Method to identify and minimize artifacts induced by fluorescent impurities in single-molecule localization microscopy

Davis, Janel L.; Dong, Biqin; Sun, Cheng

doi:10.1117/1.jbo.23.10.106501

Cited by 14 publications

(15 citation statements)

References 39 publications

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“…Autofluorescence is stronger at shorter wavelengths and can hence be alleviated using fluorophores with longer emission wavelengths and/or appropriate filters. Impurities can be reduced, although not entirely removed, by appropriate cleaning of coverslips, for example using plasma cleaners 206 .…”

Section: Sample Driftmentioning

confidence: 99%

Single-molecule localization microscopy

Lelek

Gyparaki

Beliu

et al. 2021

Nat Rev Methods Primers

472

378

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The spatial resolution of standard optical microscopy techniques is limited to roughly half the wavelength of light. As a result of diffraction 1 , the image of an arbitrarily small source of light imaged using a lens-based microscope is not a point but a point spread function (PSF), usually an Airy pattern, with a central peak approximately ~200-300 nm in width (Fig. 1a), resulting in a blurring of structures below this spatial scale. This diffraction limit restricts the ability of optical microscopy techniques to resolve the subcellular organization of individual molecules or molecular complexes, which are smaller than this limit; for example, the structure of a nuclear pore complex, which is made up of hundreds of individual proteins, with a diameter of only ~120 nm, remains obscured by conventional microscopy (Fig. 1b).Microscopy methods have emerged over the past two decades that can overcome the diffraction limit and enable the imaging of biological structures such as nuclear pores, viruses, chromatin complexes and cytoskeletal filaments at resolutions close to the molecular scale 2 . The most well known of these super-resolution microscopy methods fall into three main categories: stimulated emission depletion 3 , structured illumination microscopy 4,5 and single-molecule localization microscopy (SMLM) [6][7][8][9] , which is the focus of this Primer. SMLM methods usually employ conventional wide-field excitation and achieve super-resolution by localizing individual molecules [6][7][8][9][10][11][12][13][14][15] .They have become broadly adopted in the life sciences owing to their high spatial resolution -typically ~20-50 nm or better -and relative ease of implementation, although each super-resolution method has its unique advantages and limitations and is optimally suited for different applications (discussed elsewhere 2 ).SMLM is fundamentally based on the fact that the spatial coordinates of single fluorescent molecules (also called fluorophores, or emitters) can be determined with high precision if their PSFs do not overlap. Subpixel shifts in the coordinates of a fluorophore lead to predictable changes in pixel intensities that can be used to compute its precise location (Fig. 1c). The localization precision reflects the scatter of localizations that would be obtained if a molecule was imaged and localized many times, and is fundamentally limited by the signal to noise ratio (SNR) and not by the wavelength of light or the pixel size (Fig. 1d). To avoid overlaps between the PSFs of individual molecules, fluorescent emissions of distinct molecules are separated in time; the most common approach to obtain this temporal separation exploits the phenomenon of photoswitching, where fluorescent molecules can switch between an active 'ON' or 'bright' state, where they emit fluorescent light when excited, and one or more inactive 'OFF' or 'dark' state in which they do not fluoresce (Fig. 1e).Photoswitching for a particular molecule is a stochastic event; however, switching probabilities can be DiffractionThe be...

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Section: Sample Driftmentioning

confidence: 99%

Single-molecule localization microscopy

Lelek

Gyparaki

Beliu

et al. 2021

Nat Rev Methods Primers

472

378

View full text Add to dashboard Cite

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“…We developed sSMLM systems that utilize diffraction gratings to obtain the spatial and spectral information of the single-molecule blinking events simultaneously [16, 21–24]. Using sSMLM, we reported that Rhodamine dyes tagged microtubules assembled in vitro showed significant spectral heterogeneity.…”

Section: Introductionmentioning

confidence: 99%

Multicolor super-resolution imaging using spectroscopic single-molecule localization microscopy with optimal spectral dispersion

et al. 2019

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We developed a transmission diffraction grating-based spectroscopic single-molecule localization microscopy (sSMLM) to collect the spatial and spectral information of single-molecule blinking events concurrently. We characterized the spectral heterogeneities of multiple far-red emitting dyes in a high-throughput manner using sSMLM. We also investigated the influence of spectral dispersion on the single-molecule identification performance of fluorophores with large spectral overlapping. The carefully tuning of spectral dispersion in grating-based sSMLM permitted simultaneous three-color super-resolution imaging in fixed cells with a single objective lens at relatively low photon budget. Our sSMLM has a compact optical design and can be integrated with conventional localization microscopy to provide add-on spectroscopic analysis capability.

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“…Future updates will make RainbowSTORM compatible with a wider range of spatial analysis platforms. Further, additional spectroscopic analysis methods such as spectral unmixing (Davis, et al, 2018), machine-learning based spectral classification (Zhang, et al, 2019), and cluster analysis (Bongiovanni, et al, 2016) will be added to RainbowSTORM.…”

Section: Discussionmentioning

confidence: 99%

RainbowSTORM: an open-source ImageJ plug-in for spectroscopic single-molecule localization microscopy (sSMLM) data analysis and image reconstruction

Davis¹,

Soetikno²,

Song³

et al. 2020

Bioinformatics

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Summary Spectroscopic single-molecule localization microscopy (sSMLM) simultaneously captures the spatial locations and full spectra of stochastically emitting fluorescent single-molecules. It provides an optical platform to develop new multi-molecular and functional imaging capabilities. While several open-source software suites provide sub-diffraction localization of fluorescent molecules, software suites for spectroscopic analysis of sSMLM data remain unavailable. RainbowSTORM is an open-source ImageJ/FIJI plug-in for end-to-end spectroscopic analysis and visualization for sSMLM images. RainbowSTORM allows users to calibrate, preview, and quantitatively analyze emission spectra acquired using different reported sSMLM system designs and fluorescent labels. Availability RainbowSTORM is a java plug-in for ImageJ (https://imagej.net)/FIJI (http://fiji.sc) freely available through: https://github.com/FOIL-NU/RainbowSTORM. RainbowSTORM has been tested with Windows and Mac operating systems and ImageJ/FIJI version 1.52. Supplementary information Supplementary data are available at Bioinformatics online.

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Method to identify and minimize artifacts induced by fluorescent impurities in single-molecule localization microscopy

Cited by 14 publications

References 39 publications

Single-molecule localization microscopy

Single-molecule localization microscopy

Multicolor super-resolution imaging using spectroscopic single-molecule localization microscopy with optimal spectral dispersion

RainbowSTORM: an open-source ImageJ plug-in for spectroscopic single-molecule localization microscopy (sSMLM) data analysis and image reconstruction

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