FRAP Analysis: Accounting for Bleaching during Image Capture
نویسندگان
چکیده
The analysis of Fluorescence Recovery After Photobleaching (FRAP) experiments involves mathematical modeling of the fluorescence recovery process. An important feature of FRAP experiments that tends to be ignored in the modeling is that there can be a significant loss of fluorescence due to bleaching during image capture. In this paper, we explicitly include the effects of bleaching during image capture in the model for the recovery process, instead of correcting for the effects of bleaching using reference measurements. Using experimental examples, we demonstrate the usefulness of such an approach in FRAP analysis.
منابع مشابه
Universal Approach to FRAP Analysis of Arbitrary Bleaching Patterns
The original approach to calculating diffusion coefficients of a fluorescent probe from Fluorescence Recovery After Photobleaching (FRAP) measurements assumes bleaching with a circular laser beam of a Gaussian intensity profile. This method was used without imaging the bleached cell. An empirical equation for calculating diffusion coefficients from a rectangular bleaching geometry, created in a...
متن کاملFRAP analysis of chromatin looseness in mouse zygotes that allows full-term development
Chromatin looseness, which can be analyzed by fluorescence recovery after photobleaching (FRAP) using eGFP-tagged core histone proteins, is an important index of the differentiation potential of blastomere cells and embryonic stem cells. Whether chromatin looseness is a reliable index of the developmental potential of embryos during ontogenesis is not known. As a necessary first step toward ans...
متن کاملOn the Equivalence of Fcs and Frap: Simultaneous Lipid Membrane Measurements by Imaging Fcs
Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery after Photobleaching (FRAP) are probably the most widely used methods to determine diffusion coefficients in biological samples. However, it has already been noted in the past that the two methods do not necessarily provide the same results. This has typically been explained by the different spatial range over which the two m...
متن کاملOn the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements.
Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffus...
متن کاملSpindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching
The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allow...
متن کامل