Selecting a fluorophore

I'm assuming that anyone who reads this knows what a fluorophore is, so I'm only going to talk about how to select the right one for specific applications. Remember that the lasers at the confocal microscope have distinct wavelengths. At Brandeis, the options are: 458, 476, 488, 514, 543, and 633nm. This means for example that it is not a good idea to use Texas Red/Alexa594, since the excitation maximum is far away from both the 543 and 633 laser lines.

Single labeling

There are obviously no constraints in terms of separating signals when using single labels. However, a couple of things have to be considered. The maximum optical resolution is limited to about a half of the wavelength of the light. In theory this means that fluorophores with low wavelength (blue light) excitation are better. On the other hand, autofluorescence from endogenous fluorophores in biological tissue is decreasing with increasing wavelength, so "the redder the better". But keep in mind that fluorophores excited by red light (like Cy5 or Alexa633) emit light in the far red part of the spectrum and are barely or not at all visible for the human eye. That means you can only see your staining under the confocal microscope. For a lot of things it's just nicer to be able to eyeball your preps under a normal epifluorescence microscope first to see if the staining is any good. In addition, the fluorophores with green excitation spectra are generally brighter than ones with blue or red excitation spectra. So I would always recommend to use fluorophores that are excited by green light (e.g., tetramethylrhodamine [TRITC], Alexa568, Cy3).

Multiple labeling

The good news is that for using multiple stains we are not limited at all anymore by the kind of beamsplitters and emission filters that we have. I will not go into details here about the technology, but the beamsplitter has been replaced by an acousto-optical device and the emission filters by prism optics. What this means is that we don't have to worry about the beamsplitter at all (so forget about it), and that we can freely (with 5nm resolution) select the emission range that we want to "grab" with any of the three detectors. This basically means that now separation of signals is only a matter of the spectral properties of the fluorophores used. The image on the left shows an example of a combination of three fluorophores. As you can see, both the excitation and the emission spectra are partly overlapping. There are basically three things to consider when you try to get good signal separation into three channels here (or 2, which is less problematic):

1) You want a good combination of fluorophores. Molecular Probes offers downloadable fluorescence spectra for all of their products (which include more or less everything but the cyanine dyes [Cy2, 3, and 5]): http://www.probes.com/servlets/spectra/. You can also get a file with spectra from here, that allows you to plot combinations yourself.

2) You want to set the emission filter ranges so that you get best possible separation. Keep in mind that signal separation is not only dependent on the spectral properties but also on the relative intensities of the signals. The spectra above show normalized intensities. In a real situation the relative intensity values at a given wavelength maybe completely different between 2 fluorophores. In general, you will have more channel bleedthrough with lower NA objectives. For example, I doubt that you can get good separation for the combination above with any of the air objectives.

3) If all else fails, there is always the possibility to achieve signal separation on the level of the excitation and not the emission. The idea is to only use one of the laser line at a time and get a significantly reduced response from the fluorophore whose excitation spectrum is further away form this wavelength. Our confocal lets you do that without having to do sequential scans. It can switch back and forth between scanning frames with different settings.

Here's a fairly old reference (so out of date in terms of fluorophores), but it's still useful for general principles:

Brelje TC, Wessendorf MW, Sorenson RL (1993) Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations. Methods Cell Biol 38:97-181.