Metrics for the reliability and effective resolution in n2v-reconstructed images. (A) Representative micrographs of the DNA distribution in a nucleus in a fixed zebrafish embryo, recorded with a stimulated emission depletion (STED) super-resolution microscope. The same image plane was recorded twice at low quality, once at high quality, and two n2v-reconstructed images were prepared from the low-quality images. (B) SSIM values for pair-wise comparison (image 1 vs. image 2) and comparison against the high-quality image (image 1 vs. high-quality and image 2 vs. high-quality) for the low-quality images and the reconstructed images. (C) FRC curves calculated based on a low-quality image pair and the corresponding reconstructed image pair. (D) FRC-based effective resolution for four pairs of low-quality images and the corresponding pairs of reconstructed images.

Metric-based estimation of how far image quality can be compromised while still allowing recovery of effective resolution by denoising. (A) Representative micrographs of nuclei of human cheek cells for different camera exposure times (t_exp, as indicated), all high-quality images were acquired at the same position but with an exposure time of 200 ms. Images are maximum-intensity projections, DNA was labeled by Hoechst 33342. (B) FRC curves calculated from a pair of matched low-quality images, from a pair of reconstructed images, and a pair of high-quality images for the different t_exp. (C) Effective resolution for the indicated t_exp, n = 5 nuclei per t_exp, values are shown with mean.

A two-phase acquisition protocol to combine acquisition of quality control images with high-speed time-lapse imaging. (A) Image data were acquired at multiple positions in a sample, thus obtaining multiple viewpoints containing several objects of interest (nuclei, indicated as circles). (B) For each position, a sequence of two acquisition phases is carried out. In phase A, for each z position, a low-quality image, two high-quality reference images, and two low-quality test images are recorded. Low-quality images are recorded at a shortened exposure time (t_exp), high-quality images at a reference exposure time resulting in images of the desired quality (t_ref). Acquisition phase A obtains the images required for n2v model training as well as the assessment of effective image resolution and reconstruction errors. In phase B, only single low-quality images are recorded with the shortened exposure time (t_exp), resulting in an increased rate of acquisition compared to acquisition with full exposure time (t_ref). Acquisition phase B obtains only low-quality images, which are reconstructed after the experiment is completed.

Pseudo-time analysis of data from fixed embryos relates transient engagement and activation of a gene to the phosphorylation and shape changes observed in live embryos. (A) Example images of Pol II Ser5P (magenta signal) clusters sorted by a pseudo-time progress coordinate (s, periodic, defined on the interval [0,1)), which is calculated based on interaction with the gene klf2b (green represents oligopaint fluorescence in situ hybridization signal for klf2b). Center positions (weighted centroid) are indicated for the Pol II Ser5P cluster (white circle with black filling) and the gene (black circle with white filling) and connected with a white line for illustration. For details of the reconstruction, see Figure S8 (Supplementary Material). For an overview containing all eight genes that were assessed, see Fig. S9B (Supplementary Material). (B) Pol II Ser5P and Ser2P intensity, elongation, and solidity of Pol II Ser5P clusters sorted by pseudo-time s. A total of n = 186 clusters from N = 4 independent samples, obtained in two independent experiments, were included in the analysis. (C) Cross-correlation analyses for different register shifts in the coordinate s, the register shift is in units of data points by which the coordinate s was shifted. Gray regions indicate 95% bootstrap CI.

Work-flow for n2v reconstruction for time-lapse data with quality control.