Biology subjects

Prakash, K.

Publications and source records attributed to Prakash, K..

9 recordsLinked to original sources

bioRxiv2026

Quantifying the spatio-temporal image degradation under motion blur in fluorescence microscopy

The spatial resolution of optical imaging systems is fundamentally restricted by the diffraction limit. However, in widefield live-cell microscopy, the achievable resolution is further constrained by the specimen motion, which indicates the existence of a fundamental spatio-temporal resolution trade-off between signal accumulation during the full frame integration and the resulting motion blur. To improve the fidelity with which moving objects can be imaged, a quantitative understanding of this spatio-temporal trade-off is necessary. Here, we present a systematic analysis of motion-induced resolution dynamics measured with spectral signal-to-noise ratio (SSNR). We developed a simulation framework which models the image formation of objects undergoing arbitrary motion, to evaluate the degradation of the spatial resolution under translational and rotational dynamics. Our results demonstrate that for translating objects, the spatial resolution is anisotropically reduced as a function of the orientation of the object relative to the motion vector, leading to the spectral signal-to-noise ratio degrading by up to 50% and the resolution by up to 40% for a 90{degrees} change in the motion direction. Furthermore, we show that for rotational motion, conventional radially averaged metrics such as the Fourier Ring Correlation are not able to quantify the effects of angular blur. On the other hand, the SSNR is able to accurately quantify this degradation. These findings underscore the necessity of an object-oriented imaging approach, in which acquisition parameters such as exposure time are tuned to specific biological spatio-temporal characteristics to optimize the trade-off between motion blur and spatial fidelity.

biophysics↗

bioRxiv2024

CLEAR-IT: Contrastive Learning Enabled Accurate Registration of Immune and Tumor cells from multiplexed images with limited labels in a platform-independent manner

Accurate phenotyping of cells in the tumor microenvironment is crucial for understanding cancer biology and developing effective therapies. However, current methods require precise cell segmentations and struggle to generalize across different imaging modalities, limiting their utility in digital pathology. Here, we show that Contrastive Learning Enabled Accurate Registration of Immune and Tumor Cells (CLEAR-IT) overcomes these limitations, providing a robust and versatile tool for cell phenotyping. CLEAR-IT accurately phenotypes cells comparable to state-of-the-art methods, generalizes across multiplex imaging modalities, maintains high performance even with limited number of labels, and enables the extraction of prognostic markers. Additionally, CLEAR-IT can be combined with existing methods to boost their performance, whereas its lack of need for precise cell segmentations significantly reduces training efforts. This method enhances the robustness and efficiency of digital pathology workflows, making it a valuable tool for cancer research and diagnostics.

bioinformatics↗

bioRxiv2023

Correlative superresolution microscopy with deep UV reactivation

Correlative superresolution microscopy has the potential to accurately visualize and validate new biological structures past the diffraction limit. However, combining different superresolution modalities, such as deterministic stimulated emission depletion (STED) and stochastic single-molecule localization microscopy (SMLM), is a challenging endeavor. For correlative STED and SMLM, the following poses a significant challenge: (1) the photobleaching of the fluorophores in STED; (2) the subsequent reactivation of the fluorophores for SMLM; and (3) finding the right fluorochrome and imaging buffer for both imaging modalities. Here, we highlight how the deep ultraviolet (DBUE) wavelengths of the Mercury (Hg) arc lamp can help recover STED bleaching and allow for the reactivation of single molecules for SMLM imaging. We also show that Alexa Fluor 594 and the commercially available Prolong Diamond turn out to be excellent fluorophores and imaging media for correlative STED and SMLM.

biophysics↗

bioRxiv2021

Assessment of 3D MINFLUX data for quantitative structural biology in cells

MINFLUX is a promising new development in single-molecule localization microscopy, claiming a resolution of 1-3 nm in living and fixed biological specimens. While MINFLUX can achieve very high localisation precision, quantitative analysis of reported results leads us to dispute the resolution claim and question reliability for imaging sub-100-nm structural features, in its current state.

biophysics↗

bioRxiv2018

Single-cell in situ transcriptomic map of astrocyte cortical layer diversity

During organogenesis, patterns and gradients of gene expression underlie organization and diversified cell specification to generate complex tissue architecture. While the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells are diversified to mimic neuronal laminae or show distinct layering. To determine the molecular architecture of the mammalian cortex, we developed a high content pipeline that can quantify single-cell gene expression in situ. The Large-area Spatial Transcriptomic (LaST) map confirmed expected cortical neuron layer organization and also revealed a novel neuronal identity signature. Screening 46 candidate genes for astrocyte diversity across the cortex, we identified grey matter superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from neurons. Astrocyte layers formed in early postnatal cortex and mostly persisted in adult mouse and human cortex. Mutations that shifted neuronal post-mitotic identity or organization were sufficient to alter glial layering, indicating an instructive role for neuronal cues. In normal mouse cortex, astrocyte layer patterns showed area diversity between functionally distinct cortical regions. These findings indicate that excitatory neurons and astrocytes cells are organized into distinct lineage-associated laminae, which give rise to higher order neuroglial complexity of cortical architecture.

neuroscience↗

bioRxiv2017

Deciphering the histone code to build the genome structure

Histones are punctuated with small chemical modifications that alter their interaction with DNA. One attractive hypothesis stipulates that certain combinations of these histone modifications may function, alone or together, as a part of a predictive histone code to provide ground rules for chromatin folding. We consider four features that relate histone modifications to chromatin folding: charge neutralisation, molecular specificity, robustness and evolvability. Next, we present evidence for the association among different histone modifications at various levels of chromatin organisation and show how these relationships relate to function such as transcription, replication and cell division. Finally, we propose a model where the histone code can set critical checkpoints for chromatin to fold reversibly between different orders of the organisation in response to a biological stimulus.

cell biology↗

bioRxiv2017

High-density superresolution microscopy with an incoherent light source and a conventional epifluorescence microscope setup

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM), is an extension of single molecule localisation microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as "blinking"), detected and localised. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that STED and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution.

biophysics↗

bioRxiv2017

High-Density Super-Resolution Microscopy With An Incoherent Light Source And A Conventional Epifluorescence Microscope Setup

We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as Omnipresent Localisation Microscope (OLM), is an extension of Single Molecule Localisation Microscopy (SMLM) techniques and allows single molecules to be switched on and off (blinking), detected and localised. The use of a short burst of deep blue excitation can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, for the first time, we demonstrate that STED and OLM can be performed on the same biological sample using a simple imaging buffer. It is hoped that such a correlative imaging will provide a basis for a further enhanced resolution.\n\nScope of the findingsDespite ten years of development, superresolution microscopy is still limited to relatively few microscopy and optics groups. This is mainly due to the significant cost of the superresolution microscopes which require high-quality lasers, high NA objective lens, a very sensitive camera, a highly precise microscope stage, and a complex post-acquisition data reconstruction and analysis. We present results that demonstrate the possibility to obtain nanoscale resolution images using a conventional microscope and an incoherent light source. We show an easyto-follow protocol that every biologist can implement in the laboratory. We hope that this finding will help any scientist to generate high-density super-resolution images even with limited budget. Lastly, the new photophysical observations reported here will pave the way for more in-depth investigations on excitation, photobleaching and photoactivation of a fluorophore.

biophysics↗

bioRxiv2016

Histone code and higher-order chromatin folding: A hypothesis

Histone modifications alone or in combination are thought to modulate chromatin structure and function; a concept termed histone code. By combining evidence from several studies, we investigated if the histone code can play a role in higher-order folding of chromatin. Firstly using genomic data, we analyzed associations between histone modifications at the nucleosome level. We could dissect the composition of individual nucleosomes into five predicted clusters of histone modifications. Secondly, by assembling the raw reads of histone modifications at various length scales, we noticed that the histone mark relationships that exist at nucleosome level tend to be maintained at the higher orders of chromatin folding. Recently, a high-resolution imaging study showed that histone marks belonging to three of the five predicted clusters show structurally distinct and anti-correlated chromatin domains at the level of chromosomes. This made us think that the histone code can have a significant impact in the overall compaction of DNA: at the level of nucleosomes, at the level of genes, and finally at the level of chromosomes. As a result, in this article, we put forward a theory where the histone code drives not only the functionality but also the higher-order folding and compaction of chromatin.

biophysics↗