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Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence- based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions. [ABSTRACT FROM AUTHOR]

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non-invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two-photon microscopy to non-invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence-based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients. [ABSTRACT FROM AUTHOR]

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. Methods: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. Results: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. Conclusions: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration. [ABSTRACT FROM AUTHOR]

Axonal degeneration (AxD) is a pathological hallmark of many neurodegenerative diseases. Deciphering the morphological patterns of AxD will help to understand the underlying mechanisms and develop effective therapies. Here, we evaluated the progression of AxD in cortical neurons using a novel microfluidic device together with a deep learning tool that we developed for the enhanced-throughput analysis of AxD on microscopic images. The trained convolutional neural network (CNN) sensitively and specifically segmented the features of AxD including axons, axonal swellings, and axonal fragments. Its performance exceeded that of the human evaluators. In an in vitro model of AxD in hemorrhagic stroke induced by the hemolysis product hemin, we detected a time-dependent degeneration of axons leading to a decrease in axon area, while axonal swelling and fragment areas increased. Axonal swellings preceded axon fragmentation, suggesting that swellings may be reliable predictors of AxD. Using a recurrent neural network (RNN), we identified four morphological patterns of AxD (granular, retraction, swelling, and transport degeneration). These findings indicate a morphological heterogeneity of AxD in hemorrhagic stroke. Our EntireAxon platform enables the systematic analysis of axons and AxD in time-lapse microscopy and unravels a so-far unknown intricacy in which AxD can occur in a disease context. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to investigate the autofluorescence (AF) of the RPE with two-photon microscopy (TPM) and fluorescence lifetime imaging (FLIM) under normal and oxidative stress conditions., Methods: Porcine RPE-choroid explants were used for investigation. The RPE-choroid tissue was preserved in a perfusion organ culture system. Oxidative stress was induced by laser photocoagulation with frequency-doubled ND:YAG laser (532 nm) and by exposure to different concentrations (0, 1, 10 mM) of ferrous sulfate (FeSO4) for 1 hour. At indicated time points after exposure, the tissue was examined with TPM and FLIM. Intracellular reactive oxygen species around the photocoagulation lesion were detected with chloromethyl-2'7'-dichlorofluorescein diacetate (CM-H2DCFDA). Melanosomes were isolated from RPE cells and their fluorescence properties were investigated under normal and oxidized conditions., Results: Under normal conditions, AF in RPE cells with TPM is mostly originated from melanosomes, which has a very short fluorescence lifetime (FLT; mean = 117 ps). Under oxidative stress induced by laser irradiation and FeSO4 exposure, bright granular AF appears inside and around RPE cells, whose FLT is significantly longer (mean = 1388 ps) than the FLT of the melanosome-AF. Excitation and emission peaks are found at 710 to 750 nm and 450 to 500 nm, respectively. Oxidative stress increases the fluorescence intensity of the melanosomes but does not change their FLT., Conclusions: TPM reveals acute oxidative stress-induced bright AF granules inside and around RPE cells which can be clearly discriminated from melanosomes by FLIM. TPM combined with FLIM is a useful tool of live-cell analysis to investigate functional alterations of the RPE.

Minimally invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without actual tissue probing. For this matter, confocal microscopy (Cornea Module) is in daily use in ophthalmic practice. Multiphoton microscopy is a new optical technique that enables high-resolution imaging and functional analysis of living tissues based on tissue autofluorescence. This study was set up to compare the potential of a multiphoton microscope (DermaInspect) to the Cornea Module. Ocular surface pathologies such as pterygia, papillomae, and nevi were investigated in vivo using the Cornea Module and imaged immediately after excision by DermaInspect. Two excitation wavelengths, fluorescence lifetime imaging and second-harmonic generation (SHG), were used to discriminate different tissue structures. Images were compared with the histopathological assessment of the samples. At wavelengths of 730 nm, multiphoton microscopy exclusively revealed cellular structures. Collagen fibrils were specifically demonstrated by second-harmonic generation. Measurements of fluorescent lifetimes enabled the highly specific detection of goblet cells, erythrocytes, and nevus-cell clusters. At the settings used, DermaInspect reaches higher resolutions than the Cornea Module and obtains additional structural information. The parallel detection of multiphoton excited autofluorescence and confocal imaging could expand the possibilities of minimally invasive investigation of the ocular surface toward functional analysis at higher resolutions.

In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin-resistance cassette and transfection of microdissected, organ-cultured adult human scalp hair follicles generates specific K15 promoter-driven GFP expression in their stem cell-rich bulge region. K15-GFP+ cells can be visualized in situ by GFP fluorescence and 2-photon laser scanning microscopy. Vital K15-GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15-GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter-driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues.

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is assumed to be a key location for the generation of adaptive immune mechanisms of the ocular surface, but functional studies of CALT are still lacking. The purpose of this study was to establish an animal model that enables functional analysis of immune mechanisms going on within CALT. In addition, the use of two-photon microscopy, a new optical method, was evaluated for examining complex immunologic interactions of CALT by volume (three-dimensional [3-D]) and time-dependence (four-dimensional [4-D]) in vivo., Methods: The conjunctiva of female BALB/c mice was repeatedly challenged with topical Chlamydia trachomatis serovar C or a solution of ovalbumin and cholera toxin B. Two-photon microscopy was conducted on explanted, unfixed, and unstained eyes with adjacent nictitating membranes., Results: After three to five stimulations, CALT was detected exclusively in the nictitating membrane of 73% (C. trachomatis) or 70% (ovalbumin/ cholera toxin) of the animals. CALT mainly consisted of CD45R/B220+ B cells and CD4+ and CD8+ T cells. Electron microscopy showed intraepithelial lymphocytes and follicles consisting of lymphocytes, dendritic cells, and macrophages. Two-photon microscopy based on tissue autofluorescence allowed all components of CALT to be detected three dimensionally. High-resolution images were generated in tissue depths of 65 microm below the mucosal surface., Conclusions: This study introduces a novel mouse model for functional investigations of CALT. Topical stimulation with C. trachomatis or ovalbumin/cholera toxin B reliably leads to CALT generation at the nictitating membrane. The use of two-photon microscopy enables groundbreaking 3-D and, in the future, intravital 4-D investigations of immunologic processes initiated in CALT.

Background and Objectives: Selective treatment of the retinal pigment epithelium (RPE) by repetitively applying green micros-laser pulses is a new method for retinal diseases associated with a degradation of the RPE, which spares the neural retina. We investigated an alternative approach to realize repetitive micros-laser exposure by rapidly scanning a continuous wave (CW)-laser beam across the RPE., Study Design/materials and Methods: An Ar(+) laser beam (514 nm) with a diameter of 18.75 microm was repetitively scanned across porcine RPE samples in vitro providing an irradiation time of 1.6 micros per point on the central scan axis. RPE cell damage was investigated by means of the fluorescence viability assay Calcein-AM., Results: The ED(50) cell damage is 305 mJ/cm(2) when applying 10 scans with a repetition rate of 500 Hz. The threshold decreases with the number of scans, a saturation was found at 135 mJ/cm(2) with more than 500 exposures applied. The depth of focus in beam direction is 350 microm, defined by an increase of the threshold radiant exposure by 20%., Conclusions: Targeting of pigmented cells with high local resolution has been proved with a laser-scanning device. Looking ahead selective RPE-treatment, the adaptation of a laser-scanning device on a slit-lamp or into a modified retina angiograph seems to be an attractive alternative to the pulsed micros laser device., (Copyright 2003 Wiley-Liss, Inc.)