Highlights on selected microscopy techniques to study zebrafish developmental biology

Abstract

Bio-imaging is a tedious task when it concerns exploring cell functions, developmental mechanisms, and other vital processes in vivo. Single-cell resolution is challenging due to different issues such as sample size, the scattering of intact and opaque tissue, pigmentation in untreated animals, the movement of living organs, and maintaining the sample under physiological conditions. These factors might lead researchers to implement microscopy techniques with a suitable animal model to mimic the nature of the living cells. Zebrafish acquired its prestigious reputation in the biomedical research field due to its transparency under advanced microscopes. Therefore, various microscopy techniques, including Multi-Photon, Light-Sheet Microscopy, and Second Harmonic Generation, simplify the discovery of different types of internal functions in zebrafish. In this review, we briefly discuss three recent microscopy techniques that are being utilized because they are non-invasive in investigating developmental events in zebrafish embryo and larvae.

Keywords: Animal model; Development; Light-Sheet Microscopy; Second Harmonic Generation; Two-Photon Microscopy; Zebrafish.

Fig. 1

Schematic illustration of (a) Principle of imaging membrane order phases in zebrafish larvae by multiphoton with Laurdan dye. Titanium-Sapphire laser produces 800 nm wavelength, and the laser beam passes through a scanner unite, scan lens (SL), tube lens (TL) all focused on the zebrafish with × 63 1.4 numerical aperture (NA) of the water-immersion lens. The scattered signals from the sample focused on two photomultiplier tube detectors (PMT) with two wavelengths; 400–460 nm (for order phase) and 470–530 nm (for disorder phase) for PMT1 and PMT2, respectively. b Laurdan Fluorescence characteristics. The Laurdan dye is excited at 800 nm and its emission wavelength peaks at ~ 450 nm (violet) when existing in the ordered phase, and ~ 500 nm in the disordered phase (blue). The violet and blue boxes represent the acquisition channels conducted in the 400–460 nm and 470–530 nm wavelength bands, respectively

Fig. 2

Acquired images for zebrafish tissues (gut, kidney, and liver) during different developmental stages, from 3 dpf to 11 dpf by using a Two-Photon microscope in conjugation with Laurdan dye. Scale bar = 20 μm. Adapted with permission from ref. [18]. Copyright 2016 John Wiley and Sons Periodicals. *days post-fertilization

Fig. 3

a A presentative diagram of imaging zebrafish embryo by Second Harmonic Generation Microscopy (SHG) and Third Harmonic Generation (THG): A titanium-sapphire laser generates pulses of 200 fs at 1200 nm. The laser beam passes on galvanometric mirrors (GM) to reach the sample through the objective lens. A dichroic mirror reflects the scattered signals from the sample, and the other dichroic mirror separates them into two channels (THG) and (SHG/2PF). The signals focused on the photomultiplier tube detectors (PMT), one for THG, and the other for SHG and 2PF. b A scheme of the light spectrum in SHG and THG microscopy. The excitation wavelength is 1200 nm, while the emission spectra of THG at 400 nm, SHG at 600 nm, and Two-Photon 2PF from 600 to 800 nm with a peak at ~ 650 nm generated by the sample

Fig. 4

Presentative images of successful in toto imaging by using a combination of THG and SHG in addition to 2PEF for imaging unlabeled zebrafish embryo during the cleavage period. Adapted with permission from ref. [25]. Copyright 2010; The American Association for the Advancement of Science (AAAS) periodicals

Fig. 5

Schematic diagram of (a) LSM principle in imaging zebrafish larvae through illumination and detection at a right angle. Illumination (excitation) focused in one direction through a cylindrical lens in which a thin section of the sample is illuminated vertically to the orientation of observation. Objective lens used for fluorescence detection perpendicularly to the sample. b LSM uses a planar illumination of the focal plane from the side (selective illumination) instead of a point illumination as in confocal microscopy (equal illumination), which enables LSM to capture images at a faster speed, reducing photodamage and offering optical sectioning compared to confocal microscopy

Fig. 6

Different developmental stages of the retina of zebrafish embryo acquired by Light Sheet Microscopy (LMS) from 1.5 dpf to 3.5 dpf*. The retinal ganglion cells are shown in (magenta), the amacrine and horizontal cells are shown in (yellow), and the photoreceptors and bipolar cells are shown in (cyan). The image was created on 30 November 2015 by IchaJaroslav in the Norden lab at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, this file is licensed under the Creative Commons Attribution-Share Alike 4.0 International license. *days post-fertilization