Progress in quantitative single-molecule localization microscopy

Abstract

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).

Investigating Focal Adhesion Substructures by Localization Microscopy

“…SMLM data consist of the localizations of individual photoactivatable or photoswitchable fluorescent molecules. Therefore, a variety of methods have been developed to identify and characterize clusters of such localizations (12,13). These methods are often applied to investigate clusters of receptors in the cell membrane.…”

Challenges in quantitative single molecule localization microscopy

“…Before proceeding further, we note that it is possible that the image processing and localization algorithms used can also introduce errors in quantification, however this is treated in detail elsewhere [45,46]. A brief introduction to the quantitative measures being mentioned can be found in [7,47].…”

“…Recent reviews on the updates on the technology and its uses can be found in [4,5]. SMLM can potentially be used for quantitative measurements [6,7], e.g., in counting the number of molecules of a protein specie [8] and stoichiometry estimation of protein complexes [9][10][11], characterizing the spatial distribution of a protein specie [12][13][14][15], estimating the co-localization or co-clustering between organelles and also single molecules (SM) [16][17][18][19], estimating the relative positions of various components in a protein complex with high precision [20,21], and estimating the diffusion coefficients by means of single particle tracking (SPT) in a dense sample [22,23]. Two basic variants of SMLM are Photo-Activated Localization Microscopy (PALM) and STochastic Optical Reconstruction Microscopy (STORM).…”

“…Since the usage of fusion proteins used in PALM provides comparatively high specificity labeling as against immunolabeling (the typical labeling technique used for STORM), and since the phenomenon of photoblinking for PA-FPs is minimal (as against the photo-switchable organic dyes used in STORM, which typically blink 10 times or more before irreversible photobleaching [24]), PALM appears to be better suited for quantitative studies, and for this reason forms the focus of this article even though many of the ideas presented are applicable to SMLM in general. Yet, quantitative analysis with PALM is plagued by several sources of errors [7,70], including that of a limited detection efficiency of label molecules in the range of 40-60% [16,25], a localization uncertainty in the order of 20-50 nm [26,27], overcounting in the range of 100% due to reappearance of label molecules due to photoblinking [15,[28][29][30], errors in labeling, a sample drift in the order of 50-100 nm [1,31] and in the case of multi-color imaging, registration errors [16].…”

In this special issue

“…the topic is surveyed in two "review articles" and in seven "Original articles. "Hendrik Deschout, aleksandra radenovic, and their colleagues (Deschout et al 2014) review recent progress in single-molecule resolution microscopy with emphasis on quantitative measurements such as counting of single molecules (i.e., NMDa receptors, glycine-gated channels, and asialo-glycoprotein receptor) with PalM, analysis of heterogeneity in the spatial organization of general membrane proteins or networks of signaling receptors and nuclear pore complexes, and single-molecule colocalization involving dual-color super-resolution microscopy to analyze, for example, protein-protein interactions. Both the potential of the technique and currently existing problems (i.e., limited detection efficiency, overcounting due to photoblinking, sample drift, localization uncertainty, and photoconversion efficiency of fluorescent proteins) using these tools are evaluated.…”