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In vivo developmental biology study using noninvasive multi-harmonic generation microscopy

Shi-Wei Chu, Szu-Yu Chen, Tsung-Han Tsai, Tzu-Ming Liu, Cheng-Yung Lin, Huai-Jen Tsai, and Chi-Kuang Sun

Open Access Open Access

Abstract

Morphological changes and complex developmental processes inside vertebrate embryos are difficult to observe noninvasively with millimeter-penetration and sub-micrometer-resolution at the same time. By using higher harmonic generation, including second and third harmonics, as the microscopic contrast mechanism, optical noninvasiveness can be achieved due to the virtual-level-transition characteristic. The intrinsic nonlinearity of harmonic generations provides optical sectioning capability while the selected 1230-nm near-infrared light source provides the deep-enetration ability. The complicated development within a ~1.5-mm thick zebrafish (Danio rerio) embryo from initial cell proliferation, gastrulation, to tissue formation can all be observed clearly in vivo without any treatment on the live specimen.

©2003 Optical Society of America

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Supplementary Material (4)

Media 1: MOV (730 KB)     
Media 2: MOV (434 KB)     
Media 3: MOV (738 KB)     
Media 4: MOV (280 KB)     

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Figures (3)
Fig. 1.
Fig. 1. Mitosis processes inside a live zebrafish embryo in vivo monitored with HOM. (a) An optical section of the embryo at the dome stage (4-hpf). The imaging depth is about 400-µm from the chorion surface. THG (shown in blue throughout this paper) picks up all interfaces including external yolk syncytial layers, cell membranes, and nuclear membranes while SHG (shown in green throughout this paper) shows the microtubule-formed spindle biconical array (indicated by the arrow). (b) (730-kB) Time series of the mitosis process in the embryonic blastoderm at 1-k cell stage (2.5-hpf). The cell nuclear membrane (arrowhead) and centrosomes (arrows) can be visualized through THG and SHG respectively. (c) (434 kB) Time series of the mitosis process in the embryonic blastoderm at shield-stage (6-hpf). Scale bar: 20-µm.

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Fig. 2:
Fig. 2: In vivo HOM sectioning inside a live zebrafish embryo at the 2-somite stage. (a) A sectioning showing the whole embryo at a depth of 700-µm from the chorion surface (ventral view). PL: polster; TB: tail bud. (b) THG image of the chorion surface, showing the <1-µm diameter granular canals and demonstrating the sub-µm resolution. (c) (738 kB) Depth-resolved optical section series at depths from 300-µm to 1400-µm inside the embryo. Scale bar: 100-µm except for B: 10-µm.

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Fig. 3:
Fig. 3: In vivo HOM sectioning inside a live zebrafish larva at the 20-somite stage. (a) An optical section at the center of the larva showing the segments inside the vacuolated notochord and the distribution of somites alongside the notochord. (b) The enlarged view inside a somite showing individual muscle fiber and the sarcomeres on it through SHG as well as the interface between somites through THG. (c) (281 kB) Depth resolved optical series showing that we can visualize through a whole zebrafish larva with HOM. Scale bar: 20-µm.

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