Photobleaching event counting is a single-molecule fluorescence technique that is increasingly being used to determine the stoichiometry of protein and RNA complexes composed of many subunits in vivo as well as in vitro. time trace. Although photobleaching steps are detected by eye often, this method only works for high individual fluorophore emission signal-to-noise ratios and small numbers of fluorophores. With filtering methods or currently available algorithms, it is possible to reliably identify photobleaching steps for up to 20C30 fluorophores and signal-to-noise ratios down to 1. Here we present a new Bayesian method of counting steps in photobleaching time traces that takes into account stochastic noise variation in addition to complications such as overlapping photobleaching events that may arise from fluorophore interactions, as well as on-off blinking. Our method is capable of detecting 50 photobleaching steps even for signal-to-noise ratios as low as 0.1, can find up to 500 steps for more favorable noise profiles, and is computationally inexpensive. INTRODUCTION Fluorophores photobleach when exposed to light over time. That is, they irreversibly photochemically transition to a state no longer detectable by fluorescence (White and Stelzer, 1999 ; Lippincott-Schwartz = 10 for the single fluorophore, illustrating how noise obscures steps when many fluorophores are active at the start of the trace. Although it is Adoprazine (SLV313) easy to resolve … In addition, noise also arises from background Adoprazine (SLV313) fluorescence (Ulbrich and Isacoff, 2007 ; Coffman and Wu, 2012 ), variable fluorophore emission (Ulbrich and Isacoff, 2007 ), and fluorophore blinking (Bagshaw and Cherny, 2006 ) driven by core fluorophore instabilities (Drobizhev test (Chen is a data set consisting of fluorescence intensity measurements taken at constant time intervals and ordered in sequence by ascending acquisition time. Traces are typically obtained when fluorophore-tagged molecules of interest are illuminated at some time, say = 0, and then monitored until all fluorophores have photobleached. Photobleaching time traces present a model selection Adoprazine (SLV313) problem (Ludden {active fluorophores in a time trace, we say the trace is visiting is visited is (which may be different from 1 if fluorophores blink). We label the is the number of fluorophores initially present in the system; the number of active fluorophores can never exceed interval. {is the signal intensity at data point = {iterates over the states,|is the signal intensity at data point = iterates over the continuing states, over the number of visits to a particular state, and over the number of data points in an interval. The mean and SD of the fluorescence intensity at the interval are and = = and not the number of times that state is visited, Rabbit polyclonal to IL18 and so we can drop for notational simplicity. The quantities (where the subscript f designates single-fluorophore values and the subscript b designates background) are relatively easy to determine experimentally from the end of the photobleaching time trace. For these reasons, we will assume that these quantities are known (or, equivalently, that priors over their values are sharply peaked). Finally, the prime on the first product in Eq. 1 denotes a restricted product, meaning that does not necessarily run through all the states from 0 to I. This is because some states may not be visited at all (if two photobleaching events happen simultaneously), and thus may be 0 for some interval is drawn from a normal distribution of mean and variance into two groups, Adoprazine (SLV313) (is the number of steps (i.e., discrete jumps in the data), is the total number of events (defined as is the number of possible arrangements of single-level events (which we later ascribe to fluorescence Adoprazine (SLV313) intensity changes of magnitude steps (Figure 3), and are the mean and SD, respectively, and is a hyperparameter discussed in the refers to the total number of single fluorophore intensity level … Taking all of the above into consideration, we write the prior as (and are independent. We also dropped the hyperparameter, couples only to = 0, all fluorophores are in the active state and, subsequently, may only irreversibly photobleach. Because we have no blinking or overlapping events, the total number of events, has no meaning here and need not be considered (only one arrangement with one event per step is possible). For this simple example,.