Biology subjects

Ahluwalia, B. S.

Publications and source records attributed to Ahluwalia, B. S..

11 recordsLinked to original sources

bioRxiv2023

Super-resolution histology of paraffin-embedded samples via photonic chip-based microscopy

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.

pathology↗

bioRxiv2023

Honeycomb-inspired SERS nano-bowls for rapid capture and analysis of extracellular vesicles and liposomes in suspension

Nanoscale carriers such as liposomes and extracellular vesicles (EVs) are readily being explored for personalized medicine or disease prediction and diagnostics, respectively. Owing to their small size, such nanocarriers can undergo endocytosis or exocytosis, providing means to either transport cargo to the cells (liposomes) or to serve as a biomarker (EVs). When looking at current analysis methods, there is a growing need for detailed characterization of the content and composition of such nanocarriers in their natural state in aqueous media. This can be achieved through surface-enhanced Raman spectroscopy (SERS), which provides a molecular fingerprint of the analytes while reducing the detection limit. In this paper, we utilize a nano-structured SERS substrate to study different bio-nanoparticles such as liposomes, EVs and DNA nanogel in suspension. A silver-coated polydimethylsiloxane (PDMS) film-based honeycomb shaped nano-bowl surface passively traps and reduces the mobility of the nanosized bio-particles, improving the intensity and the reproducibility of the SERS signal. FDTD simulations are used for substrate geometry optimization, and a detection limit of 10-15 M is demonstrated for Rhodamine 6G (R6G). The potential of the proposed SERS nano-bowl is shown through distinct spectral features following surface-(polyethylene glycol) and bilayer-(cholesterol) modification of empty liposomes. For DNA nanogels, the characterization of highly crosslinked DNA specimens exhibits enhanced peaks for nitrogenous bases, sugar, and phosphate groups. EVs isolated from various cells provided spectral signatures of specific protein content, lipid components, and nucleic acids. Concluding, the findings of the spectral signatures of a wide range of molecular complexes and chemical morphology of bio-membranes in their natural state highlight the possibilities of using SERS as a sensitive and instantaneous characterization alternative.

biophysics↗

bioRxiv2023

Effect of caffeine and other xanthines on liver sinusoidal endothelial cell ultrastructure

Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as food additives. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 g/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 g/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery.

cell biology↗

bioRxiv2023

SERS nanowire chip and machine learning enabled instant identification and classification of clinically relevant wild-type and antibiotic resistant bacteria at species and strain level

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 (105 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 103 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.

biophysics↗

bioRxiv2022

Highly sensitive quantitative phase microscopy and deep learning complement whole genome sequencing for rapid detection of infection and antimicrobial resistance

The current state-of-the-art infection and antimicrobial resistance diagnostics (AMR) is based mainly on culture-based methods with a detection time of 48-96 hours. Slow diagnoses lead to adverse patient outcomes that directly correlate with the time taken to administer optimal antimicrobials. Mortality risk doubles with a 24-hour delay in providing appropriate antibiotics in cases of bacteremia. Therefore, it is essential to develop novel methods that can promptly and accurately diagnose microbial infections at both species and strain levels in clinical settings. Here, we demonstrate that the complimentary use of label-free optical assay with whole-genome sequencing (WGS) can enable high-speed culture-free diagnosis of infection and AMR. Our assay is based on microscopy methods exploiting label-free, highly sensitive quantitative phase microscopy (QPM) followed by deep convolutional neural networks (DCNNs) based classification. We benchmarked our proposed workflow on 21 clinical isolates from four WHO priority pathogens (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii) that were antibiotic susceptibility testing (AST) phenotyped, and their antimicrobial resistance (AMR) profile was determined by WGS. The proposed optical assay was in good agreement with the WGS characterization. Highly accurate classification based on the gram staining (100% for gram-negative and 83.4% for gram-positive), species (98.6%), and resistant/susceptible type (96.4%), as well as at the individual strain level (100% accurate in predicting 19 out of the 21 strains). These results demonstrate the potential of the QPM assay as a rapid and first-stage tool for species, presence, and absence of AMR, and strain-level classification, which WGS can follow up for confirmation of the pathogen ID and the characterization of the AMR profile and susceptibility antibiotic. Taken together, all this information is of high clinical importance. Such a workflow could potentially facilitate efficient antimicrobial stewardship and prevent the spread of AMR.

microbiology↗

bioRxiv2022

The complexity of keratocyte migration in salmon explant cultures: initial results and future prospects

Intact skin is of uttermost importance for fish welfare. The fish skin provides an environmental barrier and protects against invading pathogens. However, both pathogens and physical insults cause skin wounds that are of major concern in modern fish farming. The behavior and interactions between keratocyte cells and sheets of cells are not well understood. The collective migration of keratocytes (skin epithelial cells) is of central importance for wound healing in fish. In this study, we aimed to elucidate the complex wound healing process in fish skin by studying in vitro cultures of these highly motile cells. Using explant cultures from farmed Atlantic salmon (Salmo salar) and differential interference contrast microscopy (DIC), we have captured the dynamics of sheets of cells from harvested fish scales and of individual cells interacting in the cell sheet vicinity. In addition to direct contact, the cells were observed to interact through long membrane tubes, turn, rotate, merge, and/or detach. Additionally, stationary cells and cells moving on top of the cell sheets were observed. Cell sheets approaching one another from different scales did not merge but dispersed.

cell biology↗

bioRxiv2021

Photonic chip-based multimodal super-resolution microscopy for histopathological assessment of cryopreserved tissue sections

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.

pathology↗

bioRxiv2020

Nanoscopy on the Chea(i)p

Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

microbiology↗

bioRxiv2020

Visualizing ultrastructural details of placental tissue with super-resolution structured illumination microscopy

Super-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histo-pathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and demonstrate the significant contrast and resolution enhancement attainable with SIM. We show that SIM resolves ultrastructural details such as the syncytiotrophoblasts microvilli brush border, which up until now has been only resolvable by electron microscopy.

pathology↗

bioRxiv2020

Quantitative assessment of morphology and sub-cellular changes in macrophages and trophoblasts during inflammation

During inflammatory condition in pregnancy, the macrophages present at the feto-maternal junction release an increased amount of NO and pro-inflammatory cytokines such as TNF- and INF-{gamma}, which can disturb the trophoblast functions and thereby the pregnancy outcome. Measurement of the cellular and sub-cellular morphological modifications associated with inflammatory responses are important in order to quantify the extent of trophoblast dysfunction for clinical implication. With this motivation, we investigated morphological, cellular and sub-cellular changes in externally inflamed RAW264.7 (macrophage) and HTR-8/SVneo (trophoblast) using structured illumination microscopy (SIM) and quantitative phase microscopy (QPM). We monitored the production of nitric oxide (NO), changes in cell membrane and mitochondrial structure of macrophages and trophoblasts when exposed to different concentration of pro-inflammatory agents (LPS and TNF-). In vitro NO production by LPS-induced macrophages increased 22-folds as compared to controls, whereas no significant NO production was seen after TNF- challenge. Under similar conditions as with macrophages, trophoblasts did not produce NO following either LPS or TNF- challenge. Super-resolution SIM imaging showed changes in the morphology of mitochondria and plasma membrane in macrophages following LPS challenge and in trophoblasts following TNF- challenge. Label-free QPM showed a decrease in the optical thickness of the LPS-challenged macrophages while TNF- having no effect. The vice-versa is observed for the trophoblasts. We further exploited machine learning approaches on QPM dataset to detect and to classify the inflammation with an accuracy of 99.9% for LPS-challenged macrophages and 98.3% for TNF--challenged trophoblasts. We believe that the multi-modal advanced microscopy methodologies coupled with machine learning approach could be an alternative way for early detection of pregnancy related inflammation after clinical studies.

cell biology↗

bioRxiv2019

A waveguide imaging platform for live-cell TIRF imaging of neurons over large fields of view

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1, 2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGC). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.

neuroscience↗