Proximity ratio histograms

Figure 4.9 The effect of laser power upon the photobleaching of a freely diffusing spFRET labelled protein. Upon increasing the laser power from 40 to 120 /u,W (top to bottom, laser power measured before the objective, see Chapter 3), the raw data (left, donor channel grey, acceptor channel black) and the calculated proximity ratio histograms (right) both show the increase in donor only fluorescence (increase in donor channel in the raw data, increase in the relative magnitude of the zero peak) that is indicative of photobleaching.

Histograms of proximity ratios are commonly fit with single or multiple Gaussian or Lognormal curves in order to extract information on peak positions, widths and areas [76]. It is possible to describe the data with an analytic expression in some cases (see [76]), but the difference in the characteristic parameters obtained (peak position, width and area) using a Gaussian is small [ 76 ]. Care must also be taken with the construction of the histograms, with suitable numbers (and widths) of bins chosen to aid in the resolution of any components that may exist. 

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