Bruce J. Tromberg

We have developed a novel 3D visualization and guidance system for handheld optical imaging devices. In this paper, the system is applied to measurements of breast/cancerous tissue optical properties using a handheld diffuse optical spectroscopy (DOS) instrument. The combined guidance system/DOS instrument becomes particularly useful for monitoring neoadjuvant chemotherapy in breast cancer patients and for longitudinal studies where measurement reproducibility is critical. The system uses relatively inexpensive hardware components and comprises a 6 degrees-of-freedom (DOF) magnetic tracking device including a DC field generator, three sensors, and a PCI card running on a PC workstation. A custom-built virtual environment combined with a well-defined workflow provide the means for image-guided measurements, improved longitudinal studies of breast optical properties, 3D reconstruction of optical properties within the anatomical map, and serial data registration. The DOS instrument characterizes tissue function such as water, lipid and total hemoglobin concentration. The patient lies on her back at a 45-degrees angle. Each spectral measurement requires consistent contact with the skin, and lasts about 5-10 seconds. Therefore a limited number of positions may be studied. In a reference measurement session, the physician acquires surface points on the breast. A Delaunay-based triangulation algorithm is used to build the virtual breast surface from the acquired points. 3D locations of all DOS measurements are recorded. All subsequently acquired surfaces are automatically registered to the reference surface, thus allowing measurement reproducibility through image guidance using the reference measurements.

Diffuse optical spectroscopy (DOS) of breast tissue provides quantitative, functional information based on optical absorption and scattering properties that cannot be obtained with other radiographic methods. DOS‐measured absorption spectra are used to determine the tissue concentrations of deoxyhemoglobin (Hb‐R), oxyhemoglobin (Hb‐O₂), lipid, and water (H₂O), as well as to provide an index of tissue hemoglobin oxygen saturation (S_tO₂). Tissue‐scattering spectra provide insight into epithelial, collagen, and lipid contributions to breast density. Clinical studies of women with malignant tumors show that DOS is sensitive to processes such as increased tissue vascularization, hypoxia, and edema. In studies of healthy women, DOS detects variations in breast physiology associated with menopausal status, menstrual cycle changes, and hormone replacement. Current research involves using DOS to monitor tumor response to therapy and the co‐registration of DOS with magnetic resonance imaging. By correlating DOS‐derived parameters with lesion pathology and specific molecular markers, we anticipate that composite “tissue optical indices” can be developed that non‐invasively characterize both tumor and normal breast‐tissue function.

Multiphoton microscopy relies on nonlinear light–matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue “intravital” imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730–880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (in vivo characterization of cell–ECM interactions in unstained thick tissues.

NIR tissue spectroscopy was used to monitor a breast cancer patient during three cycles of presurgical chemotherapy. Lesion values of lipid, water, deoxygenated and oxygenated hemoglobin changed significantly within the first week, and continued to change throughout therapy.

A frequency-domain photon migration (FDPM) technique is developed for quantitative measurement of the absorption and reduced scattering coefficients of highly turbid samples in a small-volume (0.45-ml) reflective cuvette. We present both an analytical model for the FDPM cuvette and its experimental verification, using calibrated phantoms and suspensions of living cells. FDPM model fits to experimental data demonstrate that the reduced scattering (μ_s′) and absorption (μ_a) coefficients can be derived with accuracies of 5–10% and 10–15%, respectively. Changing the cuvette wall reflectivity alters the frequency-dependent behavior of photon density waves (PDWs). For highly reflective wall boundaries (R _eff ≥ 90–95%), PDW confinement leads to substantial enhancement in both amplitude and phase compared with identical samples in infinite media. Results from experiments on microsphere suspensions are compared with predictions from Mie theory to assess the potential of this method to interpret scattering properties in terms of scatterer size and density. Optical property measurements of biological cell suspensions are reported, and the possibility of optically monitoring cell physiology in a carefully controlled environment is demonstrated.

Near infrared diffuse optical spectroscopy and diffuse optical imaging are promising methods that eventually may enhance or replace existing technologies for breast cancer screening and diagnosis. These techniques are based on highly sensitive, quantitative measurements of optical and functional contrast between healthy and diseased tissue. In this study, we examine whether changes in breast physiology caused by exogenous hormones, aging, and fluctuations during the menstrual cycle result in significant alterations in breast tissue optical contrast. A noninvasive quantitative diffuse optical spectroscopy technique, frequency-domain photon migration, was used. Measurements were performed on 14 volunteer subjects by using a hand-held probe. Intrinsic tissue absorption and reduced scattering parameters were calculated from frequency-domain photon migration data. Wavelength-dependent absorption (at 674, 803, 849, and 956 nm) was used to determine tissue concentration of oxyhemoglobin, deoxyhemoglobin, total hemoglobin, tissue hemoglobin oxygen saturation, and bulk water content. Results show significant and dramatic differences in optical properties between menopausal states. Average premenopausal intrinsic tissue absorption and reduced scattering values at each wavelength are 2.5- to 3-fold higher and 16–28% greater, respectively, than absorption and scattering for postmenopausal subjects. Absorption and scattering properties for women using hormone replacement therapy are intermediate between premenopausal and postmenopausal populations. Physiological properties show differences in mean total hemoglobin (7.0 μM, 11.8 μM, and 19.2 μM) and water concentration relative to pure water (10.9%, 15.3%, and 27.3%) for postmenopausal, hormone replacement therapy, and premenopausal subjects, respectively. Because of their unique, quantitative information content, diffuse optical methods may play an important role in breast diagnostics and improving our understanding of breast disease.

Benzoporphyrin-derivative (BPD)–monoacid-ring A photodynamic therapy (PDT) was performed on subcutaneous tumor implants in a rat ovarian cancer model. In order to assess PDT efficacy the tumor and normal tissue optical properties were measured noninvasively prior to and during PDT using frequency-domain photon migration (FDPM). FDPM data were used to quantify tissue absorption and reduced scattering properties (given by the parameters μ_a and μ′_s, respectively) at four near-infrared (NIR) wavelengths (674, 811, 849 and 956 nm). Tissue physiologic properties, including the in vivo concentration of BPD, deoxy-hemoglobin (Hb), oxy-hemoglobin (HbO₂), total hemoglobin (TotHb), water (H₂O) and percent tissue hemoglobin oxygen saturation (%S_tO₂), were calculated from optical property data. PDT efficacy was also determined from morphometric analysis of tumor necrosis in histologic specimens. All the measured tumor properties changed significantly during PDT. [Hb] increased by 9%, while [HbO₂], [TotHb] and %S_tO₂ decreased by 18, 7 and 12%, respectively. Using histologic data we show that long-term PDT efficacy is highly correlated to mean BPD concentration in tumor and PDT-induced acute changes in [HbO₂], [TotHb] and %S_tO₂ (correlation coefficients of 0.829, 0.817 and 0.953, respectively). Overall, our results indicate that NIR FDPM spectroscopy is able to quantify noninvasively and dynamically the PDT-induced physiological effects in vivo that are highly correlated with therapeutic efficacy.