Raster Image Correlation Spectroscopy Performance Evaluation

Longfils, Marco; Smisdom, Nick; Ameloot, Marcel; Rudemo, Mats; Lemmens, Veerle; Fernández, Guillermo Solís; Röding, Magnus; Lorén, Niklas; Hendrix, Jelle; Särkkä, Aila

doi:10.1016/j.bpj.2019.09.045

Cited by 13 publications

(13 citation statements)

References 27 publications

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“…Parallel application of both techniques allow identifying and characterizing different possible mobile protein populations [41]. Essential to this is choosing imaging conditions suited to the type of diffusion process (for RICS, see [42], for TICS, see [43]).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Lemmens

Thevelein

Kankowski

et al. 2021

Preprint

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Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR alpha3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR alpha3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-alpha3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-alpha3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the alpha3K splice variant. Our data are compatible with a model where alpha3 heteropentamerization fine-tunes mobility and activity of GlyR alpha3 channels, which is important to understand and tackle alpha3 related diseases.

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Section: Resultsmentioning

confidence: 99%

“…This revealed that the species observed with RICS still exhibited faster diffusion than those observed with TICS when measured at RT, and thus indeed represents a different subpopulation. Images were collected using parameters appropriate for RICS [42], i.e. 256×256 pixels 2 with a 50 nm pixel size.…”

Section: Methodsmentioning

confidence: 99%

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Lemmens

Thevelein

Kankowski

et al. 2021

Preprint

Self Cite

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“…For example, a dwell time of 25 μs and pixel size ≤0.05 μm can measure the diffusion of a 25 kDa cytoplasmic protein by the second or third pixel scanned [115,124]. Longfils et al observed a 3-5-fold decrease in error on the diffusion constant by decreasing pixel size from 100 to 50 nm and they also observed consistent high quality RICS data over a board concentration range from nM to μM [131]. Minimum ROI of 2 μm × 2 μm is recommended by Brown et al to prevent under-sampling.…”

Section: Raster Image Correlation Spectroscopy (Rics)mentioning

confidence: 94%

Fluorescence Fluctuation Techniques for the Investigation of Structure-Function Relationships of G-Protein-Coupled Receptors

Youker¹,

Voet²

2020

Fluorescence Methods for Investigation of Living Cells and Microorganisms

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G-protein-coupled receptors (GPCRs) are seven transmembrane receptors that form the largest superfamily of signaling proteins, and the family members function in a diverse array of metabolic pathways including cardiac function, immune response, neurotransmission, smell, taste, cell differentiation and growth, and vision. It is becoming clear that alteration in the quaternary structure of the GPCR receptor can have a profound impact on signaling capabilities. Biochemical, biophysical, physiological, x-ray crystallographic, and computational methods have been used over the last 40-50 years to study the structure and function of GPCRs. Evidence from these studies confirm that GPCRs can be organized as monomers, dimers, and higher-order oligomers. However, many times, these methods require extraction of the receptor from its native environment and high levels of expression and only provide a snapshot of information. A need arose for techniques that could measure the assembly and disassembly of receptors at few-to-single molecule resolution in their native environment at fast time scales. In the last 20 years, fluorescence fluctuation techniques have filled this need and provided new insight into the dynamics of GPCR organization in the absence and presence of ligands, agonists, and antagonists. In this book chapter, we provide a brief introduction to GPCR structure and function [Section 1]. An overview of the theoretical basis for fluorescence fluctuations techniques (FFTs) and how FFTs can be used to study the oligomeric structure of GPCRs in live and fixed cells is explained [Section 2]. We discuss the advantages and limitations of FFTs [Section 3], and finally, we summarize select case studies on GPCR structure and function revealed by FFT experiments [Section 4].

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“…The most interesting feature of CLSM is the capability for single-cell microscopic spectroscopy, which allows obtaining spectroscopic information inside small regions and single cells [35]. Another group of facilities deals with the quantitative analysis of dynamic processes in living cells using techniques such as fluorescence recovery after photobleaching (FRAP) [129,130], fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM) [131], photoactivation, and photoconversion. Recently, a lot of additional techniques appear in modern CLSMs, which open new perspectives for single-cell investigation, such as white laser, which provides the ability to obtain not only fluorescence emission spectra, but also single-cell excitation and absorption spectra [132]; hyperspectral CLSM that allows more precise fluorescence spectra through the cell thickness and gives more detailed fluorescent pigments location [34,133]; and STED and multiphotonic techniques, which extend the CLSM abilities to single-molecular studies [134,135].…”

Section: Confocal Microscopic Spectroscopymentioning

confidence: 99%

Section: Raster Image Correlation Spectroscopy (Rics)mentioning

confidence: 99%

Fluorescence Methods for Investigation of Living Cells and Microorganisms

Grigoryeva¹

2020

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In the last six years, she was involved in studying cyanobacteria and developing fluorescence methods for their investigation. She developed an in vivo technique for investigating weak external actions on the physiological state of cyanobacteria. Dr. Grigoryeva recently elaborated on the base of a neural network algorithm an original method of ataxonomic classification of cyanobacteria using single-cell self-fluorescence spectra. Her research interests include both environmental and biotechnological aspects of cyanobacterial applications.

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Raster Image Correlation Spectroscopy Performance Evaluation

Cited by 13 publications

References 27 publications

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers

Fluorescence Fluctuation Techniques for the Investigation of Structure-Function Relationships of G-Protein-Coupled Receptors

Fluorescence Methods for Investigation of Living Cells and Microorganisms

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