Because of amplification with primary and fluorescent secondary antibodies, I am pretty sure that 100nm objects can be visible by confocal microscopy. I am looking for papers (maybe from physicist) that quantify and prove this. Can anybody help me ?
The maximum distinguishable size of anything viewed through microscopy is based upon the setup of your imaging system. This can be best understood by reading up on the point spread function. Here is a quick intro here by Zeiss. If you know all of the aspects of your microscope and what wavelength you are observing at you can calculate the smallest size objects that you would be able to able to distinguish in your final image.
http://zeiss-campus.magnet.fsu.edu/articles/basics/psf.html
https://en.wikipedia.org/wiki/Point_spread_function
Resolution is equal to lambda/2NA where NA is the numerical aperture of your objective lens. For the highest NA lenses (around NA=1.4) and typical wavelengths of light used for IF, this is equal to approximately 200 nm. Thus, your 100 nm object would appear to be much larger, diffracted to a spot that measures 400-500 nm wide in x-y. The measured full width half maximum (from an intensity line profile drawn across the point) would be about 200 nm corresponding to the resolution. Blame diffraction.
Many thanks for your clear answers. I dare another question : since we label these 100nm objects with 2 layers of antibodies to amplify the signal, do you think it can increase the size of a 100nm object ? to be more clear, immunolabeling has non significant effect on the size of a whole cell , but to which extend it could abherently amplify the size of nanometer objects ?
Dear Karine,
even in most Super resolution methods like STED or STORM the antibody size does not play the critical part in localization to some extend. As Christopher explain above the diffraction of the signal, the roughly 45 nm of the antibody complex (1st & 2nd) does not play an important role in conventional confocal microscopy.
In this article you can read about it and why for Super resolution microscopy where diffraction limitation is overcome the size does play a role.
http://www.leica-microsystems.com/science-lab/new-labeling-tools-can-help-to-realize-the-full-potential-of-super-resolution-microscopy/
Best regards,
Claus
Ok, so if I label a single round particle 100nm with 45nm antibodies (It is huge!), I reach 190 nm of diameter! still below 200nm resolution and indeed the particle I saw are more or less all 350-400nm in x-y. Now if I have 2 particles+antibodies stuck together it should reach ~380nm, above 200nm resolution, but it should appear bigger because of diffraction ?
thank you so much! I know, super-resolution is my dream but it is still not ready here.
Karine.
I think you also have to consider the orientation of the protein-antibody-complex.
But you do not get just one pixel of your structure. If you image according to the Nyquist criterion your pixelsize is 1/2.3 of the max resolution. So if your max resolution is 230nm your pixelsize should be 100 nm. So the pixel in the middle would be slightly brighter because it is at the center of the point spread function ..
Doing so you can deconcolve your images and get a better resolution. For more information on that: https://svi.nl/HuygensDeconvolution. Depending on the microscope software you use it has a button for optimal resolution (leica eg has it).
Maybe you have the chance to stay some time in Zurich. The center for microscopy (imaging facility) has both a GSD/STORM and a STED setup.
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