546 Quantifying Human Eosinophils Using 3-Dimensional Volumetric Images Collected With Multi-Photon Fluorescence Microscopy
Nastaran SafdarianZhongyao LiuXiaoming ZhouHenry D. AppelmanTimothy T. NostrantThomas D. WangEmily T. Wang
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Two-photon excitation microscopy
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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.
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Two-photon excitation 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.
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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.
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