Studying neuronal connections and activities in vivo is critical for understanding the brain. Optical microscopy, with the capability of specific fluorescent labeling and sub-cellular spatial resolution, has become an indispensable tool in neuroscience. However, the major limitation of optical imaging is penetration depth and imaging speed to capture neural signal dynamics in deep brain regions. Recently, by applying adaptive optics, high-energy laser, or long wavelength lasers for nonlinear imaging, penetration depth around 1mm has been achieved in living mouse brains. Nevertheless, this depth barely pierces through the mouse cortex and is far from reaching the bottom of the centimeter-thick mouse brain. For studying deeper regions of the brain, brain slice is one possible approach, yet it is invasive and cut away many neuron connections. In this study, a home-built two-photon microscope is integrated with both a gradient refractive index (GRIN) lens and a tunable acoustic gradient (TAG) lens. The GRIN lens serves as a micro-endoscope which extends the imaging depth to a centimeter while minimizing the invasiveness, and the TAG lens provides ~100kHz axial scanning which enables high-speed volumetric imaging of neuronal response. This novel high-speed volumetric endoscopy system offers an unprecedented opportunity towards studying three-dimensional neuronal dynamics in deep brains regions of a living mouse.
Drosophila is an important model animal to study connectomics since its brain is complicated and small enough to be mapped by optical microscopy with single-cell resolution. Compared to other model animals, its genetic toolbox is more sophisticated, and a connectome map with single-cell resolution has been established, serving as an invaluable reference for functional connectome study. Two-photon microscopy (2PM) is now the most popular tool to study functional connectome by taking the advantages of low photobleaching, subcellular resolution and deep penetration depth. However, using GFP-labeling with excitation wavelength ~ 920-nm, the reported penetration depths in a living Drosophila brain are limited to ~ 100-μm, which are much smaller than that in living mouse or zebrafish brains. The underlying reason is air vessels, i.e., trachea, instead of blood vessels, are responsible for oxygen exchange in Drosophila brains. The trachea structures induce extraordinarily strong scattering and aberration since the air/tissue refractive index difference is much larger than blood/tissue. By expelling the air inside trachea, whole Drosophila brain can be penetrated by 2PM without difficulty. However, the Drosophila is not alive anymore. Here, three-photon microscopy based on a 1300-nm laser is demonstrated to penetrate a living Drosophila brain with single-cell resolution. The long wavelength intrinsically reduces scattering, when combined with normal dispersion of brain tissue, aberration from trachea/tissue interface is reduced to some extent. As a result, the penetration depth is improved more than twice using 1300-nm excitation. This technique is believed to significantly contribute on functional connectome studies in the future.
In recent years, the techniques of super-resolution have generated widespread impacts in science. Stimulated emission depletion (STED) microscopy is known for achieving sub-diffraction-limit resolution by using a donut-shaped beam to deplete the fluorescence around a focal spot while leaving a central part active to emit fluorescence. However, since STED microscopy is based on fluorescence, it suffers from photo-bleaching. We recently developed a new technique and termed it as suppression of scattering imaging (SUSI) microscopy. It uses a STED-like setup and achieves super resolution imaging by utilizing the nonlinearity of scattering from gold nanoparticles. Therefore, SUSI microscopy avoids the photo-bleaching issue. Nonetheless, for fast volumetric imaging, SUSI microscopy is limited with slow axial translation of the objective or sample. Here we combine SUSI microscopy with a refractive-index-variable lens to axially move the focus at very high speed. This combination allows simultaneous observation of tissue dynamics over a three-dimensional volume within one second. The new technique paves the way toward high-speed super-resolution imaging for biological tissues.
Many biological systems are composed of chiral molecules and their functions depend strongly on their chirality. For example, most amino acids are of left-handed chirality while most polysaccharides are of right-handed chirality. Both of them are vital for human life, so it is important to perform chiral detection inside bio-tissues. Here we demonstrated second harmonic generation circular dichroism (SHG-CD) as a novel chiral imaging contrast in thick biotissue. Compared with conventional chiral detection, SHG-CD provides at least three orders higher contrast. In addition, due to the nonlinear nature of SHG, this technique provides optical sectioning capability, so the axial contrast is much better. The advantages of nonlinear optical microscopy are optical sectioning and deep penetration capabilities. The SHG-CD achieved 100% signal contrast with sub-micrometer spatial resolution. This method is expected to offer a novel contrast mechanism of imaging chirality inside complex bio-tissues.
With the aid of Maker fringe technique, we have observed two nonlinear optical (NLO) phenomena separately on diameter and
length of ZnO nanorod (NR). One is second harmonic generation (SHG) saturation in rod diameter, and the other is SHG
enhancement in rod length. Besides that, the model based on Lorentz local field is proposed for the first time to elucidate the above
phenomena. The deduced second order susceptibility <sub>χ</sub><sup>(2)</sup> with various sizes of ZnO NR matches well to our theory, demonstrating
that the size effect on <sub>χ</sub><sup>(2)</sup> is governed by Lorentz local field. Our theory provides a theoretical basis to explain the mechanism of
light-material interaction in nano-dimensions and is readily to be extended to other kind of semiconductor nanostructures when
addressing NLO properties in them.