Multi-kernel deconvolution applied to confocal fluorescence microscopy with engineered point spread function

Abstract

International audienceFluorescence 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

Improved and isotropic resolution in tomographic diffractive microscopy combining sample and illumination rotation

“…This mirror assisted tomography (which however has not yet been demonstrated experimentally), would probably constitute the easiest approach for performing isotropic, ultimate resolution tomographic diffractive microscopy. Finally, we would like to mention that other microscopy imaging techniques may benefit from specimen rotation: for example, combining fluorescence microscopy (which also exhibits anisotropic resolution in confocal as well as wide-field mode), with specimen rotation and multi-kernel deconvolution [44] would permit to significantly enhance the image quality and allow for an improved, isotropic 3-D resolution.…”

Saturated structured confocal microscopy with theoretically unlimited resolution

“…We obtain the same gain in resolution than with confocal fluorescence microscopy using laterally interfering excitation beams [21]. However, the final resolution is now isotropic with only two images, while this other method further needs recombination of several images (typically 4) in order to obtain an isotropic resolution [22]. Furthermore, producing an azimuthally polarized illumination beam from a linearly polarized beam seems to be easier than having three focused beam interfering at the focal point as proposed in Ref.…”

“…An inverse filter corresponding to the annular PSF may even permit to reduce the number of images to be taken to only one. Inverse filtering is however sensitive to noise, while multikernel deconvolution permits to even further improve the resolution, and has been proven to be very robust to both noise and kernel estimation errors [22].…”