Laser photobleaching on the LSC.
Compared to flow cytometry, laser scanning cytometry utilizes an extremely long laser "dwell time" to analyze cells and capture C-scan images (milliseconds or seconds as opposed to microseconds). It is therefore important to choose fluorochromes for LSC analysis that are more resistant to photobleaching to ensure accurate analysis, particularly if multiple scans are required. We evaluated a series of common blue-green and red-excited fluorochromes for photostability on the LSC following multiple rescans. The results are shown below.
Method. EL4 cells were labeled with biotin-conjugated CD44 antibody followed by the indicated fluorochromes conjugated to streptavidin. The cells were then fixed in 1% paraformaldehyde, washed and wet-mounted. Cells were then scanned 8-10 times on the LSC; laser power levels were 25 mW for argon-ion, 18 mW for HeNe. Scan durations were approximately 5 minutes apiece and each repetitions were started immediately after completion of the previous scan. Data is expressed as percentages derived from the mean fluorescence intensity of each scan divided by the MFI of the first scan.
(Below). Photobleach resistance of blue-green excited fluorochromes. Fluorescein, Oregon Green 488 and Alexa Fluor 488 are shown for 10 scan repetitions.
(Below). Photobleach resistance of red-excited excited fluorochromes. APC, Cy5, PBXL-3, Alexa Fluor 633 and Alexa Fluor 647 for 8 scan repetitions.
Based on these evaluations the Alexa Fluor dyes have the greatest photobleach resistance to repeated laser scanning. We strongly recommend converting your applications to these fluorochromes for laser scanning cytometry, particularly where repeated scans are required.