SOFI-based 3D superresolution sectioning with a widefield microscope

Abstract

Background: Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial.

Methods: Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope.

Results and conclusion: SOFI's inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution 'confocal widefield' microscope.

Figure 1. Lateral cross-sections

Subset of images recorded along the optical axis (in steps of 1.2um, starting with e lowest slice on the left side). Upper panels: Original widefield fluorescence images Lower panels: SOFI images. The optical sectioning capabilities are evident as well as the resolution gain along x-y. The SOFI images were processed using 2000 frames per z-position. Fluorescence images are representing the average of 2000 frames. Scalebar 20 μm.

Figure 2. Vertical cross-sections

Vertical (x-z) cross-sections of the three dimensional dataset. The scale for the z-axis is x10 times that of the x-axis. Upper panel: Original fluorescence image. Lower panel: SOFI image. Nearest neighbor smoothing was performed for the SOFI image in order to filter high frequency noise originated from long blinking intermittency of the quantum dots along the z axis (the acquisition time of each slice is 60s). Blue and red lines indicate the same cross-sections for the original and SOFI images respectively. Right Panel: the corresponding cross-sections for the original (red) and the SOFI (blue) images. The SOFI intensity profile clearly shows increased resolution along z. Scalebar: 1 μm along z and 20 μm along x.

Figure 3. FFT-Spectrum

FFT spectra along the axis k_x = k_y = 0 axis of the original fluorescence and SOFI data. Blue line: Fluorescence. Red line: SOFI. A clear cut-off frequency cannot be determined. Therefore the support of both these spectra cannot be estimated.

Figure 4. Lateral resolution gain

Estimating the resolution gain in the x-y-plane. A) Fluorescence image. B) SOFI image. The boxed regions are displayed magnified and intensity-normalized in C) and E). Blue and red lines indicate where the cross-section was taken in order to evaluate the resolution gain. D): intensity profiles along the lines of C) and E). Blue line: Cross-section of the original fluorescence image. Red line: Cross-section of the SOFI image. As can be seen the blue line displays a dip indicating a higher resolution. The distance between the vertical black lines amounts to 215 nm, which is 14% higher than the theoretically expected resolution of 188 nm. Scalebar in A) and B) 20 μm; C) and E) 1 μm.