CALIBRATION OF CONFOCAL VOLUME OF FLUORESCENCE CORRELATION SPECTROSCOPY AND DIFFUSION COEFFICIENT OF FLUORESCENCE REFERENCES AT VARIED TEMPERATURES

摘要

@@Fluorescence correlation spectroscopy (FCS) gives the access to the adequate investigation of a fluorescent probe's dynamics with single-molecule sensitivity and gains ever-increasing attention in the past two decades.1 The confocal volume of FCS, theoretically down to optical diffraction limit, depends strongly on experimental variables, especially the refractive index of the media, which can be affected by a number of factors, such as temperature, solvent type, etc.2 Therefore, it is very necessary to calibrate the confocal volume and this is usually done by using standard fluorescence references. One of the most commonly used standard molecule is Rhodamine 6G (referred as R6G) and its diffusion coefficient (D) in pure water has long been regarded as 280 μm~2/s, cited back to the original publication in the mid-1970s.3 It is until only recently that attention has been paid to re-evaluate the precise value of diffusion coefficient of R6G, by a number of methods, such as micro-fluidic techniques4,5, dual-focus FCS6,7 and scanning FCS.8 The results indicate that the D value of R6G (～400 μm~2·s~(-1)) is much higher than the commonly cited value. This brings up the important issue of the correct D value of R6G to choose. The significance of the precise calibration also lies in the confocal volume under varied temperature, which should be able to guarantee the correct determination of diffusivity under investigation, especially in in vivo biophysical study.