Fast Analysis of 2D and 3D Single-Molecule Localization Microscopy Data with Huygens Localizer
Peter J. Verveer,1 Daniel Sevilla-Sánchez,1 Paul C. Bloembergen,1
Vincent T.G. Schoonderwoert,1* and Hans T.M. van der Voort1 1Scientific Volume Imaging bv, Laapersveld 63, 1213VB, Hilversum, Te Netherlands
2Robson DNA Science Centre, Arnie Charbonneau Cancer Institute, Department of Biochemistry & Molecular Biology,
Cumming School of Medicine, University of Calgary, Calgary, AB, Canada, T2N 4N1 *
vincent@svi.nl
Abstract: Single-molecule localization microscopy (SMLM) is a fam- ily of super-resolution microscopy techniques based on localizing clusters of detected photons that are emitted by single molecules. The localization procedure is based on careful statistical analysis of long image sequences to derive the nanometer positions of the mol- ecules. By introducing additional optics, such as cylindrical lenses in the optical system, SMLM techniques have been extended to 3D super-resolution imaging. This adds a calibration step, thereby further complicating the data analysis. Here we present Huygens Localizer, a well-supported user-friendly package that carries out these tasks quickly by offloading carefully designed 2D and 3D analysis and visualization procedures to massively parallel graphical processors (GPUs).
Keywords: 3D single molecule localization microscopy (SMLM),
super-resolution, GPU acceleration, Huygens, point spread function (PSF)
Introduction Single-molecule localization microscopy (SMLM) tech-
niques are based on the localization of individual molecules in a series of images acquired over time. Well-known exam- ples are photoactivated localization microscopy (PALM [1,2]), stochastic optical reconstruction microscopy (STORM [3]) and direct STORM (dSTORM [4]), ground state depletion fol- lowed by individual molecule return (GSDIM [5]), and points accumulation for imaging in nanoscale topography (PAINT [6]). Tese approaches generate images of sparse random dis- tributions of fluorescent molecules, which can be individually distinguished and localized (Figure 1). A series of such images is statistically analyzed to generate a table of the fluorophore locations and intensities. A high-resolution image can be con- structed by rendering each location from the table as a tiny spot in the corresponding location in the image. Two-dimensional (2D) SMLM does not impose many
demands on the optical system at the detection side and is usually implemented on a standard widefield or total internal reflection fluorescence (TIRF) microscope. Te image of each fluorescent molecule is then given by the point spread func- tion (PSF) of the microscope. 2D localization amounts to find- ing the position of the images of the molecules—in principle a straightforward task that does not critically depend on the shape of the PSF. Tree-dimensional (3D) SMLM is based on fitting the
shape of the PSF. By introducing additional optical elements, such as a cylindrical lens, the shape of the PSF is made to vary strongly with the axial position. Hence, the shape of the PSF can be used to map the axial position of the fluorescent molecule. However, a calibration step that is complicated and
20 doi:10.1017/S1551929520000024
error-prone is required and thus compromises reliable and accurate 3D SMLM analysis. To address this, we have adapted the Huygens PSF Distiller [7] so that an axial calibration map is easily calculated from microsphere images. In addition to analysis tools shipped with the control
soſtware of commercial instruments, a wide selection of open-source options for localization microscopy is available [8,9]. However, the field seems to lack a generally applicable, well-supported, easy-to-use, high-performance package for 2D and 3D SMLM. Tis paper describes Huygens Localizer, the first commercially available standalone soſtware package for SMLM, which aims to provide reliable, high-performance analysis and visualization of 2D and 3D SMLM data using a user-friendly workflow to interactively optimize the result. Here we present examples of the analysis of SMLM data with Huygens Localizer, including background detection, fitting, automatic driſt correction, and visualization steps. We also describe 3D SMLM analysis, using the extended Huygens PSF Distiller to obtain an accurate PSF for calibration.
Nancy Adam,2
Figure 1: Principle of single-molecule localization microscopy (SMLM).
(A)
Example structure consisting of two closely spaced lines densely labeled with fluorophores (gray dots). (B) Because of light diffraction, the fluorescence of each fluorophore is detected as a spot in the shape of the microscope PSF, which add up to a single blurred shape where the lines cannot be resolved. (C) If only a few fluorophores are emitting, they can be localized with high precision (red dots), even though their PSF-sized image is relatively large (green). (D) By activating only a few fluorophores per image, and repeating this many times, all localizations can be determined with high precision, revealing the double-line structure.
www.microscopy-today.com • 2020 March
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