Development of in vivo Raman spectroscopy for the diagnosis of breast cancer and intra-operative margin assessment

Thesis (Ph. D.)--Harvard University--MIT Division of Health Sciences and Technology, 2005.Includes bibliographical references.Breast cancer is the most commonly diagnosed cancer among women in the United States. It is the most common cause of death in women ages 45-55. Optical techniques can potentially play a diagnostic role in several aspects of breast cancer evaluation and treatment. This thesis outlines progress on the use of Raman spectroscopy to diagnose breast cancer. Laboratory studies on fresh-frozen tissues are used to demonstrate that the detailed information provided by Raman spectroscopy yields accurate breast disease diagnosis. A Raman spectroscopic-based diagnostic algorithm was developed which classifies samples into four categories according to specific pathological diagnoses: normal, fibrocystic change, fibroadenoma, and infiltrating carcinoma. Cancerous lesions were separated from non- cancerous tissues with a sensitivity of 94% and a specificity of 95%. Further, use of a spectral model based on the morphological structures that comprise breast tissue allows increased understanding of the relationship between a Raman spectrum and tissue disease state. Based on the excellent results of our laboratory work, two clinical studies were undertaken. These studies translate Raman spectroscopy from a laboratory technique into a clinically useful tool. The first study tests the diagnostic algorithm in a prospective manner on freshly excised tissue. Preliminary results are promising. The second study is the first demonstration of in vivo data acquisition of Raman spectra of breast tissue. The culmination of this research is the demonstration of accurate intra-operative margin status assessment during partial mastectomy surgeries.(cont.) Application of our previously developed diagnostic algorithm resulted in perfect sensitivity and specificity in this small in vivo data set. These preliminary findings indicate that Raman spectroscopy has the potential to lessen the need for re-excision surgeries resulting from positive margins and thereby reduce the recurrence rate of breast cancer following partial mastectomy surgeries. The experiments and theory presented throughout this thesis demonstrate that Raman spectroscopy is a viable clinical tool that can be used to accurately diagnosis breast cancer and breast disease.by Abigail Susan Haka.Ph.D

Quantitative Analysis of Monocyte Subpopulations in Murine Atherosclerotic Plaques by Multiphoton Microscopy

<div><p>The progressive accumulation of monocyte-derived cells in the atherosclerotic plaque is a hallmark of atherosclerosis. However, it is now appreciated that monocytes represent a heterogeneous circulating population of cells that differ in functionality. New approaches are needed to investigate the role of monocyte subpopulations in atherosclerosis since a detailed understanding of their differential mobilization, recruitment, survival and emigration during atherogenesis is of particular importance for development of successful therapeutic strategies. We present a novel methodology for the <em>in vivo</em> examination of monocyte subpopulations in mouse models of atherosclerosis. This approach combines cellular labeling by fluorescent beads with multiphoton microscopy to visualize and monitor monocyte subpopulations in living animals. First, we show that multiphoton microscopy is an accurate and timesaving technique to analyze monocyte subpopulation trafficking and localization in plaques in excised tissues. Next, we demonstrate that multiphoton microscopy can be used to monitor monocyte subpopulation trafficking in atherosclerotic plaques in living animals. This novel methodology should have broad applications and facilitate new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.</p> </div

Determination of uncertainty in parameters extracted from single spectroscopic measurements

The ability to quantify uncertainty in information extracted from spectroscopic measurements is important in numerous fields. The traditional approach of repetitive measurements may be impractical or impossible in some measurements scenarios, while chi-squared analysis does not provide insight into the sources of uncertainty. As such, a need exists for analytical expressions for estimating uncertainty and, by extension, minimum detectable concentrations or diagnostic parameters, that can be applied to a single noisy measurement. This work builds on established concepts from estimation theory, such as the Cramér-Rao lower bound on estimator covariance, to present an analytical formula for estimating uncertainty expressed as a simple function of measurement noise, signal strength, and spectral overlap. This formalism can be used to evaluate and improve instrument performance, particularly important for rapid-acquisition biomedical spectroscopy systems. We demonstrate the experimental utility of this expression in assessing concentration uncertainties from spectral measurements of aqueous solutions and diagnostic parameter uncertainties extracted from spectral measurements of human artery tissue. The measured uncertainty, calculated from many independent measurements, is found to be in good agreement with the analytical formula applied to a single spectrum. These results are intended to encourage the widespread use of uncertainty analysis in the biomedical optics community.National Institutes of Health (U.S.) (Grant P41-RR-02594

Intravital multiphoton microscopy allows observation of bead-labeled monocytes within murine atherosclerotic plaques.

<p><b>A)</b> A white light image of an atherosclerotic plaque in the abdominal aorta of an ApoE^−/− mouse. <b>B)</b> A multiphoton image of the same region with excitation at 800 nm, collected from 490 to 530 nm. Dashed lines in each image demarcate the aorta. A hair which lies to the right of the atherosclerotic plaque (indicated by arrows in both A and B) is useful for orientation. <b>C)</b> Intravital images of bead-positive monocytes (green) as a function of time. Bead-positive monocytes that are circulating through the aorta may appear as lines in the image when their flow is synchronized with the raster scan of the microscope (examples are indicated by arrowheads). A stationary bead-positive monocyte was seen interacting with the endothelium of the atherosclerotic plaque (indicated by arrows). <b>D)</b> An intravital image demonstrating bead-positive monocyte-derived cell accumulation in an atherosclerotic plaque in the abdominal aorta of an ApoE^−/− mouse. <b>E and F)</b> Intravital images (excitation at 850 nm) of bead-positive monocyte-derived cells (green) interacting with fluorescently labeled low density lipoprotein (red) in an atherosclerotic plaque.</p

Verification of monocyte subpopulation bead-labeling. A

<p>–<b>C)</b> Flow cytometry gating strategy used to calculate the monocyte subpopulation bead-labeling efficiency in the blood. After gating on live cells (A), monocytes are detected by their high expression of CD115 (B). In this representative plot, 12.7% of leukocytes are monocytes. Monocytes can then be separated in 2 major populations based on their expression of Gr1 (C). In this example, 5.8% of total monocytes are bead-positive Gr1^lo. Specific labeling of monocytes can also be visualized in plaques via sectioning of the tissue and immunofluorescence. <b>D)</b> A representative image of an atherosclerotic plaque after sectioning of the aortic sinus acquired 5 days following monocyte subpopulation labeling. Bead-positive cells are indicated by arrows. <b>E)</b> Beads can be seen to associate with CD68+ macrophages.</p

Real-time Raman system for in vivo disease diagnosis

Raman spectroscopy has been well established as a powerful in vitro method for studying biological tissue and diagnosing disease. The recent development of efficient, high-throughput, low-background optical fiber Raman probes provides, for the first time, the opportunity to obtain real-time performance in the clinic. We present an instrument for in vivo tissue analysis which is capable of collecting and processing Raman spectra in less than 2 s. This is the first demonstration that data acquisition, analysis, and diagnostics can be performed in clinically relevant times. The instrument is designed to work with the new Raman probes and includes custom written LabVIEW and Matlab programs to provide accurate spectral calibration, analysis, and diagnosis along with important safety features related to laser exposure. The real-time capabilities of the system were demonstrated in vivo during femoral bypass and breast lumpectomy surgeries. Such a system will greatly facilitate the adoption of Raman spectroscopy into clinical research and practice.National Institutes of Health (U.S.) (Grant R01-HL-64675)National Center for Research Resources (U.S.) (Grant P41-RR-02594)Pfizer Inc.Cameron and Hayden Lord Foundatio

Quantification of bead-labeled monocyte-derived cells in atherosclerotic plaques. A)

<p>A sum projection of a z-stack collected from the lesser curvature of a murine aorta 1 day following subpopulation labeling. Examples of bead positive monocyte-derived cells are indicated by arrows. Automated bead counting was performed on a sum projection image generated from each z-stack in the composite multiphoton images. <b>B)</b> The result image shows segmentation of the beads identified by the algorithm (green circles). <b>C)</b> The number of bead-positive cells in each plaque at day 1 and day 5 following monocyte subpopulation labeling as determined by multiphoton microscopy. Five mice were examined per condition. Error bars ±SEM, p value = 0.02. <b>D)</b> The number of bead-positive cells in the plaques of ApoE^−/− mice treated with simvastatin or vehicle. Results show that simvastatin reduces non-classical monocyte recruitment to the plaque. Three mice were examined per condition. Error bars ±SEM, p value = 0.04.</p

Multiphoton imaging of the brachiocephalic artery facilitates localization of bead-labeled cells to distinct plaque regions.

<p>Images were collected through the entire brachiocephalic artery of an ApoE^−/− mouse at depths of up to 200 µm. <b>A)</b> A series of xy planes acquired at different depths. The tunica media is shown in red. <b>B)</b> Enlarged views at different depths, highlighted by boxes in A. At a depth of 30 µm below the vessel surface, elastin fibers, presumably comprising the internal elastic lamina, are indicated by arrows. Images at greater depths are acquired from the atherosclerotic plaque, and bead-labeled monocyte-derived cells can be seen. <b>C)</b> A three-dimensional reconstruction of the brachiocephalic artery. The artery was mounted on a glass slide with a coverslip and thus appears compressed. Significant axial distortions, consistent with the optical inhomogeneity of tissue and the instrumentation employed, can be seen. To correct for the axial broadening of fluorescence in our data, beads were identified using the automated algorithm, and their signal was replaced by a bead image at the point of brightest fluorescence along the axial vector. <b>D)</b> A three dimensional reconstruction of the brachiocephalic artery in which beads identified in this manner replace the signal collected from 490 to 530 nm and the red channel is convolved with a 7×7-pixel Gaussian filter. Bead-positive cells can be seen in the plaque (white arrows), fibrous cap (white arrow heads) and shoulder (red arrow head) of the atherosclerotic plaque. Bead-positive cells can also be seen outside of the plaque (red arrows). Red (380 to 440 nm), green (490 to 530 nm).</p

Visualization of plaque morphology and labeled monocytes.

<p><b>A and G)</b> Second harmonic scattering from collagen in an ApoE^−/− control mouse treated with vehicle. <b>B)</b> Elastin and collagen autofluorescence in an ApoE^−/− control mouse. Several bead-positive monocytes, indicated by arrows, can be seen in regions containing collagen and elastin. <b>C)</b> Overlay. <b>D and J)</b> Second-harmonic scattering from collagen in an ApoE^−/− mouse treated with simvastatin. <b>E)</b> Elastin and collagen autofluorescence in an ApoE^−/− mouse treated with simvastatin. Several bead-positive monocytes, indicated by arrows, can be seen in regions containing collagen and elastin. <b>F)</b> Overlay. <b>H)</b> Neutral lipids, visualized as ORO fluorescence, in an ApoE^−/− control mouse. <b>K)</b> Neutral lipids, visualized as ORO fluorescence, in an ApoE^−/− mouse treated with simvastatin. Bead-positive monocyte-derived cells, indicated by arrows, are present in areas of lipid accumulation. Red (380 to 440 nm), green (490 to 530 nm) and blue (530 to 650 nm).</p