Biology subjects

Alalouf, O.

Publications and source records attributed to Alalouf, O..

7 recordsLinked to original sources

bioRxiv2024

Monitoring DNA double strand break repair in Saccharomyces cerevisiae at high throughput and high resolution

DNA repair is critical for cellular function and genomic stability across organisms. Yeast mating-type switching serves as an established model for studying DNA break repair and chromatin dynamics. However, real-time tracking of mating-type switching in live cells remains challenging due to resolution limitations of existing techniques. Here, we use high-throughput methods, including three-dimensional imaging, to follow the dynamics of DNA damage and repair and to quantify mating-type switching occurrences at the single live cell level, with unprecedented resolution. We reveal chromatin reconfiguration for both single- and double-strand breaks following switching induction. Our findings provide new observation of the correlation between chromatin folding and single-strand breaks.

cell biology↗

bioRxiv2023

Large-FOV 3D localization microscopy by spatially variant point spread function generation

Accurate characterization of the microscopic point spread function (PSF) is crucial for achieving high-performance localization microscopy (LM). Traditionally, LM assumes a spatially-invariant PSF to simplify the modeling of the imaging system. However, for large fields of view (FOV) imaging, it becomes important to account for the spatially variant nature of the PSF. In this work, we propose an accurate and fast principal component analysis (PCA)-based field-dependent 3D PSF generator (PPG3D) and localizer for LM. Through simulations and experimental 3D single molecule localization microscopy (SMLM), we demonstrate the effectiveness of PPG3D, enabling super-resolution imaging of mitochondria and microtubules with high fidelity over a large FOV. A comparison of PPG3D with three other shift-invariant and shift-variant PSF generators for 3D LM reveals a three-fold improvement in accuracy and an operation speed approximately one hundred times faster. Given its user-friendliness and conciseness, we believe that PPG3D holds great potential for widespread application in SMLM and other imaging modalities.

bioengineering↗

bioRxiv2023

This microtubule does not exist: Super-resolution microscopy image generation by a diffusion model

Generative models, such as diffusion models, have made significant advancements in recent years, enabling the synthesis of high-quality realistic data across various domains. Here, we explore the adaptation and training of a diffusion model on super-resolution microscopy images from publicly available databases. We show that the generated images resemble experimental images, and that the generation process does not memorize existing images from the training set. Additionally, we compare the performance of a deep learning-based deconvolution method trained using our generated high-resolution data versus training using high-resolution data acquired by mathematical modeling of the sample. We obtain superior reconstruction quality in terms of spatial resolution using a small real training dataset, showing the potential of accurate virtual image generation to overcome the limitations of collecting and annotating image data for training. Finally, we make our pipeline publicly available, runnable online, and user-friendly to enable researchers to generate their own synthetic microscopy data. This work demonstrates the potential contribution of generative diffusion models for microscopy tasks and paves the way for their future application in this field.

bioengineering↗

bioRxiv2022

DeepOM: Single-molecule optical genome mapping via deep learning

Efficient tapping into genomic information from a single microscopic image of an intact DNA molecule fragment is an outstanding challenge and its solution will open new frontiers in molecular diagnostics. Here, a new computational method for optical genome mapping utilizing Deep Learning is presented, termed DeepOM. Utilization of a Convolutional Neural Network (CNN), trained on simulated images of labeled DNA molecules, improves the success rate in alignment of DNA images to genomic references. The method is evaluated on acquired images of human DNA molecules stretched in nano-channels. The accuracy of the method is benchmarked against state-of-the-art commercial software Bionano Solve. The results show a significant advantage in alignment success rate for molecules shorter than 50 kb. DeepOM improves yield, sensitivity and throughput of optical genome mapping experiments in applications of human genomics and microbiology.

bioinformatics↗

bioRxiv2022

DBlink: Dynamic localization microscopy in super spatiotemporal resolution via deep learning

Single molecule localization microscopy (SMLM) has revolutionized biological imaging, improving the spatial resolution of traditional microscopes by an order of magnitude. However, SMLM techniques depend on accumulation of many localizations over thousands of recorded frames to yield a single super-resolved image, which is time consuming. Hence, the capability of SMLM to observe dynamics has always been limited. Typically, a few minutes of data acquisition are needed to reconstruct a single super-resolved frame. In this work, we present DBlink, a novel deep-learning-based algorithm for super spatiotemporal resolution reconstruction from SMLM data. The input to DBlink is a recorded video of single molecule localization microscopy data and the output is a super spatiotemporal resolution video reconstruction. We use bi-directional long short term memory (LSTM) network architecture, designed for capturing long term dependencies between different input frames. We demonstrate DBlink performance on simulated data of random filaments and mitochondria-like structures, on experimental SMLM data in controlled motion conditions, and finally on live cell dynamic SMLM. Our neural network based spatiotemporal interpolation method constitutes a significant advance in super-resolution imaging of dynamic processes in live cells.

bioengineering↗

bioRxiv2020

VIPR: Vectorial Implementation of Phase Retrieval for fast and accurate microscopic pixel-wise pupil estimation

In microscopy, proper modeling of the image formation has a substantial effect on the precision and accuracy in localization experiments and facilitates the correction of aberrations in adaptive optics experiments. The observed images are subject to polarization effects, refractive index variations and system specific constraints. Previously reported techniques have addressed these challenges by using complicated calibration samples, computationally heavy numerical algorithms, and various mathematical simplifications. In this work, we present a phase retrieval approach based on an analytical derivation of the vectorial diffraction model. Our method produces an accurate estimate of the system phase information (without any prior knowledge) in under a minute.

biophysics↗

bioRxiv2018

Divergent interactions maintain the quaternary octameric structure of a new family of esterases

Protein oligomerization contributes significantly to the stability and function of enzymes, and the interacting interfaces that create the oligomers are expected to be conserved. The acetyl-xylo-oligosaccharide esterase, Axe2, from the thermophilic bacterium Geobacillus stearothermophilus represents a new family of esterases belonging to the SGNH superfamily of hydrolytic enzymes, and has a unique doughnut-like homo-octameric configuration, composed of four homo-dimers. The dimers of Axe2 are held together mainly by clusters of hydrogen bonds involving Tyr184 and Arg192, as was demonstrated by site directed mutagenesis. Dimeric mutants obtained by single amino acid replacements were inactive towards 2-naphthyl acetate, indicating the necessity of the octameric assembly for catalysis. The crystal structure of two homologous proteins (PDB 3RJT and 5JD3) reveal the same tertiary fold and octameric ring structure as of Axe2. Surprisingly, these octameric structures appear to be maintained by different sets of amino acids involving Asn183 in 3RJT and His185 in 5JD3 instead of Tyr184 in Axe2. These findings prompt us to investigate five more homologues proteins, which were found to have similar octameric structures, despite significant changes in their key residues. We revealed a conserved quaternary structure, which is maintained via non-conserved interactions.

biochemistry↗