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In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of 89Zr-trastuzumab (SKOV-3 and OE19), 89Zr-bevacizumab (SKOV-3), or 89Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment. Cancer Res; 73(11); 3347–55. ©2013 AACR.

For the rapid analysis of complicated heterogeneous mixtures, we have developed a method to acquire and intuitively display hyperspectral coherent anti-Stokes Raman scattering (CARS) images. The imaging is performed with a conventional optical setup based around an optical parametric oscillator. Color coding each frame of the resulting hyperspectral data cube according to vibrational frequency results in a two-dimensional projection wherein each compound appears with a unique hue. We demonstrate the capabilities of this system by recording a hyperspectral CARS data stack of a heterogeneous mixture composed of seven crystalline amino acids and subsequently identifying each of them by direct visual analysis of the colored projection

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 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.

We demonstrate a new technique that combines polarization sensitivity of the coherent anti-Stokes Raman scattering (CARS) response with heterodyne amplification for background-free detection of CARS signals. In this heterodyne interferometric polarization CARS (HIP-CARS), the major drawbacks of polarization and heterodyne CARS are rectified. Using a home-built picosecond optical parametric oscillator, we are able to address vibrational stretches between 600 and 1650 cm−1 and record continuous high-resolution Raman equivalent HIP-CARS spectra. Copyright © 2010 John Wiley & Sons, Ltd.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy is an important technique for spectroscopy and chemically selective microscopy, but wider implementation requires dedicated versatile tunable sources. We describe an optical parametric oscillator (OPO) based on a magnesium oxide-doped periodically poled lithium niobate crystal, with a novel variable output coupler, used as a tunable coherent light source. The OPO's signal wavelength ranges from 880 to 1040 nm and its idler wavelength from 1090 to 1350 nm. We use this OPO to demonstrate high-resolution narrowband CARS spectroscopy on bulk polystyrene from 900 to 3600 cm-1, covering a large part of the molecular fingerprint region. Recording vibrational spectra using narrowband CARS spectroscopy has several advantages over spontaneous Raman spectroscopy, which we discuss. We isolate the resonant part of the CARS spectrum and compare it to the spontaneous Raman spectrum of polystyrene using the maximum entropy method of phase retrieval; we find them to be in extremely good agreement.

The proto-oncogene tyrosine-protein kinase Src is a key ele- ment of signaling cascades involved in the invasive and meta- stasis-forming capacity of cancer cells. While membrane ty- rosine-kinase receptors are known to dimerize, Src is classified as a non-receptor kinase and assumed to remain always mono- meric. Here we demonstrate the formation of stable dimers by the first domains of myristoylated Src previously shown to be sufficient for Src trafficking. Src dimers fused to green fluo- rescent protein (GFP) on supported lipid bilayers were identi- fied using single-molecule photobleaching experiments. Com- petition with a protein containing only native Src domains without GFP confirms that dimerization is a previously over- looked intrinsic property of Src. Dimerization is concomitant to membrane binding by the myristoylated forms of Src and may constitute a new regulation layer for the Src oncogene.

Coherent anti-Stokes Raman scattering (CARS) microscopy is becoming a widely used technique for sub-micron, chemically-selective imaging at high rates of speed In this thesis I discuss three methods for increasing the specificity and selectivity of coherent Raman experiments. The first method is the application of a false-color coding of hyperspectral coherent Raman data, which enables the rapid visual analysis of complicated samples. This measurement technique has found extensive use in pharmaceutical and biomedical applications, including the characterization of the polymorphic behaviors of crystalline and semi-crystalline compounds in oral dosage forms. We have further utilized this technique to localize medicinally relevant compounds within whole, unprocessed plant material. The second method relies on the measurement of the full complex vibrational response of the sample in a heterodyne configuration, which we have combined with hyperspectral acquisition and advanced spectral unmixing algorithms to allow quantitative characterization of both heterogeneous and homogeneous mixtures. Vibrational phase contrast CARS, as we refer to it, is a powerful tool for measuring samples that have large non-resonant background signals, as the phase of the resonant vibrational response differs significantly from that of the non-resonant background and so can be easily separated. An upgraded set of infrastructure and fresh understanding the of the underlying physics enables us to acquire full phase-resolved vibrational information at high rates and in thick and/or highly scattering samples. Finally, a third method casts the heterodyne coherent Raman scattering process in a new paradigm wherein energy transfer between the optical fields and the molecule becomes the parameter of interest. Two separate optical processes are identified: parametric processes are those in which energy is merely re-arranged between the optical fields incident on the sample, leaving the molecule in its original state, while dissipative processes are those in which energy is exchanged from the optical fields into the molecule, leaving it in an excited state. In this new paradigm the pure dissipative vibrational signal can be readily measured even in the presence of a large electronic background.

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.

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.

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.

Coherent anti-Stokes Raman scattering with phase-shaped pulses exploits interference effects for optical discrimination of molecules in complex mixtures. We present our approaches combining adaptive pulse shaping and closed-loop optimization strategies for chemically selective microscopy.

Coherent anti-Stokes Raman scattering, better known as CARS, is a powerful and versatile spectroscopic and microscopic technique, with many advantages over traditional Raman and fluorescence methods. It is immune to autofluorescence, does not require labeling, and returns molecules to their ground states, which reduces sample heating. However, CARS suffers from two negative traits compared to Raman and fluorescence measurements. First, a non-resonant background is frequently more intense than the resonant contributions in a sample. Second, CARS signals are orders of magnitude weaker than Stokes Raman lines, rendering the detection of small concentrations of weakly-resonant molecules difficult. Our current research focuses on these two problems in particular: inherent polarization-sensitivity of CARS allows isolation and rejection of the non-resonant component of CARS, though at the cost of a significant reduction in resonant signal1; heterodyning techniques provide simultaneous enhancement of CARS signals and rejection of the non-resonant background, but require explicit knowledge of the phase relations in the CARS process2. Using a homebuilt optical parametric oscillator3 we have developed a method that exploits both heterodyning and polarization sensitivity that offers resonant-only enhancement of CARS signals without and specific knowledge of the phases. This “HIP-CARS” procedure significantly simplifies the detection elements of the system and provides for future development of fast, resonant-only CARS imaging.

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 Δ 9 -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.

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

The solid-state form of an active pharmaceutical ingredient (API) in an oral dosage form plays an important role in determining the dissolution rate of the API. As the solid-state form can change during dissolution, there is a need to monitor the oral dosage form during dissolution testing. Coherent anti-Stokes Raman scattering (CARS) microscopy provides rapid, spectrally selective imaging to monitor the oral dosage form during dissolution. In this study, in situ CARS microscopy was combined with inline UV absorption spectroscopy to monitor the solid-state change in oral dosage forms containing theophylline anhydrate undergoing dissolution and to correlate the solid-state change with a change in dissolution rate. The results from in situ CARS microscopy showed that theophylline anhydrate converted to theophylline monohydrate during dissolution resulting in a reduction in the dissolution rate. The addition of methyl cellulose to the dissolution medium was found to delay the theophylline monohydrate growth and changed the morphology of the monohydrate. The net effect was an increased dissolution rate for theophylline anhydrate. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring oral dosage forms undergoing dissolution and correlating changes in solid-state form with changes in dissolution rate.