Biology subjects

Manifold, B.

Publications and source records attributed to Manifold, B..

3 recordsLinked to original sources

bioRxiv2025

Effects of biological fixation methods on stimulated Raman scattering microscopy signal

Stimulated Raman Scattering (SRS) microscopy enables label-free imaging of cells and tissues in their native state with chemical specificity. However, there are often experimental advantages of chemical fixation of samples prior to imaging, which can introduce perturbations that may alter the native state of the samples, and possibly impact the SRS signal. In this study, we systematically characterize the effects of common fixatives (Paraformaldehyde, Formalin, Glutaraldehyde, Methanol, Ethanol) on the preservation of cellular integrity and molecular composition in a Hela cell model as observed by SRS microscopy. We demonstrate how the different fixatives can influence lipid and protein content, and overall cell morphology, with significant implications for the accuracy of quantitative SRS microscopy. Our findings indicate that Paraformaldehyde (PFA) imposes minimal disruption to cellular and molecular states compared to the other fixatives tested, and suggest Glutaraldehyde (GA) as a suitable alternative for SRS imaging. This study provides insights for the choice of the optimal sample preparation, enabling more reliable SRS-based studies for the evaluation of cellular processes and disease mechanisms where fixation is used.

cell biology↗

bioRxiv2025

SRS microscopy identifies inhibition of vitellogenesis as a mediator of lifespan extension by caloric restriction in C. elegans

The molecular mechanisms of aging are not fully understood. Here, we used label-free Stimulated Raman scattering (SRS) microscopy to investigate changes in proteins and lipids throughout the lifespan of C. elegans. We observed a dramatic buildup of proteins within the body cavity or pseudocoelom of aged adults that was blunted by interventions that extend lifespan: caloric restriction (CR) and the reduced insulin/insulin-like growth factor signaling (IIS) pathway. Using a combination of microscopy, proteomic analysis, and validation with mutant strains, we identified vitellogenins as the key molecular components of the protein buildup in the pseudocoelom. Vitellogenins shuttle nutrients from intestine to embryos and are homologous to human apolipoprotein B, the causal driver of cardiovascular disease. We then showed that CR and knockdown of vitellogenins both extend lifespan by >60%, but their combination has no additional effect on lifespan, suggesting that CR extends the lifespan of C. elegans in part by inhibiting vitellogenesis. The extensive dataset of more than 12,000 images stitched into over 350 whole-animal SRS images of C. elegans at different ages and subjected to different longevity intervention will be a valuable resource for researchers interested in aging.

cell biology↗

bioRxiv2021

In Vivo Simultaneous Nonlinear Absorption Raman and Fluorescence (SNARF) Imaging of Mouse Brain Cortical Structures

AO_SCPLOWBSTRACTC_SCPLOWTwo-photon excited fluorescence (TPEF) microscopy is a widely used optical imaging technique that has revolutionized neurophotonics through a diverse palette of dyes, specialized transgenic models, easy implementation, and straightforward data analysis. However, in vivo TPEF imaging is often limited in the number of contrasts available to distinguish different cells, structures, or functions. We propose using two label-free multiphoton microscopy techniques - stimulated Raman scattering (SRS) microscopy and transient absorption microscopy (TAM) - as complementary and orthogonal imaging modalities to TPEF for in vivo brain imaging. In this study, we construct a simultaneous nonlinear absorption, Raman, and fluorescence (SNARF) microscope and image several cortical structures up to 250-300 m below the pial surface, the highest reported in vivo imaging depth for SRS or TAM. We further demonstrate the capabilities of our SNARF microscope through the quantification of age-dependent myelination, hemodynamics, vessel structure, cell density, and cell identity in vivo. Using machine learning, we report the use of label-free SRS and TAM features to predict capillary-lining cell identities with 90% accuracy. The SNARF microscope and methodology outlined herein provide a powerful platform to study several research topics, including neurovascular coupling, blood-brain barrier, neuronal and axonal degeneration in aging, and neurodegenerative diseases.

neuroscience↗