|Method||Principle||Advantages||Disadvantages||Applications on zebrafish||References|
|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 |
2. Owen et al. 2010 
3. Renninger & Orger 2013 
4. Abu-Siniyeh et al. 2016 
|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 |
1. LeBert et al. 2016 
2. Hsieh et al. 2008 
|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 |
2. Kner et al. 2018 
3. Kugler et al. 2018 
4. Misha et al. 2013 
5. Ok Kyu Park et al. 2015