E.T. Garbacik

Visualizing Resonances in the Complex Plane with Vibrational Phase Contrast Coherent Anti-Stokes Raman Scattering

In coherent anti-Stokes Raman scattering (CARS), the emitted signal carries both amplitude and phase information of the molecules in the focal volume. Most CARS experiments ignore the phase component, but its detection allows for two advantages over intensity-only CARS. First, the pure resonant response can be determined, and the nonresonant background rejected, by extracting the imaginary component of the complex response, enhancing the sensitivity of CARS measurements. Second, selectivity is increased via determination of the phase and amplitude, allowing separation of individual molecular components of a sample even when their vibrational bands overlap. Here, using vibrational phase contrast CARS (VPC-CARS), we demonstrate enhanced sensitivity in quantitative measurements of ethanol/methanol mixtures and increased selectivity in a heterogeneous mixture of plastics and water. This powerful technique opens a wide range of possibilities for studies of complicated systems where overlapping resonances limit standard methodologies.

Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-stokes raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm−1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm−1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm−1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.

Phase aspects of (broadband) stimulated Raman scattering

Abstract The phase of the molecular response can be exploited to improve selectivity without sacrificing speed in both narrowband and broadband coherent anti-Stokes Raman scattering (CARS) microscopy, both of which will be considered in this review of the work that was performed in our group.

In planta imaging of Δ9-tetrahydrocannabinolic acid in Cannabis sativa L. with hyperspectral coherent anti-Stokes Raman scattering microscopy

Abstract. Nature has developed many pathways to produce medicinal products of extraordinary potency and specificity with significantly higher efficiencies than current synthetic methods can achieve. Identification of these mechanisms and their precise locations within plants could substantially increase the yield of a number of natural pharmaceutics. We report label-free imaging of Δ9-tetrahydrocannabinolic acid (THCa) in Cannabis sativa L. using coherent anti-Stokes Raman scattering microscopy. In line with previous observations we find high concentrations of THCa in pistillate flowering bodies and relatively low amounts within flowering bracts. Surprisingly, we find differences in the local morphologies of the THCa-containing bodies: organelles within bracts are large, diffuse, and spheroidal, whereas in pistillate flowers they are generally compact, dense, and have heterogeneous structures. We have also identified two distinct vibrational signatures associated with THCa, both in pure crystalline form and within Cannabis plants; at present the exact natures of these spectra remain an open question.

Hyperspectral coherent anti-Stokes Raman scattering microscopy for in situ analysis of solid-state crystal polymorphs

Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy is quickly becoming a prominent imaging modality because of its many advantages over the traditional paradigm of multispectral CARS. In particular, recording a significant portion of the vibrational spectrum at each spatial pixel allows image-wide spectral analysis at much higher rates than can be achieved with spontaneous Raman. We recently developed a hyperspectral CARS method, the driving principle behind which is the fast acquisition and display of a hyperspectral datacube as a set of intuitive images wherein each material in a sample appears with a unique trio of colors. Here we use this system to image and analyze two types of polymorphic samples: the pseudopolymorphic hydration of theophylline, and the packing polymorphs of the sugar alcohol mannitol. In addition to these solid-state form modifications we have observed spectral variations of crystalline mannitol and diprophylline as functions of their orientations relative to the optical fields. We use that information to visualize the distributions of these compounds in a pharmaceutical solid oral dosage form.

In situ dissolution analysis of pharmaceutical dosage forms using coherent anti-Stokes Raman scattering (CARS) microscopy

A custom-built intrinsic flow-through dissolution setup was developed and incorporated into a home-built CARS microscope consisting of a synchronously pumped optical parametric oscillator (OPO) and an inverted microscope with a 20X/0.5NA objective. CARS dissolution images (512×512 pixels) were collected every 1.12s for the duration of the dissolution experiment. Hyperspectral CARS images were obtained pre- and postdissolution by rapidly imaging while sweeping the wavelength of the OPO in discrete steps so that each frame in the data stack corresponds to a vibrational frequency. An image-processing routine projects this hyperspectral data into a single image wherein each compound appears with a unique color. Dissolution was conducted using theophylline and cimetidine-naproxen co-amorphous mixture. After 15 minutes of theophylline dissolution, hyperspectral imaging showed a conversion of theophylline anhydrate to the monohydrate, confirmed by a peak shift in the CARS spectra. CARS dissolution images showed that monohydrate crystal growth began immediately and reached a maximum with complete surface coverage at about 300s. This result correlated with the UV dissolution data where surface crystal growth on theophylline compacts resulted in a rapidly reducing dissolution rate during the first 300s. Co-amorphous cimetidinenaproxen didn’t appear to crystallize during dissolution. We observed solid-state conversions on the compact’s surface in situ during dissolution. Hyperspectral CARS imaging allowed visual discrimination between the solid-state forms on the compact’s surface. In the case of theophylline we were able to correlate the solid-state change with a change in dissolution rate.

Epi-detection of vibrational phase contrast coherent anti-Stokes Raman scattering.

We demonstrate a system for the phase-resolved epi-detection of coherent anti-Stokes Raman scattering (CARS) signals in highly scattering and/or thick samples. With this setup, we measure the complex vibrational responses of multiple components in a thick, highly-scattering pharmaceutical tablet in real time and verify that the epi- and forward-detected information are in very good agreement.

Dynamic process measurements in the complex plane with vibrational phase contrast CARS

In the coherent anti-Stokes Raman scattering (CARS) process, the emitted signal carries both amplitude and phase information of the molecules in the focal volume. These components form a vector in the complex plane, with the magnitude given by the amplitude and the angle between the vector and the real axis determined by the phase. Most CARS experiments ignore the phase component, but its detection allows for two advantages over amplitude-only CARS. First, the pure resonant response can be determined—and the non-resonant background rejected—by extracting the imaginary component of the complex response, enhancing the sensitivity of CARS measurements[1]. Second, selectivity is increased via determination of the phase and amplitude, allowing separation of individual molecular components of a sample even when their vibrational bands overlap[2]. The vibrational responses of individual molecular species trace different trajectories through the complex plane as functions of driving frequency. Chemically selective images can be made by locating the regions of the complex plane belonging to each substance at a given driving frequency. Dissolutions and chemical reactions can be followed by tracking the complex-plane positions, relative to the initial compounds, of each location in the sample. Furthermore, quantitative concentration measurements can be accurately performed, even on homogeneous solutions containing molecules with congested spectra. Figure 1 illustrates the CARS spectra of two common plastics. For each vibrational frequency the amplitude of the CARS signal is designated as the length of a vector in complex space, with the phase defined as the angle separating the vector from the horizontal axis. The horizontal and vertical axes correspond to the real and imaginary components of the χ (3) tensor, with vibrational frequencies plotted on the third axis. The real and imaginary components can be directly extracted from this plot. Figure 2 shows the complex plane trajectories of ethanol and methanol around 3000 cm -1 , as well as concentration measurements of five volumetric mixtures at two different driving frequencies. Line (I) indicates a driving frequency where only the amplitudes of the vibrational responses differ between the two molecular species, while line (II) lies at a frequency where the amplitudes are similar but the phases are well separated. In both cases the measurements agree with predicted values of the complex location of the mixture.