Super Temporal-Resolved Microscopy (STReM)

Wang, Wenxiao; Shen, Hao; Shuang, Bo; Hoener, Benjamin S.; Tauzin, Lawrence J.; Moringo, Nicholas A.; Kelly, Kevin F.; Landes, Christy F.

doi:10.1021/acs.jpclett.6b02098

Cited by 33 publications

(48 citation statements)

References 57 publications

(78 reference statements)

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“…6 ). Lower laser power densities can be used with the help of data analysis methods that tolerate a higher active-dye density and slight overlap of the PSFs, such as SHRIMP [ 34 , 54 ], Bayesian analysis of the blinking and bleaching (3B) [ 37 , 55 ], compressed sensing [ 56 ], and super-resolution optical fluctuation imaging (SOFI) [ 57 – 58 ], or methods that provide higher time resolution such as supertemporal-resolved microscopy (STReM) [ 59 ].…”

Section: Resultsmentioning

confidence: 99%

Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging

Pyle¹,

Chen²

2017

Beilstein J. Nanotechnol.

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Super-resolution imaging of single DNA molecules via point accumulation for imaging in nanoscale topography (PAINT) has great potential to visualize fine DNA structures with nanometer resolution. In a typical PAINT video acquisition, dye molecules (YOYO-1) in solution sparsely bind to the target surfaces (DNA) whose locations can be mathematically determined by fitting their fluorescent point spread function. Many YOYO-1 molecules intercalate into DNA and remain there during imaging, and most of them have to be temporarily or permanently fluorescently bleached, often stochastically, to allow for the visualization of a few fluorescent events per DNA per frame of the video. Thus, controlling the fluorescence on–off rate is important in PAINT. In this paper, we study the photobleaching of YOYO-1 and its correlation with the quality of the PAINT images. At a low excitation laser power density, the photobleaching of YOYO-1 is too slow and a minimum required power density was identified, which can be theoretically predicted with the proposed method in this report.

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Section: Resultsmentioning

confidence: 99%

Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging

Pyle¹,

Chen²

2017

Beilstein J. Nanotechnol.

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“…Moreover, proposed models extracted from these techniques likely oversimplify the underlying mechanism of interfacial adsorption-desorption, surface diffusion, and surfaceinduced protein unfolding effects (26,27). Single-molecule microscopy is well suited to directly visualize protein mass transport at a wide array of complex interfaces one molecule at a time with a high spatiotemporal resolution (29)(30)(31)(32)(33)(34). However, atomistic details of protein surface domains and/or substrate chemistries interacting during protein physisorption is not resolved in comparison to atomistic modeling techniques (35).…”

mentioning

confidence: 99%

A mechanistic examination of salting out in protein–polymer membrane interactions

Moringo

Bishop

Shen

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Developing a mechanistic understanding of protein dynamics and conformational changes at polymer interfaces is critical for a range of processes including industrial protein separations. Salting out is one example of a procedure that is ubiquitous in protein separations yet is optimized empirically because there is no mechanistic description of the underlying interactions that would allow predictive modeling. Here, we investigate peak narrowing in a model transferrin–nylon system under salting out conditions using a combination of single-molecule tracking and ensemble separations. Distinct surface transport modes and protein conformational changes at the negatively charged nylon interface are quantified as a function of salt concentration. Single-molecule kinetics relate macroscale improvements in chromatographic peak broadening with microscale distributions of surface interaction mechanisms such as continuous-time random walks and simple adsorption–desorption. Monte Carlo simulations underpinned by the stochastic theory of chromatography are performed using kinetic data extracted from single-molecule observations. Simulations agree with experiment, revealing a decrease in peak broadening as the salt concentration increases. The results suggest that chemical modifications to membranes that decrease the probability of surface random walks could reduce peak broadening in full-scale protein separations. More broadly, this work represents a proof of concept for combining single-molecule experiments and a mechanistic theory to improve costly and time-consuming empirical methods of optimization.

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“…Recent work published by Wang et al has provided representative examples of one such method (STReM, Figure ). , In this case, the authors employed a rotating phase mask placed in the microscope detection path to encode additional temporal information in images of fluorescent beads and a dye-labeled protein adsorbing to a surface (Figure a). The orientation of the particle image for rapidly adsorbing and desorbing species represented a snapshot of the point-spread function produced when each particle adsorbed to the surface.…”

Section: Optical Microscopic Techniques For Polymer Characterizationmentioning

confidence: 99%