QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ

Henriques, Ricardo; Lelek, Mickaël; Fornasiero, Eugenio F.; Valtorta, Flavia; Zimmer, Christophe; Mhlanga, Musa M.

doi:10.1038/nmeth0510-339

Cited by 369 publications

(341 citation statements)

References 9 publications

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“…While methods to localise molecules from the raw fluorescence data have been extensively analysed 8,10,33 , the subsequent interrogation of the point pattern data has been relatively under-studied. We have demonstrated a new, Bayesian cluster analysis algorithm for SMLM data.…”

Section: Discussionmentioning

confidence: 99%

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Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

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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...

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Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

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“…The section was incubated in imaging buffer containing oxygen scavengers and imaged using a fl uorescence microscope equipped with an EMCCD camera and appropriate laser lines and fi lter sets. For the dSTORM image, a total of 10,000 frames (30 ms/ frame) were collected and processed using ImageJ with the QuickPALM plug-in [ 27 ] 1. Twinsil (two-component silicone glue, Picodent, Wipperfürth, Germany).…”

Section: Figmentioning

confidence: 99%

“…Process the images with the PALM/STORM software (QuickPALM [ 27 ] was used to process dSTORM images in this chapter). Because fl uorescence from Alexa-647 dyes is…”

Section: Prepare Dstorm Imaging Buffer ( See Note 23 ) [ 26 ]mentioning

confidence: 99%

Nanometer-Resolution Fluorescence Electron Microscopy (Nano-EM) in Cultured Cells

Watanabe¹,

Lehmann²,

Hujber³

et al. 2013

Methods in Molecular Biology

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Nano-resolution fl uorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are fi rst imaged using a super-resolution fl uorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present fi ve technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fl uorescence, the use of ground-state depletion instead of photoactivation for temporal control of fl uorescence, the use of organic fl uorophores instead of fl uorescent proteins to obtain brighter fl uorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

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“…Some of these algorithms are freely available as standalone software (LivePalm [69], rapidStorm [70]) and ImageJ plugins (QuickPalm [71]). …”

Section: Stochastic Localisation Microscopy In 3dmentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

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Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffractionlimited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

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QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ

Cited by 369 publications

References 9 publications

Bayesian cluster identification in single-molecule localization microscopy data

Bayesian cluster identification in single-molecule localization microscopy data

Nanometer-Resolution Fluorescence Electron Microscopy (Nano-EM) in Cultured Cells

Fluorescence nanoscopy. Methods and applications

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