Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis

Sengupta, Prabuddha; Jovanovic-Talisman, Tijana; Skoko, Dunja; Renz, Malte; Veatch, Sarah L.; Lippincott‐Schwartz, Jennifer

doi:10.1038/nmeth.1704

Cited by 534 publications

(668 citation statements)

References 32 publications

Supporting

Mentioning

648

Contrasting

Unclassified

Order By: Relevance

“…mEos2 also had high blinking rate, which can be problematic in densely labeled samples since the photoactivation times become shorter obscuring the temporal separation between blinking and photoactivation 13 . Alternative analysis methods that do not rely on defining a cut-off time, such as pair correlation analysis 15,31 , may thus be preferable for quantifying super-resolution data with this fluorescent protein. PA-mCherry and Dendra2 showed moderate photoactivation and lower blinking rates and are therefore well-suited for quantitative superresolution imaging provided that the photoactivation efficiencies are taken into account.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, methods have been developed to reduce over-counting artifacts. These include pair correlation analysis (PC-PALM) 15,31 or methods that characterize fluorescence off-times and group localizations together based on a characteristic dark time 13,30 . After accounting for re-activation, the remaining localizations are a measure of the number of detected fluorescent probes.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

et al. 2014

View full text Add to dashboard Cite

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a one to one labeling ratio between the fluorescent protein and the protein of interest makes them attractive for quantitative single molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e. photoactivation efficiency) plays a critical role in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single molecule level to replicate the conditions used in super-resolution imaging. Here, we used the human Glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single molecule counting-PALM to determine the percentage of photoactivated fluorescent protein for mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

show abstract

Section: Conclusion and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Quantifying the properties of the FA substructure with SMLM clustering methods is therefore challenging. Clusters in SMLM data are often characterized using the pair correlation function (27) or Ripley's K(r) or L(r) function (28). These functions describe the density around a certain point as a function of the distance r from that point.…”

Section: Emgmmentioning

confidence: 99%

Investigating Focal Adhesion Substructures by Localization Microscopy

Deschout

Platzman

Sage

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin b3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i.e., fibronectin-coated substrates and specifically biofunctionalized nanopatterned substrates). To quantify the substructure of FAs, we developed a clustering method based on expectation maximization of a Gaussian mixture that accounts for localization uncertainty and background. Analysis of our SMLM data indicates that the structures within FAs, characterized as a Gaussian mixture, typically have areas between 0.01 and 1 mm 2 , contain 10-100 localizations, and can exhibit substantial eccentricity. Our approach based on SMLM opens new avenues for studying structural and functional biology of molecular assemblies that display substantial varieties in size, shape, and density.

show abstract

“…To investigate and quantify clustering behaviour, widely used are Ripley's K-function 14-16 and pair-correlation (PC) analysis 17,18 . Both rely on drawing a series of concentric shapes -circles in the case of the K-function and tori in the case of PC -around each localisation and counting the number of neighbours enclosed.…”

mentioning

confidence: 99%

Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

View full text Add to dashboard Cite

Correspondence should be addressed to patrick. rubin-delanchy@bristol.ac.uk and dylan.owen@kcl.ac.uk Single-molecule identification-based super-resolution microscopy techniques such as photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. While many algorithms exist for the identification and localisation of molecules from the raw image data, methods for analysing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here, we present the first model-based Bayesian approach to evaluate molecular cluster assignment proposals which, in this article, are generated by analysis based on Ripley's Kfunction. The method is also the first to take full account of the individual localisation precisions calculated for each emitter. The technique is validated using simulated and experimental data from which we characterise the clustering behaviour of CD3ζ, an important subunit of the CD3-T cell receptor complex required for T cell function, in resting and activated primary human T cells.Conventional fluorescence microscopes produce images of the distribution of fluorophores in the sample convolved with the microscope Point Spread Function (PSF). Due to diffraction, this PSF typically has a width of hundreds of nanometres meaning the resulting image has a resolution, as assessed by the Rayleigh criterion, of ~200 nm. Several strategies now exist to circumvent this resolution limit 1 . Some of these, such as Stimulated Emission Depletion (STED) microscopy, rely on narrowing the excitation spot of a confocal microscope by means of a toroidal depletion beam and the process of stimulated emission 2,3 . Despite the increased resolution, these produce conventional fluorescence images, i.e., arrays of pixels with values representing the fluorescence intensity at those locations. Quantification can be performed in the same way as for conventional microscopes.Another strategy is based on Single-Molecule Localisation Microscopy (SMLM) [4][5][6][7] . This relies on the temporal separation of the excitation of fluorophores in the sample whose PSFs would otherwise overlap at the detector.The position of each fluorophore can then be estimated from the centres of the PSFs. Many algorithms are available to extract the x-y coordinates of the molecules [8][9][10] . Each emitter can be localised to a precision between 10 and 30 nm. Common strategies for the temporal separation of molecules involve intra-molecular rearrangements to switch from dark to fluorescent states or the exploitation of non-emitting molecular radicals 11,12 . These strategies are typically pursued using photoactivatable or photoconvertible fluorescent proteins or small molecule probes coupled with a reducing buffer and immunostaining protocols 13 . We refer to all such strategies as SMLM.Unlike non-pointillist microscopy methods, SMLM imaging does not produce a conventional image. Instead, the raw data is a list of the...

show abstract

Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis

Cited by 534 publications

References 32 publications

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Investigating Focal Adhesion Substructures by Localization Microscopy

Bayesian cluster identification in single-molecule localization microscopy data

Contact Info

Product

Resources

About