Three-dimensional bright-field microscopy with isotropic resolution based on multi-view acquisition and image fusion reconstruction

Abstract

Optical Projection Tomography (OPT) is a powerful three-dimensional imaging technique used for the observation of millimeter-scaled biological samples, compatible with bright-field and fluorescence contrast. OPT is affected by spatially variant artifacts caused by the fact that light diffraction is not taken into account by the straight-light propagation models used for reconstruction. These artifacts hinder high-resolution imaging with OPT. In this work we show that, by using a multiview imaging approach, a 3D reconstruction of the bright-field contrast can be obtained without the diffraction artifacts typical of OPT, drastically reducing the amount of acquired data, compared to previously reported approaches. The method, purely based on bright-field contrast of the unstained sample, provides a comprehensive picture of the sample anatomy, as demonstrated in vivo on Arabidopsis thaliana and zebrafish embryos. Furthermore, this bright-field reconstruction can be implemented on practically any multi-view light-sheet fluorescence microscope without complex hardware modifications or calibrations, complementing the fluorescence information with tissue anatomy.

Blind deconvolution in autocorrelation inversion for multiview light‐sheet microscopy

“…For this reason, multi-view acquisitions are frequently adopted as a method to obtain isotropic resolution (Calisesi et al, 2020;Swoger et al, 2007). Moreover, in the presence of slightly absorbing diffusive specimens (Rieckher et al, 2018), multiview acquisition facilitates the reconstruction of the entire imaged volume by accessing favorable views of the object.…”

“…In Light Sheet Fluorescence Microscopy (LSFM) (Huisken & Stainier, 2009 ; Olarte et al, 2018 ), the illumination and detection optical paths are independent and orthogonal to each other: the PSF depends on both the illumination and detection optics and, typically, results in a function that is more elongated along the detection axis. For this reason, multi‐view acquisitions are frequently adopted as a method to obtain isotropic resolution (Calisesi et al, 2020 ; Swoger et al, 2007 ). Moreover, in the presence of slightly absorbing diffusive specimens (Rieckher et al, 2018 ), multiview acquisition facilitates the reconstruction of the entire imaged volume by accessing favorable views of the object.…”

Blind deconvolution in autocorrelation inversion for multi-view light sheet microscopy

“…In Light Sheet Fluorescence Microscopy (LSFM) [2], [3], the illumination and detection optical paths are independent and orthogonal to each other: the PSF depends on both the illumination and detection optics and, typically, results in a function that is more elongated along the detection axis. For this reason, multi-view acquisitions are frequently adopted as a method to obtain isotropic resolution [4], [5]. Moreover, in the presence of slightly absorbing diffusive specimens [6], multi-view acquisition facilitates the reconstruction of the entire imaged volume by accessing favourable views of the object.…”

High-Resolution Volumetric Imaging and Classification of Organisms with Standard Optical Microscopy

“…Continuously rotating the sample can achieve full 360° optical imaging with a standard optical microscope , (Figure c). Achieving multiple views of the specimen is equivalent to rotating the optical transfer function in 3D.…”