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PCH Plugins

This part of the website is incomplete. Many details of the pch plugins are similar to the FCS plugins.

When to Use PCH Analysis

A quick glance at our page on PCH reveals that there are three ways to obtain molecular brightness information: 1.) from the amplitude of the FCS curve itself, 2.) from "moment analysis" also known as N&B analysis, and 3.) from PCH analysis. So which method should be used to obtain the molecular brightness? The answer depends on the application. If the data has been obtained from a single point measurement, the answer is almost always to determine the brightness from a fit of the FCS curve. I recommend this because this method allows for visual inspection of the quality of the fit given the expected diffusion profile of the sample under study. In addition, this allows for rejection or detrending of curves which show bleaching or slow dynamics like cell motion. Of course, one can always divide the curve up into many small sections and perform N&B analysis, but it can be difficult to decide how large to make these sections. These issues are also prevalent in PCH analysis. Once bleaching is involved, the PCH curve becomes a convolution of all possible average concentration values present throughout the aquisition. In addition, both N&B and PCH contain complex corrections for detector afterpulsing and triplet blinking that are typically not present in fitting software (including mine).

So when should N&B analysis be used? There are several circumstances under which FCS cannot be used. These include circumstances under which samples are not obtained in a continuous stream (e.g. images). Here the diffusion time is much smaller than the time between frames, but much larger than the pixel dwell time. Here FCS will not work, but N&B will. Note that it may be useful to try RICS analysis for this circumstance as well. In addition, the simplicity of the N&B analysis makes it useful for simplistic analysis of many samples when automated fitting is not trivial.

Finally, when should PCH analysis be used? The major advantage and disadvantage of the PCH method is its ability to represent complexity in the sample. One must be careful not to "overfit" the PCH curve to represent more complexity than is justified in a noisy curve. Nevertheless, with high quality data, one can obtain information about multiple species in a sample. In addition, it can be much more straightforward to assess the error limits on parameters from PCH data as opposed to N&B analysis (typically requires the use of factorial cumulants). In addition, multispecies N&B models for multicolor analysis can become quite complex while 2D PCH retains a simplicity that allows for straightforward model testing.