Supplementary Figure 3: Basic characterizations of the microscope’s optical performance.

(a) Double-logarithmic plot of the fluorescence emission intensity as a function of the laser illumination intensity at the specimen plane, as determined with the laser beamlets focused and held stationary on the surface of a red fluorescent microscope slide. x-axis values denote the total laser illumination power delivered to the specimen across all 20 × 20 laser beamlets. y-axis values denote the mean fluorescence intensity across a square region of interest containing 10,000 pixels. The green line is a parametric fit to the data using the equation log(y) = A log(x) + B, where A and B are the fitting parameters. The slope, A (2.02 ± 0.06; 95% confidence interval), is an indicator of the quadratic relationship between the fluorescence emission and laser illumination intensities. (b) The microscope’s lateral optical point-spread function, as determined by focusing the laser beamlets on the surface of a red fluorescent slide, using the sCMOS camera to acquire fluorescence images of the entire set of stationary foci, and analyzing the individual images from five different foci (one at the center of the field of view and four at peripheral edges of the image). Colored data points: Cross-sectional fluorescence intensity profiles of each image (one color for each of the five selected laser foci). FWHM lateral resolution: 1.4 ± 0.1 μm (mean ± s.d; N = 5 beamlet foci), as determined from Gaussian fits to the five cross-sectional profiles. Blue curve: A Gaussian fit (for display purposes only) to the aggregated dataset created by combining the data from the five different laser foci.