High-resolution restoration of 3D structures from widefield images with extreme low signal-to-noise-ratio

Abstract

Four-dimensional fluorescence microscopy--which records 3D image information as a function of time--provides an unbiased way of tracking dynamic behavior of subcellular components in living samples and capturing key events in complex macromolecular processes. Unfortunately, the combination of phototoxicity and photobleaching can severely limit the density or duration of sampling, thereby limiting the biological information that can be obtained. Although widefield microscopy provides a very light-efficient way of imaging, obtaining high-quality reconstructions requires deconvolution to remove optical aberrations. Unfortunately, most deconvolution methods perform very poorly at low signal-to-noise ratios, thereby requiring moderate photon doses to obtain acceptable resolution. We present a unique deconvolution method that combines an entropy-based regularization function with kernels that can exploit general spatial characteristics of the fluorescence image to push the required dose to extreme low levels, resulting in an enabling technology for high-resolution in vivo biological imaging.

Keywords: 4D microscopy; low dose microscopy; noise-suppressing regularization.

Fig. 1.

Deconvolution results for Drosophila spindle. (A and E) High- and low-dose raw images; (B and F) ER-Decon output from A and E; (C and G) Huygens’ output from A and E; (D and H) DeconvolutionLab’s output from A and E. In each image, the upper part is a lateral section, and the lower part is a vertical section. (Scale bar: 4 µm.) ER-Decon’s parameters: λ = 0.05, 3 (B and F); ε = 0.01, 0.001 (B and F).

Fig. 2.

Fourier shell correlation between high- and low-dose images. Correlation plots for the Drosophila spindle displayed in Fig. 1. The vertical line is the xy theoretical resolution limit.

Fig. 3.

ER-Decon (B, F, J, and N) output from A, E, I, and M; Huygens' output (C, G, K, and O) from A, E, I, and M; DeconvolutionLab's output (D, H, L, and P) from A, E, I, and M. (Scale bar: 2 µm.) ER-Decon’s parameters: λ = 0.2, 8, 200, 650 (B, F, J, and N); ε = 0.01, 0.001, 0.0001, 0.00001 (B, F, J, and N).

Fig. 4.

Deconvolution results for a yeast Zip1 filament. (A and E) High- and low-dose raw images; (B and F) ER-Decon output from A and E; (C and G) Huygens’ output from A and E; (D and H) DeconvolutionLab’s output from A and E. (Scale bar: 2 µm.) ER-Decon’s parameters: λ = 0.05, 700 (B and F); ε = 0.01, 0.00001 (B and F).

Fig. 5.

Fourier shell correlation between high- and low-dose images. Correlation plots for the yeast ZIP1 filament displayed in Fig. 4. The vertical line is the xy theoretical resolution limit.

Fig. 6.

Depth color-coded z-projections of deconvolved live images of yeast Zip1 filaments. Upper arrow points to a filament in ER-Decon output traversing from 6 µm depth to 8 µm depth, which cannot be resolved from the outputs of the other methods. Lower arrow points to two filaments in ER-Decon output located at depths 6.5 µm and 7.5 µm, which again cannot be resolved from the output of the other two methods. ER-Decon’s parameters: λ = 12; ε = 0.001.