Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

Platzer, René; Rossboth, Benedikt K.; Schneider, Magdalena C.; Sevcsik, Eva; Baumgart, Florian; Stockinger, Hannes; Schütz, Gerhard J.; Huppa, Johannes B.; Brameshuber, Mario

doi:10.1038/s41467-020-18726-9

Cited by 24 publications

(52 citation statements)

References 62 publications

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“…However, Voronoї tessellation can be applied to a set of sub-pixel localizations obtained for a robust, non-photo-switchable emitter (e.g., Qdot) in a tracking and localization microscopy (TALM) concept [ 75 ] that complements sptPALM, wherein longer trajectories yield hidden information about subcellular morphology. One should note that a potential caveat is false-positive cluster detection due to emitter overcounting that arises from repetitive on-off blinking (especially true for immobile targets) [ 76 ]. The first step in our analysis was estimating individual Voronoї cell areas for mEos2-PH-PLCδ and Qdot-D2L localization maps, as it allows assessment of changes in emitter localization density in a parameter-free manner when compared to subjective cluster detection [ 41 ].…”

Section: Resultsmentioning

confidence: 99%

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

et al. 2021

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The role of lateral mobility and nanodomain organization of G protein-coupled receptors in modulating subcellular signaling has been under increasing scrutiny. Investigation of D2 dopamine receptor diffusion dynamics is of particular interest, as these receptors have been linked to altered neurotransmission in affective disorders and represent the primary target for commonly prescribed antipsychotics. Here, we applied our single quantum dot tracking approach to decipher intrinsic diffusion patterns of the wild-type long isoform of the D2 dopamine receptor and its genetic variants previously identified in several cohorts of schizophrenia patients. We identified a subtle decrease in the diffusion rate of the Val96Ala mutant that parallels its previously reported reduced affinity for potent neuroleptics clozapine and chlorpromazine. Slower Val96Ala variant diffusion was not accompanied by a change in receptor-receptor transient interactions as defined by the diffraction-limited quantum dot colocalization events. In addition, we implemented a Voronoї tessellation-based algorithm to compare nanoclustering of the D2 dopamine receptor to the dominant anionic phospholipid phosphatidylinositol 4,5-bisphosphate in the plasma membrane of live cells.

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Section: Resultsmentioning

confidence: 99%

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

et al. 2021

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“…Correction can also be made by measuring blinking behaviour in a separate sample of well-isolated fluorophores 7 , but this again adds complexity and experimental effort and requires the assumption that probe photophysics is maintained between the sample and the calibration. It is also possible to measure or simulate multiple-blinking using realistic photophysical models and use these to derive new CSR confidence intervals for the L-function curves 30 . It should be noted however that while all of these competing methods can be used to account for multiple-blinking, none produce a new set of corrected positions and therefore none can be used to extract rich descriptors such as those output by a clustering algorithm.…”

Section: Discussionmentioning

confidence: 99%

Correction of multiple-blinking artefacts in photoactivated localisation microscopy

Jensen

Hoh

Williamson

et al. 2021

Preprint

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Photoactivated localisation microscopy (PALM) produces an array of localisation coordinates by means of photoactivatable fluorescent proteins. However, observations are subject to fluorophore multiple-blinking and each protein is included in the dataset an unknown number of times at different positions, due to localisation error. This causes artificial clustering to be observed in the data. We present a workflow using calibration-free estimation of blinking dynamics and model-based clustering, to produce a corrected set of localisation coordinates now representing the true underlying fluorophore locations with enhanced localisation precision. These can be reliably tested for spatial randomness or analysed by other clustering approaches, and previously inestimable descriptors such as the absolute number of fluorophores per cluster are now quantifiable, which we validate with simulated data. Using experimental data, we confirm that the adaptor protein, LAT, is clustered at the T cell immunological synapse, with its nanoscale clustering properties depending on location and intracellular phosphorylatable tyrosine residues.

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“…Overcounting due to multiple blinking makes quantifying small clusters challenging (64,65). Labeling methodology generally limited to proteins.…”

Section: Methods Limitations Required Developmentsmentioning

confidence: 99%

The Role of Protein and Lipid Clustering in Lymphocyte Activation

et al. 2021

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Lymphocytes must strike a delicate balance between activating in response to signals from potentially pathogenic organisms and avoiding activation from stimuli emanating from the body's own cells. For cells, such as T or B cells, maximizing the efficiency and fidelity, whilst minimizing the crosstalk, of complex signaling pathways is crucial. One way of achieving this control is by carefully orchestrating the spatiotemporal organization of signaling molecules, thereby regulating the rates of protein-protein interactions. This is particularly true at the plasma membrane where proximal signaling events take place and the phenomenon of protein microclustering has been extensively observed and characterized. This review will focus on what is known about the heterogeneous distribution of proteins and lipids at the cell surface, illustrating how such distributions can influence signaling in health and disease. We particularly focus on nanoscale molecular organization, which has recently become accessible for study through advances in microscope technology and analysis methodology.

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Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy

Cited by 24 publications

References 62 publications

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

Correction of multiple-blinking artefacts in photoactivated localisation microscopy

The Role of Protein and Lipid Clustering in Lymphocyte Activation

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