Abstract
Fluorescence microscopy is a powerful technique in biology, because of the immense variety of markers now available. Compared to other methods, its resolution is however limited. In wide–field microscopy, the technique of structured illumination permits to improve the lateral resolution by a factor of two, even surpassing confocal microscopy, which permits a theoretical gain of about 40%. We propose an alternate technique, combining laterally interfering focused beams, which should permit the same gain of resolution in a confocal microscope. Furthermore, this technique, combined with multiple acquisition and multikernel deconvolution, permits a better object reconstruction than classical monokernel deconvolution using a regular excitation point spread function.
Highlights
At the end of 19th century, Ernst Abbe [1] introduced for conventional microscopy his resolution criterion as: RAbbe = 0.5λ/N A, (1)with λ being the wavelength of observation and N A being the numerical aperture of the objective, defined as N A = nsinα
This result about the robustness to errors in estimation of kernels, obtained in the case of asymmetric Point Spread Function (PSF), recalls those obtained by Goudail et al [24] who considered multikernel deconvolution with symmetric PSFs, but of different sizes, and confirms, for confocal fluorescence microscopy, the advantage that multikernel deconvolution with different engineered PSFs would have over averaging deconvolutions performed with a regular PSF, or over multikernel deconvolutions with the same regular PSF
We detail some advantages and drawbacks of our approach, compared to other, more better established, methods to improve the resolution in fluorescence microscopy, and conclude by mentioning some types of fluorescence microscopies, which may benefit from multikernel deconvolution
Summary
The probability of excitation Pe is modeled by the excitation PSFexc, so that the confocal PSF is in first approximation given by multiplying the illumination PSFill by the detection PSFdet (the detection PSFdet being even wider because of the Stokes shift) Multiplying both PSFs leads to a lateral resolution improvement of about 40%, for an infinitely small pinhole. Structured illumination is a wide–field technique using a sinusoidally modulated excitation, and does not require a detection pinhole As a consequence, it has the advantage (compared to confocal microscopy) that no emitted fluorescence is discarded.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Journal of the European Optical Society-Rapid Publications
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
