies on selectin-independent mechanisms. 5. Leucocyte retention in alveolar capillaries is not due solely to mechanical hindrance of leucocyte passage through narrow vessel segments, as previously hypothesized, but also depends on interaction of leucocytes with the capillary endothelium. PMID:9192309

  18. Comparison of objective lenses for multiphoton microscopy in turbid samples.

    PubMed

    Singh, Avtar; McMullen, Jesse D; Doris, Eli A; Zipfel, Warren R

    2015-08-01

    Optimization of illumination and detection optics is pivotal for multiphoton imaging in highly scattering tissue and the objective lens is the central component in both of these pathways. To better understand how basic lens parameters (NA, magnification, field number) affect fluorescence collection and image quality, a two-detector setup was used with a specialized sample cell to separate measurement of total excitation from epifluorescence collection. Our data corroborate earlier findings that low-mag lenses can be superior at collecting scattered photons, and we compare a set of commonly used multiphoton objective lenses in terms of their ability to collect scattered fluorescence, providing guidance for the design of multiphoton imaging systems. For example, our measurements of epi-fluorescence beam divergence in the presence of scattering reveal minimal beam broadening, indicating that often-advocated over-sized collection optics are not as advantageous as previously thought. These experiments also provide a framework for choosing objective lenses for multiphoton imaging by relating the results of our measurements to various design parameters of the objectives lenses used. PMID:26309771

  19. Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy

    PubMed Central

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

    2010-01-01

    We report optical-resolution photoacoustic microscopy (OR-PAM) for in vivo imaging of amyloid plaques in an Alzheimer’s disease mouse model. Validation using conventional fluorescence microscopy and multiphoton microscopy shows that OR-PAM has sufficient sensitivity and spatial resolution to identify amyloid plaques in living brains. In addition, with dual-wavelength OR-PAM, the three-dimensional morphology of amyloid plaques and the surrounding microvasculature are imaged simultaneously through a cranial window without angiographic contrast agents. OR-PAM, capable of providing both exogenous molecular contrast and endogenous hemoglobin contrast, has the potential to serve as a new technology for in vivo microscopic observations of cerebral plaque deposits. PMID:20016651

  20. 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

  1. 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

  2. Multiphoton dynamics of qutrits in the ultrastrong coupling regime with a quantized photonic field

    NASA Astrophysics Data System (ADS)

    Avetissian, H. K.; Avetissian, A. K.; Mkrtchian, G. F.; Kibis, O. V.

    2015-12-01

    Multiphoton resonant excitation of a three-state quantum system (a qutrit) with a single-mode photonic field is considered in the ultrastrong coupling regime, when the qutrit-photonic field coupling rate is comparable to appreciable fractions of the photon frequency. For ultrastrong couplings, the obtained solutions of the Schrödinger equation that reveal multiphoton Rabi oscillations in qutrits with the interference effects leading to the collapse and revival of atomic excitation probabilities at the direct multiphoton resonant transitions.

  3. 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

  4. 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

  5. The role of resonances in strong-field multiphoton processes

    SciTech Connect

    Perry, M.D.; Kulander, K.C.

    1990-10-01

    Resonantly-enhanced multiphoton ionization (REMPI) has been the subject of extensive experimental and theoretical study since the invention of the laser. Until recently, the overwhelming majority of REMPI research have been conducted at intensities less than 10{sup 12} W/cm{sup 2}. At these intensities, the strength of the applied field remains less than one percent of the atomic Coulomb field experienced by the outer electrons in a typical noble gas atom. In this regime, treatment of the applied field as a weak perturbation on the atomic system yields excellent agreement with experiment. Here, we investigate the role of resonances in multiphoton ionization at much higher intensities, specifically, we examine the behavior and influence of resonances as the strength of the applied field becomes a significant fraction of the atomic field. 33 refs., 7 figs., 2 tabs.

  6. 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.

  7. 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

  8. Nanoparticle metrology in sol-gels using multiphoton excited fluorescence

    NASA Astrophysics Data System (ADS)

    Karolin, J.; Geddes, C. D.; Wynne, K.; Birch, D. J. S.

    2002-01-01

    We have developed a method of measuring the growth of nanoparticles during sol-gel glass formation based on labelling the particle with a fluorescent dye and determining the multiphoton excited decay of fluorescence anisotropy due to Brownian rotation. Multiphoton excitation is shown to give a higher dynamic range of measurement than one-photon excitation. We illustrate the sub-nanometre resolution and stability of our approach by detecting a 0.8-1.1 nm silica particle hydrodynamic mean radius increase in a tetramethylorthosilicate sol at pH 2.3 labelled with rhodamine 6G and observed over ≈4 weeks and also with a stable silica colloid of radius 6 nm, pH 8.9, labelled with a 6-methoxyquinoline-type dye.

  9. Multiphoton Imaging of Ultrasound Bioeffects in the Murine Brain

    NASA Astrophysics Data System (ADS)

    Raymond, Scott; Skoch, Jesse; Bacskai, Brian; Hynynen, Kullervo

    2006-05-01

    The purpose of this study was to demonstrate the feasibility of multiphoton imaging in the murine brain during exposure to ultrasound. Our experimental setup coupled ultrasound through the ventral surface of the mouse while allowing imaging through a cranial window from the dorsal surface. Field attenuation was estimated by scanning the field after insertion of a freshly sacrificed mouse; beam profile and peak position were preserved, suggesting adequate targeting for imaging experiments. C57 mice were imaged with a Biorad multiphoton microscope while being exposed to ultrasound (f = 1.029 MHz, peak pressure ˜ 200 kPa, average power ˜ 0.18 W) with IV injection of Optison. We observed strong vasoconstriction coincident with US and Optison, as well as permeabilization of the blood-brain barrier.

  10. Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging.

    PubMed

    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

  11. 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.

  12. 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.

  13. 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.

  14. Waveguide characterization with multi-photon photoemission electron microscopy

    NASA Astrophysics Data System (ADS)

    Fitzgerald, J. P. S.; Word, Robert C.; Saliba, Sebastian; Koenenkamp, Rolf

    2012-10-01

    Multi-photon photoemission electron microscopy (PEEM) images surface interactions of visible light with matter, showing electromagnetic (EM) waves that propagate at or near the surface. Images are interferometric, showing where incident and surface waves are in-phase (bright) and out-of-phase (dark), with strong contrast between regions of high and low rates of photoelectron emission. Interferogram analysis can determine the amplitude, wavelength, phase evolution, and propagation decay length of the surface waves. Most multi-photon PEEM studies focus on surface plasmon polaritons. We show that this technique can also be applied to conducting thin-film waveguides, measuring the properties of confined EM waves in a two-mode slab waveguide made of indium tin oxide on glass, which are consistent with waveguide theory. This research was funded by the US Department of Energy Basic Science Office under contract DE-FG02-10ER46406.

  15. Does Infrared Multiphoton Dissociation of Vinyl Chloride Yield Cold Vinylidene?

    PubMed

    Fernando, Ravin; Qu, Chen; Bowman, Joel M; Field, Robert W; Suits, Arthur G

    2015-07-01

    Velocity map imaging of the infrared multiphoton dissociation of vinyl chloride shows the formation of HCl in rotational levels below J = 10 that are associated with the three-center elimination pathway. The total translational energy release is observed to peak at 3-5 kcal/mol, which is consistent with the low reverse barrier predicted for the formation of HCl with vinylidene coproducts. Direct dynamics trajectory studies from the three-center transition state reproduce the observed distributions and show that the associated vinylidene is formed with only modest rotational excitation, precluding Coriolis-induced mixing among the excited vibrational levels of acetylene that would lead to distribution of vinylidene character into many vibrationally mixed acetylene vibrational levels. The results suggest that infrared multiphoton dissociation of vinyl chloride is an efficient route to synthesis of stable, cold vinylidene. PMID:26266719

  16. 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.

  17. 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.

  18. Relaxation channels of multi-photon excited xenon clusters

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    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.

  19. Femtosecond Light Source for Phase-Controlled Multiphoton Ionization

    SciTech Connect

    Sokolov, A. V.; Walker, D. R.; Yavuz, D. D.; Yin, G. Y.; Harris, S. E.

    2001-07-16

    We describe a femtosecond Raman light source with more than an octave of optical bandwidth. We use this source to demonstrate phase control of multiphoton ionization under conditions where ionization requires eleven photons of the lowest frequency of the spectrum or five photons of the highest frequency. The nonlinearity of the photoionization process allows us to characterize the light source. Experiment-to-theory comparison implies generation of a near single-cycle waveform.

  20. Single- and multiphoton infrared laser spectroscopy of atomic negative ions

    NASA Astrophysics Data System (ADS)

    Scheer, Michael

    A pulsed, tunable infrared laser source (0.6-5.2 μm) has been developed on the basis of a commercial dye laser and non-linear optical conversion techniques. This laser source was combined with a keV negative ion beam apparatus in a crossed-beam geometry, with the aim to systematically study several atomic negative ions through a variety of single- and multiphoton detachment experiments. Photodetachment threshold spectra of 21 ionic species (B- , C-, O-, Al- , Si-, Cr-, Co- , Ni-, Cu-, Ge- , Mo-, Rh-, Pd- , Ag-, Sn-, Sb- , Te-, Cs-, Ir- , Pt-, and Bi-) have been recorded, in most cases resulting in very accurate determinations of ionic binding energies, marking substantial improvements over previous experimental values. In fact, several ionic states investigated here had not been observed previously. Different schemes for resonant multiphoton detachment of atomic negative ions were demonstrated for the first time. These studies were conducted with several anions (Si-, Sri- , Sb-, Te-, Ir- , and Pt-) providing highly accurate ionic energy level splittings and clearly demonstrating that multiphoton probes are generally applicable to negative ion structure.

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

    PubMed

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

    2016-02-12

    Large increases in molecular two-photon absorption, the onset of measurable molecular three-photon absorption, and record molecular four-photon absorption in organic π-delocalizable frameworks are achieved by incorporation of bis(diphosphine)ruthenium units with alkynyl linkages. The resultant ruthenium alkynyl-containing dendrimers exhibit strong multiphoton absorption activity through the biological and telecommunications windows in the near-infrared region. The ligated ruthenium units significantly enhance solubility and introduce fully reversible redox switchability to the optical properties. Increasing the ruthenium content leads to substantial increases in multiphoton absorption properties without any loss of optical transparency. This significant improvement in multiphoton absorption performance by incorporation of the organometallic units into the organic π-framework is maintained when the relevant parameters are scaled by molecular weights or number of delocalizable π-electrons. The four-photon absorption cross-section of the most metal-rich dendrimer is an order of magnitude greater than the previous record value. PMID:26797727

  2. 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.

  3. Multiphoton ionization mass spectrometry of nitrated polycyclic aromatic hydrocarbons.

    PubMed

    Tang, Yuanyuan; Imasaka, Tomoko; Yamamoto, Shigekazu; Imasaka, Totaro

    2015-08-01

    In order to suppress the fragmentation and improve the sensitivity for determination of nitrated polycyclic aromatic hydrocarbons (NPAHs), the mechanism of multiphoton ionization was studied for the following representative NPAHs, 9-nitroanthracene, 3-nitrofluoranthene, and 1-nitropyrene. The analytes were extracted from the PM2.5 on the sampling filter ultrasonically, and were measured using gas chromatography/multiphoton ionization/time-of-flight mass spectrometry with a femtosecond tunable laser in the range from 267 to 405 nm. As a result, a molecular ion was observed as the major ion and fragmentation was suppressed at wavelengths longer than 345 nm. Furthermore, the detection limit measured at 345 nm was measured to be the subpicogram level. The organic compounds were extracted from a 2.19 mg sample of particulate matter 2.5 (PM2.5), and the extract was subjected to multiphoton ionization mass spectrometry after gas chromatograph separation. The background signals were drastically suppressed at 345 nm, and the target NPAHs, including 9-nitroanthracene and 1-nitropyrene, were detected, and their concentrations were determined to be 5 and 3 pg/m(3), respectively. PMID:26048831

  4. 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

  5. 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.

  6. 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.

  7. Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy.

    PubMed

    Hanna, Gabi; Fontanella, Andrew; Palmer, Gregory; Shan, Siqing; Radiloff, Daniel R; Zhao, Yulin; Irwin, David; Hamilton, Karyn; Boico, Alina; Piantadosi, Claude A; Blueschke, Gert; Dewhirst, Mark; McMahon, Timothy; Schroeder, Thies

    2013-01-15

    Intravital microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the β1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary intravital microscopy in laboratory rats for pharmacological and physiological research. PMID:23161885

  8. 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

  9. From optical bench to cageside: intravital microscopy on the long road to rational vaccine design

    PubMed Central

    Hickman, Heather D.; Bennink, Jack R.; Yewdell, Jonathan W.

    2012-01-01

    Summary No anti-viral vaccine is perfect. For some important pathogens, there are no effective vaccines. Many current vaccines are based on the working principles of Jenner and Pasteur, i.e. empiric administration of attenuated or inactivated forms of the pathogen. Tapping the full potential of vaccination requires a thorough understanding of the mechanism of immune activation by pathogens and their individual components. Though the rate of discovery continues to accelerate, the complexity of the immune system is daunting, particularly when integrated into the overall physiology of the host. Here, we review the application of multiphoton microscopy to examine host-pathogen interactions, focusing on our recent efforts to understand mouse CD8+ T-cell responses to viruses at the level of cellular interactions in lymph nodes draining the infection site. We also discuss our recent efforts to understand the influence of the sympathetic nervous system on antiviral immunity, with the ultimate goal of appreciating the traditional elements of immunity as just one facet of the total organismal response to infection and immunization. PMID:21198674

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

    PubMed

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

    2016-05-01

    High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining. PMID:26830089

  11. 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

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

    PubMed

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

    2015-07-01

    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 3D 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 2.3-kb 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 showed 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 with host bone

  13. Dynamic Multiphoton Microscopy: Focusing Light on Acute Kidney Injury

    PubMed Central

    Molitoris, Bruce A.

    2014-01-01

    Acute kidney injury (AKI) is a major global health problem; much research has been conducted on AKI, and numerous agents have shown benefit in animal studies, but none have translated into treatments. There is, therefore, a pressing unmet need to increase knowledge of the pathophysiology of AKI. Multiphoton microscopy (MPM) provides a tool to non-invasively visualize dynamic events in real time and at high resolution in rodent kidneys, and in this article we review its application to study novel mechanisms and treatments in different forms of AKI. PMID:25180263

  14. 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.

  15. Multiphoton excitation of organic chromophores in microbubble resonators

    NASA Astrophysics Data System (ADS)

    Cohoon, Gregory A.; Kieu, Khanh; Norwood, Robert A.

    2014-03-01

    We report the observation of multiphoton excitation of organic chromophores in microbubble whispering gallery mode resonators. High-Q microbubble resonators are a formed by heating a pressurized fused silica capillary to form a hollow bubble which can be filled with liquid. In this case, the microbubble is filled with a solution of Rhodamine 6G dye. The resonator and dye are excited by evanescently coupling CW light from a 980nm laser diode using a tapered optical fiber. The two-photon fluorescence of the dye can be seen with pump powers as low as 1 mW.

  16. 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.

  17. 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

  18. Multi-photon microscope driven by novel green laser pump

    NASA Astrophysics Data System (ADS)

    Marti, Dominik; Djurhuus, Martin; Jensen, Ole Bjarlin; Andersen, Peter E.

    2016-03-01

    Multi-photon microscopy is extensively used in research due to its superior possibilities when compared to other microscopy modalities. The technique also has the possibility to advance diagnostics in clinical applications, due to its capabilities complementing existing technology in a multimodal system. However, translation is hindered due to the high cost, high training demand and large footprint of a standard setup. We show in this article that minification of the setup, while also reducing cost and complexity, is indeed possible without compromising on image quality, by using a novel diode laser replacing the commonly used conventional solid state laser as the pump for the femtosecond system driving the imaging.

  19. 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

  20. 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.

  1. Exploration of multiphoton entangled states by using weak nonlinearities

    NASA Astrophysics Data System (ADS)

    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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents

    PubMed Central

    Masedunskas, Andrius; Weigert, Roberto

    2009-01-01

    Here we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs since they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and bovine serum albumin, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context. PMID:18647170

  8. 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

  9. Development of synchrotron radiation x-ray intravital microscopy for in vivo imaging of rat heart vascular function.

    PubMed

    Umetani, Keiji; Pearson, James T; Schwenke, Daryl O; Shirai, Mikiyasu

    2011-01-01

    This study elucidates the vascular internal diameter response of coronary arterial circulation in closed-chest rats to evaluate endothelium-dependent and endothelium-independent vasodilatory ability and to investigate disease mechanisms. For this study, we developed an X-ray intravital microscopy system using a microangiography technique and a synchrotron radiation source at SPring-8. An X-ray direct-conversion type detector with 7-μm spatial resolution was used for real-time imaging. Microangiographic images were stored in a digital frame memory system at a maximum rate of 30 frame/s with a 1024 × 1024-pixel, 10-bit format. In imaging experiments, the small coronary arteries were visualized after iodine contrast agent injection into the coronary artery. PMID:22256145

  10. Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged Sickle Cell Disease mice

    PubMed Central

    Bennewitz, Margaret F.; Watkins, Simon C.; Sundd, Prithu

    2015-01-01

    Sickle cell disease (SCD) is a genetic disorder that leads to red blood cell (RBC) sickling, hemolysis and the upregulation of adhesion molecules on sickle RBCs. Chronic hemolysis in SCD results in a hyper-inflammatory state characterized by activation of circulating leukocytes, platelets and endothelial cells even in the absence of a crisis. A crisis in SCD is often triggered by an inflammatory stimulus and can lead to the acute chest syndrome (ACS), which is a type of lung injury and a leading cause of mortality among SCD patients. Although it is believed that pulmonary vaso-occlusion could be the phenomenon contributing to the development of ACS, the role of vaso-occlusion in ACS remains elusive. Intravital imaging of the cremaster microcirculation in SCD mice has been instrumental in establishing the role of neutrophil-RBC-endothelium interactions in systemic vaso-occlusion; however, such studies, although warranted, have never been done in the pulmonary microcirculation of SCD mice. Here, we show that two-photon excitation fluorescence microscopy can be used to perform quantitative analysis of neutrophil and RBC trafficking in the pulmonary microcirculation of SCD mice. We provide the experimental approach that enables microscopic observations under physiological conditions and use it to show that RBC and neutrophil trafficking is comparable in SCD and control mice in the absence of an inflammatory stimulus. The intravital imaging scheme proposed in this study can be useful in elucidating the cellular and molecular mechanism of pulmonary vaso-occlusion in SCD mice following an inflammatory stimulus. PMID:25995970

  11. Intravital imaging of SRF and Notch signalling identifies a key role for EZH2 in invasive melanoma cells.

    PubMed

    Manning, C S; Hooper, S; Sahai, E A

    2015-08-13

    The acquisition of cell motility is an early step in melanoma metastasis. Here we use intravital imaging of signalling reporter cell-lines combined with genome-wide transcriptional analysis to define signalling pathways and genes associated with melanoma metastasis. Intravital imaging revealed heterogeneous cell behaviour in vivo: <10% of cells were motile and both singly moving cells and streams of cells were observed. Motile melanoma cells had increased Notch- and SRF-dependent transcription. Subsequent genome-wide analysis identified an overlapping set of genes associated with high Notch and SRF activity. We identified EZH2, a histone methyltransferase in the Polycomb repressive complex 2, as a regulator of these genes. Heterogeneity of EZH2 levels is observed in melanoma models, and co-ordinated upregulation of genes positively regulated by EZH2 is associated with melanoma metastasis. EZH2 was also identified as regulating the amelanotic phenotype of motile cells in vivo by suppressing expression of the P-glycoprotein Oca2. Analysis of patient samples confirmed an inverse relationship between EZH2 levels and pigment. EZH2 targeting with siRNA and chemical inhibition reduced invasion in mouse and human melanoma cell lines. The EZH2-regulated genes KIF2C and KIF22 are required for melanoma cell invasion and important for lung colonization. We propose that heterogeneity in EZH2 levels leads to heterogeneous expression of a cohort of genes associated with motile behaviour including KIF2C and KIF22. EZH2-dependent increased expression of these genes promotes melanoma cell motility and early steps in metastasis. PMID:25381824

  12. Intravital imaging of SRF and Notch signalling identifies a key role for EZH2 in invasive melanoma cells

    PubMed Central

    Manning, Cerys S; Hooper, Steven; Sahai, Erik A

    2014-01-01

    The acquisition of cell motility is an early step in melanoma metastasis. Here we use intravital imaging of signalling reporter cell-lines combined with genome-wide transcriptional analysis to define signalling pathways and genes associated with melanoma metastasis. Intravital imaging revealed heterogeneous cell behaviour in vivo: less than 10% of cells were motile and both singly moving cells and streams of cells were observed. Motile melanoma cells had increased Notch- and SRF-dependent transcription. Subsequent genome-wide analysis identified an overlapping set of genes associated with high Notch and SRF activity. We identified EZH2, a histone methyltransferase in the Polycomb Repressor Complex 2, as a regulator of these genes. Heterogeneity of EZH2 levels is observed in melanoma models and co-ordinated up-regulation of genes positively regulated by EZH2 is associated with melanoma metastasis. EZH2 was also identified as regulating the amelanotic phenotype of motile cells in vivo by suppressing expression of the P-glycoprotein Oca2. Analysis of patient samples confirmed an inverse relationship between EZH2 levels and pigment. EZH2 targeting with siRNA and chemical inhibition reduced invasion in mouse and human melanoma cell lines. The EZH2 regulated SRF target genes KIF2C and KIF22 are required for melanoma cell invasion and important for lung colonisation. We propose that heterogeneity in EZH2 levels leads to heterogeneous expression of a cohort of genes associated with motile behaviour including KIF2C and KIF22. EZH2 dependent increased expression of these genes promotes melanoma cell motility and early steps in metastasis. PMID:25381824

  13. Multibeam multifocal multiphoton photon counting imaging in scattering media

    NASA Astrophysics Data System (ADS)

    Hoover, Erich E.

    Multiphoton microscopy is an invaluable technique for the neurological community, allowing for deep explorations within highly scattering tissues such as the brain. However, prior to this research multiphoton microscopy was limited in its ability to rapidly construct volumetric images deep within scattering specimens. This work establishes a technique that permits such exploration through the application of multiple beams separated in both space and time, where signal photons corresponding to those beams are demultiplexed through the use of a field programmable gate array. With this system a number of improvements are provided to research in scattering media, including the coveted ability to perform photon-counting imaging with multiple beams. The ability to perform these measurements with multiple beams permits unique quantitative measurements of fluorophores within living specimens, allowing new research into dynamic three-dimensional behavior occurring within the brain. Additionally, the ability to perform multimodal measurements without filtering allows for unique avenues of research where the harmonic generation is indistinguishable from the two-photon excited fluorescence. These improvements provide neuroscience researchers with a large assortment of technological tools that will permit them to perform numerous novel experiments within the brain and other highly-scattering specimens, which should one day lead to significant advances in our understanding of complex neuronal activity.

  14. Resonant enhanced multiphoton ionization studies of atomic oxygen

    NASA Technical Reports Server (NTRS)

    Dixit, S. N.; Levin, D.; Mckoy, V.

    1987-01-01

    In resonant enhanced multiphoton ionization (REMPI), an atom absorbs several photons making a transition to a resonant intermediate state and subsequently ionizing out of it. With currently available tunable narrow-band lasers, the extreme sensitivity of REMPI to the specific arrangement of levels can be used to selectively probe minute amounts of a single species (atom) in a host of background material. Determination of the number density of atoms from the observed REMPI signal requires a knowledge of the multiphoton ionization cross sections. The REMPI of atomic oxygen was investigated through various excitation schemes that are feasible with available light sources. Using quantum defect theory (QDT) to estimate the various atomic parameters, the REMPI dynamics in atomic oxygen were studied incorporating the effects of saturation and a.c. Stark shifts. Results are presented for REMPI probabilities for excitation through various 2p(3) (4S sup o) np(3)P and 2p(3) (4S sup o) nf(3)F levels.

  15. 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).

  16. Multiphoton Microscopy of Nonfluorescent Nanoparticles In Vitro and In Vivo.

    PubMed

    Dietzel, Steffen; Hermann, Stefanie; Kugel, Yan; Sellner, Sabine; Uhl, Bernd; Hirn, Stephanie; Krombach, Fritz; Rehberg, Markus

    2016-06-01

    Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano-bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO2 ), and silica (SiO2 ) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon-induced luminescence decaying over time. TiO2 NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano-bio interactions not only for fluorescent but also for some types of nonfluorescent NPs. PMID:27120195

  17. Spectral-resolved multifocal multiphoton microscopy with multianode photomultiplier tubes

    PubMed Central

    Cha, Jae Won; Tzeranis, Dimitrios; Subramanian, Jaichandar; Yannas, Ioannis V.; Nedivi, Elly; So, Peter T. C.

    2014-01-01

    Multiphoton excitation fluorescence microscopy is the preferred method for in vivo deep tissue imaging. Many biological applications demand both high imaging speed and the ability to resolve multiple fluorophores. One of the successful methods to improve imaging speed in a highly turbid specimen is multifocal multiphoton microscopy (MMM) based on use of multi-anode photomultiplier tubes (MAPMT). This approach improves imaging speed by using multiple foci for parallelized excitation without sacrificing signal to noise ratio (SNR) due to the scattering of emission photons. In this work, we demonstrate that the MAPMT based MMM can be extended with spectral resolved imaging capability. Instead of generating multiple excitation foci in a 2D grid pattern, a linear array of foci is generated. This leaves one axis of the 2D MAPMT available for spectral dispersion and detection. The spectral-resolved MMM can detect several emission signals simultaneously with high imaging speed optimized for high-throughput, high-contents applications. The new procedure is illustrated using imaging data from the kidney, peripheral nerve regeneration and dendritic morphological data from the brain. PMID:25321515

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Multi-photon absorption in the channeling of electrons in an external field

    NASA Astrophysics Data System (ADS)

    Yaralov, V.

    2016-07-01

    Following the methods developed for atom ionization by alternating electric field the probability of multi-photon absorption of photons of the strong external laser field by channeled electron (extraction of electron from the channel) have been calculated for different strengths of the monochromatic external field. The emission spectra of 54 MeV electron channeled in diamond crystal planes (110) are shown for different values of the resonant laser field of a frequency close to the transition frequency in the channel taking into account multi-photon absorption. It is shown that the multi-photon phenomena give some contribution to the total level width.

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

    NASA Astrophysics Data System (ADS)

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

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

  4. 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.

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

    PubMed

    König, Karsten; Andersen, Peter; Le, Tuan; Breunig, Hans Georg

    2015-12-01

    Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire-based systems for high-quality multiphoton imaging at a significantly size and weight compared to current systems will become possible. PMID:26534831

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. Multiphoton Processes: ICOMP VIII: 8th International Conference, AIP Conference Proceedings, No. 525 [APCPCS

    SciTech Connect

    DiMauro, L.F.; Freeman, R.R.; Kulander, K.C.

    2000-12-31

    Topics include: atoms in strong fields; stabilization; double ionization and multi-electron calculations; high-order harmonics; molecules in strong fields; multiphoton processes in clusters; coherent control; light sources; and relativistic effects.

  13. 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

  14. Clinical multiphoton tomography and clinical two-photon microendoscopy

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    We report on applications of high-resolution clinical multiphoton tomography based on the femtosecond laser system DermaInspectTM with its flexible mirror arm in Australia, Asia, and Europe. Applications include early detection of melanoma, in situ tracing of pharmacological and cosmetical compounds including ZnO nanoparticles in the epidermis and upper dermis, the determination of the skin aging index SAAID as well as the study of the effects of anti-aging products. In addition, first clinical studies with novel rigid high-NA two-photon 1.6 mm GRIN microendoscopes have been conducted to study the effect of wound healing in chronic wounds (ulcus ulcera) as well as to perform intrabody imaging with subcellular resolution in small animals.

  15. 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.

  16. Multiphoton-state-assisted entanglement purification of material qubits

    NASA Astrophysics Data System (ADS)

    Bernád, József Zsolt; Torres, Juan Mauricio; Kunz, Ludwig; Alber, Gernot

    2016-03-01

    We propose an entanglement purification scheme based on material qubits and ancillary coherent multiphoton states. We consider a typical QED scenario where material qubits implemented by two-level atoms fly sequentially through a cavity and interact resonantly with a single mode of the radiation field. We explore the theoretical possibilities of realizing a high-fidelity two-qubit quantum operation necessary for the purification protocol with the help of a postselective balanced homodyne photodetection. We demonstrate that the obtained probabilistic quantum operation can be used as a bilateral operation in the proposed purification scheme. It is shown that the probabilistic nature of this quantum operation is counterbalanced in the last step of the scheme where qubits are not discarded after inadequate qubit measurements. As this protocol requires present-day experimental setups and generates high-fidelity entangled pairs with high repetition rates, it may offer interesting perspectives for applications in quantum information theory.

  17. 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.

  18. Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy

    PubMed Central

    Chang, Chia-Yuan; Cheng, Li-Chung; Su, Hung-Wei; Hu, Yvonne Yuling; Cho, Keng-Chi; Yen, Wei-Chung; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

    2014-01-01

    Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved. PMID:24940539

  19. 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.

  20. Guided Homing of Cells in Multi-Photon Microfabricated Bioscaffolds.

    PubMed

    Skylar-Scott, Mark A; Liu, Man-Chi; Wu, Yuelong; Dixit, Atray; Yanik, Mehmet Fatih

    2016-05-01

    Tissues contain exquisite vascular microstructures, and patterns of chemical cues for directing cell migration, homing, and differentiation for organ development and function. 3D microfabrication by multi-photon photolithography is a flexible, high-resolution tool for generating 3D bioscaffolds. However, the combined fabrication of scaffold microstructure simultaneously with patterning of cues to create both geometrically and chemically defined microenvironments remains to be demonstrated. This study presents a high-speed method for micron-resolution fabrication of scaffold microstructure and patterning of protein cues simultaneously using native scaffold materials. By the simultaneous microfabrication of arbitrary microvasculature geometries, and patterning selected regions of the microvasculature with the homing ligand P-selectin, this study demonstrates adhesion, rolling, and selective homing of cells in defined 3D regions. This novel ability to generate high-resolution geometries replete with patterned cues at high speed enables the construction of biomimetic microenvironments for complex 3D assays of cellular behavior. PMID:27059425

  1. 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

    2014-01-01

    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. PMID:24898470

  2. Theory of multiphoton and tunnel ionization in a bichromatic field

    SciTech Connect

    Bagulov, D. S.; Kotelnikov, I. A.

    2013-01-15

    The imaginary-time method [6, 7] is used to calculate the multiphoton and tunnel ionization probabilities for atoms in a laser radiation field part of which is converted into the second harmonic. We assume that the first harmonic has a linear or elliptical polarization and the second harmonic is polarized linearly, with its polarization vector making an arbitrary angle with that of the first harmonic. The mean momentum of the photoelectrons knocked out from atoms is shown to depend on the phase shift between the first and second harmonics and their mutual polarization and to be identically equal to zero for a monochromatic field. An important difference between the case of elliptical polarization and the case of linear polarization of both harmonics is the absence of conditions under which the conditions for dominance of one of the two generation mechanisms considered here can be identified during the generation of terahertz radiation from the region of optical breakdown in a gas.

  3. 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.

  4. Reassignment of Scattered Emission Photons in Multifocal Multiphoton Microscopy

    PubMed Central

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

    2014-01-01

    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. PMID:24898470

  5. Multiphoton quantum Rabi oscillations in ultrastrong cavity QED

    NASA Astrophysics Data System (ADS)

    Garziano, Luigi; Stassi, Roberto; Macrı, Vincenzo; Kockum, Anton Frisk; Savasta, Salvatore; Nori, Franco

    2015-12-01

    When an atom is strongly coupled to a cavity, the two systems can exchange a single photon through a coherent Rabi oscillation. This process enables precise quantum-state engineering and manipulation of atoms and photons in a cavity, which play a central role in quantum information and measurement. Recently, a new regime of cavity QED was reached experimentally where the strength of the interaction between light and artificial atoms (qubits) becomes comparable to the atomic transition frequency or the resonance frequency of the cavity mode. Here we show that this regime can strongly modify the concept of vacuum Rabi oscillations, enabling multiphoton exchanges between the qubit and the resonator. We find that experimental state-of-the-art circuit-QED systems can undergo two- and three-photon vacuum Rabi oscillations. These anomalous Rabi oscillations can be exploited for the realization of efficient Fock-state sources of light and complex entangled states of qubits.

  6. Reassignment of Scattered Emission Photons in Multifocal Multiphoton Microscopy

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

    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.

  7. 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. PMID:24977374

  8. 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

  9. 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)

  10. Multiphoton gradient index endoscopy for evaluation of diseased human prostatic tissue ex vivo

    NASA Astrophysics Data System (ADS)

    Huland, David M.; Jain, Manu; Ouzounov, Dimitre G.; Robinson, Brian D.; Harya, Diana S.; Shevchuk, Maria M.; Singhal, Paras; Xu, Chris; Tewari, Ashutosh K.

    2014-11-01

    Multiphoton microscopy can instantly visualize cellular details in unstained tissues. Multiphoton probes with clinical potential have been developed. This study evaluates the suitability of multiphoton gradient index (GRIN) endoscopy as a diagnostic tool for prostatic tissue. A portable and compact multiphoton endoscope based on a 1-mm diameter, 8-cm length GRIN lens system probe was used. Fresh ex vivo samples were obtained from 14 radical prostatectomy patients and benign and malignant areas were imaged and correlated with subsequent H&E sections. Multiphoton GRIN endoscopy images of unfixed and unprocessed prostate tissue at a subcellular resolution are presented. We note several differences and identifying features of benign versus low-grade versus high-grade tumors and are able to identify periprostatic tissues such as adipocytes, periprostatic nerves, and blood vessels. Multiphoton GRIN endoscopy can be used to identify both benign and malignant lesions in ex vivo human prostate tissue and may be a valuable diagnostic tool for real-time visualization of suspicious areas of the prostate.

  11. Multiphoton fluorescence microscopy of the live kidney in health and disease.

    PubMed

    Small, David M; Sanchez, Washington Y; Roy, Sandrine; Hickey, Michael J; Gobe, Glenda C

    2014-02-01

    The structural and functional heterogeneity of the kidney ensures a diversity of response in health and disease. Multiphoton microscopy has improved our understanding of kidney physiology and pathophysiology by enabling the visualization of the living kidney in comparison with the static view of previous technologies. The use of multiphoton microscopy with rodent models in conjunction with endogenous fluorescence and exogenous infused dyes permits the measurement of renal processes, such as glomerular permeability, juxtaglomerular apparatus function, tubulointerstitial function, tubulovascular interactions, vascular flow rate, and the intrarenal renin-angiotensin-aldosterone system. Subcellular processes, including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes apoptosis and necrosis, can also be measured by multiphoton microscopy. This has allowed valuable insight into the pathophysiology of diabetic nephropathy, renal ischemia-reperfusion injury, hypertensive nephropathy, as well as inflammatory responses of the kidney. The current review presents an overview of multiphoton microscopy with a focus on techniques for imaging the kidney and gives examples of instances where multiphoton microscopy has been utilized to study renal pathophysiology in the living kidney. With continued advancements in the field of biological optics and increased adoption in experimental nephrology, multiphoton microscopy will undoubtedly continue to create new paradigms in kidney disease. PMID:24525825

  12. 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

  13. 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

  14. Reduction of Tubular Flow Rate as a Mechanism of Oliguria in the Early Phase of Endotoxemia Revealed by Intravital Imaging.

    PubMed

    Nakano, Daisuke; Doi, Kent; Kitamura, Hiroaki; Kuwabara, Takashige; Mori, Kiyoshi; Mukoyama, Masashi; Nishiyama, Akira

    2015-12-01

    Urine output is widely used as a criterion for the diagnosis of AKI. Although several potential mechanisms of septic AKI have been identified, regulation of urine flow after glomerular filtration has not been evaluated. This study evaluated changes in urine flow in mice with septic AKI. The intratubular urine flow rate was monitored in real time by intravital imaging using two-photon laser microscopy. The tubular flow rate, as measured by freely filtered dye (FITC-inulin or Lucifer yellow), time-dependently declined after LPS injection. At 2 hours, the tubular flow rate was slower in mice injected with LPS than in mice injected with saline, whereas BP and GFR were similar in the two groups. Importantly, fluorophore-conjugated LPS selectively accumulated in the proximal tubules that showed reduced tubular flow at 2 hours and luminal obstruction with cell swelling at 24 hours. Delipidation of LPS or deletion of Toll-like receptor 4 in mice abolished these effects, whereas neutralization of TNF-α had little effect on LPS-induced tubular flow retention. Rapid intravenous fluid resuscitation within 6 hours improved the tubular flow rate only when accompanied by the dilation of obstructed proximal tubules with accumulated LPS. These findings suggest that LPS reduces the intratubular urine flow rate during early phases of endotoxemia through a Toll-like receptor 4-dependent mechanism, and that the efficacy of fluid resuscitation may depend on the response of tubules with LPS accumulation. PMID:25855781

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Multiphoton microscopy of antigen presenting cells in experimental cancer therapies

    NASA Astrophysics Data System (ADS)

    Watkins, Simon C.; Papworth, Glenn D.; Spencer, Lori A.; Larregina, Adriana T.; Hackstein, Holger

    2002-06-01

    The absence of effective conventional therapy for most cancer patients justifies the application of novel, experimental approaches. One alternative to conventional cytotoxic agents is a more defined molecular approach for cancer immune treatment; promotion of the immune system specifically to target and eliminate tumor cells on the basis of expression of tumor-associated antigens (TAA). TAA could be presented to T-cells by professional antigen-presenting cells (APC) that generate a more efficient and effective anti-tumor immune response. In fact, it has been well documented that dendritic cells, the most immunologically potent APC, are capable of recognizing, processing and presenting TAA, in turn initiating a specific antitumor immune response. Results from several laboratories and clinical trials suggested significant but still limited efficacy of TAA-pulsed dendritic cells administered to tumor-bearing hosts. Following such delivery, it is fundamentally necessary to dynamically assess cell abundance within the microenvironment of the tumor in the presence of the appropriate therapeutic agent. Multiphoton microscopy was used to assess the trafficking of pulsed dendritic cells and other APC in skin, lymph nodes and brain of several animal tumor models, following different routes of administration.

  5. 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.

  6. Single and Multiphoton Infrared Laser Sectroscopy of Atomic Negative Ions

    NASA Astrophysics Data System (ADS)

    Bilodeau, René C.; Scheer, Michael; Brodie, Cicely A.; Haugen, Harold K.

    1998-05-01

    We have investigated several atomic negative ion species with the aid of a pulsed, tunable infrared laser source (M. Scheer, H.K. Haugen, and D.R. Beck, Phys. Rev. Lett. 79), 4104 (1997); M. Scheer et al, Phys. Rev. Lett. 80, 684 (1998).. In a comprehensive study of the carbon group negative ions (C^-, Si^-, Ge^-, Sn^-, Pb^-) a combination of single and multiphoton techniques was utilized to determine the bound terms and fine structure levels of the p^3 (ground state) configuration. The results comprise accurate electron affinities and the first experimental data on the fine structure of the ^2DJ terms in Si^-, Ge^-, and Sn^-. In addition, photodetachment threshold spectroscopy provided significantly impoved electron affinities for B, Cr, Mo, Ru, Rh, W, and Bi. The detachment cross section of B^-(^3P_J) appeared as a sequence of closely spaced thresholds which enabled the first experimental determination of the ionic fine structure. The detachment cross section of W^- indicates the presence of unexpected and previously unobserved resonances just below the W(5d^56s ^7S_3) threshold.

  7. 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.

  8. 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.

  9. In vivo multiphoton microscopy of deep brain tissue.

    PubMed

    Levene, Michael J; Dombeck, Daniel A; Kasischke, Karl A; Molloy, Raymond P; Webb, Watt W

    2004-04-01

    Although fluorescence microscopy has proven to be one of the most powerful tools in biology, its application to the intact animal has been limited to imaging several hundred micrometers below the surface. The rest of the animal has eluded investigation at the microscopic level without excising tissue or performing extensive surgery. However, the ability to image with subcellular resolution in the intact animal enables a contextual setting that may be critical for understanding proper function. Clinical applications such as disease diagnosis and optical biopsy may benefit from minimally invasive in vivo approaches. Gradient index (GRIN) lenses with needle-like dimensions can transfer high-quality images many centimeters from the object plane. Here, we show that multiphoton microscopy through GRIN lenses enables minimally invasive, subcellular resolution several millimeters in the anesthetized, intact animal, and we present in vivo images of cortical layer V and hippocampus in the anesthetized Thy1-YFP line H mouse. Microangiographies from deep capillaries and blood vessels containing fluorescein-dextran and quantum dot-labeled serum in wild-type mouse brain are also demonstrated. PMID:14668300

  10. 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.

  11. 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

  12. Spectroscopic analysis of skin intrinsic signals for multiphoton microscopy

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

  13. 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.

  14. 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.

  15. Tunneling dynamics in multiphoton ionization and attoclock calibration.

    PubMed

    Klaiber, Michael; Hatsagortsyan, Karen Z; Keitel, Christoph H

    2015-02-27

    The intermediate domain of strong-field ionization between the tunneling and multiphoton regimes is investigated using the strong-field approximation and the imaginary-time method. An intuitive model for the dynamics is developed which describes the ionization process within a nonadiabatic tunneling picture with a coordinate dependent electron energy during the under-the-barrier motion. The nonadiabatic effects in the elliptically polarized laser field induce a transversal momentum shift of the tunneled electron wave packet at the tunnel exit and a delayed appearance in the continuum as well as a shift of the tunneling exit towards the ionic core. The latter significantly modifies the Coulomb focusing during the electron excursion in the laser field after exiting the ionization tunnel. We show that nonadiabatic effects are especially large when the Coulomb field of the ionic core is taken into account during the under-the-barrier motion. The simple man model modified with these nonadiabatic corrections provides an intuitive background for exact theories and has direct implications for the calibration of the attoclock technique. PMID:25768761

  16. Multiphoton population transfer between rovibrational states of HF

    NASA Astrophysics Data System (ADS)

    Topcu, Turker; Robicheaux, Francis

    2011-05-01

    Efficient population transfer by adiabatically chirping through a multiphoton resonance in microwave driven and impulsively kicked Rydberg atoms has been reported both experimentally and theoretically. Previous work has demonstrated that the physical mechanism responsible for the transition can be viewed as a classical process in phase space as well as a quantum mechanical resonant transition. Here we report on our classical and quantum mechanical simulations in which we have exploited this mechanism to vibrationally excite an HF molecule up to | ν = 4 , J > from its ground state using an intense IR pulse. We compare one-dimensional quantum and classical models where there are no rotational degrees of freedom. We find that for low laser intensities, the transition is classically forbidden although it occurs quantum mechanically through tunneling. We show that for larger peak intensities, the transfer can be looked upon as a classical transition in phase space, similar to that observed in the atomic case. We extend our simulations to fully three-dimensional quantum calculations and investigate the effect of coupling between different rotational pathways. We briefly discuss the effect of thermal averaging over the final J-states. This work was supported by the Office of Basic Energy Sciences, U.S. Department of Energy.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Stabilizing 3D in vivo intravital microscopy images with an iteratively refined soft-tissue model for immunology experiments.

    PubMed

    Gómez-Conde, Iván; Caetano, Susana S; Tadokoro, Carlos E; Olivieri, David N

    2015-09-01

    We describe a set of new algorithms and a software tool, StabiTissue, for stabilizing in vivo intravital microscopy images that suffer from soft-tissue background movement. Because these images lack predetermined anchors and are dominated by noise, we use a pixel weighted image alignment together with a correction for nonlinear tissue deformations. We call this correction a poor man׳s diffeomorphic map since it ascertains the nonlinear regions of the image without resorting to a full integral equation method. To determine the quality of the image stabilization, we developed an ensemble sampling method that quantifies the coincidence between image pairs from randomly distributed image regions. We obtain global stabilization alignment through an iterative constrained simulated annealing optimization procedure. To show the accuracy of our algorithm with existing software, we measured the misalignment error rate in datasets taken from two different organs and compared the results to a similar and popular open-source solution. Present open-source stabilization software tools perform poorly because they do not treat the specific needs of the IV-2pM datasets with soft-tissue deformation, speckle noise, full 5D inter- and intra-stack motion error correction, and undefined anchors. In contrast, the results of our tests demonstrate that our method is more immune to noise and provides better performance for datasets' possessing nonlinear tissue deformations. As a practical application of our software, we show how our stabilization improves cell tracking, where the presence of background movement would degrade track information. We also provide a qualitative comparison of our software with other open-source libraries/applications. Our software is freely available at the open source repository http://sourceforge.net/projects/stabitissue/. PMID:26232672

  2. A Fluorescent Polymer Probe with High Selectivity toward Vascular Endothelial Cells for and beyond Noninvasive Two-Photon Intravital Imaging of Brain Vasculature.

    PubMed

    Mettra, B; Appaix, F; Olesiak-Banska, J; Le Bahers, T; Leung, A; Matczyszyn, K; Samoc, M; van der Sanden, B; Monnereau, C; Andraud, C

    2016-07-13

    A chromophore-engineering strategy that relies on the introduction of a ground-state distortion in a quadrupolar chromophore was used to obtain a quasi-quadrupolar chromophore with red emission and large two-photon absorption (2PA) cross-section in polar solvents. This molecule was functionalized with water-solubilizing polymer chains. It constitutes not only a remarkable contrast agent for intravital two-photon microscopy of the functional cerebral vasculature in a minimally invasive configuration but presents intriguing endothelial staining ability that makes it a valuable probe for premortem histological staining. PMID:27267494

  3. 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. PMID:24600332

  4. 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

  5. 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.

  6. Two-photon imaging of intact living plants during freezing with a flexible multiphoton tomograph

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; König, K.

    2015-02-01

    We describe the combination of a flexible multiphoton tomograph (MPTflex) with a heating and cooling stage. The stage allows temperature control in the range of (-196 °C) (77 K) to +600 °C (873 K) with selectable heating/freezing rates between 0.01 K min-1 and 150 K min-1. To illustrate the imaging capabilities of the combined system, fluorescence intensity and lifetime of intrinsic molecules from a plant leaf were imaged with submicron resolution during freezing in vivo without detaching the leaf from the plant. An increase of fluorescence intensity and decay times with decreasing temperature was observed. The measurements illustrate the usefulness of multiphoton imaging as a non-invasive online tool to investigate temperature-induced effects. The flexible multiphoton tomograph with its adjustable mechano-optical arm and scan head allows imaging at otherwise hardly accessible sample regions.

  7. 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.

  8. 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

  9. 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

  10. The effects of refractive index heterogeneity within kidney tissue on multiphoton fluorescence excitation microscopy.

    PubMed

    Young, P A; Clendenon, S G; Byars, J M; Dunn, K W

    2011-05-01

    Although multiphoton fluorescence excitation microscopy has improved the depth at which useful fluorescence images can be collected in biological tissues, the reach of multiphoton fluorescence excitation microscopy is nonetheless limited by tissue scattering and spherical aberration. Scattering can be reduced in fixed samples by mounting in a medium whose refractive index closely matches that of the fixed material. Using optical 'clearing', the effects of refractive index heterogeneity on signal attenuation with depth are investigated. Quantitative measurements show that by mounting kidney tissue in a high refractive index medium, less than 50% of signal attenuates in 100 μm of depth. PMID:21118239

  11. Mixed-Color Multiphoton Transitions as Additional Quantum Channels for Electron Photoemission

    NASA Astrophysics Data System (ADS)

    Huang, Wayne; Becker, Maria; Beck, Joshua; Batelaan, Herman

    2016-05-01

    We demonstrate mixed-color electron photoemission from tungsten nanotips. In the experiment, second-harmonic photons were introduced to modify the multiphoton emission process. A twofold increase in quantum efficiency results from the opening up of an additional three-photon quantum channel. The super-additive photoelectron signal can be controlled by input power, field polarization, and pulse overlap. The results of our study provide new prospects for quantum photonics, multiphoton microscopy, and spin-polarized electron sources. We acknowledge supports from NSF, Grant Number 1306565, 1430519. NSF Grant Number 1306565, 1430519.

  12. 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.

  13. Demonstration of structural alterations in experimental corneal infectious model using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Lo, Wen; Tan, Hsin-Yuan; Chang, Yuh-Ling; Sun, Yen; Lin, Sung-Jan; Jee, Shiou-Hwa; Dong, Chen-Yuan

    2007-02-01

    The aim of this study is to assess the application of multiphoton autofluorescence and second harmonic generation (SHG) microscopy for investigating the structural alterations and the pattern of microbial spreading during corneal infectious process in an in vitro organ culture model. The autofluorescence spectrum derived from pathogens allows us to monitoring the pattern of microbial spreading within corneal lamellae. In addition, the destruction and regeneration of second harmonic generating collagen during infectious process can also be monitored in a non-invasive fashion. Therefore we propose that multiphoton microscopy may potentially be applied as an effective monitoring tool for corneal infection studies.

  14. Fermi-coupled spherically adapted effective states in the collisionless multiphoton excitation of SF 6

    NASA Astrophysics Data System (ADS)

    Di Lauro, C.; Lattanzi, F.

    1982-10-01

    A calculation method for the collisionless multiphoton excitation of SF 6 by intense CO 2 laser light up to a chain of parallel nv3, ( n - 1) v3 + v2 + v6 … vibrational-rotational ladders linked by Fermi interaction is described. Spherically adapted effective states suitable to the purpose are defined, and matrix elements for multiphoton excitation in the rotatingwave approximation effective hamiltonian formalism are given in this basis. The method is aimed at the investigation of population transfer between the cited parallel vibrational ladders, and is suitable for computer-calculation programmation.

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

    NASA Astrophysics Data System (ADS)

    Chen, Chang-Yong

    2012-11-01

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

  16. Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging

    NASA Astrophysics Data System (ADS)

    Mancuso, James J.; Larson, Adam M.; Wensel, Theodore G.; Saggau, Peter

    2009-05-01

    The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

  17. Multiphoton ionization-fragmentation patterns of tertiary amines

    NASA Astrophysics Data System (ADS)

    Parker, D. H.; Bernstein, R. B.; Lichtin, D. A.

    1981-09-01

    Multiphoton ionization (MPI)-fragmentation patterns are reported for a series of normal and caged tertiary amines. Ionization is enhanced by two-photon resonance with the 3s and 3p Rydberg states of trimethylamine, triethylamine, and the caged amines quinuclidine and triethylenediamine. Over the wavelength region λ = 400-530 nm, N(CH3)3 ionizes to the parent ion (P) and fragments only by the loss of a H atom to yield the P-H daughter ion; N(C2H5)3 ionizes to its parent ion and fragments by the loss of a methyl to form the P-CH3 ion. The branching ratio of daughter to parent ions is found to be essentially independent of laser intensity but strongly dependent on laser wavelength. The caged amines quinuclidine [N(C2H4)3CH, or ABCO] and triethylenediamine [N(C2H4)3N, or DABCO] fragment extensively over this λ range in a manner dependent on both laser wavelength and intensity. The extent of daughter ion formation in N(CH3)3 and N(C2H5)3 can be understood by consideration of the wavelength regions in which the total available energy from the initial three- or four-photon ionization event exceeds the appearance potential of the given daughter ion. For the caged amines direct observation of this mechanism is masked by fragmentation due to sequential absorption of photons (during the ˜5 ns pulse duration) by the parent and/or daughter ions. The present results show that even for molecules with broad, unstructured UV absorption and MPI spectra such as N(CH3)3 and N(C2H5)3, considerable information on photon-molecule and photon-ion interactions can still be gained by the MPI mass spectrometry technique.

  18. Multiphoton-scattering theory and generalized master equations

    NASA Astrophysics Data System (ADS)

    Shi, Tao; Chang, Darrick E.; Cirac, J. Ignacio

    2015-11-01

    We develop a scattering theory to investigate the multiphoton transmission in a one-dimensional waveguide in the presence of quantum emitters. It is based on a path integral formalism, uses displacement transformations, and does not require the Markov approximation. We obtain the full time evolution of the global system, including the emitters and the photonic field. Our theory allows us to compute the transition amplitude between arbitrary initial and final states, as well as the S matrix of the asymptotic in and out states. For the case of few incident photons in the waveguide, we also rederive a generalized master equation in the Markov limit. We compare the predictions of the developed scattering theory and that with the Markov approximation. We illustrate our methods with five examples of few-photon scattering: (i) by a two-level emitter, (ii) in the Jaynes-Cummings model; (iii) by an array of two-level emitters; (iv) by a two-level emitter in the half-end waveguide; and (v) by an array of atoms coupled to Rydberg levels. In the first two, we show the application of the scattering theory in the photon scattering by a single emitter, and examine the correctness of our theory with the well-known results. In the third example, we analyze the condition of the Markov approximation for the photon scattering in the array of emitters. In the fourth one, we show how a quantum emitter can generate entanglement of outgoing photons. Finally, we highlight the interplay between the phenomenon of electromagnetic-induced transparency and the Rydberg interaction, and show how this results in a rich variety of possibilities in the quantum statistics of the scattering photons.

  19. 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.

  20. 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

  1. Intravital imaging of Ca2+ signals in lymphocytes of Ca2+ biosensor transgenic mice: indication of autoimmune diseases before the pathological onset

    PubMed Central

    Yoshikawa, Soichiro; Usami, Takako; Kikuta, Junichi; Ishii, Masaru; Sasano, Tetsuo; Sugiyama, Koji; Furukawa, Tetsushi; Nakasho, Eiji; Takayanagi, Hiroshi; Tedder, Thomas F.; Karasuyama, Hajime; Miyawaki, Atsushi; Adachi, Takahiro

    2016-01-01

    Calcium ion (Ca2+) signaling is a typical phenomenon mediated through immune receptors, such as the B-cell antigen receptor (BCR), and it is important for their biological activities. To analyze the signaling of immune receptors together with their in vivo dynamics, we generated stable transgenic mice with the Föster/fluorescence resonance energy transfer (FRET)-based Ca2+ indicator yellow cameleon 3.60 (YC3.60), based on the Cre/loxP system (YC3.60flox). We successfully obtained mice with specific YC3.60 expression in immune or nerve cells as well as mice with ubiquitous expression of this indicator. We established five-dimensional (5D) (x, y, z, time, and Ca2+) intravital imaging of lymphoid tissues, including the bone marrow. Furthermore, in autoimmune-prone models, the CD22−/− and C57BL/6- lymphoproliferation (lpr)/lpr mouse, Ca2+ fluxes were augmented, although they did not induce autoimmune disease. Intravital imaging of Ca2+ signals in lymphocytes may improve assessment of the risk of autoimmune diseases in model animals. PMID:26732477

  2. 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. PMID:23889391

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Photoelectron momentum spectra for multiphoton ionization of Hydrogen atoms by intense laser pulses

    NASA Astrophysics Data System (ADS)

    Ovchinnikov, Serge; Macek, Joseph

    2007-06-01

    Full three-dimensional electron momentum distribution for multiphoton ionization of Hydrogen atoms by intense laser pulses are calculated by solving the time-dependent solutions of Schr"odinger equation on a three-dimensional lattice in a scaled coordinate representation (CSLTDSE). This approach allows one to circumvent many difficulties related to the propagation of wave function to macroscopic distances.

  10. Supersonic jet/multiphoton ionization spectrometry of chemical species resulting from thermal decomposition and laser ablation of polymers

    NASA Astrophysics Data System (ADS)

    Hozumi, Masami; Murata, Yoshiaki; Cheng-Huang Lin, Imasaka, Totaro

    1995-04-01

    The chemical species resulting from thermal decomposition and laser ablation of polymers are measured by excitation/fluorescence and multiphoton ionization/mass spectrometries after supersonic jet expansion for rotational cooling to simply the optical spectrum. The signal of minor chemical species occurred is strongly enhanced by resonant excitation and multiphoton ionization, and even the isomer can be clearly differentiated. For example, p-cresol occurred by thermal decomposition of polycarbonate is detected selectively by mass-selected resonant multiphoton ionization spectrometry. Various chemical species occurred by laser ablation of even a polystyrene foam are also measured by this technique.

  11. 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

  12. 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. PMID:27375951

  13. 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

  14. 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

  15. 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

  16. 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.

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

    SciTech Connect

    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.

  18. In vitro imaging of embryonic stem cells using multiphoton luminescence of gold nanoparticles

    PubMed Central

    Nagesha, D; Laevsky, GS; Lampton, P; Banyal, R; Warner, C; DiMarzio, C; Sridhar, S

    2007-01-01

    Recent advances in nonlinear optical techniques and materials such as quantum wells, nanowires and noble-metal nanoparticles have led to advances in cellular imaging wherein various nanoparticles have been shown to improve both in vitro and in vivo visualization. In this paper, we demonstrate in vitro imaging using multi-photon photoluminescence of gold nanoparticles from two different cell types – Dictyostelium discoideum and mouse embryonic stem cells. By observing nanoparticles we show that embryonic stem cells maintained their ability to proliferate for several passages while grown in the presence of gold nanoparticles. The advantages of multi-photon luminescence using gold nanoparticles have important implications for use in stem cell proliferation experiments and in vitro experiments to monitor differentiation. PMID:18203448

  19. Statistical properties of multiphoton time-dependent three-boson coupled oscillators

    SciTech Connect

    Abdalla, M. Sebawe; Perina, Jan; Krepelka, Jaromir

    2006-06-15

    We investigate the quantum statistics of three time-dependent coupled oscillators in the presence of multiphoton processes. The system is connected with the two-atom multiphoton Tavis-Cummings model. The solution of the Heisenberg equations of the motion is obtained in a compact form. We assume that the modes are initially prepared in coherent states, and we discuss nonclassical phenomena (squeezing and sub-Poissonian behavior). Further, we examine the joint quasi-distribution functions as well as photon-number distribution and its factorial moments. The system has shown that the nonclassical effect is apparent in compound modes (1,3) and (2,3). Moreover, the superstructure phenomenon is observed when the photon transition is increased.

  20. Multiphoton ac Stark effect in a bichromatically driven two-level atom

    NASA Astrophysics Data System (ADS)

    Rudolph, T. G.; Freedhoff, H. S.; Ficek, Z.

    1998-08-01

    We study the interaction of a two-level atom with two lasers of different frequencies and amplitudes: a strong laser of Rabi frequency 2Ω1 on resonance with the atomic transition, and a weaker laser detuned by subharmonics (2Ω1/n) of the Rabi frequency of the first. We find that under these conditions the second laser couples the dressed states created by the first in an n-photon process, resulting in ``doubly dressed'' states and in a ``multiphoton ac Stark'' effect. We calculate the eigenstates of the doubly dressed atom and their energies, and illustrate the role of this multiphoton ac Stark effect in its fluorescence, absorption, and Autler-Townes spectra.

  1. 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.

  2. 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.

  3. 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

  4. Cell damage in near-infrared multimode optical traps as a result of multiphoton absorption

    NASA Astrophysics Data System (ADS)

    König, K.; Liang, H.; Berns, M. W.; Tromberg, B. J.

    1996-07-01

    We report on cell damage of single cells confined in continuous-wave (cw), near-infrared (NIR) multimode optical traps as a result of multiphoton absorption phenomena. Trapping beams at NIR wavelengths less than 800 nm are capable of damaging cells through a two-photon absorption process. Cell damage is more pronounced in multimode cw traps compared with single-frequency true cw NIR traps because of transient power enhancement by longitudinal mode beating. Partial mode locking in tunable cw Ti:sapphire lasers used as trapping beam sources can produce unstable subnanosecond pulses at certain wavelengths that amplify multiphoton absorption effects significantly. We recommend the use of single-frequency long-wavelength NIR trapping beams for optical micromanipulation of vital cells.

  5. 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

  6. Photoelectron circular dichroism of bicyclic ketones from multiphoton ionization with femtosecond laser pulses.

    PubMed

    Lux, Christian; Wollenhaupt, Matthias; Sarpe, Cristian; Baumert, Thomas

    2015-01-12

    Photoelectron circular dichroism (PECD) is a CD effect up to the ten-percent regime and shows contributions from higher-order Legendre polynomials when multiphoton ionization is compared to single-photon ionization. We give a full account of our experimental methodology for measuring the multiphoton PECD and derive quantitative measures that we apply on camphor, fenchone and norcamphor. Different modulations and amplitudes of the contributing Legendre polynomials are observed despite the similarity in chemical structure. In addition, we study PECD for elliptically polarized light employing tomographic reconstruction methods. Intensity studies reveal dissociative ionization as the origin of the observed PECD effect, whereas ionization of the intermediate resonance is dominating the signal. As a perspective, we suggest to make use of our tomographic data as an experimental basis for a complete photoionization experiment and give a prospect of PECD as an analytic tool. PMID:25492564

  7. High-Resolution Mosaic Imaging with Multifocal, Multiphoton Photon-Counting Microscopy

    SciTech Connect

    Chandler, E.; Hoover, E.; Field, J.; Sheetz, K.; Amir, W.; Carriles, R.; Ding, S. Y.; Squier, J.

    2009-04-10

    High-resolution mosaic imaging is performed for the first time to our knowledge with a multifocal, multiphoton, photon-counting imaging system. We present a novel design consisting of a home-built femtosecond Yb-doped KGdWO{sub 4} laser with an optical multiplexer, which is coupled with a commercial Olympus IX-71 microscope frame. Photon counting is performed using single-element detectors and an inexpensive electronic demultiplexer and counters.

  8. Two-color multiphoton ionization of diazabicyclooctane in a supersonic free jet

    NASA Astrophysics Data System (ADS)

    Fujii, Masaaki; Ebata, Takayuki; Mikami, Naohiko; Ito, Mitsuo

    1983-11-01

    Two-color multiphoton ionization (MPI) spectroscopy has been applied for diazabicyclooctane (DABCO) in a supersonic free jet. The MPI spectra due to transitions from the various vibronic levels of the S 1 (3s Rydberg) state which were excited by the first laser revealed the high Rydberg states above the adiabatic ionization potential. The ionization process and the vibrational potential of the ion are discussed.

  9. 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.

  10. Sample-matrix effects in infrared laser neutral desorption, multiphoton-ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Beavis, R. C.; Lindner, J.; Grotemeyer, J.; Schlag, E. W.

    1988-05-01

    Sample-matrix effects in laser evaporation of intact neutral molecules (LEIM) prior to multiphoton ionization mass spectrometry (MUPI MS) are studied. The results show that a strong influence exists in adding matrix materials to the sample upon the desorption step. Using sugars as matrix leads to a suppression of pyrolysis products in small peptides by the laser desorption. As a result mass spectrometric signals due to the pyrolysis products are avoided.

  11. Continuum generation in ultra high numerical aperture fiber with application to multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Sayler, Nicholas

    Nonlinear microscopy benefits from broadband laser sources, enabling efficient excitation of an array of fluorophores, for example. This work demonstrates broadening of a narrow band input pulse (6 nm to 40 nm) centered at 1040 nm with excellent shot-to-shot stability. In a preliminary demonstration, multiphoton imaging with pulses from the fiber is performed. In particular second harmonic imaging of corn starch is performed.

  12. 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.

  13. 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

  14. 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

  15. Multiphoton fragmentation and ionization of CF{sub 2}HCl molecules and clusters by UV radiation

    SciTech Connect

    Lokhman, V. N.; Ogurok, D. D.; Ryabov, E. A.

    2006-07-15

    The results of experimental studies of multiphoton ionization of CF{sub 2}HCl molecules and clusters by UV laser radiation in the wavelength range 217-236 nm are reported. In the case of molecules, the main reaction products are CF{sub 2}H{sup +} and CF{sup +} ions as well as atomic chlorine. It is found that the spectra of the products of ionization of free molecules and molecules condensed into clusters differ qualitatively: multiphoton ionization of clusters does not yield CF{sub 2}H{sup +} ions. The dependences of the ion yield on the intensity of laser radiation and its wavelength are measured. The effect of a constant electric field and the radiation spectral width on the multiphoton ionization process is demonstrated. The shape of the velocity distributions is determined for a number of products. A strong anisotropy is detected in the reaction of formation of CF{sub 2}H{sup +} ions. Possible mechanisms for these processes are discussed.

  16. Weak-field multiphoton femtosecond coherent control in the single-cycle regime.

    PubMed

    Chuntonov, Lev; Fleischer, Avner; Amitay, Zohar

    2011-03-28

    Weak-field coherent phase control of atomic non-resonant multiphoton excitation induced by shaped femtosecond pulses is studied theoretically in the single-cycle regime. The carrier-envelope phase (CEP) of the pulse, which in the multi-cycle regime does not play any control role, is shown here to be a new effective control parameter that its effect is highly sensitive to the spectral position of the ultrabroad spectrum. Rationally chosen position of the ultrabroadband spectrum coherently induces several groups of multiphoton transitions from the ground state to the excited state of the system: transitions involving only absorbed photons as well as Raman transitions involving both absorbed and emitted photons. The intra-group interference is controlled by the relative spectral phase of the different frequency components of the pulse, while the inter-group interference is controlled jointly by the CEP and the relative spectral phase. Specifically, non-resonant two- and three-photon excitation is studied in a simple model system within the perturbative frequency-domain framework. The developed intuition is then applied to weak-field multiphoton excitation of atomic cesium (Cs), where the simplified model is verified by non-perturbative numerical solution of the time-dependent Schrödinger equation. We expect this work to serve as a basis for a new line of femtosecond coherent control experiments. PMID:21451714

  17. 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.

  18. Multiphoton absorption in CsLiB6O10 with femtosecond infrared laser pulses

    NASA Astrophysics Data System (ADS)

    Reddy, J. N. Babu; Naik, V. B.; Elizabeth, Suja; Bhat, H. L.; Venkatram, N.; Rao, D. Narayana

    2008-09-01

    Nonlinear absorption and refraction characteristics of cesium lithium borate (CsLiB6O10) crystal have been studied using Z-scan technique. Ti:sapphire laser with 110 fs pulse width operating at 800 nm wavelength and pulse repetition rate of 1 kHz is used as the source of photons. Intensity of the laser pulse is varied from 0.541 to 1.283 T W/cm2 to estimate the intensity dependence of multiphoton absorption coefficients. Using the theory of multiphoton absorption proposed by Sutherland [Handbook of Nonlinear Optics, in 2nd ed., edited by D. G. McLean and S. Kirkpatrick, Dekker, New York (2003)], found that open aperture Z-scan data fit well for the five-photon absorption (5PA) process. 5PA coefficients are obtained by fitting the expressions into the open aperture experimental data for various peak intensities (I00). The nonlinear refractive index n2 estimated from closed aperture Z-scan experiment is 1.075×10-4 cm2/T W at an input peak intensity of 0.723 T W/cm2. The above experiment when repeated with a 532 nm, 6 ns pulsed laser led to an irreversible damage of the sample resulting in an asymmetric open aperture Z-scan profile. This indicates that it is not possible to observe multiphoton absorption in this regime of pulse width using 532 nm laser.

  19. 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.

  20. 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.

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

    PubMed

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

    2015-01-01

    Although multiphoton-pumped lasing from a solution of chromophores is important in the emerging fields of nonlinear optofluidics and bio-photonics, conventionally used organic dyes are often rendered unsuitable because of relatively small multiphoton absorption cross-sections and low photostability. Here, we demonstrate highly photostable, ultralow-threshold multiphoton-pumped biexcitonic lasing from a solution of colloidal CdSe/CdS nanoplatelets within a cuvette-based Fabry-Pérot optical resonator. We find that colloidal nanoplatelets surprisingly exhibit an optimal lateral size that minimizes lasing threshold. These nanoplatelets possess very large gain cross-sections of 7.3 × 10(-14) cm(2) and ultralow lasing thresholds of 1.2 and 4.3 mJ cm(-2) under two-photon (λexc=800 nm) and three-photon (λexc=1.3 μm) excitation, respectively. The highly polarized emission from the nanoplatelet laser shows no significant photodegradation over 10(7) laser shots. These findings constitute a more comprehensive understanding of the utility of colloidal semiconductor nanoparticles as the gain medium in high-performance frequency-upconversion liquid lasers. PMID:26419950

  2. 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.

  3. Infrared multiphoton dissociation of unsubstituted metal carbonyls at 5 μm

    NASA Astrophysics Data System (ADS)

    Au, Mei-Kuen; Hackett, P. A.; Humphries, M.; John, P.

    1984-01-01

    A frequency-doubled carbon dioxide laser of modest output energy (1 mJ) has been used to study, for the first time, the infrared multiphoton absorption by, and dissociation of, the unsubstituted carbonyls of vanadium, chromium, iron, nickel, molybdenum, and tungsten. The multiphoton absorption cross-sections measured for Ni(CO)4, Fe(CO)5, Cr(CO)6, Mo(CO)6, and V(CO)6 are high (σ˜2×10-17) and ensure facile multiphoton dissociation. In focussed beams a pressure independent reaction yield proportional to the 1.5 power of the beam energy is observed for Fe(CO)5, Cr(CO)6, and Mo(CO)6 implying threshold fluences of only 32, 25, and 26 mJ cm-2, respectively. The stoichiometry of the reaction, observed by a pressure measurement technique, is consistent with production of metal atoms and carbon monoxide as final products for Ni(CO)4, Fe(CO)5, Cr(CO)6, and Mo(CO)6. This extensive decarbonylation along the ground state surface is consistent with recent studies of the photochemistry of these molecules from excited electronic states.

  4. 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.

  5. Multiphoton Imaging of Upconverting Lanthanide Nanoparticles in Three Dimensional Models of Cancer

    PubMed Central

    Gainer, Christian F.; Romanowski, Marek

    2013-01-01

    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. PMID:24353385

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

    PubMed Central

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

    2015-01-01

    Although multiphoton-pumped lasing from a solution of chromophores is important in the emerging fields of nonlinear optofluidics and bio-photonics, conventionally used organic dyes are often rendered unsuitable because of relatively small multiphoton absorption cross-sections and low photostability. Here, we demonstrate highly photostable, ultralow-threshold multiphoton-pumped biexcitonic lasing from a solution of colloidal CdSe/CdS nanoplatelets within a cuvette-based Fabry–Pérot optical resonator. We find that colloidal nanoplatelets surprisingly exhibit an optimal lateral size that minimizes lasing threshold. These nanoplatelets possess very large gain cross-sections of 7.3 × 10−14 cm2 and ultralow lasing thresholds of 1.2 and 4.3 mJ cm−2 under two-photon (λexc=800 nm) and three-photon (λexc=1.3 μm) excitation, respectively. The highly polarized emission from the nanoplatelet laser shows no significant photodegradation over 107 laser shots. These findings constitute a more comprehensive understanding of the utility of colloidal semiconductor nanoparticles as the gain medium in high-performance frequency-upconversion liquid lasers. PMID:26419950

  7. 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.

  8. A single-photon fluorescence and multi-photon spectroscopic study of atherosclerotic lesions

    NASA Astrophysics Data System (ADS)

    Smith, Michael S. D.; Ko, Alex C. T.; Ridsdale, Andrew; Schattka, Bernie; Pegoraro, Adrian; Hewko, Mark D.; Shiomi, Masashi; Stolow, Albert; Sowa, Michael G.

    2009-06-01

    In this study we compare the single-photon autofluorescence and multi-photon emission spectra obtained from the luminal surface of healthy segments of artery with segments where there are early atherosclerotic lesions. Arterial tissue was harvested from atherosclerosis-prone WHHL-MI rabbits (Watanabe heritable hyperlipidemic rabbit-myocardial infarction), an animal model which mimics spontaneous myocardial infarction in humans. Single photon fluorescence emission spectra of samples were acquired using a simple spectrofluorometer set-up with 400 nm excitation. Samples were also investigated using a home built multi-photon microscope based on a Ti:sapphire femto-second oscillator. The excitation wavelength was set at 800 nm with a ~100 femto-second pulse width. Epi-multi-photon spectroscopic signals were collected through a fibre-optics coupled spectrometer. While the single-photon fluorescence spectra of atherosclerotic lesions show minimal spectroscopic difference from those of healthy arterial tissue, the multi-photon spectra collected from atherosclerotic lesions show marked changes in the relative intensity of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) signals when compared with those from healthy arterial tissue. The observed sharp increase of the relative SHG signal intensity in a plaque is in agreement with the known pathology of early lesions which have increased collagen content.

  9. 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.

  10. 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.

  11. Balancing Acts

    MedlinePlus

    ... Current Issue Past Issues Special Section: Focus on Communication Balancing Acts Past Issues / Fall 2008 Table of ... from the National Institute on Deafness and Other Communication Disorders (NIDCD). It involves simulated trips down the ...

  12. 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)

  13. 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 ...

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

    PubMed Central

    Jenne, Craig N.; Wong, Connie H. Y.; Petri, Björn; Kubes, Paul

    2011-01-01

    Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation. PMID:21949865

  2. 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

  3. Real-time visualization of RGD-quantum dot binding in tumor neovasculature using intravital microscopy in multiple living mouse models

    NASA Astrophysics Data System (ADS)

    Smith, Bryan Ronain; Cheng, Zhen; De, Abhijit; Koh, Ai Leen; Sinclair, Robert; Gambhir, Sanjiv Sam

    2009-02-01

    Quantum dots (Qdots) have become ubiquitous in biomedical research due to their excellent brightness, photostability, monodispersity, and fluorescent yield. Furthermore, they have become increasingly useful as imaging agents which are valuable for answering molecular questions in living subjects. However, little is currently known about how nanoparticles such as Qdots interact at the microscale within the vasculature and tumor microenvironments in living subjects. In order to further our understanding of the dynamic processes involved in Qdot targeting in the intact tumor, we developed an in vivo binding assay to visualize and fully elucidate this approach using a variety of animal models and tumor types. We employed argine-glycine-aspartic acid (RGD) peptides to specifically target the αvβ3 integrins which are expressed on the surface of endothelial cells comprising newly formed or forming blood vessels; RGD peptides were conjugated to the Qdot surface. Exploiting intravital microscopy with subcellular-level resolution, we directly observed and recorded the binding of nanoparticle conjugates in two different murine models, using three different tumor cell lines. Using this generalizable approach, we learned that RGD-qdots unexpectedly bind to tumor blood vessels in all models tested only as aggregates rather than individually. Understanding these issues on the microscale using such techniques will provide a platform for the rational design of molecularly-targeted nanoparticles including Qdots. This is critical for nanoparticles to become a valuable research tool with the potential to become clinically valuable imaging and therapeutic agents, particularly for ensuring regulatory approval of such nanoparticles.

  4. An intravital microscopy study of radiation-induced changes in permeability and leukocyte-endothelial cell interactions in the microvessels of the rat pia mater and cremaster muscle.

    PubMed

    Gaber, M Waleed; Yuan, Hong; Killmar, John T; Naimark, Michael D; Kiani, Mohammad F; Merchant, Thomas E

    2004-04-01

    Using intravital microscopy and a closed window method, we measured irradiation-induced changes in the vascular permeability and cell interactions in microcirculation networks of the rat pia mater; the same effects were monitored in the cremaster muscle as a control. The closed cranial window has many advantages, including long-term direct visualization of microcirculation. The method allows for repeated testing of the same vessel or network, thereby reducing variability. The method also allows for measurement of permeability changes and the accompanying leukocyte-endothelial cell interactions in the same network or vessel, which permits correlative studies of these phenomena. However, this method is not without challenges. The optical conditions are difficult, because the brain is three-dimensional and its parenchyma is more complex than the thinner, flatter peripheral tissues. To overcome this limitation, we performed a dynamic background subtraction. The background is dynamically related to vessel intensity, and changes in intensity were determined by eliminating the effects of neighboring and underlying vessels. We applied this method to studying the effects of ionizing radiation on the blood-brain barrier (BBB) permeability and cell interactions and the modulation of these effects by anti-ICAM-1 antibodies. Our results demonstrate that this method is sensitive to changes in these properties of the BBB. PMID:15063835

  5. 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

  6. 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

  7. Calculations of hydrogen atom multiphoton energy level shifts, transition amplitudes and ionization probabilities

    NASA Astrophysics Data System (ADS)

    Whitney, K. G.; Chang, C. S.

    2008-07-01

    Analyses of the resonant multiphoton ionization of atoms require knowledge of ac Stark energy shifts and of multiphoton, bound-to-bound state, transition amplitudes. In this paper, we consider the three-photon photoionization of hydrogen atoms at frequencies that are at and surrounding the two-photon 1s to 2s resonance. AC energy shift sums of both the 1s and 2s states are calculated as a function of the laser frequency along with two-photon 1s → 2s resonant transition amplitude sums. These quantities are calculated using an extended version of a method, which has often been employed in a variety of ways, of calculating these sums by expressing them in terms of solutions to a variety of differential equations that are derived from the different sums being evaluated. We demonstrate how exact solutions are obtained to these differential equations, which lead to exact evaluations of the corresponding sums. A variety of different cases are analysed, some involving analytic continuation, some involving real number analysis and some involving complex number analysis. A dc Stark sum calculation of the 2s state is carried out to illustrate the case where analytic continuation, pole isolation and pole subtraction are required and where the calculation can be carried out analytically; the 2s state, ac Stark shift sum calculations involve a case where no analytic continuation is required, but where the solution to the differential equation produces complex numbers owing to the finite photoionization lifetime of the 2s state. Results from these calculations are then used to calculate three-photon ionization probabilities of relevance to an analysis of the multiphoton ionization data published by Kyrala and Nichols (1991 Phys. Rev. A 44, R1450).

  8. 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

  9. Characterizing germania concentration and structure in fiber soot using multiphoton microscopy and spectroscopy technology

    NASA Astrophysics Data System (ADS)

    Chen, Minghan; Li, Ming-Jun; Liu, Anping

    2015-02-01

    Germania doping is commonly used in the core of optical fiber due to its advantages compared to other materials such as superior transparency in near-infrared telecommunication wavelength region. During fiber preform manufacturing using the outside vapor deposition (OVD) process, Ge is doped into a silica soot preform by chemical vapor deposition. Since the Ge doping concentration profile is directly correlated with the fiber refractive index profile, its characterization is critical for the fiber industry. Electron probe micro-analyzer (EPMA) is a conventional analysis method for characterizing the Ge concentration profile. However, it requires extensive sample preparation and lengthy measurement. In this paper, a multiphoton microscopy technique is utilized to measure the Ge doping profile based on the multiphoton fluorescence intensity of the soot layers. Two samples, one with ramped and another with stepped Ge doping profiles were prepared for measurements. Measured results show that the technique is capable of distinguishing ramped and stepped Ge doping profiles with good accuracy. In the ramped soot sample, a sharp increment of doping level was observed in about 2 mm range from soot edge followed by a relative slow gradient doping accretion. As for the stepped doping sample, step sizes ranging from around 1 mm (at soot edge) to 3 mm (at soot center) were observed. All the measured profiles are in close agreement with that of the EPMA measurements. In addition, both multiphoton fluorescence (around 420 nm) and sharp second harmonic generations (at 532 nm) were observed, which indicates the co-existence of crystal and amorphous GeO2.

  10. In situ multiphoton microscopy for monitoring femtosecond laser eye surgery in the human cornea and sclera

    NASA Astrophysics Data System (ADS)

    Plamann, Karsten; Albert, Olivier; Giulieri, Damien; Donate, David; May, Frank; Giraud, Jean-Marie; Legeais, Jean-Marc

    2005-08-01

    We present a multiphoton imaging system mounted on a microsurgery experimental set-up using a Nd:glass femtosecond laser. The system permits to induce laser incisions in human cornea and sclera and to perform nonlinear imaging during the intervention. The laser is a chirped pulse amplification (CPA) system with a regenerative amplifier delivering pulses at a wavelength of 1.06 μm, pulse durations of 400 fs and a maximum energy of 60 μJ at repetition rates up to 10 kHz. The delivery system provides spot sizes down to the micron range. The samples are human corneas retracted from the transplant circuit mounted on a moveable anterior chamber system. Photons generated by non-linear processes in the cornea travel backwards through the beam delivery optics and are captured by a photomultiplier tube behind a dichroic mirror. The signal is filtered by a lock-in amplifier tuned to the laser repetition rate. Scanning the sample permits the acquisition of three-dimensional microscopic images. Above the incision threshold the set-up permits to induce laser cuts in human cornea following complex geometries. Below the threshold the laser pulses create secondary photons by the stimulation of non-linear optical processes in the samples which could be identified as being predominantly second harmonic generation (SHG). The in situ images obtained from the multi-photon module permit to control and optimise the surgical intervention. The combination of multiphoton imaging and corneal surgery necessitates only minimal modifications of the optical system of a femtosecond surgical laser system. A combined system significantly improves parameter control and permits the monitoring of the surgical procedure.

  11. 5-HT spatial distribution imaging with multiphoton excitation of 5-HT correlative visible fluorescence in live cells

    NASA Astrophysics Data System (ADS)

    Zhang, Zhihong; Zeng, Shaoqun; Liu, Yafeng; Zhou, Wei; Chen, Tongsheng; Luo, Qingming

    2002-04-01

    The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) is imaged with multiphoton excitation laser scanning microscope (MPELSM). 5-HT correlative visible fluorescence (Fco-vis) excited with 740-nm multiphoton excitation is observed in live cells for the first time, and the generating mechanism of 5-HT Fco-vis is studied. The spatial distribution of 5-HT in live cells is imaged at high spatial resolution in our experiment, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.

  12. 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.

  13. Imaging the bone marrow stem cells morphogenesis in PGA scaffold by multiphoton autofluorescence and second harmonic (SHG) imaging

    NASA Astrophysics Data System (ADS)

    Lee, Hsuan-Shu; Teng, Shu-Wen; Chen, Hsiao-Ching; Lo, Wen; Sun, Yen; Lin, Tze-Yu; Chiou, Ling-Ling; Jiang, Ching-Chuan; Dong, Chen-Yuan

    2006-02-01

    The ability to image tissue engineering products without damaging histological procedures is important for the understanding of the dynamics of tissue reorganization and formation. In this work, we test the ability of multiphoton autofluorescence and second harmonic generation microscopy to image engineered tissues following chrondrogenic induction. The system we used is human bone marrow stem cells seeded in the scaffold polyglycolic acid (PGA). Our results show that autofluorescence can be used to image cells while second harmonic generation signal can be used to visualize the synthesis of extracellular matrix. This approach demonstrates the ability of multiphoton imaging in the study of tissue engineering products.

  14. Spin-statistic selection rules for multiphoton transitions: Application to helium atoms

    NASA Astrophysics Data System (ADS)

    Zalialiutdinov, T.; Solovyev, D.; Labzowsky, L.; Plunien, G.

    2016-01-01

    A theoretical investigation of the three-photon transition rates 2 1P1→2 1S0,1 1S0 and 2 3P2→2 1S0,1 1S0 for the helium atom is presented. Photon energy distributions and precise values of the nonrelativistic transition rates are obtained with employment of correlated wave functions of the Hylleraas type. The possible experiments for the tests of the Bose-Einstein statistics for multiphoton systems are discussed.

  15. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

  17. Characterizing collagen-based materials modified by glycation: a multiphoton optical image guided spectroscopy method

    NASA Astrophysics Data System (ADS)

    Hwang, Yu-Jer; Granelli, Joseph; Flores, Christina; Lyubovitsky, Julia

    2011-02-01

    In spite of the adverse ageing effects of glycation in vivo, in vitro this process is widely employed to increase stiffness and strength of tissues' and artificial scaffolds'. In-situ optical characterization methods that report on the structures within these materials could clarify the effects of glycation. We employed one-photon fluorescence and multiphoton microscopy method that combined two-photon fluorescence and second harmonic generation signals to characterize collagen hydrogels modified with glyceraldehyde, ribose and glucose. We observed an increase in the in situ fluorescence as well as structural alterations within the materials during the course of glycation.

  18. 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.

  19. 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

  20. 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.

  1. Ultrasensitive detection of atmospheric constituents by supersonic molecular beam, multiphoton ionization, mass spectroscopy.

    PubMed

    Syage, J A; Pollard, J E; Cohen, R B

    1987-09-01

    An ultrasensitive detection method for atmospheric monitoring has been developed based on the technique of supersonic molecular beam, resonance enhanced multiphoton ionization, and time-of-flight mass spectroscopy (MB/REMPI/TOFMS). Several organophosphonate and organosulfide compounds, representing simulants to a class of toxic compounds, were studied. Detection levels as low as 300 ppt (dimethyl sulfide) were obtained. Single-vibronic-level REMPI of the cooled molecules in conjunction with TOFMS provided selectivity of ~10(4) against chemically similar compounds in humid air expansions. The fragment ions formed by REMPI excitation are shown for diisopropyl methylphosphonate to depend strongly on the resonant intermediate state of the neutral molecule. PMID:20490096

  2. Identification of dirty necrosis in colorectal carcinoma based on multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Li, Lianhuang; Jiang, Weizhong; Yang, Yinghong; Chen, Zhifen; Feng, Changyin; Li, Hongsheng; Guan, Guoxian; Chen, Jianxin

    2014-06-01

    Dirty necrosis within glandular lumina is often considered as a characteristic of colorectal carcinomas (CRCs) that is a diagnostically useful feature of CRCs with DNA microsatellite instability (MSI). Multiphoton microscopy (MPM), which is based on the second-harmonic generation and two-photon excited fluorescence signals, was used to identify dirty necrosis. Our results demonstrated that MPM has the ability to exhibit the microstructure of dirty necrosis and the signal intensity as well as an emission spectrum that can help to differentiate dirty necrosis from cancer cells. These findings indicate that MPM may be helpful in distinguishing MSI colorectal carcinoma via the identification of dirty necrosis.

  3. In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems.

    PubMed

    Huland, David M; Brown, Christopher M; Howard, Scott S; Ouzounov, Dimitre G; Pavlova, Ina; Wang, Ke; Rivera, David R; Webb, Watt W; Xu, Chris

    2012-05-01

    We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically. PMID:22567597

  4. 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.

  5. 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

  6. Combined multiphoton imaging-pixel analysis for semiquantitation of skin penetration of gold nanoparticles.

    PubMed

    Labouta, Hagar I; Kraus, Tobias; El-Khordagui, Labiba K; Schneider, Marc

    2011-07-15

    Interaction of nanoparticles with the skin barrier is a recent area of research that draws a lot of attention from the researchers. However, monitoring nanoparticles in or through the skin is mainly based on qualitative microscopical techniques. Yet, a quantitative approach is required for a better basic understanding. In response, a combined "multiphoton-pixel analysis" method was developed in this study for semiquantitation of gold nanoparticles penetration into different skin layers. The developed approach provides a useful tool for future studies focusing on skin penetration of nanoparticles for the aim of health risk assessment or for the design of topical and transdermal drug delivery systems. PMID:21515347

  7. Multiphoton ionization/dissociation of cyclopentanone at the lower Rydberg states

    NASA Astrophysics Data System (ADS)

    Philis, John G.; Kosmidis, Constantine; Tzallas, Paraskevas

    1998-12-01

    The 2-photon excitation of the 3p and 3d Rydberg states in jet-cooled cyclopentanone has been investigated by resonance enhanced multiphoton ionization (REMPI) in a time of flight mass spectrometer. The three 3px,y,z components are clearly resolved while the case for the 3di excitations is obscure due to the S1 one-photon resonance. The ns laser induced mass spectra are characteristic of hard ionization while the fs laser induced mass spectrum is very similar to the Electron Impact one.

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  9. New electronic states of NH and ND observed by resonance enhanced multiphoton ionization spectroscopy

    NASA Technical Reports Server (NTRS)

    Johnson, Russell D., III; Hudgens, Jeffrey W.

    1990-01-01

    Resonance Enhanced MultiPhoton Ionization (REMPI) spectra of NH and ND, which reveal four new electronic states are presented. Transitions from NH a 1 delta to 3s and 3p Rydberg states in both NH and ND have been observed and rotationally analyzed. The transitions were observed in the wavelength range of 258 to 288 nm. The state assignments are: e 1 pi (3s sigma) at 82857/cm, f 1 pi (3p sigma) at 86378/cm, g 1 delta (3p pi) at 88141/cm and h 1 sigma (3p pi) at 89151/cm.

  10. Multiphoton dissociation of electrosprayed megadalton-sized DNA ions in a charge-detection mass spectrometer.

    PubMed

    Doussineau, Tristan; Paletto, Pierre; Dugourd, Philippe; Antoine, Rodolphe

    2015-01-01

    Charge detection mass spectrometry in combination with a linear electrostatic ion trap coupled to a continuous wavelength infrared CO2 laser has been used to study the multiphoton dissociation of DNA macromolecular ions. Samples, with masses ranging from 2.23 to 31.5 MDa, include single strand circular M13mp18, double strand circular M13mp18, and double strand linear LambdaPhage DNA fragments. Their activation energies for unimolecular dissociation were determined. Activation energy values slightly increase as a function of the molecular weight. The most important result is the difference between the fragmentations observed for hybridized double-strands and dimers of single strands. PMID:25348472

  11. Second and third harmonic generation in few-layer gallium telluride characterized by multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Susoma, Jannatul; Karvonen, Lasse; Säynätjoki, Antti; Mehravar, Soroush; Norwood, Robert A.; Peyghambarian, Nasser; Kieu, Khanh; Lipsanen, Harri; Riikonen, Juha

    2016-02-01

    We report on the nonlinear optical properties of few-layer GaTe studied by multiphoton microscopy. Second and third harmonic generation from few-layer GaTe flakes were observed in this study with the laser pump wavelength of 1560 nm. These processes were found to be sensitive to the number of GaTe layers. The second- and third-order nonlinear susceptibilities of 2.7 × 10-9 esu (1.15 pm/V) and 1.4 × 10-8 esu (2 × 10-16 m2/V2) were estimated, respectively.

  12. Multi-photon microscopy based on resonant four-wave mixing of colloidal quantum dots

    NASA Astrophysics Data System (ADS)

    Masia, F.; Langbein, W.; Borri, P.

    2009-02-01

    We demonstrate a novel multi-photon imaging modality based on the detection of four-wave mixing (FWM) from colloidal nanoparticles. Four-wave mixing is a third-order signal which can be excited and detected in resonance with the ground-state excitonic transition of CdSe/ZnS quantum dots. The coherent FWM signal is detected interferometrically to reject incoherent backgrounds for improved image contrast compared to fluorescence methods. We measure transversal and axial resolutions of 140nm and 590nm respectively, significantly beating the one-photon diffraction limit. We also demonstrate optical imaging of quantum-dot-labeled Golgi structures of HepG2 cells.

  13. 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.

  14. Complete QED theory of multiphoton Trident pair production in strong laser fields.

    PubMed

    Hu, Huayu; Müller, Carsten; Keitel, Christoph H

    2010-08-20

    Electron-positron pair creation by multiphoton absorption in the collision of a relativistic electron with a strong laser beam is calculated within laser-dressed quantum electrodynamics. Total production rates, positron spectra, and relative contributions of different reaction channels are obtained in various interaction regimes. We study the process in a manifestly nonperturbative domain which is shown accessible to future experiments utilizing the electron beam lines at novel x-ray laser facilities or all-optical setups based on laser acceleration. Our theory moreover allows us to add further insights into the experimental data from SLAC [D. Burke, Phys. Rev. Lett. 79, 1626 (1997).]. PMID:20868080

  15. Compressor optimization with compressor-based multiphoton intrapulse interference phase scan (MIIPS).

    PubMed

    Hou, B; Easter, J H; Nees, J A; He, Z; Thomas, A G R; Krushelnick, K

    2012-04-15

    The multiphoton intrapulse interference phase scan (MIIPS) technique is modified to optimize the compressor settings of a chirped pulse amplification (CPA) laser system. Here, we use the compressor itself to perform the phase scan inherent in MIIPS measurement . A frequency-resolved optical gating measurement shows that the pulse duration of the compressor optimized using the modified MIIPS technique is 33.8 fs with a 2.24 rad temporal phase variation above 2% intensity. The measured time-bandwidth product is 0.60, which is close to that of transform-limited Gaussian pulse (0.44). PMID:22513694

  16. Coherent control of radiation patterns of nonlinear multiphoton processes in nanoparticles

    PubMed Central

    Papoff, Francesco; McArthur, Duncan; Hourahine, Ben

    2015-01-01

    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. PMID:26155833

  17. 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.

  18. 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.

  19. Ab initio molecular dynamics of protonated dialanine and comparison to infrared multiphoton dissociation experiments.

    PubMed

    Marinica, D C; Grégoire, G; Desfrançois, C; Schermann, J P; Borgis, D; Gaigeot, M P

    2006-07-20

    Finite temperature Car-Parrinello molecular dynamics simulations are performed for the protonated dialanine peptide in vacuo, in relation to infrared multiphoton dissociation experiments. The simulations emphasize the flexibility of the different torsional angles at room temperature and the dynamical exchange between different conformers which were previously identified as stable at 0 K. A proton transfer occurring spontaneously at the N-terminal side is also observed and characterized. The theoretical infrared absorption spectrum is computed from the dipole time correlation function, and, in contrast to traditional static electronic structure calculations, it accounts directly for anharmonic and finite temperature effects. The comparison to the experimental infrared multiphoton dissociation spectrum turns out very good in terms of both band positions and band shapes. It does help the identification of a predominant conformer and the attribution of the different bands. The synergy shown between the experimental and theoretical approaches opens the door to the study of the vibrational properties of complex and floppy biomolecules in the gas phase at finite temperature. PMID:16836443

  20. 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.

  1. 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

  2. THREE-DIMENSIONAL RANDOM ACCESS MULTIPHOTON MICROSCOPY FOR FAST FUNCTIONAL IMAGING OF NEURONAL ACTIVITY

    PubMed Central

    Reddy, Gaddum Duemani; Kelleher, Keith; Fink, Rudy; Saggau, Peter

    2009-01-01

    The dynamic ability of neuronal dendrites to shape and integrate synaptic responses is the hallmark of information processing in the brain. Effectively studying this phenomenon requires concurrent measurements at multiple sites on live neurons. Significant progress has been made by optical imaging systems which combine confocal and multiphoton microscopy with inertia-free laser scanning. However, all systems developed to date restrict fast imaging to two dimensions. This severely limits the extent to which neurons can be studied, since they represent complex three-dimensional (3D) structures. Here we present a novel imaging system that utilizes a unique arrangement of acousto-optic deflectors to steer a focused ultra-fast laser beam to arbitrary locations in 3D space without moving the objective lens. As we demonstrate, this highly versatile random-access multiphoton microscope supports functional imaging of complex 3D cellular structures such as neuronal dendrites or neural populations at acquisition rates on the order of tens of kilohertz. PMID:18432198

  3. 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.

  4. 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

  5. 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.

  6. Evidence for excitation of fluorescence in RPE melanin by multiphoton absorption

    NASA Astrophysics Data System (ADS)

    Glickman, Randolph D.; Rockwell, Benjamin A.; Noojin, Gary D.; Stolarski, David J.; Denton, Michael L.

    2002-06-01

    Previously, we reported that ultrashort, near infrared (NIR) laser pulses caused more DNA breakage in cultured retinal pigment epithelial (RPE) cells than did CW, NIR laser radiation delivering a similar radiant exposure. We hypothesized that this difference was due to multiphoton absorption in an intracellular chromophore such as the RPE melanin. We investigated two-photon excitation of fluorescence in a suspension of isolated bovine RPE melanosomes exposed to a 1-KHz train of approximately 50- fsec laser pulses at 810 nm from a Ti:Sapphire laser, and compared this to the fluorescence excited by CW exposures at 406 nm from a Krypton ion laser. Fluorescence was measured with a PC-based spectrometer. The CW sources excited fluorescence with a peak at 525 nm. The fluorescence intensity depended on the irradiance of the sample, as well as the melanosome concentration. Peak fluorescence was obtained with a suspension of ~2 x 107 melanin granules/ml. The 810-nm, ultrashort pulses also excited fluorescence, but with a broader, lower-amplitude peak. The weaker fluorescence signal excited by the 810-nm ultrashort pulse laser for a given melanosome concentration, compared to 406-nm CW excitation, is possibly due to the smaller two- photon absorption cross-section. These results indicate the involvement of multiphoton absorption in DNA damage.

  7. 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.

  8. 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

  9. Comparison Study of Atomic and Molecular Single Ionization in the Multiphoton Ionization Regime

    SciTech Connect

    Wu Jian; Zeng Heping; Guo Chunlei

    2006-06-23

    In this Letter, we report, for the first time in the multiphoton ionization regime, a comparison study of single-electron ionization of diatomic molecules versus rare gas atoms with virtually the same ionization potentials. In comparing N{sub 2}{sup +} to Ar{sup +}, a higher ion signal is seen in N{sub 2}{sup +} compared to Ar{sup +} for linear polarization but the difference vanishes in circularly polarized light. In comparing O{sub 2}{sup +} to Xe{sup +}, we observe a suppression in O{sub 2}{sup +} compared to Xe{sup +} for both linear and circular polarization but this suppression exhibits an intensity dependence; i.e., there is little suppression for O{sub 2}{sup +} at the lowest intensity range, but the suppression becomes increasingly stronger as the laser intensity increases. The multielectron screening model is used to discuss possible mechanisms of this intensity dependent suppression in O{sub 2}{sup +} in the multiphoton ionization regime.

  10. Use of multiphoton microscopy to diagnose liver cancer and lung metastasis in an orthotopic rat model.

    PubMed

    Yan, Jun; Zhuo, Shuangmu; Chen, Gang; Tan, Changjun; Zhu, Weifeng; Lu, Jianping; Fan, Jia; Chen, Jianxin; Zhou, Jian

    2012-01-01

    Liver or lung biopsy for suspicious lesions has several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure. The ability to directly observe cellular and subcellular details and then perform "optical biopsy" is a major goal in the development of new interventional techniques. Multiphoton microscopy (MPM) enables real-time noninvasive visualization of tissue architecture and cell morphology in live tissue. We performed a study to evaluate whether MPMcan make real-time optical diagnosis for liver cancer and lung metastasis using an orthotopic rat model with Morris hepatoma. We found that real-time high-resolution MPMimaging could clearly show tissue architecture and cell morphology. In the normal liver tissue, MPMimaging clearly revealed the blood-filled sinusoids and cords of hepatocytes. In the cancerous tissue, MPMimaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. MPMimages were comparable to golden standard hematoxylin-eosin staining images. Moreover, MPMimaging had deep penetration with the capability of optical sectioning. In short, MPMcan make real-time optical diagnosis for liver cancer and lung metastasis. This study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive "optical biopsy" for liver cancer and lung metastasis in the near future. PMID:22331704

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

    NASA Astrophysics Data System (ADS)

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

    2006-03-01

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

  12. 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.

  13. In vivo multiphoton NADH fluorescence reveals depth-dependent keratinocyte metabolism in human skin.

    PubMed

    Balu, Mihaela; Mazhar, Amaan; Hayakawa, Carole K; Mittal, Richa; Krasieva, Tatiana B; König, Karsten; Venugopalan, Vasan; Tromberg, Bruce J

    2013-01-01

    We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 μmoles/10(6) cells/h. PMID:23332078

  14. In Vivo Multiphoton NADH Fluorescence Reveals Depth-Dependent Keratinocyte Metabolism in Human Skin

    PubMed Central

    Balu, Mihaela; Mazhar, Amaan; Hayakawa, Carole K.; Mittal, Richa; Krasieva, Tatiana B.; König, Karsten; Venugopalan, Vasan; Tromberg, Bruce J.

    2013-01-01

    We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 μmoles/106 cells/h. PMID:23332078

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Preparation of metallo-dielectric photonic crystals by multi-photon direct laser writing

    NASA Astrophysics Data System (ADS)

    Kuebler, Stephen M.; Tal, Amir; Chen, Yun-Sheng

    2008-02-01

    Metallo-dielectric photonic crystals (MDPCs) can exhibit intriguing and potentially useful optical properties, including ultra-wide photonic bandgaps, engineered thermal emission, and negative refractive index. But access to such materials has been limited by the lack of suitable methods for their preparation. We have developed a route to three-dimensional (3D) MDPCs that involves fabricating a polymeric pre-form by multi-photon direct laser writing and then conformally depositing metal onto the pre-form by electroless metallization. We use the approach to prepare silver- and copper-plated "woodpile" PCs having face-centered tetragonal symmetry and unit-cell period of several micrometers. The resulting 3D metallized structures exhibit mid-infrared reflectance that is consistent with theory and experimental observations obtained for MDPCs prepared by other routes. These data indicate that multi-photon direct laser writing coupled with electroless metallization is a viable route to complex 3D MDPCs of many symmetries and basis sets and provides a path for integrating such structures with other micron-scale optical elements.

  20. Nonlinear Absorption and Low-Threshold Multiphoton Pumped Stimulated Emission from All-Inorganic Perovskite Nanocrystals.

    PubMed

    Wang, Yue; Li, Xiaoming; Zhao, Xin; Xiao, Lian; Zeng, Haibo; Sun, Handong

    2016-01-13

    Halide perovskite materials have attracted intense research interest due to the striking performance in photoharvesting photovoltaics as well as photoemitting applications. Very recently, the emerging CsPbX3 (X = Cl, Br, I) perovskite nanocrystals have been demonstrated to be efficient emitters with photoluminescence quantum yield as high as ∼90%, room temperature single photon sources, and favorable lasing materials. Herein, the nonlinear optical properties, in particular, the multiphoton absorption and resultant photoluminescence of the CsPbBr3 nanocrystals, were investigated. Notably, a large two-photon absorption cross-section of up to ∼1.2 × 10(5) GM is determined for 9 nm sized CsPbBr3 nanocrystals. Moreover, low-threshold frequency-upconverted stimulated emission by two-photon absorption was observed from the thin film of close-packed CsPbBr3 nanocrystals. The stimulated emission is found to be photostable and wavelength-tunable. We further realize the three-photon pumped stimulated emission in green spectra range from colloidal nanocrystals for the first time. Our results reveal the strong nonlinear absorption in the emerging CsPbX3 perovskite nanocrystals and suggest these nanocrystals as attractive multiphoton pumped optical gain media, which would offer new opportunities in nonlinear photonics and revive the nonlinear optical devices. PMID:26652773

  1. Multiphoton ionisation and dissociation of NO 2 by 50 fs laser pulses

    NASA Astrophysics Data System (ADS)

    Singhal, R. P.; Kilic, H. S.; Ledingham, K. W. D.; Kosmidis, C.; McCanny, T.; Langley, A. J.; Shaikh, W.

    1996-04-01

    Multiphoton ionisation and dissociation of NO 2 has been studied experimentally at 375 nm for laser pulse widths of 10 ns and 50 fs. The parent NO 2 ion peak is not seen in the ns data. In all spectra, the main peak observed is due to the ionisation of the NO molecule which results from the dissociation of excited NO 2 formed after absorbing a 375 nm photon. The intensity dependencies of both NO and NO 2 ion peaks have also been measured. The data has been analysed within the context of a rate equation model using published cross-sections and dissociation rates except for the two-photon ionisation cross-section for NO 2 which was chosen to reproduce the NO 2/NO ion signal ratios at 50 fs. The rate equation model provides a good description of the complete set of data. Indirectly, it may be concluded that coherence effects do not play an important role in the multiphoton excitation/ionisation of NO 2. The data also rules out the importance of above-ionisation dissociation in NO 2 — a conclusion which is consistent with previous data at 496 and 248 nm for laser pulse widths ⩾ 300 fs.

  2. Visualization of basement membranes in normal breast and breast cancer tissues using multiphoton microscopy

    PubMed Central

    WU, XIUFENG; CHEN, GANG; QIU, JINGTING; LU, JIANPING; ZHU, WEIFENG; CHEN, JIANXIN; ZHUO, SHUANGMU; YAN, JUN

    2016-01-01

    Since basement membranes represent a critical barrier during breast cancer progression, timely imaging of these signposts is essential for early diagnosis of breast cancer. A label-free method using multiphoton microscopy (MPM) based on two-photon excited fluorescence signals and second harmonic generation signals for analyzing the morphology of basement membrane in normal and cancerous breast tissues is likely to enable a better understanding of the pathophysiology of breast cancer and facilitate improved clinical management and treatment of this disease. The aim of this study was to determine whether MPM has the potential for label-free assessment of the morphology of basement membrane in normal and cancerous breast tissues. A total of 60 tissue section samples (comprising 30 fresh breast cancer specimens and 30 normal breast tissues) were first imaged (fresh, unfixed and unstained) with MPM and are then processed for routine hematoxylin and eosin (H&E) histopathology. Comparisons were made between MPM imaging and gold standard sections for each specimen stained with H&E. Simply by visualizing morphological features appearing on multiphoton images, cancerous lesions may be readily identified by the loss of basement membrane and tumor cells characterized by irregular size and shape, enlarged nuclei and increased nuclear-cytoplasmic ratio. These results suggest that MPM has potential as a label-free method of imaging the morphology of basement membranes and cell features to effectively distinguish between normal and cancerous breast tissues. PMID:27313695

  3. Monitoring photoaging by use of multiphoton fluorescence and second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Sung-Jan; Jee, Shiou-Hwa; Chan, Jung-Yi; Wu, Ruei-Jr; Lo, Wen; Tan, Hsin-Yuan; Lin, Wei-Chou; Chen, Jau-Shiuh; Young, Tai-Horng; Hsu, Chih-Jung; Dong, Chen-Yuan

    2006-02-01

    It is a field of great interest to develop therapies to rejuvenate photoaged skin. However, the treatment response can not be ideally determined due to lack of a reliable non-invasive method to quantify photoaging. In this study, the photoaging process of skin is investigated by use of a multiphoton fluorescence and second harmonic generation microscopy. We obtain the autofluorescence and second harmonic generation images of superficial dermis from facial skin of individuals of different ages. The results show that autofluorescence signals increase with age while second harmonic generation signals decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. In the case of severe photoaging, solar elastosis can be clearly demonstrated by the presence of thick curvy autofluorescent materials in the superficial dermis. We propose a second harmonic generation to autofluorescence aging index of dermis to quantify the photoaging changes. This index is shown to be a good indicator of photoaging. Our results suggest that multiphoton fluorescence and second harmonic generation microscopy can be developed into a non-invasive imaging modelity for the clinical evaluation of photoaging.

  4. 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.

  5. 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.

  6. Measurements of serotonin and related indoles using capillary electrophoresis with multiphoton-induced hyperluminescence.

    PubMed

    Gostkowski, M L; Wei, J; Shear, J B

    1998-07-01

    We report the use of multiphoton-excited photochemistry to generate highly fluorescent products from hydroxyindoles fractionated in submicron capillary electrophoresis channels. In this approach, the near-infrared (750 nm) output from a modelocked titanium:sapphire laser is focused at the outlet of a 0.6-micron i.d. capillary, producing pulse intensities of approximately 10(12) W cm-2 within a femtoliter focal volume. Hydroxyindole molecules migrating through the outlet aperture of the capillary intersect the beam focus, where absorption of three to four photons (approximately 1.65 eV photon-1) initiates a photobleaching reaction. The resultant hydroxyindole photoproducts produce broadband visible emission (lambdamax approximately 500 nm) when excited with two additional near-IR photons and appear substantially more resistant to photobleaching than the parent hydroxyindoles. This multiphoton-induced conversion of analytes to hyperluminescent derivatives thus offers a more sensitive approach than UV fluorescence for detecting extremely small quantities of material. Mixtures of the hydroxyindoles serotonin (5-hydroxytryptamine), 5-hydroxytryptophan, and 5-hydroxyindole acetic acid are reliably characterized (relative error approximately 10%) in 100 s, with detection limits as low as approximately 70 zmol (approximately 42,000 molecules). The sensitivity of this measurement strategy improves on the best previously reported results for capillary separations of indoles by more than one order of magnitude. PMID:9657885

  7. Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks

    NASA Astrophysics Data System (ADS)

    Dana, Hod; Marom, Anat; Paluch, Shir; Dvorkin, Roman; Brosh, Inbar; Shoham, Shy

    2014-06-01

    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 bioengineered 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 per second of structures with mm-scale dimensions containing a network of over 1,000 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.

  8. Label-free monitoring of colorectal adenoma-carcinoma sequence based on multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Chen, J. X.; Li, H. S.; Chen, Z. F.; Feng, C. Y.; Yang, Y. H.; Jiang, W. Z.; Guan, G. X.; Zhu, X. Q.; Zhuo, S. M.; Xu, J.

    2014-06-01

    The monitoring and evaluation of colorectal adenoma-carcinoma sequence during endoscopy are important for endoscopic resection of precursor lesions to disrupt the adenoma-carcinoma sequence and halt progression to invasive neoplastic disease. In this study, multiphoton microscopy (MPM) was used to identify different stages during the development of colorectal adenocarcinoma including adenoma with low-grade and high-grade dysplasia, and adenocarcinoma invading the submucosa. It was found that by combining two-photon excited fluorescence (TPEF) imaging and second harmonic generation (SHG) imaging, MPM can reveal the morphological changes of the epithelial cells and glands, identify the invasive position and depth of atypical glands and quantitatively describe the change of the cellular nucleus and the nuclear-to-cytoplasmic ratio during the stepwise progression of colorectal adenocarcinoma. These are important pathological findings for pathologists when diagnosing colorectal lesions. With the advancement of a compact and flexible multiphoton endoscope for in vivo imaging and clinical applications, MPM has the potential to provide immediate histological diagnosis for the monitoring and evaluation of the colorectal adenoma-carcinoma sequence during endoscopy.

  9. Multiphoton microscopy using intrinsic signals for pharmacological studies in unstained cardiac and vascular tissue

    NASA Astrophysics Data System (ADS)

    Beaurepaire, Emmanuel; Boulesteix, Thierry; Pena, Ana-Maria; Pages, Nicole; Senni, Karim; Godeau, Gaston; Sauviat, Martin-Pierre; Schanne-Klein, Marie-Claire

    2005-03-01

    We report two novel applications of multiphoton microscopy for pharmacological studies of unstained cardiovascular tissue. First, we show that second harmonic generation (SHG) microscopy of unstained cardiac myocytes can be used to determine the sarcomere length with sub-resolution accuracy, owing to the remarkable contrast of the SHG signal originating from myosin filaments. A measurement precision of 20 nm is achieved, taking the sample variability into account. We used this technique to measure sarcomere contracture in the presence of saxitoxin, and results were in agreement with mechanical measurements of atrial tissue contracture. Second, we characterized multiphoton microscopy of intact unlabeled arteries. We performed simultaneous detection of two-photon-excited fluorescence (2PEF) from elastin laminae and SHG from collagen fibers upon 860 nm excitation. Combined 2PEF/SHG images provide a highly specific, micron scale description of the architecture of these two major components of the vessel wall. We used this methodology to study the effects of lindane (a pesticide) on the artery wall structure and evidenced structural alteration of the vessel morphology.

  10. 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.

  11. 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.

  12. 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.

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

    PubMed

    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. PMID:25950644

  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. 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

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

    PubMed

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

    2016-01-01

    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. PMID:27138688

  17. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27138688

  19. Evidence for Multi-photon transitions between energy levels in a large Current-Biased Magnesium Diboride Josephson Heterojunction

    NASA Astrophysics Data System (ADS)

    Ramos, Roberto; Carabello, Steven; Lambert, Joseph; Cunnane, Daniel; Dai, Wenqing; Chen, Ke; Li, Qi; Xi, Xiaoxing

    2013-03-01

    When photons are strongly coupled to a quantum system, multiphoton transitions can be observed between two energy levels when the quantum energy of the exciting radiation, multiplied by an integer, matches the level spacing. This phenomenon can be observed in Josephson junction qubits exposed to weak microwave radiation at very low temperatures. At microwave resonance, the transition probability of a junction from superconducting to normal state is enhanced and these are used to map multiphoton transitions. We report observation of single- and multi-photon transitions between ground and first excited states in current-biased MgB2 thin film junctions by applying RF with frequencies between 0.5 and 3 Ghz. These large (up to 0.2mm x 0.3 mm) junctions consist of an MgB2 electrode insulated by native oxide from a lead (Pb) or tin (Sn) counter-electrode, and have areas at least 600 times bigger than Nb junctions previously shown to exhibit multiphoton transitions. The data is consistent with theoretical models of junctions behaving in the quantum limit and show anharmonicity of the junction potential when biased near the critical current.

  20. Quantitative evaluation of the effect of multiphoton ionization in laser Thomson scattering diagnostics of low-temperature plasmas

    NASA Astrophysics Data System (ADS)

    Kono, Akihiro; Matsuda, Yukitaka; Okada, Ken; Aramaki, Mitsutoshi

    2011-10-01

    Laser Thomson scattering technique allows one very high spatial-resolution measurements of electron density and temperature as required in diagnostics of microplasmas or measurement of regions very close to the wall. In such high-resolution measurements, laser energy density in the scattering volume must be very high for obtaining sufficient amount of scattering photons; therefore, electron production via multiphoton ionization by the laser beam may significantly affect Thomson scattering due to plasma electrons. In order to clarify the effect of multiphoton ionization in Thomson scattering diagnostics, we have been measuring absolute efficiency of multiphoton ionization caused by frequency-doubled Nd:YAG laser for various gases including rare gases, N2, O2, CF4 and SF6. It has been found that electrons produced via multiphoton ionization can reach a significant fraction of plasma electrons even at low pressures, depending on the gas species and laser focusing conditions. Details will be given in the presentation, including simulation results about how the escape of photoelectrons from the scattering volume affects the situation.

  1. First-principles calculation of multiphoton absorption cross section of α-quartz under femtosecond laser irradiation

    NASA Astrophysics Data System (ADS)

    Yu, Dong; Jiang, Lan; Wang, Feng; Qu, Liangti; Lu, Yongfeng

    2016-05-01

    Time-dependent density functional theory-based first-principles calculations have been used to study the ionization process and electron excitation. The results show that the number of excited electrons follows the power law σ k I k at peak intensities of I < 5 × 1013 W/cm2, indicating that the multiphoton ionization plays a key role. The multiphoton absorption cross section of α-quartz σ k is further calculated to be 3.54 × 1011 cm-3 ps-1 (cm2/TW)6. Using the plasma model, the theoretical results of the damage threshold fluences are consistent with the experimental data, which validates the calculated value of multiphoton absorption cross section. By employing the calculated cross section value in the plasma model, the damage threshold fluences are theoretically estimated, being consistent with the experimental data, which validates the calculated value of multiphoton absorption cross section. The preliminary multiscale model shows great potential in the simulation of laser processing.

  2. 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.

  3. 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. PMID:22147676

  4. 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...

  5. 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...

  6. 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.

  7. 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...

  8. 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

  9. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

    PubMed

    Kumar, Devender; Ristow, Laura C; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

    2015-12-01

    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

  10. 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

  11. Fiber-optic multiphoton flow cytometry in whole blood and in vivo

    NASA Astrophysics Data System (ADS)

    Chang, Yu-Chung; Ye, Jing Yong; Thomas, Thommey P.; Cao, Zhengyi; Kotlyar, Alina; Tkaczyk, Eric R.; Baker, James R.; Norris, Theodore B.

    2010-07-01

    Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.

  12. 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

  13. Multi-photon ionization of atoms in intense short-wavelength radiation fields

    NASA Astrophysics Data System (ADS)

    Meyer, Michael

    2015-05-01

    The unprecedented characteristics of XUV and X-ray Free Electron Lasers (FELs) have stimulated numerous investigations focusing on the detailed understanding of fundamental photon-matter interactions in atoms and molecules. In particular, the high intensities (up to 106 W/cm2) giving rise to non-linear phenomena in the short wavelength regime. The basic phenomenology involves the production of highly charged ions via electron emission to which both sequential and direct multi-photon absorption processes contribute. The detailed investigation of the role and relative weight of these processes under different conditions (wavelength, pulse duration, intensity) is the key element for a comprehensive understanding of the ionization dynamics. Here the results of recent investigations are presented, performed at the FELs in Hamburg (FLASH) and Trieste (FERMI) on atomic systems with electronic structures of increasing complexity (Ar, Ne and Xe). Mainly, electron spectroscopy is used to obtain quantitative information about the relevance of various multi-photon ionization processes. For the case of Ar, a variety of processes including above threshold ionization (ATI) from 3p and 3s valence shells, direct 2p two-photon ionization and resonant 2p-4p two-photon excitations were observed and their role was quantitatively determined comparing the experimental ionization yields to ab-initio calculations of the cross sections for the multi-photon processes. Using Ar as a benchmark to prove the reliability of the combined experimental and theoretical approach, the more complex and intriguing case of Xe was studied. Especially, the analysis of the two-photon ATI from the Xe 4d shell reveals new insight into the character of the 4d giant resonance, which was unresolved in the linear one-photon regime. Finally, the influence of intense XUV radiation to the relaxation dynamics of the Ne 2s-3p resonance was investigated by angle-resolved electron spectroscopy, especially be observing

  14. 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

  15. 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

  16. Act resilient.

    PubMed

    Joseph, Genie; Bice-Stephens, Wynona

    2014-01-01

    Attendees have reported changing from being fearful to serene, from listless to energized, from disengaged to connected, and becoming markedly less anxious in a few weeks. Anecdotally, self-reported stress levels have been reduced by over 50% after just one class. Attendees learn not to be afraid of their feelings by working with emotions in a playful manner. When a person can act angry, but separate himself from his personal story, the emotional energy exists in a separate form that is not attached to specific events, and can be more easily dealt with and neutralized. Attendees are taught to "take out the emotional trash" through expressive comedy. They become less intimated by their own emotional intensity and triggers as they learn how even metaphorical buckets of anger, shame, guilt and hurt can be emotionally emptied. The added benefit is that this is accomplished without the disclosure of personal information of the requirement to reexperience past pain which can trigger its own cascade of stress. PMID:24706248

  17. Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe.

    PubMed

    Liu, Gangjun; Kieu, Khanh; Wise, Frank W; Chen, Zhongping

    2011-01-01

    We report on the development of a compact multiphoton microscopy (MPM) system that integrates a compact and robust fiber laser with a miniature probe. The all normal dispersion fiber femtosecond laser has a central wavelength of 1.06 μm, pulse width of 125 fs and average power of more than 1 W. A double cladding photonic crystal fiber was used to deliver the excitation beam and to collect the two-photon signal. The hand-held probe included galvanometer-based mirror scanners, relay lenses and a focusing lens. The packaged probe had a diameter of 16 mm. Second harmonic generation (SHG) images and two-photon excited fluorescence (TPEF) images of biological tissues were demonstrated using the system. PMID:20635426

  18. Time and spectrum-resolving multiphoton correlator for 300–900 nm

    SciTech Connect

    Johnsen, Kelsey D.; Thibault, Marilyne; Jennewein, Thomas; Kolenderski, Piotr; Scarcella, Carmelo; Tosi, Alberto

    2014-10-14

    We demonstrate a single-photon sensitive spectrometer in the visible range, which allows us to perform time-resolved and multi-photon spectral correlation measurements at room temperature. It is based on a monochromator composed of two gratings, collimation optics, and an array of single photon avalanche diodes. The time resolution can reach 110 ps and the spectral resolution is 2 nm/pixel, limited by the design of the monochromator. This technique can easily be combined with commercial monochromators and can be useful for joint spectrum measurements of two photons emitted in the process of parametric down conversion, as well as time-resolved spectrum measurements in optical coherence tomography or medical physics applications.

  19. 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.

  20. 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.