I used a conventional fluorescence microscopy (mercury burner) to image the Calcium influx of a leukocyte. Firstly, the leukocyte were pre-loaded with Fluo-4 AM, and then treated with Thapsigargin and A23187 to observe changes in the cytosolic Calcium concentration. I take pictures every 1 minute (by switching the shutter). However, I have encountered some problems in the results. I have seen a gradual decrease in the fluorescence intensity of all samples (including both A23187 and Thapsigargin -treated cells). It did not increase as I expected. Importantly, I also observed such gradual decrease in the intensity of the untreated sample (cells with the medium only). So I am wondering that does this problem comes from my fluorescence microscope? Or is there any problem in my procedure?
This is my first time to perform the experiment with Calcium influx. I am very grateful for all your comments.
The gradual decrease of fluorescence could be due to photobleaching of Fluo-4 AM under your microscope. I am not experienced with this particular marker, but I would employ the same imaging procedure for all samples (same exposure times, temperutre, etc..) and compare treated samples with untreated at each timepoint (so that photobleaching is the same for both).
You could also try, to confirm this is due to bleaching, to image treated and untreated samples at the time of expected maximum effect, without pre-exposure to light. They should be brighter than samples imaged every minute.
prolonged exposure to UV light tends to degrade the fluorescent compounds, causing photobleaching. you can use 10% glycerol containing0.1% p-phenylene diamine to minimize the photobleaching
Dear Silvia Torchio ,
Thank you so much for your reply
I did not observe such decrease in background intensity.
I have not understand what was the speed that you mentioned about? My samples were exposure to UV light to take pictures every 1 minute. I started observe this gradual decrease right after the first 3 minutes.
In addition to such decrease in fluorescence intensity, I further recognized an excretion from a number of cells. (When all the pictures were displayed continuously over time, it was like some fluorophore bubbles released from the cells). So I think maybe there are two main reasons for my problems: the first is photobleaching as Gabriele Casirati and Sharad Purohit mentioned, and the second is the excretion from the cells.
If you have any recommend please let me know. I will try to solve my problem and show you the results.
Dear Gabriele Casirati
Thank you so much for your suggestion!
I will try to figure out the problem of photobleaching.
About my procedure, I do not tend to compare the difference of the samples at each time point. I just want to check the effect of A23187 or Thapsigargin after treatment with cells independently. As I predicted, the fluorescence intensity will increase after treatment during a certain period of time. But unfortunately, it decreased over time. I will try to solve it and tell you the result, thank you so much!
Dear Sharad Purohit
Thank you so much for your comment!
I will try to use glycerol and p-phenylene diamine . Further, I recognized an excretion from a number of cells (not all the cells). When all the pictures were displayed continuously over time, it was like some fluorophore bubbles released from the cells. So in addition to dealing with photobleaching, I will try to use Probenecid to slow the speed of excretion.
If you have any idea, please suggest me.
The problem was my buffer that I used to load Fluo-4. At first, I used RPMI (-FBS, 1%P/S). Then I have changed to use HBSS (-) without Calcium and Magnesium. Now I could observe the appropriate results. Although this work has been resolved for a long time, but hopefully this update can help those who have the same problem.
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