Image reconstruction enables high resolution imaging at large penetration depths in fluorescence microscopy

Abstract

Imaging thick specimen at a large penetration depth is a challenge in biophysics and material science. Refractive index mismatch results in spherical aberration that is responsible for streaking artifacts, while Poissonian nature of photon emission and scattering introduces noise in the acquired three-dimensional image. To overcome these unwanted artifacts, we introduced a two-fold approach: first, point-spread function modeling with correction for spherical aberration and second, employing maximum-likelihood reconstruction technique to eliminate noise. Experimental results on fluorescent nano-beads and fluorescently coated yeast cells (encaged in Agarose gel) shows substantial minimization of artifacts. The noise is substantially suppressed, whereas the side-lobes (generated by streaking effect) drops by 48.6% as compared to raw data at a depth of 150 μm. Proposed imaging technique can be integrated to sophisticated fluorescence imaging techniques for rendering high resolution beyond 150 μm mark.

The elegance of localization microscopy and the promise it holds

“…In tissue sample, effects such as, scattering, noise, absorption, background and autofluorescence are quite prominent. This leads to limited penetration depth as the PSF distorts at large depths (Dilipkumar et al, ). Localization techniques integrated with orthogonal detection and light‐sheet illumination has the ability to reduce the artifacts caused by these effects, facilitate selectivity and high numerical aperture (NA) at long working distance.…”

“…These factors are critical and severely hamper the image quality if not corrected for. Although image reconstruction techniques (Dilipkumar et al, ; Mondal and Diaspro, ; Mondal et al, ) are helpful, all‐optical solutions are preferred. The importance of localization technique that is essentially based on single molecule imaging has better selectivity and large field of view.…”