Molecule counts in complex oligomers with single-molecule localization microscopy

Baldering, Tim N.; Bullerjahn, Jakob Tómas; Hummer, Gerhard; Heilemann, Mike; Malkusch, Sebastian

doi:10.1088/1361-6463/ab3b65

Cited by 12 publications

(17 citation statements)

References 38 publications

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“…However, the quantitative analysis of fluorescent protein emission kinetics allows to extract the oligomeric state of proteins within these nanoclusters ( Dietz and Heilemann, 2019 ). Here, we used quantitative PALM (qPALM) ( Fricke et al., 2015 ; Hummer et al., 2016 ; Baldering et al., 2019a ) and aimed to determine the oligomeric state of MET-mEos4b before and after ligand stimulation.…”

Section: Resultsmentioning

confidence: 99%

“…Since individual cells do not contain enough statistics, we randomly mixed the data of each condition ten times and analyzed 80 percent of the respective data. Histograms of the relative frequency of number of blinking events were plotted and fitted with the qSMLM software (Baldering et al, 2019a) yielding the corrected monomer/dimer fractions. The mean values were determined for each condition and the error is given as standard deviation.…”

Section: Resource Availabilitymentioning

confidence: 99%

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CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

et al. 2021

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Summary Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

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Section: Resultsmentioning

confidence: 99%

Section: Resource Availabilitymentioning

confidence: 99%

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

et al. 2021

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“…12 In this technique the stoichiometry of protein complexes, typically non-resolvable spatially, can in principle still be determined by counting fluorescence bursts from each complex. [12][13][14][15][16] However, a limited PCE results in severe undercounting errors.…”

Section: Toc Graphicsmentioning

confidence: 99%

mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

Wulffele

Thédié

Glushonkov

et al. 2022

Preprint

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Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state.

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“…Fluorescent proteins, on the other hand, may be directly co-expressed with a protein of interest, but θ must now account for incomplete maturation and the photoconversion efficiency. Fortunately, for many fluorescent proteins, θ may be measured a priori and appears to be a fairly robust property of the fluorophore [24][25][26]. Now let us incorporate the labeling efficiency into our mixture model of Eq.…”

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confidence: 99%

Quantitative Single-Molecule Imaging with Statistical Machine Learning

Boonkird

Nino

Milstein

2021

Preprint

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Single-molecule localization microscopy (SMLM) is a super-resolution technique capable of rendering nanometer scale images of cellular structures. Recently, much effort has gone into developing SMLM into a quantitative method capable of determining the abundance and stoichiometry of macromolecular complexes. These methods often require knowledge of the complex photophysical properties of photoswitchable flourophores. We previously developed a simpler method built upon the observation that most photswitchable fluorophores emit an exponentially distributed number of blinks before photobleaching, but its utility was limited by the need to calibrate for the blinking distribution. Here we extend this method by incorporating a machine learning technique known as Expectation-Maximization (EM) and apply it to a statistical mixture model of monomers, dimers and trimers. We show that the protomer fractions and the underlying single-fluorophore blinking distributions can be inferred, simultaneously, from SMLM datasets, obviating the need for an additional calibration and greatly expanding the applicability of this technique. To illustrate the utility of our approach, we benchmark the method on both simulated datasets and experimental datasets assembled from dSTORM images of Alexa-647 labeled DNA nanostructures.

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Molecule counts in complex oligomers with single-molecule localization microscopy

Cited by 12 publications

References 38 publications

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

Quantitative Single-Molecule Imaging with Statistical Machine Learning

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