spectral-reflectometric-microscopy-on-myelinated-axons-in-situ

Spectral Reflectometric Microscopy on Myelinated Axons <em>In Situ</em>

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

tongue-microfluidics-based-functional-imaging-platform-for-tongue

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue <em>In Vivo</em>

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

SungKyunKwan University

Institute for Basic Science (IBS)

Seoul National University

Although most nanofabrication techniques can control nano/micro particle (NMP) size over a wide range, the majority of NMPs for biomedical applications exhibits a diameter of ~100 nm. Here, the vascular distribution of spherical particles, from 10 to 1,000 nm in diameter, is studied using intravital microscopy and computational modeling. Small NMPs (≤100 nm) are observed to move with Red Blood Cells (RBCs), presenting an uniform radial distribution and limited near-wall accumulation. Larger NMPs tend to preferentially accumulate next to the vessel walls, in a size-dependent manner (~70% for 1,000 nm NMPs). RBC-NMP geometrical interference only is responsible for this behavior. In a capillary flow, the effective radial dispersion coefficient of 1,000 nm particles is ~3-fold larger than Brownian diffusion. This suggests that sub-micron particles could deposit within diseased vascular districts more efficiently than conventional nanoparticles. 

On the near-wall accumulation of injectable particles in the microcirculation: smaller is not better.

Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo. 

Intravital microscopic interrogation of peripheral taste sensation.

A biocompatible step-index optical fiber made of poly(ethylene glycol) and alginate hydrogels is demonstrated. The fabricated fiber exhibits excellent light-guiding efficiency in biological tissues. Moreover, the core of hydrogel fibers can be easily doped with functional molecules and nanoparticles for localized light emission, sensing, and therapy. 

Step-Index Optical Fiber Made of Biocompatible Hydrogels.

Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans-cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm(-2) . Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell-cell interactions in vivo. 

Bioorthogonal Click Chemistry-Based Synthetic Cell Glue.

Advances in photonics have stimulated significant progress in medicine, with many techniques now in routine clinical use. However, the finite depth of light penetration in tissue is a serious constraint to clinical utility. Here we show implantable light-delivery devices made of bio-derived or biocompatible, and biodegradable polymers. In contrast to conventional optical fibres, which must be removed from the body soon after use, the biodegradable and biocompatible waveguides may be used for long-term light delivery and need not be removed as they are gradually resorbed by the tissue. As proof of concept, we demonstrate this paradigm-shifting approach for photochemical tissue bonding (PTB). Using comb-shaped planar waveguides, we achieve a full thickness (>10 mm) wound closure of porcine skin, which represents ∼ 10-fold extension of the tissue area achieved with conventional PTB. The results point to a new direction in photomedicine for using light in deep tissues.

Bioabsorbable polymer optical waveguides for deep-tissue photomedicine.

The advent of phototransformable fluorescent proteins has led to significant advances in optical imaging, including the unambiguous tracking of cells over large spatiotemporal scales. However, these proteins typically require activating light in the UV-blue spectrum, which limits their in vivo applicability due to poor light penetration and associated phototoxicity on cells and tissue. We report that cyanine-based, organic dyes can be efficiently photoconverted by nonlinear excitation at the near infrared (NIR) window. Photoconversion likely involves singlet-oxygen mediated photochemical cleavage, yielding blue-shifted fluorescent products. Using SYTO62, a biocompatible and cell-permeable dye, we demonstrate photoconversion in a variety of cell lines, including depth-resolved labeling of cells in 3D culture. Two-photon photoconversion of cyanine-based dyes offer several advantages over existing photoconvertible proteins, including use of minimally toxic NIR light, labeling without need for genetic intervention, rapid kinetics, remote subsurface targeting, and long persistence of photoconverted signal. These findings are expected to be useful for applications involving rapid labeling of cells deep in tissue. 

Two-photon excited photoconversion of cyanine-based dyes.

Manipulating neural activity is crucial for studying the neural connectivity and the pathophysiology of neurodegenerative disease. Among various techniques for neural activation, direct optical stimulation method with femtosecond-pulsed laser is simple and can be specifically applied on a single neuron. Brief irradiation of femtosecond laser pulses on a neuron elevates intracellular calcium, and it propagates to adjacent neurons. However, the mechanisms of laser-induced neural activation are still unclear. In this report, we have elucidated the mechanism of laser-induced neural activation which could be mediated by superoxide, specifically blocked by diphenyleneiodonium chloride, and depletion in intracellular calcium storage. Furthermore, we also showed that the propagation of calcium initiated by laser stimulation is dependent on the presence of extracellular calcium as well as electrical and chemical synapses. We verified the applicability of such mechanism for the assessment of neuronal functionality, by measuring calcium elevation, intracellular calcium propagation, ROS increase, and performing cell death assay in vehicle and Aβ-treated neurons. This work suggests promising applications of the potential for implementing such laser-induced neural activation for rapid and reliable drug screening.

Induction of neuronal activation by femtosecond-pulsed laser irradiation and its potential application for amyloid-β-induced toxicity assessment.

Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. We discover that  Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in  was mediated only by the TRPA1(A) isoform, despite the TRPA1(A) and TRPA1(B) isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (HO). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1(A)-specific residues dispensable for electrophile detection. Collectively, TRPA1(A) rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in .

Nucleophile sensitivity of  TRPA1 underlies light-induced feeding deterrence.

The limited penetration depth of light in skin tissues is a practical bottleneck in dermatologic applications of light-induced therapies, including anti-microbial blue light therapy and photodynamic skin cancer therapy. Here, we demonstrate a novel device, termed optical microneedle array (OMNA), for percutaneous light delivery. A prototype device with a 11 by 11 array of needles at a spacing of 1 mm and a length of 1.6 mm was fabricated by press-molding poly-(lactic acid) (PLA) polymers. The device also incorporates a matched microlens array that focuses the light through the needle tips at specific points to achieve an optimal intensity profile in the tissue. In experiments done with bovine tissues, the OMNA enabled us to deliver a total of 7.5% of the input photons at a wavelength of 491 nm, compared to only 0.85% without the device. This 9-fold enhancement of light delivery was close to the prediction of 10.8 dB by ray-tracing simulation and is expected to increase the effective treatment depth of anti-microbial blue light therapy significantly from 1.3 to 2.5 mm in human skin.

Optical lens-microneedle array for percutaneous light delivery.

Two-photon polymerization has enabled precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without perturbation of the surrounding non-irradiated regions. 2P-CXL induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissueat high resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.

Selective two-photon collagen crosslinkingmeasured by Brillouin microscopy.

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.

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

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 Cu 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.

Color of Copper/Copper Oxide.

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.

Myunghwan Choi

Department of Biomedical Engineering

SungKyunKwan University

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Myunghwan Choi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

SungKyunKwan University

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Department of Biomedical Engineering