Before purchasing reagents or staining samples, it is imperative to make sure you have a microscope with hardware that can image the probes. In general, microscopes have fixed wavelength filter blocks for imaging fluorescence. Typically, these do not include Alexa Fluor 594 and a new generation of near infra-red probes. But some microscopes do have these capabilities. Check before doing the experiment.
This table shows the most common filter block availability and, in general, probes that work well.
Before you label your samples, it is best to check that the microscope you need to use has the hardware to match the reagents. Most probes are excited at 405, 488, 568, and 633 nm with short Stokes shifts such that the emissions are detected in the next longer wavelength range (violet to blue, blue to green, etc.). This is standard and the table lists many of the common probes.
This is complicated by the 405 nm lasers on confocals. Some of the blue probes for widefield fluorescence that work well with Dapi filter block do not work on the confocal. For instance Alexa Fluor 350 works well on widefield fluorescent scopes, but does not work at all on the confocal microscopes. Alexa Fluor 405 is ok with widefield scopes whereas ideal with 405 nm laser using confocal.
A few probes have longer Stokes shifts. For instance, look at Brilliant Violet series of probes. They all excite in the 400-420 nm range but emit at different colors all across the spectrum. One wavelength, such as a 405 nm line on the confocal, can excite multiple emitting dyes that have easily separable emissions such as blue (Brilliant Violet 421), red (Brilliant Violet 570) and near infra-red (Brilliant Violet 711). Used in combination with the shorter Stokes shifts probes, specific labelling of more than 4 molecules can be imaged in sequence without complex spectral deconvolution. I would recommend trying one that emits within the window of 550 to 740 nm. Beware trying to use with Dapi as emissions shorter than 600 nm might have dapi spillover. Also, standard confocal cannot detect longer than 750 nm.
Please consult with us for other special combinations, for instance dyes that excite at 490 nm but have a long Stokes shift to emit in the red or far-red. Also, there are a lot of options not listed here and a few other ways of generating contrast such as reflection and harmonics in multi-photon.
Other useful tools:
Our favorite fluorescent probe spectra resource as of Aug 2018:
If you have experience with these, please let us know!
You may want to try recombinant single-domain antibodies (sdAbs) derived from llamas or alpacas. http://nano-tag.com/products
Multiple rounds of staining is a tedious way to do multiple probes, but here is one method:
We occasionally get questions about Alexa 700 and similar near-IR dyes. We do not have the filter blocks for any of the widefield epifluorescent scopes. The confocals (depending which one) only detect below 700 to 740 nm. And even if they did, users would have to change focus due to chromatic aberration. Also, we do not have the correct excitation lasers.
This is how the author of this web page perceives the colors.