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Table 1 Summary of three microscopy techniques used in zebrafish studies

From: Highlights on selected microscopy techniques to study zebrafish developmental biology

MethodPrincipleAdvantagesDisadvantagesApplications on zebrafishReferences
Two-Photon Microscopy• Based on nonlinear optical processes.
• Image fluorescent dyes or endogenous molecules.
• Near-infrared light is used instead of visible.
• Two-photon excited fluorescence is based on the simultaneous absorption of two lower-energy photons.
• Suitable for imaging optically thick specimens.
• Less scattering and absorption in biological tissue, permitting for deeper penetration.
• No pinhole aperture and minimizes signal loss.
• Photobleaching of fluorescent molecules outside the focus is almost abolished.
• High resolution in imaging intact biological samples without spatial filtering.
• Photobleaching within the focal volume, with laser power levels typically used in biological imaging.
• Induce considerable photodamage at the focal volume where photochemical interactions occur.
1. Study morphogenetic movements during early zebrafish embryonic development
2. Measure membrane order in tissues of zebrafish larvae.
3. Neural Population Activity in Zebrafish
4. Studying membrane order polarity proteins in the gut, kidney, and liver during vertebrate organogenesis.
1. Carvalho & Heisenberg 2009 [1]
2. Owen et al. 2010 [12]
3. Renninger & Orger 2013 [17]
4. Abu-Siniyeh et al. 2016 [18]
Second Harmonic Generation• Used to image non-centrosymmetric structures such as collagen fibers and
• A nonlinear optical process where two photons are converted to a single photon without losing any energy.
• Visualizes the tissue structure directly because the contrast is produced from endogenous species.
• Significantly reduced photobleaching and phototoxicity compared to fluorescence methods.
• It can reach high-resolution imaging to several hundred microns depths.
• Limited penetration depths 100–300 μm with laser excitation in the 800–1000 nm range to increase image resolution.
• Micrometer depths are often inadequate for in vivo applications.
1. Collagen organization in zebrafish during wound healing.
2. Gene expression observation in zebrafish embryo nerve systems.
1. LeBert et al. 2015 [27]
1. LeBert et al. 2016 [28]
2. Hsieh et al. 2008 [40]
Light-Sheet Microscopy• The defining feature of LSM is the planar illumination of the focal plane from the side.
• Only a thin section of the sample is illuminated at any given time.
• Rapid imaging with high frame rates
• High signal-to-noise ratios.
• Minimum rates of photo-bleaching and toxicity.
• Three-dimensional imaging of live samples.
• Minimized photodamage.
• Deep optical sectioning.
• Faint excitation intensities.
• Moderate mounting techniques.
• Extra optics are required to produce the light sheet.
• Adding the extra lens introduces steric constraints to the imaging system and sample mounting.
1. Image zebrafish eye development
2. Imaging a seizure model in zebrafish
3. Zebrafish vascular development
4. Brain functional imaging
5. 3D imaging of cranial neurons and vasculature during zebrafish embryogenesis.
1. Icha et al. 2016 [34]
2. Kner et al. 2018 [35]
3. Kugler et al. 2018 [36]
4. Misha et al. 2013 [41]
5. Ok Kyu Park et al. 2015 [42]