Fluorescence Recovery Curve
Fluorescence Recovery Curve - Related Videos
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Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching
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Cited by 4 •
2010
In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.
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Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching
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Cited by 16 •
2012
This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.
07:54
Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer
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Cited by 2 •
2015
We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.
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Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons
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Cited by 33 •
2011
FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.
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Calibration Curves
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2023
Source: Laboratory of Dr. B. Jill Venton - University of Virginia Calibration curves are used to understand the instrumental response to an analyte and predict the concentration in an unknown sample. Generally, a set of standard samples are made at various concentrations with a range than includes the unknown of interest and the instrumental response at each concentration is recorded. For more accuracy and to understand the error, the response at each concentration can be repeated so an error...
FAQs
The rate and extent of fluorescence return provide clues about how readily labeled molecules move within the bleached region. Rapid recovery indicates faster redistribution, whereas slower recovery reflects slower molecular movement or exchange. The recovered and unrecovered portions help distinguish mobile molecules from an immobile fraction, allowing researchers to quantify mobility rather than relying only on a visual image.
Bleached molecules no longer contribute detectable fluorescence in the targeted area, while unbleached molecules outside that area can move inward. Their redistribution changes the measured intensity with time. This exchange produces the recovery signal and links the curve to molecular movement, including the balance between molecules that can enter the region and molecules that remain effectively stationary.
Curve kinetics can provide evidence about whether fluorescence changes mainly reflect diffusion, binding-related exchange, or both. Diffusion describes movement through the cellular environment, whereas exchange reflects molecules leaving and entering a region through molecular interactions. Examining recovery behavior over time helps characterize these processes and can reveal how molecular binding influences apparent mobility.
The mobile fraction represents labeled molecules capable of contributing to recovery, while the immobile fraction indicates molecules that do not readily redistribute during the observation. Their relative contributions can reveal whether a cellular component is freely mobile or retained within a stable structure. This distinction helps investigate intracellular organization and the degree of molecular constraint in living cells.
A labeled cellular region is first selected and exposed to focused laser light to remove its fluorescence temporarily. Fluorescence intensity is then measured as the region recovers over time. Plotting those measurements produces the recovery curve, which can be analyzed for molecular mobility, diffusion behavior, mobile and immobile fractions, and exchange kinetics.
Researchers can apply recovery-curve analysis when they need quantitative information about movement within living cells. For membrane studies, the measurement helps characterize fluidity. For protein studies, it can indicate how binding affects mobility and exchange. The same approach supports investigation of cytoplasmic transport and intracellular organization by connecting fluorescence changes with molecular behavior.