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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. This research describes the invention of a new method to color the surface of stainless steel with brown to blue hues at micron resolution using a low‐power laser. This method is safer and lower cost than previous methods. The study determines that the main coloring compound is chromium oxide. A laser‐induced chemical reaction that does not involve dissolved diatomic oxygen is proposed.

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Single-molecule fluorescence microscopy with “turn-on” dyes that change fluorescent state after a reaction report on the chemistry of interfaces relevant to analytical and bioanalytical chemistry. Paramount to accurately understanding the phenomena at the ultimate detection limit of a single molecule is ensuring fluorophore properties such as diffusion do not obscure the chemical reaction of interest. Here, we develop Monte Carlo simulations of a dye that undergoes reduction to turn-on at the cathode of a corroded iron surface taking into account the diffusion of the dye molecules in a total internal reflection fluorescence (TIRF) excitation volume, location of the cathode, and chemical reactions. We find, somewhat counterintuitively, that a fast diffusion coefficient of D  = 10 8 nm 2 /s, corresponding to the dye in aqueous solution, accurately reports the location of single reaction sites. The dyes turn on and are present for the acquisition of a single frame allowing for localization before diffusing out of the thin TIRF excitation volume axially. Previously turned-on (i.e., activated) dyes can also randomly hit the surface surrounding the reaction site leading to a uniform increase in the background. Using concentrations that lead to high turnover rates at the reaction site can achieve signal-to-background ratios of  ~100 in our simulation. Therefore, the interplay between diffusion, turn-on reaction rate, and concentration of the dye must be strategically considered to produce accurate images of reaction locations. This work demonstrates that modeling can assist in the design of single-molecule microscopy experiments to understand interfaces related to analytical chemistry such as electrode, nanoparticle, and sensor surfaces.

Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction 1 . Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts 2 , is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes 3 . Current models postulate a large pore formed by transmembrane proteins 4 ; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae , the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid–liquid phase separation (LLPS) with Pex5–cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as ‘stickers’ in associative polymer models of LLPS 5 , 6 . Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP–Pex13 and GFP–Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5–cargo. Our findings lead us to suggest a model in which LLPS of Pex5–cargo with Pex13 and Pex14 results in transient protein transport channels 7 . A study presents evidence to support a model in which liquid–liquid phase separation of components of the transport machinery mediates formation of transient protein transport channels on peroxisomes.

As the pharmeceutical industry moves away from traditional small organic molecules towards biologically-based treatments, ion-exchange separation methods must be investigated to improve the cost and time required for protein purification. Several new single molecule, super-resolution techniques are presented to offer a mechanistic experimental understanding of chromatography unachievable through traditional ensemble-averaged methods. Super-resolution analysis visualizes single protein adsorption kinetics to single, super-resolved ligands, allowing for the first experimental validation of the statistical mechanical stochastic theory of chromatography. Imperative results on the spatial charge-distribution of ligands, reduction of heterogeneity by ionic strength, and tuning of protein/stationary phase interfacial interactions by pH are observed. A common finding that the sterics of the agarose support induces separation heterogeneity leads to super-resolution imaging of the agarose structure and diffusion properties. Finally, the single molecule techniques are applied to several applications beyond protein chromatography to demonstrate the potential for future materials research. Overall, we have shown that single molecule spectroscopy can aid in the mechanistic experimental and theoretical understanding of the ion-exchange chromatographic separation of proteins.

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.

Correlation signal processing of optical three-dimensional (x, y, t) data can produce super-resolution images. The second order cross-correlation function has been documented to produce super-resolution imaging with static and blinking emitters but not for diffusing emitters. Here, we both analytically and numerically demonstrate cross-correlation analysis for diffusing particles. We then expand our fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) analysis to use cross-correlation as a post-processing computational technique to extract both dynamic and structural information of particle diffusion in nanoscale structures simultaneously. We further show how this method increases sampling rates and reduces aliasing for spatial information in both simulated and experimental data. Our work demonstrates how fcsSOFI with cross-correlation can be a powerful signal-processing tool to resolve the nanoscale dynamics and structure in samples relevant to biological and soft materials.

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 /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 across 1.3 . 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.

We present a comprehensive guide to light-sheet microscopy (LSM) to assist scientists in navigating the practical implementation of this microscopy technique. Emphasizing the applicability of LSM to image both static microscale and nanoscale features, as well as diffusion dynamics, we present the fundamental concepts of microscopy, progressing through beam profile considerations, to image reconstruction. We outline key practical decisions in constructing a home-built system and provide insight into the alignment and calibration processes. We briefly discuss the conditions necessary for constructing a continuous 3D image and introduce our home-built code for data analysis. By providing this guide, we aim to alleviate the challenges associated with designing and constructing LSM systems and offer scientists new to LSM a valuable resource in navigating this complex field.