Figure 1

Optical performance of SDCLM. (a) Schematic principle of SDCLM. Arrayed microlenses split the excitation laser beam (green) and focus each of the split beams into its corresponding pinhole. The laser beam passing the pinhole focuses through the objective lens to the corresponding spot and excites fluorophores. Emitted fluorescence (red) passes the corresponding pinhole and focuses onto the corresponding photosensor. Each pinhole prevents out-of-focus fluorescence from reaching the corresponding photosensor. Right bottom panel, a representative image of non-rotating pinholes at the sample plane. Scale bar, 200 µm. (b) To measure the spatial resolution in SDCLM imaging (c,d), fluorescent beads were embedded in 0.5% agarose gel and Z-stacks of the beads located around a depth of 20 µm from the coverslip were acquired. (c) Representative normalized XY (left) and XZ (right) SDCLM images of fluorescent beads with a diameter of 0.5 µm. Scale bar, 2 µm. (d) The FWHMs of the 0.5 µm beads along X (top) and Z (bottom) axes (n = 5 for each axis). A Gaussian curve (blue) was fitted to the intensity profile of the bead along each axis. (e) Representative normalized XZ images of fluorescent beads with a diameter of 0.5 µm (bottom) at three locations along the X axial centerline in a 1 mm² FOV (top). Scale bar, 5 µm. (f) Lateral and axial FWHMs and intensity of the 0.5 µm beads at six locations along the X axial centerline in the FOV (n = 5 beads for each point). The FWHMs at the center of the FOV were longer than those in d. This was probably because the pixel width of the camera image that was used to calculate the FWHMs was larger in f (1.05 µm at the sample) than in d (0.21 µm at the sample). (g–j) Performance of SDCLM compared with OEFM in a tissue phantom. (g) Tissue phantom preparation. Fluorescent beads with a diameter of 2.0 µm (green circles) were embedded in 0.5% agarose gel with non-fluorescent polystyrene beads (open circles) as a scattering agent, and were imaged with OEFM or SDCLM. Volumes of 1 × 1 × 0.3 mm were imaged at 2 µm spacing along the Z axis. (h) Representative images of the 2.0 µm fluorescent beads at two depths (top, 5 µm depth, and bottom, 210 µm depth) with OEFM (left) and SDCLM (right). The images were cropped (300 × 300 µm) from the original images (1 × 1 mm). Scale bar, 100 µm. (i) Signal-to-background ratios as a function of the imaging depth, where the signal was the maximum fluorescence intensity in a 30 × 30 µm square that had an in-focus fluorescent bead at the center and did not appear to have any other one, and the background was the average intensity of this square excluding a circle with a diameter of 10 µm at the center of the square. Data were presented as mean ± SEM. **p < 0.01, n = 5 beads, Wilcoxon rank sum test. (j) Axial resolution as a function of imaging depth. The values were normalized to the mean value at a 5 µm depth in the SDCLM image and plotted as mean ± SEM (n = 5 beads for each point). *p < 0.1, **p < 0.01, Wilcoxon rank sum test.