M. Jurna

Chemical Imaging of Oral Solid Dosage Forms and Changes upon Dissolution Using Coherent Anti-Stokes Raman Scattering Microscopy

Dissolution testing is a crucial part of pharmaceutical dosage form investigations and is generally performed by analyzing the concentration of the released drug in a defined volume of flowing dissolution medium. As solid-state properties of the components affect dissolution behavior to a large and sometimes even unpredictable extent there is a strong need for monitoring and especially visualizing solid-state properties during dissolution testing. In this study coherent anti-Stokes Raman scattering (CARS) microscopy was used to visualize the solid-state properties of lipid-based oral dosage forms containing the model drug theophylline anhydrate during dissolution in real time. The drug release from the dosage form matrix was monitored with a spatial resolution of about 1.5 microm. In addition, as theophylline anhydrate tends to form the less soluble monohydrate during dissolution, CARS microscopy allowed the solid-state transformation of the drug to be spatially visualized. The results obtained by CARS microscopy revealed that the method used to combine lipid and active ingredient into a sustained release dosage form can influence the physicochemical behavior of the drug during dissolution. In this case, formation of theophylline monohydrate on the surface was visualized during dissolution with tablets compressed from powdered mixtures but not with solid lipid extrudates.

Shot noise limited heterodyne detection of CARS signals

We demonstrate heterodyne detection of CARS signals using a cascaded phase-preserving chain to generate the CARS input wavelengths and a coherent local oscillator. The heterodyne amplification by the local oscillator reveals a window for shot noise limited detection before the signal-to-noise is limited by amplitude fluctuations. We demonstrate an improvement in sensitivity by more than 3 orders of magnitude for detection using a photodiode. This will enable CARS microscopy to reveal concentrations below the current mMolar range.

Background free CARS imaging by phase sensitive heterodyne CARS

In this article we show that heterodyne CARS, based on a controlled and stable phase-preserving chain, can be used to measure amplitude and phase information of molecular vibration modes. The technique is validated by a comparison of the imaginary part of the heterodyne CARS spectrum to the spontaneous Raman spectrum of polyethylene. The detection of the phase allows for rejection of the non-resonant background from the data. The resulting improvement of the signal to noise ratio is shown by measurements on a sample containing lipid.

Noncritical phase-matched lithium triborate optical parametric oscillator for high resolution coherent anti-Stokes Raman scattering spectroscopy and microscopy

An efficient, widely tunable, narrow-bandwidth, green-pumped, noncritical phase-matched lithium triborate based optical parametric oscillator (OPO) is applied to coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy. The tunable signal beam (740–930 nm) of the OPO is combined with the fundamental of a Nd:YVO4 pump laser (1064 nm, 15 ps) to obtain high resolution vibrational spectra of molecules around the CH vibrational stretch (2700–3100 cm−1). The straightforward and convenient tunability of the OPO is demonstrated by CARS microscopy for the identification of different polymer microparticles on the same substrate.

COHERENT ANTI-STOKES RAMAN SCATTERING MICROSCOPY TO MONITOR DRUG DISSOLUTION IN DIFFERENT ORAL PHARMACEUTICAL TABLETS

Coherent anti-Stokes Raman scattering (CARS) microscopy is used to visualize the release of a model drug (theophylline) from a lipid (tripalmitin) based tablet during dissolution. The effects of transformation and dissolution of the drug are imaged in real time. This study reveals that the manufacturing process causes significant differences in the release process: tablets prepared from powder show formation of theophylline monohydrate on the surface which prevents a controlled drug release, whereas solid lipid extrudates did not show formation of monohydrate. This visualization technique can aid future tablet design.

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.

Implementation of vibrational phase contrast coherent anti-Stokes Raman scattering microscopy

Detection of molecules using vibrational resonances in the fingerprint region for narrowband coherent anti-Stokes Raman scattering (CARS) is challenging. The spectrum is highly congested resulting in a large background and a reduced specificity. Recently we introduced vibrational phase contrast CARS (VPC-CARS) microscopy as a technique capable of detecting both the amplitude and phase of the CARS signal, providing background-free images and high specificity. In this paper we present a new implementation of VPC-CARS based on a third-order cascaded phase-preserving chain, where the CARS signal is generated at a single (constant) wavelength independent of the vibrational frequency that is addressed. This implementation will simplify the detection side considerably.

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.