Lydia Kisley

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.

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.

The cellular cytosol is a crowded environment. Biomolecular Förster resonance energy transfer (FRET) sensors have been developed to measure crowding in cytosol mimics composed of synthetic polymers such as polyethylene glycol (PEG) and Ficoll that impart an excluded volume effect. We investigate the PEG-driven phase separation of a protein crowding sensor, AcGFP1/mCherry-FRET crowding helix 2 (CrH2), into fluorescent puncta compared to a DNA-based crowding sensor (CrD) with an Alexa488/Cy5 FRET pair that did not form observable puncta under the same crowding conditions of 100-400 mg/mL 8 kDa PEG. Using fluorescence recovery after photobleaching imaging, we uncover the liquid-like physical properties of the PEG-induced puncta. Two-color fluorescence microscopy imaging reveals crowder-induced inhomogeneity, concentration variations, and partition coefficients across the dilute and dense phases of the liquid puncta, which remain largely underexplored in bulk fluorometry measurements. Thus, the average crowding sensor response may originate from an aqueous biphasic system, reporting an erroneous average response instead of distinct levels of crowdedness. A comparison of excluded volume effects conferred by Ficoll and PEGs of various molecular weights shows the influence of size, concentration, excluded volume, and chemical composition on the CrH2 sensor response. We demonstrate that ≥18% PEG (w/v) was sufficient to enable the phase separation of CrH2 and alter sensor response through a mechanism that may be driven by polymer interactions with the flexible hinge region. We also show that CrD can form fluorescence puncta upon charge-neutralization with poly-l-lysine. Thus, our study emphasizes the need to probe crowding environments with orthogonal sensors.

Green chemistry is a set of principles for assessing, developing, and implementing methods that are safer, more efficient, and less detrimental to the environment. The analytical method greenness score (AMGS) is one of many metrics that attempt to evaluate traditional liquid chromatography (LC) based on the energy consumption of the instrument and the safety, health risks, and environmental impact of the solvents employed. Unfortunately, in practice, the AMGS is primarily focused on traditional separation methods in the pharmaceutical industry and is not amenable to cutting-edge separation science, including miniaturization. To broaden this scope, the unified Analytical Method Greenness Score (uAMGS) is presented here, which clarifies and expands on the underlying mathematics and incorporates both dimensional and uncertainty analysis, enabling its application to a broader range of analytical techniques. The uAMGS is used to compare the greenness of two distinct methods: single-molecule microscopy (SMM) and high-performance liquid chromatography (HPLC), which were used to collect equivalent data. uAMGS determines that SMM is significantly greener than HPLC due primarily to decreased solvent consumption. Overall, the uAMGS should allow chemists ranging from undergraduates to industrial PhDs to assess the greenness of a wide range of separations.

The stochastic model of chromatography mathematically links microscopic, molecular adsorption times to chromatographic elution. The nonlinear effects of the number of adsorption events and number of modeling points on elution profiles are explored.

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- Ca ²⁺ /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 3.3× with distortions less than 12 µm across 1.3 mm ² . 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 3.2× 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.

The cellular cytosol is a crowded environment. Biomolecular Förster resonance energy transfer (FRET) sensors have been developed to measure crowding in cytosol mimics comprised of synthetic polymers such as polyethylene glycol (PEG) and Ficoll that impart an excluded volume effect. In the current study, we explore the unsolicited role of PEG in driving the phase separation of a protein crowding sensor, AcGFP1/mCherry-FRET crowding helix 2 (CrH2), into fluorescent puncta. In contrast, a DNA-based crowding sensor (CrD), with an Alexa488/Cy5 FRET pair, does not form puncta under the same crowding conditions. Using fluorescence recovery after photobleaching imaging, we uncover the liquid-like physical properties of the PEG-induced puncta. Two-color fluorescence microscopy imaging reveals crowder-induced inhomogeneity, concentration variations, and partition coefficient across the dilute and dense phases of the liquid puncta, which remain largely underexplored in bulk fluorometry measurements. Thus, the average crowding sensor response may originate from an aqueous biphasic system, reporting an erroneous average response instead of distinct levels of crowdedness. A comparison of excluded volume effects conferred by Ficoll and PEGs of various molecular weight ranges shows the influence of size, concentration, excluded volume, and chemical composition on the CrH2 sensor response. We demonstrate that PEGs enable phase separation and alter sensor response through a mechanism that may be driven by polymer interactions with the flexible hinge region of CrH2. Overall, we determine the biophysical mechanisms underlying PEG-induced condensation of CrH2 and demonstrate a CrD sensor as an alternative that does not undergo phase separation.

Redox-active colloids (RACs) represent a novel class of energy carriers that exchange electrical energy upon contact. Understanding contact-mediated electron transfer dynamics in RACs offers insights into physical contact events in colloidal suspensions and enables quantification of electrical energy transport in nonconjugated polymers. Redox-based electron transport was directly observed in monolayers of micron-sized RACs containing ethyl-viologen side groups via fluorescence microscopy through an unexpected nonlinear electrofluorochromism that is quantitatively coupled to the redox state of the colloid. Via imaging studies, using this electrofluorochromism, the apparent charge transfer diffusion coefficient D _CT of the RAC was easily determined. The visualization of energy transport within suspensions of redox-active colloids was also demonstrated. Our work elucidates fundamental mechanisms of energy transport in colloidal systems, informs the development of next-generation redox flow batteries, and may inspire new designs of smart active soft matter including conductive polymers for applications ranging from electrochemical sensors and organic electronics to colloidal robotics.

Laser color marking produces nearly permanent, environmentally friendly, vibrant colors on surfaces. However, previous work has used high‐power‐density pulsed lasers to induce the physicochemical reactions for marking. Here, laser color marking on stainless steel 304 (SS304) is performed with a less expensive continuous wave (CW) laser and a power density five orders of magnitude below that previously reported by combining an electrochemical cell with a fluorescence microscope. Using a combination of optical microscopy, x‐ray photoelectron spectroscopy, and bulk electrochemistry, it is demonstrated that the laser‐induced luminescence and colors are due to enrichment (32 ± 9% increase) of Cr₂O₃ in the SS304 passive film. It is shown that the enrichment proceeds by a different chemical mechanism than the oxygen pyrolysis that occurs in typical laser color marking. The technique provides a new pathway for laser color marking of metals in industrial settings with applications as diverse as solar absorbers or corrosion prevention.

Laser color marking produces nearly permanent, environmentally friendly, vibrant colors on surfaces. However, previous work has used high power density pulsed lasers to induce the physicochemical reactions for marking. Here, we perform laser color marking on stainless steel 304 (SS304) with a less expensive continuous wave (CW) laser and a power density five orders of magnitude below previously reports by combining an electrochemical cell with a fluorescence microscope. Using a combination of optical microscopy, x-ray photoelectron spectroscopy, and bulk electrochemistry, we demonstrate that the laser-induced luminescence and colors are due to enrichment (32±9% increase) of Cr2O3 in the SS304 passive film. We show that the enrichment proceeds by a different chemical mechanism than the oxygen pyrolysis that occurs in typical laser color marking. Our technique provides a new pathway for laser color marking of metals in industrial settings with applications as diverse as solar absorbers or corrosion prevention.