Fluorescence Spectrum Viewers Tool.

                Spectra Viewers are the fluorochrome tools which help in understanding the wavelength ranges of excitation and emission of fluorochromes.. These tools are very helpful in determining specific laser, detectors and range of fluorescence spectra. When Multi-Color Flow Cytometry is the experiment, then these must be used to analyze emission spectrum, fluorescence spill over and compensation values.

How these tools help..

First, Lets consider a simple example of analyzing the spectrum for Green Fluorescence Protein (GFP) and you want to know the excitation wavelength and emission wavelength range for GFP.
By knowledge, you know that GFP will excite with 488nm Blue laser and emits the green fluorescence at apporximately 510nm.. Now, just open a spectrum viewer and select the GFP as shown below.

Now, you have analyzed GFP fluorescence and received the knowledge that It excites with 488nm Blue Laser and emission maximum is at 510nm.. So, in this case, the detector will be 510/15 BP filter. If the cytometer does not have this detector, then you can also use 530/30 BP Filter to analyze the GFP Fluorescence. (In case, why 530/30 BP Filter? It means, the detector is BP filter and it will collect the data between 515nm and 545nm wavelength range).

Just like GFP fluorescence, you can analyze whichever fluorescence of your interest and know the excitation and emission wavelengths.. (List of Fluorochromes Excitation & Emission).



The above example is for single color experiments.

Now, Consider an example of Multi-Colro Flow Cytometry experiment, in this case, you have more than one fluorescence. For Examplyou have tagged the cells with FITC, PE, PerCP and APC fluorochrome antibodies. Your aim is to analyze all these fluorescence in a single tube.. But, you should consider the required controls and compensation values for this experiment. (Compensation in flow cytometry).

Open the Spectrum Viewer, and select all these fluorochromes and choose the best excitation lasers and emission detectors for these 4 fluorochromes.
Example shown below.

                                     Source: Thermofisher Spectral Viewer

Now you have the perfect excitation sources and emission detectors for this example. here, FITC, PE, PerCP excite with 488nm laser and APC excites with 640nm laser. and you know the emission detectors for these fluorochromes in spectrum viewer. The percentages shown in the table represent how much fluorescence is collected in those detectors and how much spillover of fluorescence from detector into other detectors. These values are important for compensation purpose and accurately representing a multi-color data. 

Example (Explanation of those percentages). Consider FITC and PE fluorescence and these fluorescence molecules can be excited by 488nm blue laser and the respective detectors for these two are 530/30 BP and 585/29 BP detectors.

In this case, 530/30 detector is detecting 45.9% FITC and it is also detecting 0.4% PE. That means 0.4% of PE Fluorescence is spilling in FITC Detector. So, you will exclude that 0.4% of PE in compensation matrix. and 585/29 detector is detecting 53.4% PE and but it is also detecting 7.5% of FITC. That means 7.5% of FITC is spilling in PE Detector. So, you will exclude that 7.5% FITC in compensation matrix which in turn makes sure that PE in PE Channel and FITC is in FITC Channel.

To understand it practically and completely.. Go to "Compensation in Flow Cytometry".

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