What type of instrumentation are you using? It may be different for flow cytometry, fluorescence plate readers, microscopes, etc.

microscopes. But for my question it shouldn't matter actually. You might want to think of mult-spectral area detectors, i.e., PMTs in confocal or two-photon scanning microscopy, or flow cytomety or even plate readers. I am basically looking for a tool to assess excitation cross-talk and emission bleed-through

Martin-- thanks for the clarification. I'm not aware of any software that does this (other than FlowJo for flow cytometry) for most applications. That doesn't mean it doesn't exist, but I've always had to do it the hard way: know what my excitation filter's range is, what the dichroic mirror (if used) transmits/reflects, and what the emission filter's range is. Some are simply cutoff filters, others have a defined range (bandpass and longpass filters). Not all instruments use an excitation filter--some use lasers of defined wavelength and bandwith. Both wavelength and bandwidth are important criteria. Since most fluorescence microscopy relies on broad-spectrum, white-light illumination (often from an Hg bulb source), filters are the most common setup. You want the most specific excitation you can reasonably achieve so that you can minimize photobleaching of other fluorophores in the same sample, and to give the best signal-to-noise ratio (you do not want excitation light bleeding into your emission detector).

Depending on your microscope's CCD camera, it may and SHOULD record light intensity on three channels: red, green, blue, independently. The images you see are actually pseudocolour based on the intensity of light for each channel--and then the channels get composited into one image. Most microscopy systems I've worked with save images as uncompressed TIFFs, in which the R, G, B channels actually DO have separate information. You can use Adobe Photoshop to determine the intensity of light for each color channel--so if you're using GFP, for example, the green channel should have the highest signal, while the red and blue channels should have relatively lower background. You can subtract that background. There are tutorials online--if anyone is interested, I can try to find a good one that's still up--but it's the old and difficult way. Most newer camera software can do that for you (so can MetaMorph).

Hi, Christina, thanks for your detailed reply. This is very kind of you. I was thinking to formalize the problem so as to provide a software tool that would do the job conveniently, but I couldn't believe that there was nothing on the market. It is such an obvious problem and, much as you say, most if not all folks seem to do it "the hard way"...

Martin:

That would be very helpful! The biggest problem I foresee is that the setups vary so much from lab to lab. In addition, I know my microscope has a GFP filter cube, but I honestly do not know which filters are in it--I will have to disassemble the microscope to remove the cube and check (it has been in the microscope since before I began working here and nobody knows exactly which filters they are). It seems that the best option would be something like a plugin for Photoshop that could quantify the "bleed-through" fluorescence from ANY image and display it separately. The other issue is the sensitivity range of the cameras. Most CCD cameras don't see in colour to start wtih--they just see light-bright and light-dim (I know that is an oversimplification), but their response range varies, too--how sensitive are they to red vs. yellow light? Or UV light? I am actually getting a new microscope camera next week and this has been on my mind lately!