Biology subjects

Silvestri, L.

Publications and source records attributed to Silvestri, L..

3 recordsLinked to original sources

bioRxiv2017

Increasing sensitivity and accuracy of brain-wide quantitative studies in light-sheet microscopy

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution. LSM is employed either in combination with tissue clearing to reconstruct the cyto-architecture over the entire mouse brain or with intrinsically transparent samples like zebrafish larvae for functional imaging. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data. Furthermore, using Bessel beams, we demonstrate a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Our results demonstrate the contamination of data by systematic and random errors through Gaussian illumination and furthermore quantify the increase in fidelity of such data when using Bessel beams.

neuroscience↗

bioRxiv2017

Whole-brain vasculature reconstruction at the single capillary level

The distinct organization of the brains vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network.

neuroscience↗

bioRxiv2017

RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems

Autofocus methods used in biomicroscopy are either based on the search of an optimal focus position - which requires suspending data collection during the optimization process - or on the continuous monitoring of the position of a fiducial plane - which may not coincide with the sample itself. Here, we introduce RAPID (Rapid Autofocus via Pupil-split Image phase Detection), a method for real-time image-based focus stabilization, applicable in all wide-field microscopy systems. We demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to light-sheet microscopy. RAPID provides a universal autofocus solution for automated microscopy, and enables quantitative assays otherwise impossible in a standard microscope, such as 3D tracking of fast-moving organisms.

bioengineering↗