Label-free photoacoustic nanoscopy.

Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

Noise-equivalent sensitivity of photoacoustics.

The fundamental limitations of photoacoustic microscopy for detecting optically absorbing molecules are investigated both theoretically and experimentally. We experimentally demonstrate noise-equivalent detection sensitivities of 160,000 methylene blue molecules (270 zeptomol or 2.7×10-19 mol) and 86,000 oxygenated hemoglobin molecules (140 zeptomol) using narrowband continuous-wave photoacoustics. The ultimate sensitivity of photoacoustics is fundamentally limited by thermal noise, which can present in the acoustic detection system as well as in the medium itself. Under the optimized conditions described herein and using commercially available detectors, photoacoustic microscopy can detect as few as 100s of oxygenated hemoglobin molecules. Realizable improvements to the detector may enable single molecule detection of select molecules.

Optical polarimetry for noninvasive glucose sensing enabled by Sagnac interferometry.

Optical polarimetry is used in pharmaceutical drug testing and quality control for saccharide-containing products (juice, honey). More recently, it has been proposed as a method for noninvasive glucose sensing for diabetic patients. Sagnac interferometry is commonly used in optical gyroscopes, measuring minute Doppler shifts resulting from mechanical rotation. In this work, we demonstrate that Sagnac interferometers are also sensitive to optical rotation, or the rotation of linearly polarized light, and are therefore useful in optical polarimetry. Results from simulation and experiment show that Sagnac interferometers are advantageous in optical polarimetry as they are insensitive to net linear birefringence and alignment of polarization components.

In vivo, dual-modality OCT/LIF imaging using a novel VEGF receptor-targeted NIR fluorescent probe in the AOM-treated mouse model.

PURPOSE: Increased vascular endothelial growth factor (VEGF) receptor expression has been found at the sites of angiogenesis, particularly in tumor growth areas, as compared with quiescent vasculature. An increase in VEGF receptor-2 is associated with colon cancer progression. The in vivo detection of VEGF receptor is of interest for the purposes of studying basic mechanisms of carcinogenesis, making clinical diagnoses, and monitoring the efficacy of chemopreventive and therapeutic agents. In this study, a novel single chain (sc)VEGF-based molecular probe is utilized in the azoxymethane (AOM)-treated mouse model of colorectal cancer to study delivery route and specificity for disease. PROCEDURES: The probe was constructed by site-specific conjugation of a near-infrared fluorescent dye, Cy5.5, to scVEGF and detected in vivo with a dual-modality optical coherence tomography/laser-induced fluorescence (OCT/LIF) endoscopic system. A probe inactivated via excessive biotinylation was utilized as a control for nonreceptor-mediated binding. The LIF excitation source was a 633-nm He:Ne laser, and red/near-infrared fluorescence was detected with a spectrometer. OCT was used to obtain two-dimensional longitudinal tomograms at eight rotations in the distal colon. Fluorescence emission levels were correlated with OCT-detected disease in vivo. OCT-detected disease was verified with hematoxylin and eosin stained histology slides ex vivo. RESULTS: High fluorescence emission intensity from the targeted probe was correlated with tumor presence as detected using OCT in vivo and VEGFR-2 immunostaining on histological sections ex vivo. The inactivated probe accumulated preferentially on the surface of tumor lesions and in lymphoid aggregate tissue and was less selective for VEGFR-2. CONCLUSION: The scVEGF/Cy probe delivered via colonic lavage reaches tumor vasculature and selectively accumulates in VEGFR-2-positive areas, resulting in high sensitivity and specificity for tumor detection. The combination of OCT and LIF imaging modalities may allow the simultaneous study of tumor morphology and protein expression for the development of diagnostic and therapeutic methods for colorectal cancer.

Quantitative tool for rapid disease mapping using optical coherence tomography images of azoxymethane-treated mouse colon.

Optical coherence tomography (OCT) can provide new insight into disease progression and therapy by enabling nondestructive, serial imaging of in vivo cancer models. In previous studies, we have shown the utility of endoscopic OCT for identifying adenomas in the azoxymethane-treated mouse model of colorectal cancer and tracking disease progression over time. Because of improved imaging speed made possible through Fourier domain imaging, three-dimensional imaging of the entire mouse colon is possible. Increased amounts of data can facilitate more accurate classification of tissue but require more time on the part of the researcher to sift through and identify relevant data. We present quantitative software for automatically identifying potentially diseased areas that can be used to create a two-dimensional "disease map" from a three-dimensional Fourier domain OCT data set. In addition to sensing inherent changes in tissue that occur during disease development, the algorithm is sensitive to exogeneous highly scattering gold nanoshells that can be targeted to disease biomarkers. The results of the algorithm were compared to histological diagnosis. The algorithm was then used to assess the ability of gold nanoshells targeted to epidermal growth factor receptor in vivo to enable functional OCT imaging.

Laparoscopic optical coherence tomography imaging of human ovarian cancer.

OBJECTIVES: Ovarian cancer is the fourth leading cause of cancer-related death among women in the US largely due to late detection secondary to unreliable symptomology and screening tools without adequate resolution. Optical coherence tomography (OCT) is a recently emerging imaging modality with promise in ovarian cancer diagnostics, providing non-destructive subsurface imaging at imaging depths up to 2 mm with near-histological grade resolution (10-20 microm). In this study, we developed the first ever laparoscopic OCT (LOCT) device, evaluated the safety and feasibility of LOCT, and characterized the microstructural features of human ovaries in vivo. METHODS: A custom LOCT device was fabricated specifically for laparoscopic imaging of the ovaries in patients undergoing oophorectomy. OCT images were compared with histopathology to identify preliminary architectural imaging features of normal and pathologic ovarian tissue. RESULTS: Thirty ovaries in 17 primarily peri- or post-menopausal women were successfully imaged with LOCT: 16 normal, 5 endometriosis, 3 serous cystadenoma, and 4 adenocarcinoma. Preliminary imaging features developed for each category reveal qualitative differences in the homogeneous character of normal post-menopausal ovary, the ability to image small subsurface inclusion cysts, and distinguishable features for endometriosis, cystadenoma, and adenocarcinoma. CONCLUSIONS: We present the development and successful implementation of the first laparoscopic OCT probe. Comparison of OCT images and corresponding histopathology allowed for the description of preliminary microstructural features for normal ovary, endometriosis, and benign and malignant surface epithelial neoplasms. These results support the potential of OCT both as a diagnostic tool and an imaging modality for further evaluation of ovarian cancer pathogenesis.

Inherent homogenous media dispersion compensation in frequency domain optical coherence tomography by accurate k-sampling.

Depth dependent broadening of the axial point spread function due to dispersion in the imaged media, and algorithms for postprocess correction, have been previously described for both time domain and frequency domain optical coherence tomography. We show that homogeneous media dispersion artifacts disappear when frequency domain samples are acquired with uniform spacing in circular wavenumber, as opposed to uniform sampling in optical frequency. We further explicate the source of this point spread broadening and simulate its magnitude in aqueous media. We experimentally demonstrate media dispersion compensation in high dispersion glass by choosing sample frequencies at equal intervals of media index of refraction divided by vacuum wavelength, and we recover unbroadened reflections without an additional postprocessing step.