Real-time 3D stabilization of a super-resolution microscope using an electrically tunable lens

Tafteh, Reza; Abraham, Libin; Seo, Denny; Lu, Henry Y.; Gold, Michael R.; Chou, Kuang-Chao

doi:10.1364/oe.24.022959

Cited by 14 publications

(20 citation statements)

References 37 publications

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“…The latter was obtained by biopanning an optimized M13 bacteriophage phagemid library presenting ∼10 9 different scFv clones against Rv1747 (FHA-1 and partial linker). SMLM imaging was carried out using a custom-built microscope with a real-time sample drift-stabilization system as described previously (30,31). Custom software written in MATLAB (MathWorks) was used to reconstruct the SMLM images (30).…”

Section: Methodsmentioning

confidence: 99%

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Heinkel

Abraham

et al. 2019

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

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Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

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Section: Methodsmentioning

confidence: 99%

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Heinkel

Abraham

et al. 2019

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

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“…S1 for additional data on blob features). Relative to the PC3 data 26 , we observed a doubling in molecular localizations for S1B scaffolds relative to S1A scaffolds and increased modularity of S1B scaffolds in the HeLa data set that we attribute to the improved resolution obtained with the real-time drift control SMLM 29,33 . Increased Cav1 localization number in S1B scaffolds parallels the increased size (X-range) and reduced network density of these clusters relative to S1A scaffolds, reflecting differences between these structures that led to their classification as distinct cluster groups in this and our previous analysis 26 .…”

Section: Group Matchingmentioning

confidence: 68%

“…A 405 nm laser line (Laserglow Technologies) was used to activate Alexa Fluor 647. The detailed optical setup and the imaging acquisition procedure were described previously 29,33 .…”

Section: Smlm Imagingmentioning

confidence: 99%

“…We then matched the blobs with the sub-modules of the various Cav1 domains based on the similarity (i.e., smaller Euclidean or L2 norm) between their biosignatures. To enhance localization precision, we used an SMLM microscope equipped with real-time nanometer-scale drift correction hardware 29 and included only high-precision localizations to improve localization accuracy 30,31 . With this inhouse built SMLM microscope, the localization precision approaches 10 nm 32 , and the drift is limited to 1 nm in the x-y plane and 3 nm in the z axis 29,33 .…”

Section: Introductionmentioning

confidence: 99%

“…To enhance localization precision, we used an SMLM microscope equipped with real-time nanometer-scale drift correction hardware 29 and included only high-precision localizations to improve localization accuracy 30,31 . With this inhouse built SMLM microscope, the localization precision approaches 10 nm 32 , and the drift is limited to 1 nm in the x-y plane and 3 nm in the z axis 29,33 . Modularity analysis and group matching show that S1A scaffolds can dimerize to form S1B scaffolds and oligomerize to form hemispherical S2 scaffolds.…”

Section: Introductionmentioning

confidence: 99%

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Super-resolution modularity analysis shows polyhedral caveolin-1 oligomers combine to form scaffolds and caveolae

Khater

Liu

Chou

et al. 2018

Preprint

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Caveolin-1 (Cav1), the coat protein for caveolae, also forms non-caveolar Cav1 scaffolds. Single molecule Cav1 super-resolution microscopy analysis previously identified caveolae and three distinct scaffold domains: smaller S1A and S2B scaffolds and larger hemispherical S2 scaffolds. Application here of network modularity analysis of SMLM data for endogenous Cav1 labeling in HeLa cells shows that small scaffolds combine to form larger scaffolds and caveolae. We find modules within Cav1 blobs by maximizing the intra-connectivity between Cav1 molecules within a module and minimizing the inter-connectivity between Cav1 molecules across modules, which is achieved via spectral decomposition of the localizations adjacency matrix. Features of modules are then matched with intact blobs to find the similarity between the module-blob pairs of group centers. Our results show that smaller S1A and S1B scaffolds are made up of small polygons, that S1B scaffolds correspond to S1A scaffold dimers and that caveolae and hemi-spherical S2 scaffolds are complex, modular structures formed from S1B and S1A scaffolds, respectively. Polyhedral interactions of Cav1 oligomers therefore leads progressively to the formation of larger and more complex scaffold domains and the biogenesis of caveolae.

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Simple, non‐mechanical and automatic calibration approach for axial‐scanning microscopy with an electrically tunable lens

Zhao

Wen

et al. 2023

Microscopy Res & Technique

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We describe a simple and robust calibration approach for axial‐scanning microscopy that realizes axial focus shifts with an electrically tunable lens (ETL). We demonstrate the calibration approach based on a microscope with an ETL placed close to the rear stop of the objective lens. By introducing a target—consisted of repeating lines at one known frequency and placed at a ~45° angle to the imaging path, the calibration method captures multiple images at different ETL currents and calibrates the dependence of the axial focus shift on the ETL current by evaluating the sharpness of the captured images. It calibrates the dependence of the magnification of the microscope on the ETL current by measuring the period of the repeating lines in the captured images. The experimental results show that different from the conventional calibration procedure, the proposed scheme does not involve any mechanical scanning and can simultaneously calibrate the dependence of the axial focus shift and the magnification on the ETL current. This might facilitate imaging studies that require the measurement of fine structures in a 3D volume. We also show the calibration procedure can be used to estimate the radius of a conner‐arc sample, fabricated using laser micromachining. We believe that this easy‐to‐use calibration approach may facilitate use of ETLs for a variety of imaging platforms. It may also provide new insights for the development of novel 3D surface measurement methods.Research HighlightsThe proposed calibration scheme does not involve any mechanical scanning and can simultaneously calibrate the dependence of the axial focus shift and the magnification on the electrically tunable lens (ETL) current. It might facilitate imaging studies that require the measurement of fine structures in a 3D volume, and the use of ETLs for a variety of imaging platforms. It may also provide new insights for the development of novel 3D surface measurement methods.

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Real-time 3D stabilization of a super-resolution microscope using an electrically tunable lens

Cited by 14 publications

References 37 publications

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Super-resolution modularity analysis shows polyhedral caveolin-1 oligomers combine to form scaffolds and caveolae

Simple, non‐mechanical and automatic calibration approach for axial‐scanning microscopy with an electrically tunable lens

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