I've heard that filtering of the results based on the uncertainty value should be done to all the images and the treshold value for uncertainty depends on fluorophore. For Alexa Fluor 647 I have filtered the data followingly: uncertainty < 15.0. However, I have not managed to find any literature about this issue, i.e. which uncertainty tresholds should be used for different fluorophores. Could you help me with this? I'm currently setting up a multicolour STORM system and have to find a way to treat the data from different fluorophores.
Thanks a lot!
-Miina
and thanks a lot for your answer! I was also thinking the option of determining the uncertainty level based on the data, so after your recommendation I will definitely try that. However, if I want to do some quantitative image analysis and compare different samples, I have to use the same level throughout the whole sample set to keep them comparable. But I guess I can just evaluate several good images of certain fluorophore and imaging buffer, and then end up using a fixed uncertainty level value obtained from these images for all the images with this same fluorophore in the same buffer, independent of the target. Do you agree?
Do you have any rule of thumb how you choose the ROI? Or any statistical justifications, e.g. 95% of the datapoints have lower values than the chosen level etc.?
Thanks again!
-Miina