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 Zr-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. Ó2013 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.

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.