Laser line selection

In conventional HBO mercury light or Xenon light, besides peaks at certain wavelength, there is also continuous low percentage light available for the spectra outside the peaks, refer Part 1, Fluorescence Microscope. Taking the famous FITC as example, its Ex peak is 490 nm,  HBO light provides only less the 10% intensity relative to their peak position at 350 nm. But it still enough for FITC due to the fact that FITC has high molar extinct coefficient and high quantum yielding.

In case of laser, wavelength selection is critical since laser does not provide continuous wavelength, instead, it offer monochromatic, discrete narrow peaks.

New fluorophores on the market are ever growing but laser lines available are limited, and to be worse, the installed laser lines on a given system are even more limited. So, it is common that you can not find a perfect match of laser line for your fluorophores.
How to choose appropriate laser line is a concern that need more discussion.

When you buy a new fluorophore, the datasheet included usually mention the peak (100%) value for its excitation and emission wavelength. This is a relative scale indicates at which wavelength the fluorophore absorbs light and emits fluorescence most. This does not means you have to use the peak value since at certain range of wavelength, you get less percentage but still effective excitation.

As a thumb of rule, 25% is regarded as satisfactory, 30-50% is good, 50-80% is very good, and more than 80% is excellent. This is just a general guide line, it varies with fluorophore' extinct coefficient and quantum yielding.

If no perfect match between the fluorophore' peak value with existing laser line, you need to know how many percentage it will be excited at other wavelength. Unfortunately, most data sheet included with the package does not provide that information. Molecular Probe provides text files listing spectra data for almost all of their products, which can be download from their website.

To help user to choose excitation laser lines for fluorophores, here I made a table listing some commonly used fluorophores and available laser lines on this system and their corresponding percentage of excitation and emission.

Table 1. Fluorophore Spectral data and corresponding laser for Excitation (PDF file)

Note:
 1. The percentage in table 1 should not be used as the setting for AOTF percentage (as more than one user asked). They speak totally different things. value in table 1 indicate the percent of fluorophore excited under one laser line. AOTF percentage means the percent of light from laser source delivered to the specimen. Maybe, the low value in table 1 implies you have to use high AOTF percentage and vice versa.
2. The range in parenthesis indicates the 20% boundary on both side, at that wavelength, the intensity drop to 20% of its peak value. Value with * means until there, it has not dropped below 20%, further data is not available.

Special attention at choosing fluorophores for this SP2 confocal microscope setup:
1. For green excitation, due to the Krypton  568 nm line used in stead of the traditional HeNe 546 nm line, Alexa 568 is more suitable than TRITC for this laser: 81.53% vs.51.77%.
2. For Red excitation, Texas Red is not a good choice because we do not have HeNe 590 nm line, Kr 568 excites it only 35% while HeNe 633 excites it at a negligible level. Of course, 35% can still be regarded as effective excitation.

Both TRITC and Texas Red can still be used here, but for best results, if there is no special reason for you to stick on them, please avoid them when you prepare specimen for this confocal microscope.