Biology subjects

Zammali, M.

Publications and source records attributed to Zammali, M..

3 recordsLinked to original sources

bioRxiv2026

The Tensile Expansion Microscopy (TExM) cell stretcher: an iris expansion device integrated with automated real-time autofocus and tracking to super-resolve cells

Mechanical stretchers are used to physically expand biological samples for microscopic studies of mechanobiology. Existing stretchers suffer from limited strain capacity and non-quantifiable forces, along with focus drift and feature-tracking limitations during microscopy. We develop an iris-based cell stretcher that applies isotropic equibiaxial force to achieve aerial strain of 1664% that corresponds to 4.2x linear expansion for Tensile Expansion Microscopy (TExM), a super-resolution method which increases sample size above the diffraction limit of light. The TExM stretcher uses 3D-printed, printed circuit board (PCB) and cost-effective parts, is portable and automated. We verify the performance of the TExM cell stretcher using image analysis, achieving ~90% mechanical precision, a maximum of four degrees of arm angular deviation, precise speed control down to 0.01 cm/sec, and an average expansion resolution of 3.98 x 10^-3 x. Integrated strain gauge sensors confirm equal application of force by each arm of the stretcher throughout expansion and finite-element simulation and planar-polariscope photoelastic imaging characterize the uniform substrate stress distribution while indicating high stress at the substrate gripping area. An AutoTracking software in communication with the stretcher hardware and microscope enables continuous autofocus and feature tracking during TExM and fiducial markers verify uniform equibiaxial stretch. The stretcher is demonstrated with fixed NIH 3T3 fibroblasts and live HeLa cells, observing ~4x cellular expansion of fixed cell size and separation of live cell clusters, highlighting the potential of TExM cell stretcher for biological imaging.

biophysics↗

bioRxiv2026

Tensile Expansion Mass Spectrometry for single cell metabolomics imaging

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables the spatial mapping of endogenous biomolecules within native biological specimens; however, it remains limited in achieving single-cell resolution. While advances in instrument modifications, computational processing methods, and tissue-based sample preparation have facilitated high lateral resolutions and cellular level imaging, resolving metabolic heterogeneity at the single-cell level remains challenging for users without specific expertise or custom instrumentation. Here, we present tensile expansion mass spectrometry (TExMS), a cost-effective approach for single-cell MALDI-MSI that is compatible with commercial MSI instrumentation. TExMS utilizes highly stretchable hydrogels as a substrate for live-cell seeding, attachment, and desiccation, avoiding the need for chemical fixation and enabling the retention of both intracellular and extracellular metabolites, including media-derived components that are lost during fixation and washing. We used TExMS to expand individual cells of a human high-grade serous ovarian cancer (HGSOC) cell line and spatially map their small molecule (<800 Da) production. TExMS enabled [~]4-fold linear expansion of the hydrogel, translating to a [~]1.7-fold increase in average cell area and [~]1.3-fold increase in nuclear area and resulting in improved lateral resolution of metabolite distributions. Benchmarking against other platforms for high resolution MALDI-MSI, TExMS offered comparable spatial resolution to microgrid-enabled MALDI-MSI with 15 to 20-fold shorter acquisition times. We then used TExMS to map numerous intermediates from glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid biosynthesis and probe the effects of serum starvation conditions on metabolic flux through these pathways, demonstrating a powerful use case for single-cell MALDI-MSI through TExMS. Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/745024v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@18f8c09org.highwire.dtl.DTLVardef@132bc4aorg.highwire.dtl.DTLVardef@1e7c2caorg.highwire.dtl.DTLVardef@a586cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Tensile Expansion Microscopy Applies Mechanical Force to Super-resolve Fixed and Image Live Cellular Samples

Understanding biophysical phenomena requires techniques that access biologically relevant spatial and temporal scales. Expansion Microscopy (ExM) is a sample preparation approach which achieves super-resolution spatial scales by leveraging osmotic forces in a swellable hydrogel to physically separate structures to distances larger than the diffraction limit of light. Yet, in traditional osmotic ExM only pre- and post- expanded samples can be imaged. Further, fragmentation, hydrogel deformation, and signal loss are common while requiring samples to be chemically fixed. Therefore, there is little control of the expansion, reproducibility can be challenging, and dynamics of biological samples at applicable temporal scales cannot be observed. Here, we develop Tensile Expansion Microscopy (TExM) to mechanically expand fixed and, notably, living cellular samples. Highly-stretchable and tough double network alginate- Ca2+/polyacrylamide hydrogels are expanded by tensile forces applied using an electromechanical iris expansion device during continuous imaging on a fluorescence microscope. We incorporate two-photon polymerized microscale fluorescent fiducial markers to track samples and distortion during expansion. The hydrogels controllably and repeatedly expand up to 3.3x with distortions less than 12 {micro}m across 1.3 mm2. TExM is first applied to fixed NIH 3T3 fibroblast cells with immunohistochemistry-stained microtubules, achieving super-resolutions of 100 nm. Then, TExM is demonstrated with living HeLa cells with internal fluorescent reporters showing increased cell size and cell-to-cell separation under 3.2x linear expansion. Overall, TExM allows for continuous, stepwise, and precise temporal modulation of lateral substrate strain, enabling real time monitoring of dynamics of both fixed and viable live cell processes at higher spatial resolutions. TExM can further investigate broad biophysical questions due to its compatibility with other analytical imaging methods that are sensitive to water or fixatives used in traditional osmotic ExM.

biophysics↗