A simple introduction to multiphoton microscopy
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Abstract:
Multiphoton microscopy is a powerful technique based on complex quantum mechanical effects. Thanks to the development of turnkey mode-locked laser systems, multiphoton microscopy is now available for everyone to use without extreme complexity. In this short introduction, we describe qualitatively the important concepts underlying the most commonly used type of multiphoton microscopy (two-photon excitation). We elucidate how those properties lead to the powerful results that have been achieved using this technique. As with any technique, two-photon excitation microscopy has limitations that we describe, and we provide examples of particular classes of experiments where two-photon excitation microscopy is advantageous over other approaches. Finally, we briefly describe other useful multiphoton microscopy approaches, such as three-photon excitation and second harmonic generation imaging.We report on recent advances in multiphoton light-sheet microscopy to perform fast multimodal imaging combining fluorescence with second-harmonic generation and to mitigate photodamage during in vivo imaging of embryos.
Two-photon excitation microscopy
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Two-photon microscopy is a key method for biomedical imaging but time consuming for three-dimensional and fluorescence lifetime images. To speed up acquisitions, we have developed a time resolved multifocal multiphoton microscope.
Fluorescence-lifetime imaging microscopy
Two-photon excitation microscopy
Photon Counting
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This purpose of this study is to demonstrate the feasibility of using multiphoton microscopy in ophthalmologic imaging. Without the introduction of extrinsic fluorescence molecules, multiphoton induced autofluorescence and second harmonic generation signals can be used to obtain useful structural information of normal and diseased corneas. Our work can potentially lead to the in vivo application of multiphoton microscopy in investigating corneal physiology and pathologies.
Autofluorescence
Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
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Two-photon microscopy is a key method for biomedical imaging but leads to a long recording time for three-dimensional and FLIM images. To speed up acquisitions, we have developed a time resolved multifocal multiphoton microscope.
Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
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Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
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In this talk a brief review on the non linear laser imaging techniques will be displayed. In particular, two photon fluorescence microscopy, lifetime imaging, multispectral imaging, second harmonic generation microscopy principles will be described.
Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
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In recent years, non-linear imaging techniques such as multiphoton fluorescence and harmonic generation microscopy have been successfully applied to dermatological imaging. Confocal-like image quality, enhanced depth penetration, and non-linear spectral signatures are among the main advantages of this family of techniques. In this presentation, we will focus on the applications of multiphoton microscopy to skin specimens in different physiological states. Images of normal and diseased tissue specimens will be presented and spectrally characterized. Our work has potential applications in developing multiphoton microscopy into a clinically applicable diagnostic tool.
Fluorescence-lifetime imaging microscopy
Two-photon excitation microscopy
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Two-photon excitation 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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