Single-molecule counting applied to the study of GPCR oligomerization

Fluorescence-Based Detection of Proteins and Their Interactions in Live Cells

“…While conceptually straightforward, a major hurdle in the PSC approach is the presence of noise due to various factors, such as background fluorescence, variable fluorophore emission, fluorophore blinking, and other photophysical effects exhibited by fluorescent molecules, whether subjected to single- or two-photon excitation . A second major hurdle in PSC is the manner in which steps are identified and counted. , Visual inspection is the primary method to analyze stepwise photobleaching intensity traces with some assistance from noise reduction via edge-preserving signal filtering. However, with an increasing number of fluorophores, the process can become unreliable and subjective …”

“…The above-described effects that ligand binding has on the receptor oligomerization are important, as they add important details to the body of evidence available until recently (see next section). A discussion of earlier references on the topic of oligomerization studies using PSC can be found in the review by Milstein et al…”

“…Photobleaching step counting (PSC) is a method used in fluorescence microscopy to determine the number of fluorescent molecules present in an oligomeric complex. 17 In this method, a small region of the sample is repeatedly excited with light, and the fluorescence intensity is monitored over time. As the excitation light causes the fluorescence to diminish (photobleaching), the fluorescence intensity will decrease until no fluorescence can be detected.…”

“…18 A second major hurdle in PSC is the manner in which steps are identified and counted. 17,19 Visual inspection is the primary method to analyze stepwise photobleaching intensity traces with some assistance from noise reduction via edge-preserving signal filtering. However, with an increasing number of fluorophores, the process can become unreliable and subjective.…”

Strategies and experimental tips for optimized quantitative single-molecule studies of membrane and membrane-associated proteins

“…SMLM, based on fluorescence localization microscopy (PALM) [1,2], stochastic optical reconstruction microscopy (STORM) [3,4], or (PAINT) [5] provides intrinsic information at the molecular scale thanks to the ability to separately record the emission from spatially separated molecules. SMLM techniques have been proposed to study membrane molecular structures, such as membrane receptors and ionic channels, and to understand their molecular mechanisms [6][7][8][9]. In particular stepwise photobleaching (SP) [7,[10][11][12] and quantitative singlemolecule localization microscopy (qSMLM) [13][14][15][16][17][18][19] have recently emerged as powerful tools for quantitative insights into membrane proteins stoichiometry and organization [7,8,10,20,21].…”