A correlation analysis framework for localization-based super-resolution microscopy

Abstract

Super-resolution images reconstructed from single-molecule localizations can reveal cellular structures close to the macromolecular scale and are now being used routinely in many biomedical research applications. However, because of their coordinate-based representation, a widely applicable and unified analysis platform that can extract a quantitative description and biophysical parameters from these images is yet to be established. Here, we propose a conceptual framework for correlation analysis of coordinate-based super-resolution images using distance histograms.We demonstrate the application of this concept in multiple scenarios including image alignment, tracking of diffusing molecules, as well as for quantification of colocalization. Significance statementCorrelation analysis is one of the most widely used image processing method. In the quantitative analysis of localization-based super-resolution images, there still lacks a generalized coordinatebased correlation analysis framework to take fully advantage of the super-resolution information.We show a coordinate-based correlation analysis framework for localization-based superresolution microscopy. This framework is highly general and flexible in that it can be easily extended to model the effect of localization uncertainty, to the time domain and other distance definitions, enabling it to be adapted for a wide range of applications. Our work will greatly benefit the quantitative interpretation of super-resolution images and thus the biological application of super-resolution microscopy.

A hierarchical pathway for assembly of the distal appendages that organize primary cilia

“…This averaged image was then cropped into the same fields of view as used for data acquisition in the two channels, and the MATLAB function imregtform was used to find the affine transformation that mapped the beads in the green channel onto the beads in the red channel. As the registration data was acquired at the coverslip while the single-molecule data could be acquired multiple microns above the coverslip through the cell, the registration was fine-tuned in an additional step by adapting a 2D cross-correlation approach (Schnitzbauer et al, 2018) to 3D and using it to account for any residual nanoscale offsets caused by aberrations when imaging higher up in the sample and to correct for any offset in the z-direction. The protein FOP is known to localize to the region close to the subdistal appendages of the mother centriole and daughter centriole, and forms ring-like structures (T. Kanie et al, 2017).…”

KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in P. falciparum-infected erythrocytes

“…Two-dimensional cross-correlation between two color channels was computed by calculating the pairwise inter-molecular distance distribution (PDD) from the images (Schnitzbauer et al, 2018). The shortest distance between the points on two images is limited by the size of a pixel (15 nm), which, in turn, limits the PDD at short distances by the size of a pixel.…”

“…This is possible because single fluorescence signals cover multiple pixels. The image interpolation step smoothens the image and improves the PDD (Schnitzbauer et al, 2018). We chose one tenths of a pixel, corresponding to ~1.5 nm, as the width of the interpolated grid.…”

“…Recent advances in super-resolution fluorescence microscopy and image processing (Hell and Wichmann, 1994;Schnitzbauer et al, 2018) have offered new opportunities to interrogate the membrane cytoskeletal organization of erythrocytes at the nanometer scale (Pan et al, 2018). To gain insights into the structural organization of knobs, we implemented several high-resolution imaging techniques, including two-color stimulated emission depletion (STED) microscopy with two-dimensional pairwise cross-correlation analysis, photo-activated localization microscopy (PALM) with single molecule counting, and electron tomography labelling with specific nanoprobes and stereological computer simulations.…”