Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

Patel, Lekha; Owen, Dylan M.; Cohen, Edward

doi:10.1101/834572

Cited by 6 publications

(16 citation statements)

References 29 publications

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“…Considering that the numbers of molecules counted was mostly <10 per cluster, it is important to note that these figures should be considered as rough estimates and not true numbers of molecules. The accurate determination of molecule numbers using SMLM methods remains a challenge due to labelling efficiency, re-emitting fluorophores, variable imaging conditions and differences in analytical approaches [ 66 ]. A potential undercounting in our experiments may arise from low labelling efficiency.…”

Section: Discussionmentioning

confidence: 99%

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

Yuan

Jacobs

Llorente-Garcia

et al. 2021

Viruses

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The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

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Section: Discussionmentioning

confidence: 99%

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

Yuan

Jacobs

Llorente-Garcia

et al. 2021

Viruses

View full text Add to dashboard Cite

show abstract

“…Considering that the numbers of molecules counted was mostly <10 per cluster, it is important to note that these figures should be considered as rough estimates and not true numbers of molecules. The accurate determination of molecule numbers using SMLM methods remains a challenge due to labelling efficiency, re-emitting fluorophores, variable imaging conditions and differences in analytical approaches (60). A potential undercounting in our experiments may arise from low labelling efficiency.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule super-resolution imaging of T-cell plasma membrane CD4 redistribution upon HIV-1 binding

Yuan

Jacobs

Llorente-Garcia

et al. 2021

Preprint

View full text Add to dashboard Cite

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For the first time, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.

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“…The on state was accessible from any of the dark states, while dark state 0 was accessible only from the on state, and dark states 01 and 02 were accessible only from the previous dark states, 0 and 01, respectively. We used the following parameters (using the notation described in (16, 18)) to capture essential elements of our experimental observations: λ (0 → 1) = 1.2 Hz; λ (0 → 01) =0.05 Hz; λ (01 → 02) =0.0033 Hz; λ (01 → 1) =0.02 Hz; λ (02 → 1) =0.0005 Hz; 2(1 → 0) = 5 Hz; μ(1 → B ) =0.05 Hz. The continuous time Markov process was simulated with the MATLAB File Exchange code “simCTMC.m” (19).…”

Section: Methodsmentioning

confidence: 99%

A method to estimate the effective point spread function of static single molecule localization microscopy images

Fazekas

Kim

Flanagan-Natoli

et al. 2022

Preprint

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Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins.STATEMENT OF SIGNIFICANCESingle molecule localization microscopy (SMLM) is a class of imaging methods that resolve fluorescently labeled structures beyond the optical resolution limit of visible light. SMLM detects stochastically blinking labels over time, and localizes each blink with precision of order 10 nm. The effective resolution depends on factors such as signal-to-noise ratio, localization algorithm, and several post-processing steps such as stage drift correction. We present a method to evaluate this effective resolution by taking advantage of temporal correlations of fluorophore blinking to separate the distribution of pairs of localizations from the same molecule from those from different molecules. The method is robust on useful timescales for a range of SMLM probes.

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Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

Cited by 6 publications

References 29 publications

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

Single-molecule super-resolution imaging of T-cell plasma membrane CD4 redistribution upon HIV-1 binding

A method to estimate the effective point spread function of static single molecule localization microscopy images

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