Estimating the dynamic range of quantitative single-molecule localization microscopy (original) (raw)
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2016
Super-resolved localization microscopy (SLM) has the potential to serve as an accurate, single-cell technique for counting the abundance of intracellular molecules. However, the stochastic blinking of single fluorophores can introduce large uncertainties into the final count. Here we provide a theoretical foundation for applying SLM to the problem of molecular counting based on the distribution of blinking events from a single fluorophore. We show that by redundantly tagging single-molecules with multiple blinking fluorophores, the accuracy of the technique can be enhanced by harnessing the central limit theorem. The coefficient of variation (CV) then, for the number of molecules M estimated from a given number of blinks B , scales like ∼ 1/√ N l , where N l is the mean number of labels on a target. As an example, we apply our theory to the challenging problem of quantifying the cell-to-cell variability of plasmid copy number in bacteria.
Proceedings of the National Academy of Sciences, 2012
We present a single molecule method for counting proteins within a diffraction-limited area when using photoactivated localization microscopy. The intrinsic blinking of photoactivatable fluorescent proteins mEos2 and Dendra2 leads to an overcounting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe blinking and show that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra2 a better photoactivated localization microscopy tag than mEos2 for molecular counting. The simultaneous activation of multiple molecules is another source of error, but it leads to molecular undercounting instead. We propose a photoactivation scheme that maximally separates the activation of different molecules, thus helping to overcome undercounting. We also present a method that quantifies the total counting error and minimizes it by balancing over-and undercounting. This unique method establishes that Dendra2 is better for counting purposes than mEos2, allowing us to count in vitro up to 200 molecules in a diffraction-limited spot with a bias smaller than 2% and an uncertainty less than 6% within 10 min. Finally, we demonstrate that this counting method can be applied to protein quantification in vivo by counting the bacterial flagellar motor protein FliM fused to Dendra2.
Histochemistry and cell biology, 2015
Single-molecule localization microscopy has been widely applied to count the number of biological molecules within a certain structure. The percentage of molecules that are detected significantly affects the interpretation of data. Among many factors that affect this percentage, the polarization state of the excitation light is often neglected or at least unstated in publications. We demonstrate by simulation and experiment that the number of molecules detected can be different from -40 up to 100 % when using circularly or linearly polarized excitation light. This is determined mainly by the number of photons emitted by single fluorescent molecule, namely the choice of fluorescence proteins, and the background noise in the system, namely the illumination scheme. This difference can be further exaggerated or mitigated by various fixation methods, magnification, and camera settings We conclude that the final choice between circularly or linearly polarized excitation light should be ma...
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2019
Single-molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here we present a method for molecular counting with SMLM built upon the exponentially distributed blinking statistics of photoswitchable fluorophores, with a focus on organic dyes. We provide a practical guide to molecular counting, highlighting many of the challenges and pitfalls, by benchmarking the method on fluorescently labeled, surface mounted DNA origami grids. The accuracy of the results illustrates SMLM's utility for optical '-omics' analysis.
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Fluorescence microscopy has become a standard tool within the life sciences but its major drawback has been its maximum achievable resolution (≈ 250 nm) given by the diffraction limit. The advent of super-resolution (SR) microscopy could overcome this limitation by bringing fluorescence microscopy into the nanoscale, reaching resolutions on the molecular level. The SR methods summarized as Single-Molecule Localization Microscopy (SMLM) circumvent the diffraction limit by acquiring image sequences of stochastically activated subsets of 'blinking' target structures. The subsequent fitting of the recorded fluorescent bursts from individual emitters allows localization of individual fluorophore positions. This concept is similarly applied in Single Particle Tracking (SPT) to monitor the motion of individual biomolecules with nanometer precision. However, a quantitative interpretation of both SMLM and SPT data is often not straightforward since it requires exact modeling of the p...
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Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially repla...
A Platform To Enhance Quantitative Single Molecule Localization Microscopy
Journal of the American Chemical Society, 2018
Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell l...
Analysis of super-resolution single molecule localization microscopy data: A tutorial
AIP Advances
The diffraction of light imposes a fundamental limit on the resolution of light microscopes. This limit can be circumvented by creating and exploiting independent behaviors of the sample at length scales below the diffraction limit. In super-resolution single molecule localization microscopy (SMLM), the independence arises from individual fluorescent labels stochastically switching between dark and fluorescent states, which in turn allows the pinpointing of fluorophores post experimentally using a sequence of acquired sparse image frames. Finally, the resulting list of fluorophore coordinates is utilized to produce high resolution images or to gain quantitative insight into the underlying biological structures. Therefore, image processing and post-processing are essential stages of SMLM. Here, we review the latest progress on SMLM data processing and post-processing.
Time-correlated single molecule localization microscopy enhances resolution and fidelity
Scientific Reports
Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samples down to ~ 10–20 nm and in single molecule detail. However, common SMLM reconstruction largely disregards information embedded in the entire intensity trajectories of individual emitters. Here, we develop and demonstrate an approach, termed time-correlated-SMLM (tcSMLM), that uses such information for enhancing SMLM reconstruction. Specifically, tcSMLM is shown to increase the spatial resolution and fidelity of SMLM reconstruction of both simulated and experimental data; esp. upon acquisition under stringent conditions of low SNR, high acquisition rate and high density of emitters. We further provide detailed guidelines and optimization procedures for effectively applying tcSMLM to data of choice. Importantly, our approach can be readily added in tandem to multiple SMLM and related superresolution reconstruction algorithms. Thus, we expect that our approach will become an effective an...