Fluorescence photobleaching method

Petersen N. O. and Elson E. L. (1986) Measurements of Diffusion and Chemical Kinetics by Fluorescence Photobleaching Recovery and Fluorescence Correlation Spectroscopy Methods Enzymol. 130, 454-84. 

Fig. 3. Schematic diagram of the spot photobleaching method of FRAP. (A) Darkened circles represent fluorescently labeled molecules evenly distributed over a two-dimensional surface (assumed to be an infinite plane). (B) White and light gray circles represent the initial postbleach distribution of photobleached molecules within a 1-pm diameter spot. (C) Redistribution of photobleached and unbleached molecules as a consequence of random diffusion over time. (D) Curve representing the fluorescence intensity within the l-pm diameter spot monitored over time arrows a, b, and c indicate the time-points that correspond to their respective panels. The rate of recovery from point b to point c is used to determine the diffusion constant. The magnitude of the recovery is determined by comparing the fluorescence intensity at point c with the initial intensity at point a, and is used to determine the mobile fraction.

Compared to fluorescence, SERRS has some significant advantages. Unlike fluorescence-based methods, SERRS measurements are unaffected by quenching by oxygen or other species, are less sensitive to photobleaching, and are inherently... 

In another approach, the interfacial diffusion of the nanoparticles was determined using two photobleaching methods fluorescence loss induced by photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP). It was found that the lateral diffusion of the nanoparticles at the interface as well as the diffusion normal to and from the interface deviated by about four orders of magnitude from the values obtained in free solution [46],... 

W.R. Zipfel, W.W.W. Webb, In vivo Diffusion Measurements Using Multiphoton Excitation Fluorescence Photobleaching Recovery and Fluorescence Correlation Spectroscopy, in A. Periasamy (ed.), Methods in Cellular Imaging, Oxford University Press, 216 (2001)... 

Another method, called photobleaching, works on robust soHds but may cause photodecomposition in many materials. The simplest solution to the fluorescence problem is excitation in the near infrared (750 nm—1.06 pm), where the energy of the incident photons is lower than the electronic transitions of most organic materials, so fluorescence caimot occur. The Raman signal can then be observed more easily. The elimination of fluorescence background more than compensates for the reduction in scattering efficiency in the near infrared. Only in the case of transition-metal compounds, which can fluoresce in the near infrared, is excitation in the midvisible likely to produce superior results in practical samples (17). 

Patel, R. C., Lange, D. C. and Patel, Y. C. (2002). Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes. Methods 27, 340-8. 

The advantage of fluorescent dyes coupled to primary antibodies is the fast result obtained with the direct method. Another advantage is the possibility to label more than one antigen at the same time. In plant material, it is important to take into account the possible autofluorescence of the tissue. Disadvantages can be the lack of orientation in the tissue and photobleaching of the dyes. The latter problem can often be... 

When the fluorescence spectra of the probe shifts on protonation, two emission wavelengths with opposite proton-sensitive response are chosen to give a pH-dependent emission intensity ratio. In this ratio method a number of ion-independent factors that affect the signal intensity like photobleaching, variations in probe concentration, and illumination instability are eliminated. 

In this chapter, we demonstrate the implementation of the acceptor photobleaching FRET method, which is the simplest, robust image cytometric FRET technique that can be performed using any confocal microscopes (15). It involves measuring the donor fluorescence in the presence of the acceptor, and then repeating the measurement after having photobleached the acceptor fluorophore. The result is independent of donor and acceptor concentration and stoichiometry and requires relatively simple image mathematics. 

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