All-optical observation on activity-dependent nanoscale dynamics of myelinated axons.

Significance: In the mammalian brain, rapid conduction of neural information is supported by the myelin, the functional efficacy of which shows steep dependence on its nanoscale cytoarchitecture. Although previous in vitro studies have suggested that neural activity accompanies nanometer-scale cellular deformations, whether neural activity can dynamically remodel the myelinated axon has remained unexplored due to the technical challenge in observing its nanostructural dynamics in living tissues. Aim: We aim to observe activity-dependent nanostructural dynamics of myelinated axons in a living brain tissue. Approach: We introduced a novel all-optical approach combining a nanoscale dynamic readout based on spectral interferometry and optogenetic control of neural excitation in an acute brain slice preparation. Results: In response to optogenetically evoked neuronal burst firing, the myelinated axons exhibited progressive and reversible spectral redshifts, corresponding to the transient swelling at a subnanometer scale. We further revealed that the activity-dependent nanostructural dynamics was localized to the paranode. Conclusions: Our all-optical studies substantiate that myelinated axon exhibits activity-dependent nanoscale swelling, which potentially serves to dynamically tune the transmission speed of neural information.

Robust functional imaging of taste sensation with a Bessel beam.

Functional imaging of intact taste cells in response to various tastant solutions poses a technical challenge since the refractive index of the immersion medium dynamically changes during tastant delivery. Critically, the focal shift introduced by high-index tastant solutions has been the fundamental limit in experimental design. Here we seek to address this issue by introducing an axially elongated Bessel beam in two-photon microscopy. Compared to the conventional Gaussian beam, the Bessel beam provides superior robustness to the index-induced focal shift, allowing us to acquire near artifact-free imaging of taste cells in response to a physiological taste stimulus.

Spectral Reflectometry in Biomedical Imaging and Sensing.

Spectral reflectometry is a spectroscopic measurement technique based on thin-film interference, which has been widely applied in industries to measure thicknesses of thin dielectric layers at the nanoscale. Recent advances in the understanding of biological nanostructures have opened a new field of spectral reflectometry in biomedicine from molecular level sensing to biomedical imaging. This chapter comprehensively covers the relevant topics on spectral reflectometry in biomedicine from its principle to applications.

Color of Copper/Copper Oxide.

Stochastic inhomogeneous oxidation is an inherent characteristic of copper (Cu), often hindering color tuning and bandgap engineering of oxides. Coherent control of the interface between metal and metal oxide remains unresolved. Coherent propagation of an oxidation front in single-crystal Cu thin film is demonstrated to achieve a full-color spectrum for Cu by precisely controlling its oxide-layer thickness. Grain-boundary-free and atomically flat films prepared by atomic-sputtering epitaxy allow tailoring of the oxide layer with an abrupt interface via heat treatment with a suppressed temperature gradient. Color tuning of nearly full-color red/green/blue indices is realized by precise control of the oxide-layer thickness; the samples cover ≈50.4% of the standard red/green/blue color space. The color of copper/copper oxide is realized by the reconstruction of the quantitative yield color from the oxide "pigment" (complex dielectric functions of Cu2 O) and light-layer interference (reflectance spectra obtained from the Fresnel equations) to produce structural color. Furthermore, laser-oxide lithography is demonstrated with micrometer-scale linewidth and depth through local phase transformation to oxides embedded in the metal, providing spacing necessary for semiconducting transport and optoelectronics functionality.

Microsphere-based interferometric optical probe.

Fluorescent optical probes have rapidly transformed our understanding of complex biological systems by providing specific information on biological targets in the natural living state. However, their utility is often limited by insufficient brightness, photostability, and multiplexing capacity. Here, we report a conceptually new optical probe, termed 'reflectophore', which is based on the spectral interference from a dielectric microsphere. Reflectophores are orders-of-magnitudes brighter than conventional fluorophores and are free from photobleaching, enabling practically unlimited readout at high fidelity. They also offer high-degree multiplexing, encoded in their optical size, which can be readily decoded through interferometric detection with nanoscale accuracy, even in turbid biological media. Furthermore, we showcase their biological applications in cellular barcoding and microenvironmental sensing of a target protein and local electric field.

Millisecond cellular labelling in situ with two-photon photoconversion.

In situ labeling of cells within living biological tissues using photoconversion has provided valuable information on cellular physiology in their natural environments. However, current photoconvertible probes typically require seconds to minutes of light exposure, limiting their uses in rapid biological processes such as intracellular diffusion and circulating cells. Here, we report that two-photon photoconversion of cyanine-based dyes offers unprecedentedly rapid photoconversion down to millisecond timescales per cell. We demonstrate potential biological applications including measuring intracellular diffusion kinetics in a spinal nerve, labeling of rapidly flowing cells in a microfluidic channel, and photoconversion of a circulating cell in vivo.

Label-free nanoscale optical metrology on myelinated axons in vivo.

In the mammalian nervous system, myelin provides electrical insulation for the neural circuit by forming a highly organized, multilayered thin film around the axon fibers. Here, we investigate the spectral reflectance from this subcellular nanostructure and devise a new label-free technique based on a spectroscopic analysis of reflected light, enabling nanoscale imaging of myelinated axons in their natural living state. Using this technique, we demonstrate three-dimensional mapping of the axon diameter and sensing of dynamic changes in the substructure of myelin at nanoscale. We further reveal the prevalence of axon bulging in the brain cortex in vivo after mild compressive trauma. Our novel tool opens new avenues of investigation by creating unprecedented access to the nanostructural dynamics of live myelinated axons in health and disease.