Make sure the Edit › Options › Profile Plot Options setting “Do not save x-values” is off.The k value can be calculated in ImageJ by: Note that the plugin is expecting the k value to be “per-slice” rather than per-second, per-minute, etc. It may be worth performing a background subtraction prior to running the plugin. If you know the decay constant k, you can use the plugin Image › Adjust › Bleach Correction with the exponential fitting method. A mono-exponential decay is described by the equation:Ĭorrected intensity = (Intensity at time t) ÷ exp -k× t where k = decay constant bleach_correction_window.png The process of bleaching and decrease in image intensity can be fitted with a mono-exponential decay, although it often follows a bi-exponential. A stretch in contrast at the first time point may not be adequate 100 time points later. This can make it harder to discern events at the end of the sequence. Often, during acquisition of a time-course, the fluorophore may bleach and the intensity of the image is reduced. If you’d like to help, check out the how to help guide! Correcting for bleaching I hope this was somewhat helpful, and maybe others can pitch in their thoughts as well.The content of this page has not been vetted since shifting away from MediaWiki. I don’t really think there’s a way around that though, except by just making sure all of the step sizes are equivalent for the various z-stacks you compare (so if it’s happening in one, it’s happening in all of them). This is because he felt like it was possible for the same molecules to show up as fluorescence in more that one slice, and thus their colocalization could count double or triple. Someone from Nikon told me though that they felt it was important to have the incremental steps in the z-stack be the same for all of the z-stacks being compared (so even though one might be 30 slices and another 45, the step size for both should be the same). So a single slice is just like one quadrant of an image, but the value given is for the image in its entirety, if that makes sense. What I determined from all the sources I read and people I talked to was that the JaCOP plugin is NOT calculating an average, but instead just giving the coefficients as proportions relative to the total amounts of fluorescence that is there within the entire z-stack as a whole. Sorry it took me a couple days to see this and respond. I still can’t find the exact answer I’m looking for though… I have read that paper as well as a few others over the past few months, though I have to admit some of the information goes over my head. I have to give my thesis seminar pretty soon, and I’m afraid someone on my committee will end up asking me just how exactly these values are calculated from a z-stack…specifically if they are an average or not, but I just can’t seem to find that information anywhere! Thank you for the paper suggestion. The ones I have now range from about 35 to 50 slices. I’m glad to hear you think it is acceptable!īut since all of my images are z-stacks, I was trying to figure out how important it is for them to all be the exact number of slices. I’m trying to be as objective and consistent as possible in setting the threshold values, but I was also unsure just how much that is frowned upon. It seems like most of the papers I’ve read strongly caution against setting your own threshold values though, and instead advocate using the Costes method…but every time I’ve used that method it sets the threshold values way too low. The process you are describing sounds like what I have been doing with the JaCOP plugin which allows me to set my own threshold values and get a Manders M1 and M2 coefficients (red coloc/ total red and green coloc/ total green, which I think is the same as what you described, right?). Thank you for your reply! I agree about the intensity-based colocalization methods, which is also why I chose the Manders method because it was my understanding that it ignores intensity differences…but someone correct me if I’m wrong.
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