Chao-Yu Chung

Characterization of Cholesterol Crystals in Atherosclerotic Plaques Using Stimulated Raman Scattering and Second-Harmonic Generation Microscopy

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue.

Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy

Optical imaging with spectroscopic vibrational contrast is a label-free solution for visualizing, identifying, and quantifying a wide range of biomolecular compounds in biological materials. Both linear and nonlinear vibrational microscopy techniques derive their imaging contrast from infrared active or Raman allowed molecular transitions, which provide a rich palette for interrogating chemical and structural details of the sample. Yet nonlinear optical methods, which include both second-order sum-frequency generation (SFG) and third-order coherent Raman scattering (CRS) techniques, offer several improved imaging capabilities over their linear precursors. Nonlinear vibrational microscopy features unprecedented vibrational imaging speeds, provides strategies for higher spatial resolution, and gives access to additional molecular parameters. These advances have turned vibrational microscopy into a premier tool for chemically dissecting live cells and tissues. This review discusses the molecular contrast of SFG and CRS microscopy and highlights several of the advanced imaging capabilities that have impacted biological and biomedical research.

Enhancement of Deuterated Ethane on Jupiter

We report laboratory measurements of cross sections of CH3D and C2H5D in the extreme ultraviolet. The results are incorporated in a photochemical model for the deuterated hydrocarbons up to C2 in the upper atmosphere of Jupiter, taking into account the fast reactions for exchanging H and D atoms between H2 and CH4 ,H , . Since there is no reliable kinetics measurement for the reaction, HD ↔ D HC H D ↔ CH D H 23 2 , we use Yung et al.’s estimate for its rate constant. The strong temperature dependence CH D H r CH D 23 for this reaction leads to large isotopic fractionation for CH 3D and C2H5D in the upper atmosphere of Jupiter, where their production rates depend on the abundance of deuterated methyl radical. The model predicts that the D/H ratio in deuterated ethane is about 15 times that of the bulk atmosphere. A confirmation of this result would provide a sensitive test of the photochemistry of hydrocarbons in the atmosphere of Jupiter.

Probing the Dynamics of Doxorubicin-DNA Intercalation during the Initial Activation of Apoptosis by Fluorescence Lifetime Imaging Microscopy (FLIM)

Doxorubicin is a potent anthracycline antibiotic, commonly used to treat a wide range of cancers. Although postulated to intercalate between DNA bases, many of the details of doxorubicin’s mechanism of action remain unclear. In this work, we demonstrate the ability of fluorescence lifetime imaging microscopy (FLIM) to dynamically monitor doxorubicin-DNA intercalation during the earliest stages of apoptosis. The fluorescence lifetime of doxorubicin in nuclei is found to decrease rapidly during the first 2 hours following drug administration, suggesting significant changes in the doxorubicin-DNA binding site’s microenvironment upon apoptosis initiation. Decreases in doxorubicin fluorescence lifetimes were found to be concurrent with increases in phosphorylation of H2AX (an immediate signal of DNA double-strand breakage), but preceded activation of caspase-3 (a late signature of apoptosis) by more than 150 minutes. Time-dependent doxorubicin FLIM analyses of the effects of pretreating cells with either Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)-hydrazine (a histone acetyltransferase inhibitor) or Trichostatin A (a histone deacetylase inhibitor) revealed significant correlation of fluorescence lifetime with the stage of chromatin decondensation. Taken together, our findings suggest that monitoring the dynamics of doxorubicin fluorescence lifetimes can provide valuable information during the earliest phases of doxorubicin-induced apoptosis; and implicate that FLIM can serve as a sensitive, high-resolution tool for the elucidation of intercellular mechanisms and kinetics of anti-cancer drugs that bear fluorescent moieties.

Mapping molecular orientation with phase sensitive vibrationally resonant sum-frequency generation microscopy

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue. The presence of collagen microdomains in tendon tissue may have implications for the interpretation of the mechanical properties of the tissue.

Quantitative spectroscopic and theoretical study of the optical absorption spectra of H2O, HOD, and D2O in the 125–145 nm region

The room temperature absorption spectra of water and its isotopomers D2O and HOD have been determined in absolute cross section units in the 125 to 145 nm wavelength region using synchrotron radiation. The experimental results for these B band spectra are compared with results from quantum mechanical calculations using accurate diabatic ab initio potentials. A Monte Carlo sampling over the initial rotational states of the molecules is applied in order to calculate the cross sections at a temperature of 300 K. The overall rotation of the water molecule is treated exactly. Both for the experimental and for the theoretical spectrum an analysis is made in terms of a component attributed to rapid direct dissociation processes and a component attributed to longer-lived resonances. The agreement between the results from experiment and theory is excellent for H2O and D2O. In the case of HOD in the results of theory two more resonances are found at low energy. It is demonstrated that the width of the resonances of 0.04 eV is the result of overlapping and somewhat narrower resonances in the spectra of molecules differing in rotational ground state.

ABSORPTION CROSS SECTIONS OF HCl AND DCl AT 135-232 NANOMETERS: IMPLICATIONS FOR PHOTODISSOCIATION ON VENUS

Cross sections for photoabsorption of HCl and DCl are determined in the spectral region of 135-232 nm using radiation from a synchrotron light source. At wavelengths near the onset of absorption (λ > 200 nm), cross sections of HCl are approximately 5-10 times larger than those of DCl. These data are used to calculate rates of photodissociation of HCl and DCl in the Venusian atmosphere. For the entire wavelength region measured, the rate of photodissociation of DCl is only 16% that of HCl. The difference in rates of photodissociation contributes to the exceptionally large [D]/[H] ratio of the Venusian atmosphere.

Three-center versus four-center elimination of haloethene: Internal energies of HCl and HF on photolysis of CF2CHCl at 193 nm determined with time-resolved Fourier-transform spectroscopy

Following photodissociation of 2-chloro-1,1-difluoroethene CF2CHCl) at 193 nm, vibration–rotationally resolved emission spectra of HCl(v⩽3) and HF(v⩽4) in spectral regions 2000–2900 and 3050–4410 cm−1, respectively, are detected with a step-scan time-resolved Fourier-transform spectrometer. All vibrational levels of HCl and HF show Boltzmann-type rotational distributions. HCl has an average rotational energy of 23±4 kJ mol−1 and a vibrational energy of 25±5 kJ mol−1, whereas HF has an average rotational energy of 20±4 kJ mol−1 and a vibrational energy of 48±6 kJ mol−1. The observed internal energy distribution indicates that HCl is produced via the three-center (α,α), but HF via the four-center (α,β) elimination. A modified separate statistical ensemble model predicts an internal energy distribution of HCl slightly greater than experimental observation. A modified impulse model taking into account geometries and displacement vectors of transition states during bond breaking predicts satisfactorily the rot...

Quantitative spectral analysis of HCl and DCl in 120–220 nm: Effects of singlet–triplet mixing

Photoabsorption cross sections of HCl and DCl in the spectral region 120–220 nm were measured using synchrotron radiation as a light source. The absorption contour of the A 1Π←X 1Σ+ band of HCl in the 134–195 nm region is slightly asymmetric, whereas that of DCl is symmetric, with wavelength of absorption maximum shifted from ∼153.9 nm for HCl to ∼155.8 nm. The asymmetry in HCl and the atypical direction of shift in peak wavelengths upon deuteration are attributed to singlet–triplet mixing in its ground X 1Σ+ state of HCl; this triplet component contributes to the oscillator strengths of the A–X band of HCl via transition to the repulsive triplet state 1 3Σ+. The small triplet character in HCl is further supported by an observation that the oscillator strength of the (0,0) band for the b 3Π–X 1Σ+ transition of DCl is ∼17% smaller than that of HCl. This triplet component may account for greater values of spin–orbit branching ratios for products, Cl*(2P1/2)/Cl(2P3/2), observed upon photolysis of HCl in the ...

Fate of Intravenously Administered Gold Nanoparticles in Hair Follicles: Follicular Delivery, Pharmacokinetic Interpretation, and Excretion

Gold nanoparticles (GNPs) are intravenously administered to mice. Deposition at the pilosebacious unit and whiskers is visualized with X-ray fluorescence after 30 minutes and 14 days. After 30 minutes the dermal papilla, bulge region, and root sheath all contain NPs. GNPs are driven externally out from follicles, counteractive to transfollicular delivery. After 14 days, gold bands in hairs reflect pharmacokinetic profiles indicating blood concentration kinetics. Elimination rate constants infer half-lives from 3 hairs from an individual mouse within reasonable agreement (6.08, 7.15, and 8.66 hours). 3D reconstruction of NP distributions with confocal microscopy identifies aggregates within the medullary canal. Intermittent NP deposition continues randomly over the two week period demonstrating prolonged NP mobility in vivo. NPs are still retained at the hair bulb after 14 days. The observations further account for the excretory mechanisms of NPs and their behavior in the pilosebacous unit, and demonstrate monitoring pharmacokinetic behavior in individual animals.