Faculty Opinions recommendation of Combined non-linear laser imaging (two-photon excitation fluorescence microscopy, fluorescence lifetime imaging microscopy, multispectral multiphoton microscopy) in cutaneous tumours: first experiences.
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Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
Two-photon excitation microscopy
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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.
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Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
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Image formation in three-photon fluorescence microscopy is analysed. The point spread function, three-dimensional optical transfer function and axial resolution are considered. The results are generalized to multiphoton fluorescence. Three-photon fluorescence is particularly amenable to resolution improvement by use of pupil filters.
Two-photon excitation microscopy
Point spread function
Fluorescence-lifetime imaging microscopy
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Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.
Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
Fluorescence Correlation Spectroscopy
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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
Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
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Two-photon excitation microscopy
Fluorescence-lifetime imaging 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.
Fluorescence-lifetime imaging microscopy
Two-photon excitation microscopy
Second-harmonic imaging microscopy
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Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
Fluorescence cross-correlation spectroscopy
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Efficient two-photon (2PA) absorbing dyes and bioconjugates were used in two-photon fluorescence microscopy (2PFM) of cells, tissue sections, and excised tumors. Results show the utility of these dyes in studying biological processes.
Two-photon excitation microscopy
Ex vivo
Fluorescence-lifetime imaging microscopy
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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
Photoactivated localization microscopy
Fluorescence-lifetime imaging microscopy
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