Biomechanical Dependence of SARS-CoV-2 Infections

Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, that is, the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly

Comparing lipid remodeling of brown adipose tissue, white adipose tissue, and liver after one-week high fat diet intervention with quantitative Raman microscopy

Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance

Nanographenes: Ultrastable, Switchable, and Bright Probes for Super-Resolution Microscopy

Super-resolution fluorescence microscopyh as enabled important breakthroughs in biology and materials science.Implementations such as single-molecule localization microscopy(SMLM) and minimal emission fluxes (MINFLUX) microscopyinthe localization mode exploit fluorophores that blink, i.e., switch on and off,stochastically.Here, weintroducenanographenes,namelylargepolycyclicaromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots,asanew class of fluorophores for super-resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties:intrinsic blinking even in air,excellent fluorescence recovery,and stability over several months.Asaproof of concept for super-resolution applications,weuse nanographenes in SMLM to generate 3D super-resolution images of silica nanocracks.O ur findings open the door for the widespread application of nanographenes in super-resolution fluorescence microscopy

Multiscale Effects of Interfacial Polymer Confinement in Silica Nanocomposites

Dispersing hydrophilic nanofillers in highly hydrophobic polymer matrices is widely used to tune the mechanical properties of composite material systems. The ability to control the dispersion of fillers is closely related to the mechanical tunability of such composites. In this work, we investigate the physical–chemical underpinnings of how simple end-group modification to one end of a styrene–butadiene chain modifies the dispersion of silica fillers in a polymer matrix. Using surface-sensitive spectroscopies, we directly show that polymer molecular orientation at the silica surface is strongly constrained for silanol functionalized polymers compared to nonfunctionalized polymers because of covalent interaction of silanol with silica. Silanol functionalization leads to reduced filler aggregation in composites. The results from this study demonstrate how minimal chemical modifications of polymer end groups are effective in modifying microstructural properties of composites by inducing molecular ordering of polymers at the surface of fillers

Loading History Determines the Velocity of

aracterized by Fv relationships, which describe velocity in a given biochemical system as a function of only the instantaneous applied force 9,10 . Multiple theoretical models posit such Fv relationships for growing actin networks 11--15 , but the predictions of these models vary widely because of different assumptions about the effects of filament branching and capping, network elasticity and network -load tethering. Fv relationships using reconstituted motility assays of Listeria and beads 17 have been reported where the visco-elasticity of the medium was varied to control the drag force. More recently, an Fv relationship for UCSF/UC Berkeley Joint Graduate Group in Bioengineering, University of California at Berkeley and San Francisco; Department of Bioengineering, University of California at Berkeley, Berkeley, CA 94720. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305. These authors contributed equally to this work. Corr

Molecular Microscopy of Oil Body and Lipid Droplet Chemistry In Situ with Physiologically-Relevant Readouts

Spatial heterogeneity at the molecular scale is a ubiquitous feature of all biological tissues, which is fundamentally linked to their native functions and to pathology. Probing the local chemistry of complex biological tissues requires the development and application of imaging tools that can identify the intrinsic molecular features in a sample without sacrificing high spatial resolution. In this talk, I will describe our efforts to tackle this challenge for measuring lipid inclusion chemistry and morphology in situ. We have developed nonlinear label-free microscopy and associated analytical tools to determine the biochemistry of lipid droplets and oil bodies with high spatial resolution in a variety of samples. Importantly, our method yields physiologically-relevant quantities (chain length and saturation) as opposed to physical chemical ratios. I will show how we have used this ability to map how lipid droplet chemical composition in brown and white adipose tissue adapts to high fat dietary intervention. Going forward, we want to expand the disease pathologies studied and I will present early results on our work on lipid droplets in neurological diseases

Automated cell segmentation in FIJI® using the DRAQ5 nuclear dye

Abstract Background Image segmentation and quantification are essential steps in quantitative cellular analysis. In this work, we present a fast, customizable, and unsupervised cell segmentation method that is based solely on Fiji (is just ImageJ)®, one of the most commonly used open-source software packages for microscopy analysis. In our method, the “leaky” fluorescence from the DNA stain DRAQ5 is used for automated nucleus detection and 2D cell segmentation. Results Based on an evaluation with HeLa cells compared to human counting, our algorithm reached accuracy levels above 92% and sensitivity levels of 94%. 86% of the evaluated cells were segmented correctly, and the average intersection over union score of detected segmentation frames to manually segmented cells was above 0.83. Using this approach, we quantified changes in the projected cell area, circularity, and aspect ratio of THP-1 cells differentiating from monocytes to macrophages, observing significant cell growth and a transition from circular to elongated form. In a second application, we quantified changes in the projected cell area of CHO cells upon lowering the incubation temperature, a common stimulus to increase protein production in biotechnology applications, and found a stark decrease in cell area. Conclusions Our method is straightforward and easily applicable using our staining protocol. We believe this method will help other non-image processing specialists use microscopy for quantitative image analysis

Tissue harvest with a laser microbiopsy (Erratum)

[This corrects the article DOI: 10.1117/1.JBO.27.12.125001.]

Tissue harvest with a laser microbiopsy

SignificanceTraditional pathology workflow suffers from limitations including biopsy invasiveness, small fraction of large tissue samples being analyzed, and complex and time-consuming processing.AimWe address limitations of conventional pathology workflow through development of a laser microbiopsy device for minimally invasive harvest of sub-microliter tissue volumes. Laser microbiopsy combined with rapid diagnostic methods, such as virtual hematoxylin and eosin (H&E) imaging has potential to provide rapid minimally invasive tissue diagnosis.ApproachLaser microbiopsies were harvested using an annular shaped Ho:YAG laser beam focused onto the tissue surface. As the annulus was ablated, the tissue section in the center of the annulus was ejected and collected directly onto a glass slide for analysis. Cryogen spray cooling was used before and after laser harvest to limit thermal damage. Microbiopsies were collected from porcine skin and kidney. Harvested microbiopsies were imaged with confocal microscopy and digitally false colored to provide virtual H&E images.ResultsMicrobiopsies were successfully harvested from porcine skin and kidney. Computational and experimental results show the benefit of cryogen pre- and post-cooling to limit thermal damage. Virtual H&E images of microbiopsies retained observable cellular features including cell nuclei.ConclusionsLaser microbiopsy with virtual H&E imaging shows promise as a potential rapid and minimally invasive tool for biopsy and diagnosis