Figures and data
AMATERAS-2, a centimeter-FOV cell-imaging system
(A) Schematics of the design of the objective and tube lenses for AMATERAS-2. (B) Appearance of the objective and tube lenses with the width and length. (C) Diagram of the wide-field fluorescence imaging system, AMATERAS-2w, using the two lenses in (A-B). See Materials and Methods for detail.
Optical performance of AMATERAS-2w
(A),(B) Experimentally obtained PSF of green fluorescent beads with a 0.2-µm diameter in the xy and xz planes, captured using CMOS cameras with (A) 120 megapixels (pixel size = 2.2 µm) and (B) 250 megapixels (pixel size = 1.5 µm). The representative line profiles in the x and z directions on the beads are shown (black lines with circular markers) along with Gaussian fit curves (red lines) and FWHMs, and the mean and standard deviation (SD) calculated for 100 beads are described beside each profile. (C) Fluorescence image of a cardiomyocyte sheet in the entire FOV. (D) Digitally magnified view of the area indicated by a magenta square at the center of (C); scale bar, 100 µm. (E) Same area as (D) but observed by AMATERAS-1 for comparison. (F) Line profiles of the lines in (D) and (E) for comparison of spatial resolution.
AMATERAS-2c, a multipoint confocal imaging system for 3D volume imaging.
(A) Design of our pinhole-array disk. (B) Schematic of the optical configuration using the pinhole-array disk. (C) Effect of background light rejection by the presence of the pinhole-array disk in a 292 × 202 µm2 area at the FOV center corresponding to 1/502 of the entire FOV. (D),(E) Experimentally obtained PSF of green fluorescent beads with a diameter of 0.5 µm in the xy and xz planes, captured using relay lens with (D) 1× magnification (NA = 0.079) and (E) 2× magnification (NA = 0.12). The representative line profiles in the x and z directions on the beads are also shown (black lines with circular markers) along with Gaussian fit curves (red lines) and FWHMs, and the mean and standard deviation (SD) calculated for 100 beads are described beside each profile.
Volumetric imaging with computational sectioning.
(A),(B) Two-color images of the cavity chamber structure of a myocardial organoid (A) before and (B) after computational sectioning. Both (A) and (B) are MIP images of the z-stack data, captured using the 250-megapixel CMOS camera; here, magenta and cyan represent the distribution of immunostained cardiac troponin T and Hoechst-labeled nuclei, respectively. (C),(D) Six z-layers with a difference of 40 µm in the yellow square region indicated in (A),(B); scale bars, 200 µm. (E) Three-dimensional isosurface representation in the dashed square region indicated in (D).
Volumetric imaging of a mouse brain section.
(A) Schematic of a mouse brain showing the placement of the brain sample in the inverted configuration. (B) Schematic of a single z-layer in the coronal plane with size of the effective area covering the entire brain section. (C) Fluorescence images of three orthogonal cross sections of the dotted square indicated in (B); here, the yellow, cyan, and magenta lines represent the z-, x-, and y-positions of the cross sections, respectively. (D) Cross-sectional images of raw data in the same region as (C), where the computational sectioning is not applied. (E),(F) Magnified images of the hippocampal region (dashed square in (B)) in the (E) xy-plane and (F) xz-plane. For comparison, images obtained with 2× (left) and 4× (right) magnification systems are shown together. Note that the 3D regions observed at different magnifications do not perfectly match owing to the difficulty in optical alignment. (G-I) Cell detection by ELEPHANT in three brain regions, the cerebral cortex (G), medial habenula (H), and choroid plexus (I), where pairs of intersecting xy-and xz-planes are arranged vertically. The detected cells within each plane are marked with green and cyan ovals, and the straight line shared by the two planes is represented by a yellow dashed line, with certain cells along this line highlighted by magenta ovals. The xy-planes overlay the oval markers within a range of ± 25 µm above and below the focal plane, while the xz-plane shows only oval markers on the plane.
Dynamics of vascular endothelial cells in quail embryo captured by time-lapse observation.
(A) Schematic showing how the specimen is mounted (left) and a photograph of a specimen placed in a glass-bottom dish. (B) Representative images obtained at t = 0 h, 8 h, and 16 h; scale bars, 2 mm. (C) Enlarged view of the image corresponding at t = 24 h; the pseudo-colors represent the z-position of the cells and fluorescence intensity by their hue and brightness, respectively. (D) Magnified views of the anterior side of ventral aortae (left dashed square in (C)) at the four time-points with intervals of 8 h; scale bars, 200 µm. (E) Three-dimensional isosurface representation of the dorsal aorta region (right dashed square in (C)) viewed from the left side. (F),(G) Cell position trajectories and spatial mapping of features related to the cell dynamics in the two time-regions of (F) t = 8–10 h and (G) t = 16– 18 h. Left panels show enlarged views in the white square regions (292 × 202 µm2) indicated in the right panels. In both the panels, the rainbow-colored trajectories of the cell movement are overlaid on the grayscale MIP images at the first frame in the time regions (F: t = 8 h, G: t = 16 h). The scale bars are 50 µm. The rainbow color table represents the meandering index of the cell movement in the two time-regions.
