The simple ratio method seems to be the most widely used method.
Simple ratio method and the second method Estimation of bleach ratio could either be calculated directly Several different algorithms have been developed and used by various researchers.Ī conventional method for correcting the bleaching has been done by multiplying the inverse of the ratio of intensity loss compared to a reference image frame. We call such restoration procedure “bleach correction”. To do this, the amount of the fluorescence loss is estimated and then the none-bleached condition is restored by image processing. After the experiment, time-lapse sequence image data can be processed to compensate for the loss of intensity. Before the experiment, one can tune instruments and sample environment, such as careful choice of the fluorophore, use of anti-fading reagents, decreasing the power of laser irradiation, increasing the detector gain, and increasing the time interval of capturingġ. To overcome the problem of photobleaching, improvements can be made either before or after the experiment. Since bleaching attenuates total signal intensity even when the density of labeled protein is unchanged, precise estimation of the amount of protein or the boundary of the biological structure becomes difficult. Photobleaching not only degrades the visual quality of the results but also interferes with the measurement of molecular kinetics and the quality of the segmentation of target objects. This effect is called “photobleaching” and is a widely-known problem in bioimage analysis. This is because, with a certain probability, fluorophores irreversibly lose the ability to fluoresce. Emitted fluorescence gradually decreases by time. This irradiation is necessary to detect the position of proteins but it also has a side effect.
In biological fluorescence microscopy, cells are irradiated with excitation light that causes the emission of fluorescence from protein markers.