#Isolating z and t plane imaris software
#Isolating z and t plane imaris trial

If you define a (nearly) diffraction limited P1G spot, I'd be looking for what's in that exact area in the other channel P2R. You have to consider that it's biologically possible for the proteins/areas of interest to co-localize, because it's subjective as to how you define your 'overlapping' area.ДЄbsolutely, I'd be concerned about overlap. I would say don't use Elements - it's too much trouble to figure out this sort of thing on there.

I wouldn't necessarily recommend it for what you're doing, but I know there are ImageJ scripts for machine learning if you have very irregular looking images. Maybe this steers you in the right direction? They have some pretty good online tutorials on these sorts of things, as well. Z-stacks have always worked well for me, and if you do a max intensity projection or something of the like, you'll lose the 3D aspect of your image and it will be more difficult to determine co-localization. You should be able to shift-click or left click to add/delete. If you auto-generate the spot ROI's, they'll all be the same size, but you can add spots manually, as well. You should be able to subtract background and add spots for each channel independently. It's important you try to eliminate background, but you should do it separately from the spots tool. I'm not really sure what you mean by your question about background subtraction. When you auto-generate spots, you can specify their dimensions, but also the shape, and when you co-localize those, you have to consider how close that is to what you're seeing.

Some caveats, you have to consider that it's biologically possible for the proteins/areas of interest to co-localize, because it's subjective as to how you define your 'overlapping' area. I've used Imaris several times for the exact thing you're mentioning, and it has worked pretty well for me. What are your spot-finding algorithms in other programs? ImageJ? Icy? Elements?

#Isolating z and t plane imaris software

So, these are things I'm thinking about and no one selling this $20,000 software package wants to spend ten minutes explaining to me:ДЎ) Is there background subtraction (based on the 8/9 diameter thing) in BOTH the Green and Red channels, or just the selection channel?Дў) Are all spot ROIs the same size in a 2D image? When I try to filter on X or Y diameter, why are dim and bright vesicles ALL the same area? That doesn't make any sense.ДЈ) Is the 'Quality' filter basically circularity?Д¤) Would I be better off taking Z-stacks? Is that going to change the way area/volume are measured to my benefit? This means, in the most basic sense, I'm interested in answering, 'If there is a P1G spot, how much P2R is there?' I'm interested in how P1G and P2R co-vary. I'm not interested in knowing about colocalization in the Manders/Pearson or yes/no sense. I'm looking at < 400 nm vesicles that contain Protein1-Green (P1G) and Protein2-red (P2R). That said, I need to know what the 'spots' functions are capable of describing.

#Isolating z and t plane imaris trial

Are there any Imaris users out there? I can't recommend the BitPlane/Imaris package since I've had extremely poor contact with sales/customer support during my 10-day free trial period.