In order to obtain optimal results from the flow cytometry analysis, especially when performing multidimensional analysis, it is vital that the appropriate combination of fluorochromes is used. When choosing fluorochromes there are a number of different parameters to take into account. Each fluorochrome has distinct properties and is characterized by specific excitation and emission wavelengths. First, the fluorochrome must be excited by the lasers available on the instrument. Second, the emission wave lengths are read by different detectors or photomultiplier tubes and the range of detection is limited by optical filters.
Currently, the Gallios has a five + one configuration with five photomultiplier tubes for detecting light emitted from a 488 nm excitation source and one for detecting light emitted by the 638 nm excitation source. For example the FL-1 photomultiplier tube is preceded by a 525/40 bandpass filter, this allows wavelengths of 525 +/- 20 nm to pass through to the FL-1 photomultiplier tube.
Similarly, the FL-2, FL-3 and FL-4 photomultiplier tubes are preceded by specific bandpass filters, while the FL-5 is preceded by a 755 nm long pass filter that will allow any wavelength >755nm through. A similar configuration is available on the MoFlo XDP cell sorter with four detectors for light emitted by the 488 nm excitation source and one each for light emitted from the 355 nm UV and 635 nm excitation sources. Furthermore, the individual filters on the MoFlo XDP are interchangeable and can be further optimized.
Gallios filter configuration
MoFlo filter configuration*
*The filter configuration on the MoFlo is interchangeable.
Some of the most commonly used fluorochromes and their excitation, emission wavelengths and other properties are listed below:
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