Optical microscopic imaging, manipulation, and analysis methods for morphogenesis research

Abstract

Morphogenesis is a developmental process of organisms being shaped through complex and cooperative cellular movements. To understand the interplay between genetic programs and the resulting multicellular morphogenesis, it is essential to characterize the morphologies and dynamics at the single-cell level and to understand how physical forces serve as both signaling components and driving forces of tissue deformations. In recent years, advances in microscopy techniques have led to improvements in imaging speed, resolution and depth. Concurrently, the development of various software packages has supported large-scale, analyses of challenging images at the single-cell resolution. While these tools have enhanced our ability to examine dynamics of cells and mechanical processes during morphogenesis, their effective integration requires specialized expertise. With this background, this review provides a practical overview of those techniques. First, we introduce microscopic techniques for multicellular imaging and image analysis software tools with a focus on cell segmentation and tracking. Second, we provide an overview of cutting-edge techniques for mechanical manipulation of cells and tissues. Finally, we introduce recent findings on morphogenetic mechanisms and mechanosensations that have been achieved by effectively combining microscopy, image analysis tools and mechanical manipulation techniques.

Keywords: mechanobiology; morphogenesis; optical microscopy; segmentation tool; tracking tool.

Fig. 1.

Comparison of microscopy (a) Schematic of spinning-disk confocal microscopy. A rotating spinning-disk (illustrated in black) with numerous pinholes is placed in the equivalent sample plane, which scans the sample plane with multiple points (blue lines). (b) Schematic of two-photon excitation microscopy. The excitation laser, illustrated in red, is focused in the sample and excites the fluorophore when two photons are simultaneously absorbed by fluorophores. The emitted fluorescence, illustrated in green, has a shorter wavelength than that of the excited laser. (c) Schematic of the light-sheet microscopy. A sample (not illustrated), usually embedded in gel, is located at the intersecting point of the optical axes of two objective lenses. One objective illuminates a single plane in the sample (illustrated in blue plane), and another objective lens observes this plane in a single shot. The upper right panel shows a schematic of axially swept light-sheet microscopy. The lower right panel shows the comparison of the Gaussian beam and the Bessel beam. The blue line represents the outline of light rays, and the light blue regions in the Bessel beam represent the side lobes.

Fig. 2.

Illustration of segmentation and tracking concepts (a) Typical image processing workflow for single-cell scale segmentation and tracking analysis. The expected outcome is presented below each illustration. This workflow implicitly uses the tracking-by-detection approach (see main text). (b) Illustration of the semantic and instance segmentation. The colors represent the values obtained by the segmentation algorithms. In semantic segmentation, close objects are detected as a connected region (white arrow). The original image is taken from Ref. [188], licensed under the Creative Commons 0 license (See https://github.com/CellProfiler/examples/issues/41 for discussion). (c) Illustration of the tracking-by-detection approach. The segmented/detected cells are temporally connected to find tracks. The image is taken from Ref. [88], licensed under the Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0/).

Fig. 3.

Comparison of the manipulation techniques (a) Schematic of a probe of magnetic tweezers. Typically, a solenoid (red line) wraps around a rod (black). The distance between the solenoid and the sample is commonly controlled by a manipulator (data not shown). A 1–10 µm-diameter magnetic particle is usually used for the probe (brown). (b) Schematic of optical tweezers. A laser beam is focused by an objective lens (illustrated as an ellipsoid), trapping a particle (green) with a high refractive index in the medium. The 0.05–5 µm diameter polystyrene particle is usually used for the probe (green). (c) Schematic of an AFM probe, using a fine probe that typically moves along the z-direction. AFM can manipulate the specific point in the sample. Generally, the specimen is fixed on the flat stage, such as the mica surface (data not shown), and the probe scans the surface.

Fig. 4.

Mechanical signals involved in the L–R determination (a) Steps in the establishment of L–R asymmetry (left panel). The step of symmetry breaking includes three smaller steps (right gray box). (b) Measurement of the 3D deformation of the nodal immotile cilia through optical regulation of the nodal flow. The left and right panels show 3D reconstructed images of the left-side (L-side) and the right-side (R-side) cilium. Red and green colors represent the same cilium with and without nodal flow, respectively. The middle panel shows the schematic of the cross-section of the node. The L- and R-side cilia show ventral and dorsal bending by the nodal flow (illustrated in an orange arrow), respectively. (c) Manipulation of nodal immotile cilia by optical tweezers and measurement of the Dand5 mRNA degradation. A polystyrene bead (white dotted circle) was trapped and oscillated along the z-axis by optical tweezers and contacted a cilium (left panel). Dand5 mRNA degradation, the earliest marker for the L-side determination, was activated through mechanical stimuli of optical tweezers (right panel). To evaluate the response of the mechanical stimuli while excluding any influence from chemical cues, the authors used the iv/iv mutant, which lacks the nodal flow and flow-derived chemical cues. Even without chemical cues, mechanical signals activated mRNA degradation, which meant mechanical stimuli were sufficient to initiate the L–R determination. The authors measured the mRNA level by whole-cell fluorescence recovery after photobleaching [14]. The intensity was linearly correlated to the Dand5 mRNA level. (d) Model of the initial L–R determination by the nodal immotile cilia. The cross-section of the node is shown. By the nodal flow (orange arrow), the L- and R-side cilia illustrated by the pink rods are bent to the ventral (V-bend) and dorsal (D-bend) sides, respectively. The Pkd2 channels are cation channels and one of the candidates of the mechanosensor on the cilia are localized on the dorsal side of both side cilium. On the L-side cilia, the membrane tension of the dorsal side is increased, which activates the dorsally localized Pkd2 channel. The Dand5 mRNA degradation occurs only on the L-side and determines the L–R axis. (a–d) Modified from Ref. [14].