The Role of Probe Photophysics in Localization-Based Superresolution Microscopy

Pennacchietti, Francesca; Gould, Travis J.; Hess, Samuel T.

doi:10.1016/j.bpj.2017.11.116

Cited by 3 publications

(3 citation statements)

References 46 publications

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“…Up to 20,000 frames were collected per field of view with an exposure time of 25 ms. Exposure times of 25 ms were used for all experiments to maximize the fluorescent signal to background ratio (32). A flip mirror in the emission pathway enables toggling the microscope between fluorescence imaging and phase contrast imaging modes without having to change the objective lens of the microscope.…”

Section: Introductionmentioning

confidence: 99%

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

Rocha

Corbitt

Yan

et al. 2019

Biophysical Journal

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The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate wellsampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in two-dimensional or three-dimensional singlemolecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.

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Section: Introductionmentioning

confidence: 99%

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

Rocha

Corbitt

Yan

et al. 2019

Biophysical Journal

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“…It has previously been shown for the use of mEOS2 31 , 41 – 44 , PA-GFP, and PA-mCherry1 45 that detailed fluorophore characterization prior to conducting SMLM is instrumental for quantitative readout and maximally achievable resolution 46 . As is outlined in Fig.…”

Section: Discussionmentioning

confidence: 99%

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

et al. 2020

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Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

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“…This will depend on the photophysical properties of the fluorophore. Pennacchietti et al (2017) have made a detailed study of a Dendra2. In selecting a fluorophore to use under cryo-FM, we could use their methodology as a model for evaluating candidate fluorophores.…”

Section: What Avenues Are Available To Us?mentioning

confidence: 99%

Where in the cell is my protein?

DeRosier

2021

Quart. Rev. Biophys.

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The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253-278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.How well can we align the fluorescence map to the cryo-EM map? 9Concluding remarks and outstanding questions 10

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The Role of Probe Photophysics in Localization-Based Superresolution Microscopy

Cited by 3 publications

References 46 publications

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

Where in the cell is my protein?

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