Vishesh Dubey

HIV-1 latency in myeloid cells remains a major obstacle to viral eradication. Here, we applied a systems biology approach combining transcriptomics, proteomics, metabolic modeling, targeted metabolite profiling, and advanced imaging to investigate metabolic alterations in a pre-monocytic latent cell model (U1). Upon latency reversal, we identified disrupted α-ketoglutarate (AKG) homeostasis driven by mitochondrial biogenesis and glutamine/glutamate metabolism, supporting energy production and M2-like macrophage polarization. Reporter metabolite analysis predicted cytoplasmic amino acid accumulation, and functional assays showed that tryptophan suppressed HIV reactivation by promoting mitochondrial and antioxidant metabolism. Additionally, we observed enhanced formation of tunneling nanotubes (TNTs), which facilitated intercellular transfer of mitochondria and viral components, potentially aiding viral persistence. Our study reveals cell-type–specific metabolic reprogramming and intercellular communication mechanisms underlying HIV-1 persistence. Our findings highlight the glutamine-AKG axis and TNTs as promising targets for strategies aimed at eliminating long-lived macrophage-associated HIV reservoirs.

This review covers the advancements of optical super-resolution microscopy (SRM) on formalin-fixed paraffin-embedded (FFPE) histological samples. We cover the implementation of various SRM strategies in histology, including wide field methods such as structured illumination microscopy, single-molecule localization microscopy and fluorescence fluctuations-based SRM, as well as the point-scanning stimulated emission depletion microscopy. We also cover the recent developments in FFPE-based expansion microscopy. The review highlights the advantages and challenges of these SRM methods in FFPE histology, and provides insights into emerging optical and computational techniques that can potentially open avenues for understanding disease mechanisms, tailoring treatments, and advancing personalized medicine across disciplines. This review article is intended for a broad audience, including histopathologists, biologists, physiologists, and physicists.

This paper presents the development of a single-shot, partially spatially coherent quantitative differential phase contrast microscopy (Q-DPCM) system. The optical scheme comprises a polarizer, lenses, calcite beam displacer, and analyzer, which can be seamlessly integrated to an existing bright-field microscopy system, transforming it into a Q-DPCM system. It utilizes a partially spatially coherent light source, enabling single-shot quantitative differential phase recovery of the specimens with high spatial phase sensitivity. It generates highly sensitive quantitative differential phase images of the specimens along one direction, like a gradient light interference microscopy (GLIM) system, using only a single interferogram. First, we validated the differential phase measurement capability of the system through experiments on polystyrene spheres (diameter 5.2 µm) and HeLa cells. Next, the system is utilized to generate quantitative phase maps of human red blood cells using two orthogonal differential interferograms recorded at two orientations of the calcite beam displacer. Further, the Q-DPCM system is implemented for 1-h time-lapse live cell monitoring, revealing the dynamics of intracellular granules such as nucleolus and lipids in U2OS cells.

Fluorescence-based super-resolution optical microscopy (SRM) techniques allow the visualization of biological structures beyond the diffraction limit of conventional microscopes. Despite its successful adoption in cell biology, the integration of SRM into the field of histology has been deferred due to several obstacles. These include limited imaging throughput, high cost, and the need for complex sample preparation. Additionally, the refractive index heterogeneity and high labeling density of commonly available formalin-fixed paraffin-embedded (FFPE) tissue samples pose major challenges to applying existing super-resolution microscopy methods. Here, we demonstrate that photonic chip-based microscopy alleviates several of these challenges and opens avenues for super-resolution imaging of FFPE tissue sections. By illuminating samples through a high refractive-index waveguide material, the photonic chip-based platform enables ultra-thin optical sectioning via evanescent field excitation, which reduces signal scattering and enhances both the signal-to-noise ratio and the contrast. Furthermore, the photonic chip provides decoupled illumination and collection light paths, allowing for total internal reflection fluorescence (TIRF) imaging over large and scalable fields of view. By exploiting the spatiotemporal signal emission via MUSICAL, a fluorescence fluctuation-based super-resolution microscopy (FF-SRM) algorithm, we demonstrate the versatility of this novel microscopy method in achieving superior contrast super-resolution images of diverse FFPE tissue sections derived from human colon, prostate, and placenta. The photonic chip is compatible with routine histological workflows and allows multimodal analysis such as correlative light-electron microscopy (CLEM), offering a promising tool for the adoption of super-resolution imaging of FFPE sections in both research and clinical settings.

The world health organization considers antimicrobial resistance (AMR) to be a critical global public health problem. Conventional culture-based methods that are used to detect and identify bacterial infection are slow. Thus, there is a growing need for the development of robust, cost-effective, and fast diagnostic solutions for the identification of pathogens. Surface-enhanced Raman spectroscopy (SERS) can be used to identify target analytes with sensitivity down to the single-molecule level. Here, we developed a SERS chip by optimizing the entire fabrication pipeline of the metal-assisted chemical etching (MACE) method. The MACE approach offers a large-scale, densely packed silver (Ag) nanostructure on top of silicon nanowires (Si-NWs) with a large aspect ratio that significantly enhances the Raman signal due to localised surface plasmonic enhancement. The optimised SERS chips exhibited sensitivity down to 10 ^-12 M concentration of R6G molecule and detected reproducible Raman spectra of bacteria down to a concentration of 100 colony forming units (CFU)/ml, which is a thousand times lower than the clinical threshold of bacterial infections like UTI (10 ⁵ CFU/ml). A Siamese neural network model was used to classify SERS Raman spectra from bacteria specimens. The trained model identified 12 different bacterial species, including those which are causative agents for tuberculosis and urinary tract infection (UTI). Next, the SERS chips and another Siamese neural network model were used to differentiate antibiotic-resistant strains from susceptible strains of E. coli . The enhancement offered by SERS chip enabled acquisitions of Raman spectra of bacteria directly in the synthetic urine by spiking the sample with only 10 ³ CFU/ml E. coli . Thus, the present study lays the ground for the identification and quantification of bacteria on SERS chips, thereby offering a potential future use for rapid, reproducible, label-free, and low limit detection of clinical pathogens.

Treatment options for infections caused by antimicrobial-resistant bacteria are rendered ineffective, and drug alternatives are needed—either from new chemical classes or drugs with new modes of action. Historically, natural products have been important contributors to drug discovery. In a recent study, the dimeric naphthopyrone lulworthinone produced by an obligate marine fungus in the family Lulworthiaceae was discovered. The observed potent antibacterial activity against Gram-positive bacteria, including several clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, prompted this follow-up mode of action investigation. This paper aimed to characterize the antibacterial mode of action (MOA) of lulworthinone by combining in vitro assays, NMR experiments and microscopy. The results point to a MOA targeting the bacterial membrane, leading to improper cell division. Treatment with lulworthinone induced an upregulation of genes responding to cell envelope stress in Bacillus subtilis. Analysis of the membrane integrity and membrane potential indicated that lulworthinone targets the bacterial membrane without destroying it. This was supported by NMR experiments using artificial lipid bilayers. Fluorescence microscopy revealed that lulworthinone affects cell morphology and impedes the localization of the cell division protein FtsZ. Surface plasmon resonance and dynamic light scattering assays showed that this activity is linked with the compound‘s ability to form colloidal aggregates. Antibacterial agents acting at cell membranes are of special interest, as the development of bacterial resistance to such compounds is deemed more difficult to occur.

Contrast in fluorescence microscopy images allows for the differentiation between different structures by their difference in intensities. However, factors such as point-spread function and noise may reduce it, affecting its interpretability. We identified that fluctuation of emitters in a stack of images can be exploited to achieve increased contrast when compared to the average and Richardson-Lucy deconvolution. We tested our methods on four increasingly challenging samples including tissue, in which case results were comparable to the ones obtained by structured illumination microscopy in terms of contrast.

Histopathological assessment involves the identification of anatomical variations in tissues that are associated with diseases. While diffraction-limited optical microscopes assist in the diagnosis of a wide variety of pathologies, their resolving capabilities are insufficient to visualize some anomalies at subcellular level. Although a novel set of super-resolution optical microscopy techniques can fulfill the resolution demands in such cases, the system complexity, high operating cost, lack of multimodality, and low-throughput imaging of these methods limit their wide adoption in clinical settings. In this study, we interrogate the photonic chip as an attractive high-throughput super-resolution microscopy platform for histopathology. Using cryopreserved ultrathin tissue sections of human placenta, mouse kidney, and zebrafish eye retina prepared by the Tokuyasu method, we validate the photonic chip as a multi-modal imaging tool for histo-anatomical analysis. We demonstrate that photonic-chip platform can deliver multi-modal imaging capabilities such as total internal reflection fluorescence microscopy, intensity fluctuation-based optical nanoscopy, single-molecule localization microscopy, and correlative light-electron microscopy. Our results demonstrate that the photonic chip-based super-resolution microscopy platform has the potential to deliver high-throughput multimodal histopathological analysis of cryopreserved tissue samples.

1. Histopathological assessment involves the identification of anatomical variations in tissues that are associated with diseases. While diffraction-limited optical microscopes assist in the diagnosis of a wide variety of pathologies, their resolving capabilities are insufficient to visualize some anomalies at subcellular level. Although a novel set of super-resolution optical microscopy techniques can fulfill the resolution demands in such cases, the system complexity, high operating cost, lack of multimodality, and low-throughput imaging of these methods limit their wide adoption in clinical settings. In this study, we interrogate the photonic chip as an attractive high-throughput super-resolution microscopy platform for histopathology. Using cryopreserved ultrathin tissue sections of human placenta, mouse kidney, and zebrafish eye retina prepared by the Tokuyasu method, we validate the photonic chip as a multi-modal imaging tool for histo-anatomical analysis. We demonstrate that photonic-chip platform can deliver multi-modal imaging capabilities such as total internal reflection fluorescence microscopy, intensity fluctuation-based optical nanoscopy, single-molecule localization microscopy, and correlative light-electron microscopy. Our results demonstrate that the photonic chip-based super-resolution microscopy platform has the potential to deliver high-throughput multimodal histopathological analysis of cryopreserved tissue samples.