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Narayanasamy, K. K.

Publications and source records attributed to Narayanasamy, K. K..

5 recordsLinked to original sources

One-click image reconstruction in single-molecule localization microscopy via deep learning

Deep neural networks have led to significant advancements in microscopy image generation and analysis. In single-molecule localization based super-resolution microscopy, neural networks are capable of predicting fluorophore positions from high-density emitter data, thus reducing acquisition time, and increasing imaging throughput. However, neural network-based solutions in localization microscopy require intensive human intervention and computation expertise to address the compromise between model performance and its generalization. For example, researchers manually tune parameters to generate training images that are similar to their experimental data; thus, for every change in the experimental conditions, a new training set should be manually tuned, and a new model should be trained. Here, we introduce AutoDS and AutoDS3D, two software programs for reconstruction of single-molecule super-resolution microscopy data that are based on Deep-STORM and DeepSTORM3D, that significantly reduce human intervention from the analysis process by automatically extracting the experimental parameters from the imaging raw data. In the 2D case, AutoDS selects the optimal model for the analysis out of a set of pre-trained models, hence, completely removing user supervision from the process. In the 3D case, we improve the computation efficiency of DeepSTORM3D and integrate the lengthy workflow into a graphic user interface that enables image reconstruction with a single click. Ultimately, we demonstrate superior performance of both pipelines compared to Deep-STORM and DeepSTORM3D for single-molecule imaging data of complex biological samples, while significantly reducing the manual labor and computation time.

bioengineering↗

Fast DNA-PAINT imaging using a deep neural network

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we trained the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-color super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule microscope and enables fast single-molecule super-resolution microscopy.

biophysics↗

Visualizing multi-protein patterns at the synapse of neuronal tissue with DNA-assisted single-molecule localization microscopy

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.

biophysics↗

Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

Stochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the off-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at [~] 3-times the speed and [~] 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

biophysics↗

Effect of PEI-coated MNPs on the Regulation of Cellular Focal Adhesions and Actin Stress Fibres

The biocompatibility of surface coated/functionalised magnetic nanoparticles (MNPs) is key to their successful incorporation and application in biological systems. Polyethylene imine (PEI) -coated MNPs provide improved in vitro transfection efficiency compared to conventional chemical methods such as Lipofectamine and cationic polymers, and are also safer than viral transduction. Commercial cell toxicity assays are useful for end-point and high-throughput screening, providing fast results and an overview of cell health. However these assays only take into account cells that have undergone an extreme toxic response leading to cell death. Cell toxicity is a complex process which can be expressed in many forms, through morphological, metabolic, and epigenetic changes. A common indicator of cell stress and toxic response is increased cell adhesion and stress fibre formation. It is important to identify these changes in cells as it may affect downstream results and applications in biomedicine. This study explores the effect of the nanomagnetic transfection agent PEI-coated MNPs (MNP-PEIs) and an external magnetic field on cell behaviour, by studying particle internalization, changes in cellular morphology, and cell adhesion. We found that MNP-PEIs induced cell stress through a dose-dependent increase in cell adhesion via the overexpression of vinculin and formation of actin stress fibres. While the presence of PEI was the main contributor to increased cell stress, free PEI polyplexes induced higher toxicity compared to PEI bound to MNPs. MNPs without PEI coating however did not adversely affect cells suggesting a chemical effect instead of a mechanical one. In addition, genes identified as being associated with actin fibre regulation and cell adhesion, showed significant increases in expression due to the internalization of the MNP-PEI complex. From these results, we identify anomalous cell behaviour, morphology, and gene expression after interaction with MNP-PEIs, as well as a safe dosage to reduce acute cell toxicity.

bioengineering↗