Tetra-color superresolution microscopy based on excitation spectral demixing

Abstract

Multicolor imaging allows protein colocalizations and organelle interactions to be studied in biological research, which is especially important for single-molecule localization microscopy (SMLM). Here, we propose a multicolor method called excitation-resolved stochastic optical reconstruction microscopy (ExR-STORM). The method, which is based on the excitation spectrum of fluorescent dyes, successfully separated four spectrally very close far-red organic fluorophores utilizing three excitation lasers with cross-talk of less than 3%. Dyes that are only 5 nm apart in the emission spectrum were resolved, resulting in negligible chromatic aberrations. This method was extended to three-dimensional (3D) imaging by combining the astigmatic method, providing a powerful tool for resolving 3D morphologies at the nanoscale.

Fig. 1. Working principle of ExR-STORM.

a Scheme of the optical setup. Three excitation lasers (620, 639, and 671 nm) were combined and then modulated by an AOTF prior to the illumination of the sample. The laser switching was synchronized with the scanner, resulting in three subregions with different excitation wavelengths within one exposure time. b Absorption spectra of the eight fluorescent dyes. The three black lines correspond to the three excitation wavelengths. c Scatter plot of the photon number of Alexa Fluor 647 in the three excitation channels. d 2D projections of the photon number spatial occupation along the 671 path, 620 path, 639 path, and the isometric projection (which is an axonometric projection in which the three coordinate axes appear equally foreshortened and the angle between any two of them is 120°) for the investigated dyes, where the half-maximum contour of the distribution was selected as the boundary of the investigated dye; more than one million single molecules of each dye were selected for a statistical examination of the molecular behaviors

Fig. 2. Workflow of ExR-STORM.

a One frame of the raw image stack. b Split channels of the raw image based on the ROI of the three light paths, and transformed by the affine matrices to the 639 nm light path. c Diagram of dye assignment along the projections of the 671 path, 620 path, 639 path, and the isometric projection (which is an axonometric projection in which the three coordinate axes appear equally foreshortened and the angle between any two of them is 120 degrees), with the yellow regions between the dashed lines, rejected. d Single-molecule of each dye in the three light paths from (b) based on the classification process shown in (c)

Fig. 3. Image registration and verification of co-fitting method.

100 nm fluorescent microspheres were used for image registration and verification. a–d Transformed images of the three light paths and merged image. e Positions of independent fitting in each transformed channel relative to the co-fitting results in the X and Y directions. Error bars are presented as the mean ± standard deviation. Scale bars: 10 μm (a–d)

Fig. 4. Tetra-color imaging based on ExR-STORM.

CF660C, Alexa Fluor 647, Dyomics 654, and DyLight 633 were selected to label the outer mitochondrial membrane (cyan), intermediate filaments (magenta), endoplasmic reticulum (green), and microtubules (yellow), respectively, in a fixed COS-7 cell. a Four-color superresolution reconstruction image in which each detected single molecule was classified based on the excitation spectrum of the fluorophores. b-e Split channels of the white box region in (a). f–i 2D projections of the photon number distributions along the 671 path, 620 path, 639 path, and the isometric projection (which is an axonometric projection in which the three coordinate axes appear equally foreshortened and the angle between any two of them is 120°). j Cross-talk and rejected fraction for the four dyes. Scale bars: 2 μm (a); 1 μm (b-e)

Fig. 5. 3D Tetra-color imaging based on ExR-STORM.

a-e CF660C, Alexa Fluor 647, Dyomics 654, and DyLight 633 were selected to label the outer mitochondrial membrane (cyan), intermediate filaments (magenta), endoplasmic reticulum (green), and peroxisomes (yellow), respectively, in a fixed COS-7 cell. a Reconstructed image of the four merged channels. b–e Color-coded four channels in (a). f–k Colocalization of peroxisomes and endoplasmic reticulum. f Zoomed-in view of boxed region i in (a). g XZ (120 nm in Y) cross-section of the boxed region in (f). h Intensity profile along the boxed region in (f). i Zoomed-in view of the boxed region ii in (a). j YZ (120 nm in X) cross-sections of the region between the two dashed lines in (i), where the distance between adjacent cross-sections in the X direction is 120 nm. k Intensity profiles along the Y direction in (i), with each profile corresponding to the cross-section image in (j). l The ER–mitochondria–peroxisome tripartite contact site in the boxed region iii in (a). m, n XZ (120 nm in Y) and YZ (120 nm in X) cross-sections of the boxed regions in (l). Scale bars: 2 μm (a–e); 1 μm (f–g); 500 nm (i, j, l–n)