HARMONIC IN VIVO IMAGING MICROSCOPY OF THE ZEBRAFISH VASCULATURE
2Laboratory of Condensed Matter Physics, Ecole Polytechnique, Palaiseau, France
3Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland
4IGBMC, Universite De Strasbourg, Illkirch, France
Conventional two-photon microscopy (2PM) enables deep fluorescence imaging of live tissues with reduced photodamage levels1. 2PM generates non-fluorescent second-harmonic generation (SHG) and third-harmonic generation (THG) signals from endogenous tissue structures. Since SHG and THG are generated without any chemical or fluorescence labeling, these harmonic signals are also termed “label-free” signals2.
2PM has been extended to 2P light-sheet microscopy (2P-LSM)3. In 2P-LSM, kHz-fast fluorescence imaging of large fields-of-view is possible because of the configuration of the imaging setup, where the excitation objective and the detection objective are orthogonal one in respect to the other3. As harmonic signals propagate mainly along the illumination direction, this raises the question of whether the orthogonal configuration of 2P-LSM can be used to detect harmonic signals. Detecting such signals in 2P-LSM in addition to fluorescence will expand the repertoire of available imaging modalities that when combined together, could generate much more information from a single imaging experiment.
Here I will present the suitability of 2P-LSM for fast imaging of SHG nanoprobes4, which are nanocrystal point-like SHG-sources that can efficiently scatter light in multiple directions and can thus be detected using 2P-LSM. I will describe parameters that govern the detection efficiency of nanocrystals using 2P-LSM and I will discuss how to optimize SHG detection to enable fast light-sheet imaging combining SHG and 2P-excited fluorescence. I will present single particle tracking of SHG nanoprobes in-vivo in blood vessels at high temporal resolution of up to 180 frames/s in zebrafish embryos, which enables re-construction of velocity flow profiles along the vessel`s radius. This is the first demonstration of single-particle microangiography using light-sheet microscopy.
I will also present novel data of our recent efforts to utilize harmonic signals to quantitatively characterize zebrafish embryos in-vivo. I will demonstrate how these signals can be efficiently used to reconstruct the entire zebrafish heart in three dimensions at a sub-micron scale.
Altogether, our recent advances highlight harmonic imaging microscopy as a useful tool to study biological tissue structure and dynamics, and by combining harmonic imaging with fluorescence microscopy, valuable information about the sample can be furthermore obtained.
References:
1. Zipfel W.R., Williams R.M. and Webb W.W, Nat Biotech, 21, 1369-1377 (2003).
2. James D.S. and Campagnola P.J., BME Frontiers, Volume 2021 (2021).
3. Truong T.V. et al., Nat Meth, Jul 17; 8(9): 757-760 (2011).
4. Malkinson G. et al., ACS Photonics, 7, 4, 1036-1049 (2020).