25 years of Confocal Imaging

Saw this on BioTechniques…

A nice review of confocal imaging is available at Biotechniques website.

– Austin


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2 responses to “25 years of Confocal Imaging”

  1. Aditya Avatar
    Aditya

    Hello,
    I am Aditya working as a JRF at NCCS. I am working on primary mice macrophage obtained form injection of thioglycolate. I have to stain the CD40 receptor of these primary macrophages.

    I have tried to stain these cells but I am not getting clear and sharp images more over the cells are showing very week florescence of my target molecule, CD40 with a large back ground. If I try reducing the background by more number of washing then I am losing the cells from the chamber slide and if I reduce the concentration of the antibody the cells are not getting stained at all. I have gone as low as 1:20 dilution but still it is not working. If I increase the pinhole the background signals are high and also the image is not so sharp and crisp. I am using 0.5% PFA and 0.5 M NH4Cl, 0.01% PBSA for washing, Blocking 1% BSA + 1:100 Fc block, all at 4 degrees. To make a note when I stain for CD11b, a Macrophage marker, it is getting stained well but the same is not happening when I stain for CD40 in same condition. Can you please help me in troubleshooting this problem?

    Regards

    1. Austin Avatar
      Austin

      Hi Aditya,

      I think the answer to your question is actually found in your statement that, “…when I stain for CD11b…it is getting stained well…”
      Consider the possibilities:
      1. something wrong with the instrument, (somewhat disproved by the correct performance of CD11b).
      2. something wrong in the protocol, (again disproved if things are working on 11b).
      3. Another agent/compound present in the specimen which is increasing the background. Now this is a possibility if the fluorophore has been attached in a different way than when setting it up to label CD11b. i.e. is there some difference in the chemistry which might be increasing the background when targeting CD40, what is not exhibited when using -11b? One way to determine at least the background problem would be to compare background intensity levels between specimens loaded with similar dilutions of CD40 and CD11b. If they are equal, this indicates that the issue is related to the tagging. If the 40 background is much higher than 11b, it indicates something in the chemistry which is washing out the otherwise good signal in the 40 specimens.

      One other possibility is that the energy performance of the fluorophore may shift when set to tag for CD40. i.e. it may excite and emit at longer, or shorter wavelengths. This basically does happen in many specimens but the shift is not so sufficient that different excitation or emission filters are required. This can also be confirmed by loading a sample with the fluorophore as set for -40 and then running it on a fluorometer. If you see a large discrepancy in the peak excitation and emission spectra between the prepped label and the raw fluorescent molecule you know this is the issue.

      I think the most likely possibilities are as follows, in order of likelihood:
      1. Label is not tagging to it’s target – this is confirmed if the background intensities of the (working) CD11b and (not working) CD40 specimens are roughly equivalent, i.e. signal is not present but noise is equal between specimens.
      2. High background due to some additional or different molecular structure between the -40 and -11b preps.
      3. Spectral shift only found in -40 but not found in -11b due to either the protocol (washing/blocking etc) or the actual binding to the target.

      I hope these notes help you find the cause of the problem! Also – are the molecular labels you are using for CD40 the same as CD11b? (IE are these all tagged with GFP or RFP etc) I only ask because if they are different there could be numerous issues with different label wavelengths on the instrument side…(Laser power, alignment, filter selection, objective performance and so forth). I Hope this helps!

      -Austin