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Quantitative Photoactivated Localization Microscopy of Membrane Receptor Oligomers

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Fluorescence Spectroscopy and Microscopy in Biology

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 20))

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Abstract

Optical super-resolution microscopy allows the visualization of cellular structures with a spatial resolution of a few tens of nanometers and has revolutionized our understanding in cell biology. However, the spatial resolution achieved with to-date super-resolution microscopy methods in cells is not sufficient to optically resolve proteins within densely packed protein clusters, which themselves represent relevant functional assemblies in cells. Single-molecule localization microscopy (SMLM) offers an opportunity to retrieve this information by analyzing the kinetics of on-off-switching (“blinking”) observed in the fluorescence emission signatures of single fluorophores. We report the theoretical background of kinetics-based molecular quantification of SMLM data, discuss fluorescent probes and methods for protein labeling, and showcase applications in biology.

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Abbreviations

DNA-PAINT:

DNA-based point accumulation for imaging in nanoscale topography

dSTORM:

Direct stochastic optical reconstruction microscopy

FCS:

Fluorescence correlation spectroscopy

FPALM :

Fluorescence photoactivated localization microscopy

FRET:

Förster resonance energy transfer

HGF:

Hepatocyte growth factor

InlB:

Internalin B

N&B:

Number and brightness analysis

paFP:

Photoactivatable fluorescent protein

PAINT:

Point accumulation for imaging in nanoscale topography

PALM:

Photoactivated localization microscopy

pcFP:

Photoconvertible fluorescent protein

PC-PALM:

Pair-correlation photoactivated localization microscopy

PLAD :

Preligand assembly domain

PSF:

Point spread function

QAFKA :

Quantitative algorithm for fluorescence kinetics analysis

qPAINT:

Quantitative point accumulation for imaging in nanoscale topography

qPALM:

Quantitative photoactivated localization microscopy

qSMLM:

Quantitative single-molecule localization microscopy

RTK:

Receptor tyrosine kinase

SMLM:

Single-molecule localization microscopy

STORM:

Stochastic optical reconstruction microscopy

TIRF:

Total internal reflection fluorescence

TLR4:

Toll-like receptor 4

TNFR1:

Tumor necrosis factor receptor 1

TNFα:

Tumor necrosis factor alpha

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Acknowledgments

The authors gratefully acknowledge funding by the German Science Foundation (grants SFB 902, SFB 1177, HE 6166/17-1, GRK 2566 project number 414985841), LOEWE (Frankfurt Cancer Insititute), and the Volkswagen Foundation (grant 91067-9). We thank all past and present members of the Single-Molecule Biophysics group and our collaborators for their valuable contributions to this research.

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  1. Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Frankfurt, Germany

    Marina S. Dietz & Mike Heilemann

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  1. Marina S. Dietz
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Correspondence to Mike Heilemann .

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  1. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Radek Šachl

  2. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Mariana Amaro

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Dietz, M.S., Heilemann, M. (2022). Quantitative Photoactivated Localization Microscopy of Membrane Receptor Oligomers. In: Šachl, R., Amaro, M. (eds) Fluorescence Spectroscopy and Microscopy in Biology. Springer Series on Fluorescence, vol 20. Springer, Cham. https://doi.org/10.1007/4243_2022_37

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