I recently changed the Hg Lamp in our epifluorescent microscope and now for some reason the CFP and YFP channels do not work, they seem over-exposed even at exposure times as short as 10ms. However, the GFP and TRITC channels are fine. Any suggestions on how to fix this problem?
Without knowing the setup...have you checked to see that your excitation filters and / or the dichroic filter block is in the correct position. Did you have a neutral density filter in the light path somewhere to attenuate the excitation light? Is it still there? I presume the lamp was the same wattage?
Thank you very much for your response. I think the excitation filters are in the correct positions because the color of the light I see is the correct one for each channel, CFP and YFP. Yes, I am using an ND8 filter which is still there. I think the lamp wattage is the same but I will double check, however if this was the problem wouldn't all channels be affected? I am puzzled by the fact that only the CFP and YFP channels seem to be affected, all the others are fine.
Hi Virginia, again, I'm guessing as I don't know your system. However, the Hg spectrum is not constant across all wavelengths...if you google it, you will see that its actually very spikey. This means that depending on which band of wavelengths you choose with your bandpass excitation filters, the intensity of excitation light you get out will likely be different. Most groups have switched to xenon arc lamps for this very reason, because the xenon spectrum is pretty flat. Anyway, the upshot is, it is possible to have different excitation intensities using a Hg source. What you could do is just try to fix the situation that you now have. Is your light detected using a PMT or a CCD camera? If its a PMT, you could reduce the PMT voltage. Obviously, this would make all wavelengths less bright. However, on the system I used previously (fura-2), I had adjustable gains on the PMT outputa for each of 2 wavelengths, allowing them to be tweaked before dividing the signal to get the ratio. If its a CCD, this might not be possible.
Derek is right, moreover, your new lamp may be just brighter in then the old one. Can you just put a ND filter to reduce the intensity? Or adjust the position of the lamp that less light is coming to the sample.
I fixed it, the problem was that for some reason the filter wheels were going to the wrong positions. Thank you very much for your comments and suggestions.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence