Superresolution Microscopy Imaging Based on Wide-Field Stochastic Fluorescent Bleaching
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Photoactivated localization microscopy
Fluorescence-lifetime imaging microscopy
Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.
Autofluorescence
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
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Development of non-invasive techniques for early stage diagnostics of melanoma metastasis is great importance to improve survival rates. Multiphoton microscopy together with fluorescence lifetime imaging is here explored for finding metastasis in lymph node tissues.
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One-photon fluorescence microscopy is an important biological and biomedical imaging technique. This chapter provides a comprehensive introduction of one-photon microscopy to help researchers maximize the effectiveness of their imaging experiments. This chapter first introduces fluorescence generation and the diffraction limit as background. It then outlines the basic operating principles of multiple one-photon microscopy configurations. Specific configurations include wide-field microscopy, light-field microscopy, confocal microscopy, light-sheet microscopy, and super-resolution microscopy. This chapter concludes by discussing multiple specific applications of one-photon fluorescence microscopy in neuroscience, matching the capabilities of the various microscope configurations with their role in obtaining novel information from biological samples.
Photoactivated localization microscopy
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Two-photon excitation microscopy
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Photoactivated localization microscopy
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Two-photon excitation microscopy
Photoactivated localization microscopy
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The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.
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Abstract An overview on fluorescence microscopy with high spatial, spectral and temporal resolution is given. In addition to 3D microscopy based on confocal, structured or single plane illumination, spectral imaging and fluorescence lifetime imaging microscopy (FLIM) are used to probe the interaction of a fluorescent molecule with its micro‐environment. Variable‐angle total internal reflection fluorescence microscopy (TIRFM) permits selective measurements of cell membranes or cell‐substrate topology in the nanometre scale and is also combined with spectral or time‐resolved detection. In addition to single cells or cell monolayers, 3‐dimensional cell cultures are of increasing importance, since they are more similar to tissue morphology and function. All methods reported are adapted to low dose of illumination, which is regarded as a key parameter to maintain cell viability. Applications include cancer diagnosis and cell tomography under different physiological conditions. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
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Abstract Two‐photon fluorescence microscopy allows three‐dimensional imaging of biological specimens in vivo . Compared with confocal microscopy, it offers the advantages of deeper tissue penetration and less photodamage but has the disadvantage of slightly lower resolution.
Two-photon excitation microscopy
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