Super-resolution imaging with stochastic single-molecule localization: Concepts, technical developments, and biological applications (original) (raw)
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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.
Superresolution Imaging using Single-Molecule Localization
Annual Review of Physical Chemistry, 2010
Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (∼10-20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-moleculebased imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales. 345 Annu. Rev. Phys. Chem. 2010.61:345-367. Downloaded from arjournals.annualreviews.org by National Institutes of Health Library on 07/22/10. For personal use only.
Minimizing Structural Bias in Single-Molecule Super-Resolution Microscopy
Scientific Reports, 2018
Single-molecule localization microscopy (SMLM) depends on sequential detection and localization of individual molecular blinking events. Due to the stochasticity of single-molecule blinking and the desire to improve SMLM's temporal resolution, algorithms capable of analyzing frames with a high density (HD) of active molecules, or molecules whose images overlap, are a prerequisite for accurate location measurements. Thus far, HD algorithms are evaluated using scalar metrics, such as root-mean-square error, that fail to quantify the structure of errors caused by the structure of the sample. Here, we show that the spatial distribution of localization errors within super-resolved images of biological structures are vectorial in nature, leading to systematic structural biases that severely degrade image resolution. We further demonstrate that the shape of the microscope's point-spread function (PSF) fundamentally affects the characteristics of imaging artifacts. We built a Robust Statistical Estimation algorithm (RoSE) to minimize these biases for arbitrary structures and PSFs. RoSE accomplishes this minimization by estimating the likelihood of blinking events to localize molecules more accurately and eliminate false localizations. Using RoSE, we measure the distance between crossing microtubules, quantify the morphology of and separation between vesicles, and obtain robust recovery using diverse 3D PSFs with unmatched accuracy compared to state-of-the-art algorithms. Since its invention, fluorescence imaging has been an indispensable tool for biological studies of cells, tissues, and organisms because of its ability to visualize specific molecules of interest against a dark background in a relatively noninvasive manner. Tagging a biological molecule with a small organic fluorophore or fluorescent protein enables a fluorescence microscope to produce pictures of structures and movies of interactions between molecules within living cells. The optical detection of individual fluorescent molecules in condensed matter 1 is the basis for an entire family of super-resolved fluorescence microscopy techniques 2-5. These methods rely upon the blinking of fluorescent molecules in time to reduce the concentration of active emitters and resolve each molecule in a microscope image 6-8. Repeated cycles of molecular blinking and measurement of molecular positions from their point spread functions (PSFs) by an image analysis algorithm result in reconstructed images of a biological structure with resolution beyond the Abbé diffraction limit (~λ/2NA ≈ 250 nm for visible light, where NA is the numerical aperture of the fluorescence microscope). Here, we refer to these techniques collectively as single-molecule localization microscopy (SMLM). Although the experimenter often chooses imaging conditions to minimize the probability of image overlap between two molecules, the stochasticity of molecular blinking often leads to some overlap in SMLM datasets, especially for complex biological structures with high fluorophore labeling density 9. One may even purposefully increase the density of active fluorescent probes in any given camera acquisition, such that images of neighboring molecules frequently or regularly overlap, in order to improve the temporal resolution of SMLM. Consequently, fewer imaging frames are needed to reconstruct a target structure, thereby leading to decreased phototoxicity as well as a reduction in motion-blur artifacts 10,11. From a statistical perspective, super-resolution imaging in the presence of significant image overlap poses two major problems: (i) identifying the underlying molecules and (ii) estimating their positions and brightnesses. Strategies for resolving overlapping molecules are primarily based on two aspects of prior knowledge: molecules are sparsely distributed in space and they repeatedly and independently blink over time 12. The first strategy recasts the estimation of molecular positions as a sparse recovery optimization problem, where a sparsity prior regulates the solution 13,14. The second approach exploits molecular emission characteristics (e.g., uncorrelated and
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...
Localization-Based Super-Resolution Light Microscopy
Microscopy Today, 2011
Fluorescence microscopy is an essential and flexible tool for the study of biology, chemistry, and physics. It can provide information on a wide range of spatial and temporal scales. However, since the inception of light microscopy, diffraction has limited the size of the smallest details that could be imaged in any sample using light. Because much of biology occurs on molecular length scales, interest in circumventing the diffraction limit has been high for many years. Recently, several techniques have been introduced that can bend or break the diffraction limit. Localization-based methods introduced in 2006 have reached this goal and are now rapidly growing in popularity.
PloS one, 2015
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative ima...
Doubling the resolution of single-molecule localization microscopy with image scanning microscopy
Single-molecule localization microscopy (SMLM) is a widely used super-resolution microscopy technique, renowned for its simplicity and impressive achievable resolution. It is typically based on a wide-field fluorescence microscope and relies on emitter photoswitching to capture individual snapshots of a sample with sparse distributions of fluorescent labels. These labels can then be precisely identified and localized. Recently, we demonstrated that SMLM can also be realized with a fast confocal laser-scanning microscope (CLSM), opening the door to fluorescence lifetime SMLM. This technique has found applications in lifetime-based image multiplexing and metal-induced energy transfer SMLM.In this work, we present an extension of CLSM-based SMLM by incorporating a single-photon detector array into the CLSM. This enables the combination of CLSM-based SMLM with Image Scanning Microscopy (ISM), a powerful technique for doubling the lateral resolution of a laser-scanning confocal microscop...
Synergizing superresolution optical fluctuation imaging with single molecule localization microscopy
Methods and Applications in Fluorescence
biological samples well beyond the diffraction limit of light, but they vary significantly in their spatial and temporal resolutions. High-order statistical analysis of temporal fluctuations as in superresolution optical fluctuation imaging (SOFI) also enable imaging beyond diffraction limit, but usually at a lower resolution as compared to SMLM. Since the same data format is acquired for both methods, their algorithms can be applied to the same data set, and thus may be combined synergistically to improve overall imaging performance. Here, we find that SOFI converges much faster than SMLM, provides additive information to SMLM, and can efficiently reject background. We then show how SOFI-assisted SMLM imaging can improve SMLM image reconstruction by rejecting common sources of background, especially under low signal-to-background conditions. The performance of our approach was evaluated using a realistic simulation of fluorescence imaging we developed and further demonstrated on experimental SMLM images of the plasma membrane of activated fixed and live T cells. Our approach significantly enhances SMLM performance under demanding imaging conditions and could set an example for synergizing additional imaging techniques.
Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy
PLoS ONE, 2013
Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based superresolution microscopy.
Journal of Physics: Photonics
Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for sand p-polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient.