Multiphotonic microscopy as a processing tool for optical data storage and imaging in biophysics (original) (raw)
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New developments in multiphoton microscopy
Current Opinion in Neurobiology, 2002
2PE two-photon excitation 2PM two-photon-excited fluorescence microscopy AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid FCS fluorescence correlation spectroscopy FPs fluorescent proteins GFP green fluorescent protein NA numerical aperture NLOM nonlinear optical microscopy NMDA N-methyl-D-aspartate SHG second harmonic generation SHIM second harmonic imaging microscopy STED stimulated-emission depletion
Multiphoton microscopes are one of the most essential imaging tools for 3D, nonin- vasive imaging of the living organisms. These microscopes work by exploiting second harmonic generation from the biological samples under intense pulsed lasers. This innovation was enabled by the development of critical techniques such as ecient u- orescent labeling and novel laser types. In this essay, author aims to introduce the reader to the multiphoton microscopy along with the key techniques involved in its operation with a special emphasis on the laser illumination techniques.
Multiphoton Microscopy in the Biomedical Sciences VI
Multiphoton Microscopy in the Biomedical Sciences XX, 2002
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Nonlinear magic: multiphoton microscopy in the biosciences
Nature Biotechnology, 2003
Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 µm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in subfemtoliter volumes.
Nonlinear magic: multiphoton microscopy in the biosciences : Optical imaging
Nature Biotechnology, 2003
Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 µm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in subfemtoliter volumes.
Label-Free Multiphoton Microscopy: Much More Than Fancy Images
International Journal of Molecular Sciences
Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and param...
Multiphoton Fluorescence Microscopy
Methods, 2001
In the context of single-point measurements in con-Multiphoton fluorescence microscopy has now become a relatively junction with image analysis, the most striking difference common tool among biophysicists and biologists. The intrinsic secbetween a conventional confocal system and a multiphotioning achievable by multiphoton excitation provides a simple ton microscope is that in the latter case the emitted light means to excite a small volume inside cells and tissues. Multiphoton does not need to travel through the confocal pinhole to microscopes have a simplified optical path in the emission side due to the lack of an emission pinhole, which is necessary with normal reach the detector. This different optical configuration confocal microscopes. This article illustrates examples in which this makes it possible, with relatively simple optics, to use advantage in the simplified optics is exploited to achieve a new type several colors in the emission arm without being affected of measurements. First, dual-emission wavelength measurements by color aberrations of the objective. This alone is a disare used to identify regions of different phase domains in giant tinct advantage. We also note that multiphoton excitation vesicles and to perform fluctuation experiments at specific locations is particularly good at exciting several common fluoroin the membrane. Second, we show how dual-wavelength measurephores using a single excitation wavelength. In addition, ments are used in conjunction with scanning fluctuation analysis to the collection of the emitted light with a minimum of measure the changes in the geometry of the domains and the optical components in the emission path increases light incipient formation of gel domains when the temperature of the collection efficiency. We performed experiments to comgiant vesicles is gradually lowered. ᭧ 2001 Academic Press