Basics of Lasers, Excitation and Emission

Most flow cytometers use single wavelength laser light as their excitation source. Many  machines have more than one laser which are used to excite specific fluorchromes. These can be co-linear or non-co-linear, fixed angle or non-fixed angle.

We have several machines with different co-linear lasers (375, 405, 488, 561,640-nm) and the user should contact the Core if they are unsure of which laser to use. The configuration of the lasers can be found under the individual description of the machines on this website and iLabs.

Two lasers which users may be unfamiliar with are:

1. THE YELLOW GREEN LASER  on the BD FACSAria III and FACSAria IIIU, and the SORP BD LSR FORTESSA has an excitation line 561-nm.

Which fluorochromes are excited at this wavelength?

– there are several new commercial fluorescent proteins available (e.g. mCherry, tdTomato, mStrawberry, mOrange, DSRed). Dr. Robert Campbell at U of A is an expert in protein engineering and has an comprehensive resource on these proteins here.

– this laser is reported (BD application note and Telford et al’s paper) to give a better excitation of PE so if you have really dim signals you  may now be able to better discriminate the signal from the background.

– this laser allows the ability to combine multiple FPs in one experiment e.g. CFP (violet excitation), GFP (blue excitation)  and tdTomato (yellow-green excitation) without compensation.

Further details are presented in this article in Nature Methods.

2. THE UV  LASER  on the BD FACSAria III and FACSAria IIIU, SORP BD LSR FORTESSA has an excitation line 375-nm.

Which fluorochromes are excited at this wavelength?

– lasers in the near-UV are attractive for exciting fluorophores, such as Hoechst Blue, for use in DNA analysis. They are also well suited for analysis of stem cell side population with Hoechst populations as described here.

Please note the new Brilliant Violet dyes offered by BD Biosciences and Biolegend are well suited for a variety of  lasers and can help expand your cocktail. They have the advantage of being very bright and more stable than tandem dyes, which is an advantage for low expressing proteins .

Spectra Viewer and Fluorescence

Many times users come to us and say they have a blue dye. This means nothing to us as there are many blue dyes and they need to be exited by different lasers and many probes will have long Stokes shift (e.g DAPI, PI).

Therefore we need to know the excitation wavelength (i.e. the laser) and the emission wave length.

We recommend that you become familiar with fluorescence and make use of Spectra Viewers such as the one from eBioscience- FluorPlan.

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