Quantification of LITE spatial resolution. (A) Image of a fluorescent 100-nm bead, visualized by using LITE. Image is maximal-intensity projected along y axis to show lateral (x) and axial (z) resolution. (B) Image of the same bead from A, visualized with epi-illumination. (C) Pixel intensity values for line scans across the x axis and z axis for LITE and epi-illumination images of the bead in A and B, respectively. Gaussian fits of intensity for each dimension are overlaid in corresponding colored lines. (D) Plots of FWHM for Gaussian fits to fluorescence intensity of all beads (n = 12) in x (circles), y (squares), and z (triangles) dimensions for LITE (blue) and epi-illumination (orange). Statistical significance assessed by Student’s t test (ns, P > 0.05). Error bars represent mean ± SEM. Upper and lower black dotted lines indicate theoretical axial and lateral (0.211 and 0.568 µm, respectively) resolution for this objective. (E) Scatterplot of each bead’s measured lateral (x/y) resolution as a function of the measured distance from the coverslip surface. Dotted line corresponds to predicted resolution from D. (F) Scatterplot of each bead’s measured axial (z) resolution as a function of measured distance from coverslip surface. Dotted line corresponds to predicted resolution from D.