Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS

Altered cellular biomechanics have been implicated as key photogenic triggers in age-related diseases. An aberrant liquid-to-solid phase transition, observed in in vitro reconstituted droplets of FUS protein, has been recently proposed as a possible pathogenic mechanism for amyotrophic lateral sclerosis (ALS). Whether such transition occurs in cell environments is currently unknown as a consequence of the limited measuring capability of the existing techniques, which are invasive or lack of subcellular resolution. Here we developed a non-contact and label-free imaging method, named background-deflection Brillouin microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling, to the best of our knowledge, the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our findings provide fundamental insights on the critical aggregation step underlying the neurodegenerative ALS disease

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds

Real-Time analysis and visualization for single-molecule based super-resolution microscopy

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy

3D + time blood flow mapping using SPIM-microPIV in the developing zebrafish heart

We present SPIM-μPIV as a flow imaging system, capable of measuring in vivo flow information with 3D micron-scale resolution. Our system was validated using a phantom experiment consisting of a flow of beads in a 50 μm diameter FEP tube. Then, with the help of optical gating techniques, we obtained 3D + time flow fields throughout the full heartbeat in a ∼3 day old zebrafish larva using fluorescent red blood cells as tracer particles. From this we were able to recover 3D flow fields at 31 separate phases in the heartbeat. From our measurements of this specimen, we found the net pumped blood volume through the atrium to be 0.239 nL per beat. SPIM-μPIV enables high quality in vivo measurements of flow fields that will be valuable for studies of heart function and fluid-structure interaction in a range of small-animal models

Simulated single molecule microscopy with SMeagol

SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation, and optimization of advanced analysis methods for live cell single molecule microscopy data. Availability and Implementation: SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction-diffusion simulations. Documentation, source code, and binaries for recent versions of Mac OS, Windows, and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net.Comment: v2: 14 pages including supplementary text. Pre-copyedited, author-produced version of an application note published in Bioinformatics following peer review. The version of record, and additional supplementary material is available online at: https://academic.oup.com/bioinformatics/article-lookup/doi/10.1093/bioinformatics/btw10

Efficient Bayesian-based Multi-View Deconvolution

Light sheet fluorescence microscopy is able to image large specimen with high resolution by imaging the sam- ples from multiple angles. Multi-view deconvolution can significantly improve the resolution and contrast of the images, but its application has been limited due to the large size of the datasets. Here we present a Bayesian- based derivation of multi-view deconvolution that drastically improves the convergence time and provide a fast implementation utilizing graphics hardware.Comment: 48 pages, 20 figures, 1 table, under review at Nature Method

Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. 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 quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams