China
Africa
Explore chapters and articles related to this topic
Nanoscopic Imaging to Understand Synaptic Function
Published in Francesco S. Pavone, Shy Shoham, Handbook of Neurophotonics, 2020
Daniel Choquet, Anne-Sophie Hafner
In the last 40 years, our understanding of protein dynamics improved tremendously with the development of strategies allowing real-time imaging of protein diffusion. Initially, a method was developed to study protein and lipid dynamics at the cell surface, fluorescence recovery after photobleaching (FRAP) (Axelrod et al., 1976). For this method, protein or lipids of interest have to be specifically labeled with fluorophores. The core idea of FRAP is to photobleach a specific area of a living cell and analyze fluorescence recovery in the bleached region. Since the emergence of GFP-like variants fluorescent proteins, FRAP and related methods such as fluorescent loss in photobleaching (FLIP) or photoactivation, have been extensively used to study the population of proteins’ diffusion properties, stability, and turnover. For a uniform population of molecules, measurements of average parameters versus an ensemble of unique elements should yield identical values. However, for most biological systems, populations of molecules do not display a homogenous behavior. For instance, synaptic PSD-95 molecules are highly stable whereas cytoplasmic PSD-95 are highly mobile (Sturgill et al., 2009).
Selection Criteria for Optical Microscopy
Published in John Girkin, A Practical Guide to Optical Microscopy, 2019
Fluorescence recovery after photobleaching (FRAP) is a method in which an area within the sample is deliberately photobleached and then the recovery of the fluorescence monitored to provide information on cell dynamics and local diffusion rates. The typical protocol here is to image an area using a low excitation power and then to zoom in to a small area within the original field of view. This area is then imaged with a very high level of excitation for a period of time until the fluorescence intensity has been significantly reduced (different protocols use different values). The microscope is then “un-zoomed” and the original area imaged at low excitation intensity again. Over time the fluorescence returns to the area due to either the production of new fluorophores, in the case of fluorescent proteins, or the diffusion of new fluorescent molecules into the bleached area. Various mathematical models can then be applied to determine the rate of return of fluorescence.
Advanced Fluorescence Techniques: FRAP, iFRAP, FLIP, FLAP, Photoconvertible, Photoactivatable, and Photoswitchable Proteins
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
FRAP operates on the basis that if a fluorescent molecule outside the ROI in the cytoplasm, organelle, or membrane is mobile, it will diffuse or be actively pumped into the bleached area. The rate of fluorescence recovery into the bleached area is then recorded in a time-lapse sequence using low laser intensity (down to 1% or below that of the laser power used for bleaching). A graph is then constructed of the temporal changes in fluorescence intensity within the bleached region (Fig. 11.1). FRAP depends on the principle that recovery of fluorescence within the bleached region is highly dependent on factors such as (i) the molecular weight of the fluorescent molecule or hybrid protein, (ii) its interaction with binding partners, (iii) whether or not the protein of interest is anchored to a less mobile structure (e.g., membrane protein), and (iv) whether or not the protein has restricted movement within a subcellular domain due to other structures, like membranes (Reits and Neefjes 2001).
Silica-based diffusion probes for use in FRAP and NMR-diffusometry
Published in Journal of Dispersion Science and Technology, 2019
Maria Pihl, Krzysztof Kolman, Antiope Lotsari, Marie Ivarsson, Erich Schüster, Niklas Lorén, Romain Bordes
In the FRAP technique, the fluorescence of a probe is deactivated by fast laser beam irradiation. The recovery of the fluorescence of the bleached area, resulting from the diffusion-in of the non-bleached diffusion probe and the diffusion-out of the deactivated probe, is monitored as function of time. When the probe diffuses freely in the sample, that is without interaction with the structure of the soft matrix, the recovery curves allows the determination of the diffusion coefficient and by use of the Stokes-Einstein relationship, the probe size.[13] Because of the principle of measurement, the probe has of course to bear a chromophore which fluorescence can be annihilated by a laser intensity that does not alter the material structure. Furthermore the material studied must present a certain transparency at the laser wavelength to allow the laser beam penetration. One important feature of FRAP is the fact that it allows local diffusion measurements at the micrometer scale, thus introducing spatial resolution in the nature of the measurements.