Hi James, I'm a service engineer with BD in Australia. Regarding red diode laser reliability. That this is an issue is surprising to me. I've never found a faulty red diode laser (with or without cooling fins) in FACS Caliburs or FACSorts in the last ten months working for BD. Although I have replaced a blue tube laser in that time. Perhaps I've been lucky and you've been unlucky, and there is nothing more to the issue than that. I think it is worth pointing out that, if I encounter any of the following problems: - Higher than reasonable FL4-FL3 compensation - Time delay calibration failure - Reduced (or loss) of FL4 signal I've found most (~90%) are due to fluidics problems, not red diode laser failure. However the symptoms can appear identical. I'll try to briefly explain why this happens, however this does require a good depth of knowledge about how the Calibur works. Normal operation - Sheath flows at a constant rate through the flow cell. Cells take 20us to travel the distance between the red and blue laser intercept points, red diode laser first. The signals that have been exited by the red laser, into FL4, are held back electronically 20us so that FL4-FL3 compensation can be accomplished. (The 20us time can be changed a little, this is done indirectly by users when a time delay calibration is performed.) Fault condition - A partial blockage occurs in the waste line (I'm sure you clean your instrument regularly so this never occurs). The flow rate has reduced in the flow cell. Cells now take longer to travel the same distance between the red and blue laser intercepts, perhaps 60us. The blockage and resulting flow rate change 'appears' on the computer screen as if the red laser diode has failed. Assuming you are triggering on one of the signals derived from the blue laser only FL4 will disappear, the reason is all the blue laser signals, FSC, SSC, FL1, FL2 and FL3 are still coherently occurring and thus will be A-D converted, FL4 will not be coherent. To compensate for the fault (but the wrong thing to do to fix the instrument), the red laser intercept point would need to be moved closer to the blue laser intercept point, thereby restoring the 20us delay and restoring coherent signals from individual cells to the instruments electronics. I'm not suggesting a fluidics blockage is what caused the problem that started this thread. It is a fairly simple matter to put a laser power meter up to the red laser and check it's output. The point I want to make is that appearances can be deceptive. A note about the above, laser alignment is something service engineers only should do on the FACS Caliburs. Checking the sheath flow rate prior to a laser alignment would be a much wiser thing to do. Most well trained engineers will know to check flow rate before looking at deeper problems. >Typically, one will observe a decline in sensitivity in fluorescence >in channels that rely on the use of the diode laser, A minor point: In a properly aligned FACS Calibur, the aperture for the FL4 PMT is the only one that is focused on the red diode laser intercept. If any other "channels" are failing then the red diode laser is not at fault. I'm sorry about the inconvenience you have obviously suffered, I hope you are up and running again soon. I hope the above is of help to you and others. Regards, Malcolm Vickers Disclaimer: Please disregard all I've said if you believe working for BD somehow makes me biased. Opinions expressed are my own not those of BD.
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