Hi guys, we can break the diffraction limit by narrowing the Point Spread Function (PSF), I want to know that what are the possible ways to narrow the PSF ??
The minimum point spread function of your optical system is limited by the diffraction limit, so the only means of changing it would be to change your aperture sizes and/or illumination wavelength.
In super resolution microscopy, sub diffraction limit imaging is possible but this is achieved given understanding of the PSF. Florescent markers within the sample are activated, in such a way that a low number will statistically fluoresce . Each will be diffracted when imaged, however given the low number of objects within the image, and by quantifying the PSF of each, the centre of each object can be defined with high precision. Repeating this a number of time allows a high resolution image to be ascertained.
Point spread function can be defined as the image obtained from an imaging system for a point object. Using this definition, modern day superresolving microscopes, such as STED, PALM and STORM, have PSF's no smaller than the underlying optical system can afford. The improvement in resolution is achieved by manipulating the illumination, either in the intensity level (as in STORM and PALM), or the shape of the illumination (STED). Imagine if the illumination is point like, with a diameter of say 5nm, that would be the imaging resolution of the system, regardless of what the numerical aperture of the imaging system is. Or as explained in ANS (1), by using low illumination intensity, the number of fluorescent particles excited is kept very small, the location of individual particles can be determined much more precise that the resolution limit.
@Chung Wah See and Alexander Malm Thanks for your reply, I mean by post-processing by using some mathematical formulas we can also shrink the PSF is not it?
You can, actually, improve your diffraction limited resolution by altering the point spread function optically. I have published two articles on this if you are interested. I believe though that you are referring to deconvolution, a post processing technique which attempts to remove the effect of a point spread function on an image. There are lots of different ways of deconvolving an image which are specialised for different applications.
Article Single-image far-field subdiffraction limit imaging with axicon
Article Imaging performance of Bessel beam microscopy
@Craig Snoeyink thanks for your reply can you tell me please how much axial resolution we can get by using BEM?
@Kaleem Ullah, it depends. If you are localizing a particle in three dimensions as in 3D STORM, PALM, or the Double-Helix method then we have experimentally gotten around 7 nm axial resolution at 3000 photons collected and we predict that we can do better than that if we solve some spherical aberration problems (we are currently working on this manuscript). This requires specialized algorithms though which I am happy to share after we publish on them.
If you are interested in using BBM as you would when doing 3D deconvolutions (take a stack of images, run an algorithm that removes the 3D effect of the PSF and returns a 3D reconstruction of the object) then the axial resolution is *much* worse than for a traditional microscope. Using a 1.45 NA 100x oil immersion objective would result in an axial resolution of greater than a micron, the base microscope by itself would have better than 500 nm axial resolution.
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