Dear all,
I am using confocal microscopy to visualise membrane proteins of oleosomes (in any case proteins surrounding the oleosome) extracted from rapeseed.
The oleosomes have been stained with Nile red to detect the oil, and Fast Green FCF (0.013%) to detect the proteins.
The core oil is easily visualised but I am struggling to clearly visualise the proteins which would surround the oil droplet.
Do you have any advice on how to improve the visualisation? It may depend on the microscope settings I guess.
Thank you very much to anybody who could help me
Filippo
Filippo Bramante Not enough info. It is not really clear what the problem is, could you share a picture and say exactly what is wrong and what you want to do?
Hi Yulia, many thanks for your reply. I am trying to visualise oilseed rape oleosomes by confocal microscopy. I stained the core oil with Nile Red, and the surface proteins with Fast green FCF. The oil is clearly visualised as a red droplet. The surface proteins should appear as a green ring around the red droplet, or as green spots on the red droplet, according (I believe) to which section of the droplet is visualised.
Image 1 has been taken from De Chirico et al. (2020; https://doi.org/10.1016/j.foodchem.2020.126355), which shows the distribution of the proteins (green) around the oil droplet (red). The separated channels are also showed.
Image 2 is another example from the same paper, where the protein can be seen as a ring surrounding the red oil disk.
My image is instead Image 3. I can still observe the red and green components, but the green areas do not seem to be correctly distributed.
I am using the same microscope (Zeiss LSM880) and same method of detection for nile red and fast green FCF (images acquired with an x63 oil objective, with Argon and HeNe laser for nile red and fast green, respectively). For fast green, excitation was at 633 nm, and a band pass filter between 640 and 700 nm for the detection of the dye was used.
The only difference I can imagine is that I am diluting the oleosomes in buffer at pH 9.5 rather than water. This high pH does not affect protein stability, but may it degrade the stain?
I hope all makes sense. Please let me know if you need further info.
Many thanks
Filippo
Filippo Bramante I see. So, is the problem the "tail" or the lack of "crispy clarity" in the image? If 1st, then there are 2 scenarios: 1) this is ACTUALLY what your samples look like, this is valid data and should be reported as such, or 2) your sample preparation is a problem, in this case try following the protocol from the paper you cited EXACTLY. If you still get same "tail" result, IT IS YOUR DATA. It is true and you should believe it.
But personally, I don't see a "tail", I think you can see that where there is a "tail", there is also some red, only at other depth, so to speak, so I don't see a problem.
If you are talking about clarity of the image, it usually depends on numeric aperture of the lense VERY much.
https://www.leica-microsystems.com/science-lab/collecting-light-the-importance-of-numerical-aperture-in-microscopy/#:~:text=Numerical%20aperture%20(abbreviated%20as%20'NA,are%20collected%20by%20a%20lens.
It even matters MORE than the lense's x-times magnification. NA is EXTREMELY important.
So, if you have good NA, then you may have a problem with adjustment. Any lens in a microscope has like, a ridge-ring around it, approximately in the middle. You can rotate upper part of the lens against lower part. Sometimes it can improve the image dramatically. If you have manual access to the lens, try rotating it while looking into the microscope.
There is like a "the finest point" when the image becomes crisp-clear, due to rotation.
I know it is hard to explain in words, try going to the microscope and looking at the lens. The ridge should look like this
https://www.microscope.healthcare.nikon.com/products/confocal-microscopes/a1hd25-a1rhd25/objectives
See? There are numbers, such as 0.17 etc. You can rotate it. But you have to look in the oculars at the same time, so you can see where you are rotating and what is happening.
Thank you Yulia Panina, I did more tests and the results are better (no tail and green ring around the red droplet), thus it seems to be matter of sample preparation.
Probably if pH is too high, the FCF dye loses its property.
But I beieve that playing with the lens could improve the image even more. I may repeat the tests considering your advices.
Many thanks for your interest.
Best wishes,
Filippo
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