sample preparation and 100X strong background

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  • #4640

    vane
    • New User

    Hello, I am want to observe the cell exposed to the nano carbon(less than 100 nm).But now I have 2 questions:
    1.When I am preparing the samples, I just culture the cell with carbon on the coverslip for several days, the wash for 4 times and fix them with 4% PFA. And them discard the PFA and wait for coverslip to dry out and then add 4 uL Glycerol Jelly Mounting Medium on the glass slide and then attach the coverslip on it. Is there some problem about my preparation? Should I wash the sample before I fix them? And the coverslip should be wet or completely dry our?
    2.If I suspended my nano carbon on the cell culture medium, how should I prepare them on the coverslip?
    3.I have no problem with observation under 10X and 40X magnification, but when I turn to 100X, the light are so strong and I could not find my cell, so in this case I turn down the light but the picture have a strong background and looked wield like this. Could this picture be used in the paper?And does this have sth to do with my sample preparation? Thanks~

    picture1: 10 ug/mL carbon under 100X
    picture2: 10 ug/mL carbon exposed with cell under 100X
    picture3: 0.1 ug/mL carbon exposed with cell under 100X

    #4644

    Stewart Mills
    Keymaster
    • Sophomore Member
    • ★★

    Yes, your images do look very bright. I am curious if you are using reflected optics and not the CytoViva illuminator which provides transmitted darkfield illumination from the bottom of the sample? I would expect much more contrast in the image with darkfield ilumination.

    You mentioned adjusting the light, but did you check the adjustable iris on the 100x objective? If this is opened too far it will let excessive light in.

    If you are using darkfield illumination and have checked the adjustable iris and are not getting better image contrast, I then have to suspect that your sample may have something in the medium reflecting excess light or possible an extremely high confluence of cells which can also lend to excessive light scatter. The goal with these types of samples in darkfield is to have somewhat dispersed cells and not a thick confluence.

    #4645

    vane
    • New User

    Thank you so much Mills. Those are the picture I took under 40X, but when I switch to 100X, it seems the sample are little over exposed and I couldn’t find my samples under microscope. So does this means there is some problem with my sample? By the way I used the oil under 100X and change no setup when I switch from 40X to 100X. Should I change some options?

    picture1: carbon under 40X
    picture2: cell exposed with carbon under 40X

    #4646

    vane
    • New User

    pictures are here~

    #4649

    Stewart Mills
    Keymaster
    • Sophomore Member
    • ★★

    Great. The 40x images look as we would expect for darkfield. The set-up from 40x air to 100x oil requires no special set-up other than adding oil to the top of the sample for the 100x oil objective. Sometimes you may have to adjust the fine focus a half or full turn (estimated) when going up to 100x to find the precise focal plane as the two objectives are usually not on the exact same focal plane.

    Seeing your 40x images (they look as expected) makes me think the iris on the 100x objective is opened too much allowing in excess light. Have you tried adjusting the iris on the 100x objective? There are ‘grooves’ just above the white ring that you grip lightly with your fingers and twist left or right. If your 100x objective does not have this white ring and adjustable iris, you may not be using the correct objective with the NA designed for darkfield imaging.

    #4650

    vane
    • New User

    OK~thanks a lot.I will try to find the iris and adjust it.
    By the way, when I am preparing samples, should I dry out the coverlip or keep it wet?
    And how should I prepare my carbon sample suspended in the medium? Could you give me some advice?
    Thanks~

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