Quantitative Image Restoration in Bright Field Optical Microscopy Braulio Gutiérrez-Medina, Manuel de Jesús Sánchez Miranda  Biophysical Journal  Volume 113, Issue 9, Pages 1916-1919 (November 2017) DOI: 10.1016/j.bpj.2017.09.002 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 QRBF effectively restores cellular morphology in BF images of single E. coli. (A) Shown here is the x-z intensity distribution of the theoretical PSF used for deconvolution. z = 0 corresponds to in-focus, where image contrast is least. (B–M) Shown here is a cell undergoing division imaged using BF (B–D), QRBF (E–G), DIC (H–J), and fluorescence (F) (K–M) microscopy. The x-z and z-y intensity profiles correspond to the dashed cyan lines of their respective x-y image. QRBF and F images are displayed in false color. z = 0.3 μm in (B). (N–Q) Shown here is validation of QRBF. A cell is observed before (N) and after deconvolution (O). In (O), the cellular contour (black line) and width (straight line) were automatically determined from the QRBF image. (P) The cellular contour identified in (O) was superimposed to the corresponding fluorescence image of the same cell. (Q) Shown here is distribution of cellular widths (W) for a bacterial population (n = 108), measured from BF images deconvolved using a theoretical (black bars) or experimental (gray bars, red online) PSF. The solid line is a Gaussian fit to the theoretical-PSF width distribution (653 ± 73 nm, mean ± SD). The inset displays the width values, showing coincidence between the two types of data. The scale bar represents 2 μm. To see this figure in color, go online. Biophysical Journal 2017 113, 1916-1919DOI: (10.1016/j.bpj.2017.09.002) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 High-throughput, automated quantification of bacterial width and length during hyperosmotic shock. (A) Shown here is a 2D-QRBF image of unstained E. coli cells immobilized on a coverslip at [NaCl] = 0.5 M. Insets show enlarged views of cells, evidencing plasmolysis (white arrows). The contours of cells (black lines) were determined automatically. The scale bar represents 5 μm (1 μm, insets). (B) Cell samples exposed to increasing concentrations of NaCl were imaged and processed using 2D-QRBF. The cellular width (W) was plotted against length (L) and 2D histograms were created for four different [NaCl] datasets (0, 0.25, 0.5, and 1.0 M). Histograms were normalized with respect to the maximum bin count. (C) Given here are histograms (P) of width (W) and length (L) for increasing [NaCl], normalized by the number of cells measured in each case. (Insets) Width (Wn) and length (Ln) values were normalized with respect to W and L values at [NaCl] = 0 M, (mean ± SD). For (B) and (C): n = 2283 (0 M); 4596 (0.25 M); 6657 (0.5 M); and 6079 (1 M). To see this figure in color, go online. Biophysical Journal 2017 113, 1916-1919DOI: (10.1016/j.bpj.2017.09.002) Copyright © 2017 Biophysical Society Terms and Conditions