Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Pisapati, Avani V.; Wang, Yi; Blauch, Megan E; Wittenberg, Nathan J.; Cheng, Xuanhong; Zhang, X. Frank

doi:10.3791/60784-v

Cited by 2 publications

(1 citation statement)

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“…[8][9][10][11][12][13] However, intravital microscopy methods are highly costly, and require intensive effort in both setup and analysis, and therefore have limited applicability for the broader research community. 14,15 Total internal reflection fluorescence microscopy is a potential in vitro alternative for visualizing cellular interactions under whole blood flow (and examples with buffer flow already exist 16,17 ). But fluorophore labeling in whole blood can be challenging.…”

Section: Mainmentioning

confidence: 99%

Motion Blur Microscopy

Goreke,

Gonzales,

Shipley

et al. 2023

Preprint

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Imaging and characterizing the dynamics of cellular adhesion in blood samples is of fundamental importance in understanding biological function.In vitromicroscopy methods are widely used for this task, but typically require diluting the blood with a buffer to allow for transmission of light. However whole blood provides crucial mechanical and chemical signaling cues that influence adhesion dynamics, which means that conventional approaches lack the full physiological complexity of living microvasculature. We propose to overcome this challenge by a newin vitroimaging method which we call motion blur microscopy (MBM). By decreasing the source light intensity and increasing the integration time during imaging, flowing cells are blurred, allowing us to identify adhered cells. Combined with an automated analysis using machine learning, we can for the first time reliably image cell interactions in microfluidic channels during whole blood flow. MBM provides a low cost, easy to implement alternative to intravital microscopy, thein vivoapproach for studying how the whole blood environment shapes adhesion dynamics. We demonstrate the method’s reproducibility and accuracy in two example systems where understanding cell interactions, adhesion, and motility is crucial—sickle red blood cells adhering to laminin, and CAR-T cells adhering to E-selectin. We illustrate the wide range of data types that can be extracted from this approach, including distributions of cell size and eccentricity, adhesion times, trajectories and velocities of adhered cells moving on a functionalized surface, as well as correlations among these different features at the single cell level. In all cases MBM allows for rapid collection and processing of large data sets, ranging from thousands to hundreds of thousands of individual adhesion events. The method is generalizable to study adhesion mechanisms in a variety of diseases, including cancer, blood disorders, thrombosis, inflammatory and autoimmune diseases, as well as providing rich datasets for theoretical modeling of adhesion dynamics.

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Section: Mainmentioning

confidence: 99%

Motion Blur Microscopy

Goreke,

Gonzales,

Shipley

et al. 2023

Preprint

View full text Add to dashboard Cite

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DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

2022

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Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

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Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Cited by 2 publications

References 0 publications

Motion Blur Microscopy

Motion Blur Microscopy

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

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