Biology subjects

Kapishnikov, S.

Publications and source records attributed to Kapishnikov, S..

10 recordsLinked to original sources

bioRxiv2026

Multimodal 3D imaging reveals a central role for lysosomes indissolution of cholesterol crystals by macrophages

Formation of cholesterol crystals (CCs) is a key event during the development of atherosclerosis, but the molecular mechanisms of their degradation within cells are poorly understood. By incorporating the fluorescent cholesterol analogue TopFluor-Cholesterol (TF-Chol) into CCs, we were able to visualize the uptake of CCs in macrophages using correlative fluorescence and soft X-ray microscopy. Using quantitative 3D live-cell imaging, we show that CCs are processed in late endosomes and lysosomes (LE/Lys), resulting in formation of TF-Chol containing lipid droplets (LDs) over time. Inhibition of lysosomal sterol export with U18666A caused accumulation of TF-Chol in LE/Lys, and inhibition of lysosomal acidification with bafilomycin A1 led to reduced dissolution of the CCs. Using a novel assay combined with 3D image processing, we show that large CCs in contact with macrophages are processed via lysosomal exocytosis followed by extracellular and intracellular degradation of CCs. Treating macrophages with a fluorescent version of cyclodextrin (CD) promoted the dissolution of CCs and enhanced the formation of LDs enriched with TF-Chol. The majority of fluorescent CD co-localized with a marker for LE/Lys during this process, suggesting that intracellular delivery to LE/Lys may contribute to the dissolution of CCs. Dehydroergosterol (DHE) is an intrinsically fluorescent sterol closely mimicking the properties and behavior of cholesterol. DHE is known to self-associate into aggregates and crystals, and by using fluorescence spectroscopy and specialized ultraviolet (UV) microscopy, we found that CD enhances the dissolution of DHE crystals in vitro and in cells. Together, our findings highlight the lysosomal pathway as responsible for dissolution of CCs, cholesterol trafficking, and efflux in macrophages.

biochemistry↗

bioRxiv2026

Physics-aware measurement-supervised deep learning enables point spread function inversion in soft X-ray tomography

Soft X-ray tomography (SXT) is an emerging modality for whole-cell 3D imaging in near-native states. However, the effective spatial resolution is limited by optical artifacts characterized by the point spread function (PSF). Standard reconstruction methods force a compromise between structural sharpness and noise, failing to fully resolve these depth-dependent artifacts. By embedding experimentally measured, depth-variant PSFs into a differentiable forward model, we demonstrate a physics-aware computational optimization that bypasses these limitations to recover high-frequency cellular ultrastructure. The structural fidelity was validated using split-tilt Fourier ring correlation (FRC), alongside an experimental bead phantom tomogram, providing supporting evidence that the recovered high-frequency features reflect genuine specimen structure rather than fabricated artifacts. Our method effectively increases FRC spatial resolution and recovers cellular ultrastructure. Furthermore, under sparse-angular subsampling, the framework maintained spatial resolution using half the projection angles, a computational proxy pointing toward the potential for reduced radiation exposure in future acquisitions. This hardware-free, computational approach offers a route toward mitigating the optical and dosimetric constraints that currently limit nanoscale soft X-ray tomography.

Cell Biology↗

bioRxiv2025

Self-Supervised Missing Wedge Correction in Soft X-Ray Tomography: Towards Accurate Cellular Morphology and Volume Quantification

Soft X-Ray tomography (SXT) is a non-invasive bio-imaging technique that enables 3D imaging of cellular structures in large volume, with a unique resolution range that bridges the gap between fluorescence and transmission electron microscopy. However, a fundamental limitation, the missing wedge artefact caused by incomplete tilt-series acquisition, introduces systematic structural elongation in the reconstructed tomograms. This artifact compromises accurate quantitative biological analysis by overestimating cellular and organelle volumes. To overcome this persistent issue, we introduce a novel, self-supervised missing wedge correction model that learns key sine-wave patterns from existing SXT sinograms of the tilt series stacks. This model can be applied to recover the missing-angle region of the sinogram, reducing distortions and elongations in the reconstructed tomograms. We demonstrate a significant quantitative improvement in artifact removal, achieving faithful recovery of the spherical morphology of lipid droplets. We further applied this method to Plasmodium falciparum hemozoin crystals, a biomarker in antimalarial drug efficacy studies. Our model successfully reduced volume overestimation, achieving up to a 16% decrease in distorted volume. This level of precision is paramount for correctly interpreting the mode of action of antimalarial drugs.

cell biology↗

bioRxiv2025

Bridging the resolution gap in cryo-CLEM by introducing cryo-SXT: cryo-CLXEM

Cryo-imaging in cellular biology provides the means to visualize the cellular interieur at close-to-native conditions. A cornerstone in the field has been cryo-correlative light and electron microscopy (cryo-CLEM), with cryo-visible light fluorescent microscopy (cryo-VLFM) providing the specificity by tagging macromolecules or structures and cryo-electron tomography (cryo-ET) for the structural details at molecular level. The large resolution gap between these techniques, however, is limiting this correlative workflow as cryo-ET targets are often smaller than the resolution limit of cryo-VLFM. Here we introduce cryo-soft X-ray tomography (cryo-SXT) as an intermediate step that can compensate for the partial view and limited resolution of cryo-VLFM by providing invaluable cellular context information in 3D to the cryo-ET dataset within an integrated workflow.

cell biology↗

bioRxiv2025

SXTractor: A Self-Supervised Feature Extractor of Soft X-Ray Images That Enables Few-Shot Tomogram Segmentation

Soft X-ray tomography (SXT) is a powerful, non-invasive bio-imaging technique that enables visualization of cellular structures in near-native states. Despite its potential, the development of dedicated image analysis tools -- particularly deep-learning-based models -- has been limited, largely due to the limited accessibility of soft X-ray microscopes and the scarcity of labeled SXT data. To address this deficit, in this work, we present SXTractor, a self-supervised SXT feature extractor based on the DINO framework. SXTractor can be fine-tuned with minimal labeled data and effectively adapted to various downstream tasks. We demonstrate its utility on few-shot tomogram segmentation, where it significantly outperforms the model when trained from scratch. Furthermore, it achieves few-shot segmentation performance comparable to that of the Segment Anything Model (SAM), despite SAM being a segmentation-specific model pretrained on millions of labeled images with a significantly larger model size. Most importantly, SXTractor enables a diverse range of downstream applications of deep learning to SXT, thus offering a practical and scalable solution for SXT image analysis in data-constrained settings.

bioengineering↗

bioRxiv2025

Robust Deep Denoising of Soft X-Ray Tomography Data for Biological Research: Targeting Tilt Series Versus Reconstructed Tomograms

1.Soft X-ray microscopy (SXM) is a powerful tool for nanoscale 3D imaging of hydrated, intact cells. However, its application is limited by pixel-wise-correlated noise inherent in the imaging process. While the deep-learning-based framework Noise2Inverse has been proposed for tomography denoising, the lack of robust validation for newly revealed features raises concerns regarding their practical biological utility. We argue that denoising the raw tilt series, instead of on the reconstructed tomogram, offers a significant advantage by leveraging the tomographic reconstruction process to mitigate local prediction errors through averaging. Consequently, features present in the tomogram reconstructed from a denoised tilt series exhibit higher credibility, as falsely predicted details on certain tilt series slices are likely to be averaged out. Nevertheless, denoising the tilt series remains challenging due to the spatially correlated noise that occurs in bioimaging. Existing self-supervised methods relying on a single noisy image struggle with such noise, while the Noise2Noise framework necessitates paired noisy datasets for training. This study addresses these challenges by investigating practical imaging workflows in SXM and related bioimaging modalities to identify existing imaging resources as training data for tilt series denoising. We compare the denoising performance when applied to tilt series versus reconstructed tomograms and evaluate the efficacy of chosen methods on real biological specimens to assess their applicability within practical SXM research. Our findings establish a robust and efficient denoising strategy for SXM by directly addressing the complexities of noise in the raw tilt series. HighlightsO_LITargeting tilt series for denoising inherently validates the correctness of the denoised tomogram via the reconstruction process. C_LIO_LIExisting multi-frame imaging schemes provide necessary resources for training Noise2Noise framework for tilt series denoising. C_LIO_LINoise2Noise applied to tilt series reveals finer and more reliable details than direct denoising on reconstructed tomograms (Noise2Inverse). C_LIO_LIDenoising tilt series minimizes inference time compared to Noise2Inverse in soft X-ray tomography. C_LI

bioengineering↗

bioRxiv2024

Demonstrating Soft X-Ray Tomography in the lab for correlative cryogenic biological imaging using X-rays and light microscopy

Soft X-ray tomography (SXT) enables native-contrast three-dimensional (3D) imaging of fully hydrated, cryogenically preserved biological samples, revealing ultrastructural details without the need for staining, embedding, or sectioning. Traditionally available only at synchrotron facilities, recent advances in laser-driven plasma sources have led to the development of compact soft X-ray microscopes, such as the SXT-100. The SXT-100 achieves imaging resolutions down to 54 nm full-pitch, with tomograms acquired in 30 minutes to two hours. Integrated with an epifluorescence microscope, the SXT-100 facilitates correlative workflows by bridging fluorescence and electron microscopy while preserving the structural integrity of vitrified samples. We demonstrate the capabilities of the SXT-100 through various use cases, including imaging Euglena gracilis, Saccharomyces cerevisiae yeast cells, and nanoparticles in mammalian cells. The relatively short tomogram acquisition times, the virtually non-destructive nature of soft X-ray tomography, and its quantitative imaging capabilities underscore its potential as a powerful tool for advanced biological imaging. Future developments promise enhanced throughput and deeper integration with emerging correlative imaging modalities, and a wider variety of sample types including tissue.

biophysics↗

bioRxiv2023

Progression of herpesvirus infection remodels mitochondrial organization and metabolism

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized at the perinuclear area, a significant increase in the number and clustering of ER-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

microbiology↗

bioRxiv2020

A novel calcium-concentrating compartment drives biofilm formation and persistent infections

In nature, bacteria reside in biofilms - multicellular differentiated communities held together by extracellular matrix. In this work, we identified a novel subpopulation essential for biofilm formation - mineral-forming cells. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. The molecular mechanisms promoting calcite scaffold formation were conserved between three distant phyla - the Gram-positive Bacillus subtilis, Gram-negative Pseudomonas aeruginosa and the actinobacterium Mycobacterium abscessus. Biofilm development of all three species was similarly impaired by inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization both significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. The evolutionary conserved cellular pathway controlling the fundamental feature of biofilm development uncovered in this work offers novel druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

microbiology↗

bioRxiv2018

Analysis of cholesterol export from endo-lysosomes by Niemann Pick C2 protein using combined fluorescence and X-ray microscopy

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in late endosomes and lysosomes (LE/LYSs). How its capacity to transport cholesterol between membranes is linked to endo-lysosomal membrane trafficking is not known. Using quantitative fluorescence imaging combined with soft X-ray tomography (SXT); we show that NPC2 mediated sterol efflux is accompanied by large changes in distribution, size and ultrastructure of endocytic organelles. We observed clearance of intra-luminal lipid deposits, a decrease in number of autophagosomes, formation of membrane contact sites (MCSs) to the endoplasmic reticulum and extensive tubulation of LE/LYSs in three-dimensional SXT reconstructions of NPC2 treated human fibroblasts. The cells could recycle the cholesterol analog dehydroergosterol (DHE) from LE/LYSs slowly also in the absence of NPC2 protein but internalized NPC2 synchronized and accelerated this process significantly. Most fluorescent NPC2 was retained in LE/LYSs while DHE was selectively removed from these organelles, at least partially by non-vesicular exchange with other membranes. During sterol efflux LE/LYSs were reallocated to the cell periphery, where they could fuse with newly formed endosomes. Surface shedding of micro-vesicles was found, suggesting a pathway for cellular sterol release. We conclude that NPC2 mediated sterol efflux from LE/LYSs controls membrane traffic through the endo-lysosomal pathway.

cell biology↗