(A)Considerable emphasis was placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan and continuous scanning interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array and linear array detectors. The integration of high performance digital imaging with noninvasive, high resolution infrared spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. As one example of the utility of the infrared imaging technique in diagnostic pathology, we applied this approach to both supervised and unsupervised (observer independent) prostate histopathology involving large numbers of tissue samples examined in the form of tissue microarrays. This infrared spectroscopic method eliminates the necessity for chemically stained tissue. Further, the high throughput approach inherent in the use of tissue microarrays allows the efficient and effective acquisition of a samples vibrational spectral signature for pathologic analyses of biopsied specimens representative of controls, prostatic intraepithelial neoplasia, benign prostatic hyperplasia and adenocarcinoma. In continuing our research along these lines, we demonstrate the application of automated histologic segementation for a series of archival tissue samples; well-defined tests of statistical significance were incorporated. This approach demonstrates that histopathologic changes can now be defined by biochemistry-based, objective spectroscopic criteria that do not necessarily require a pathologist's intervention or interpretation. In these examples, our imaging instrumentation incorporated highly sensitive linear array and focal plane array detection for rapidly recording hypercube spectral data. For spectroscopically elucidating the various histologic features present in prostate tissue, large spectral training sets and appropriate spectroscopic metrics were developed for distinguishing ten morphological entities occurring in this specific tissue. Both the use of the Maximum Gaussian Likelihood Method and, separately, a probabilistic classification model based on Beyesian statistics, allowed an objective, automated delineation of the ten histologic categories to be correct to the order of 95-99%. Fine tuning of the tissue segmentation process was developed. Additionally, receiver operating characteristic curves were used to explore relationships between sensitivity and specificity of the high throughput, spectroscopic delineations for distinguishing adenocarcinoma. These procedures are entirely compatible with current tissue processing procedures. With regard to our general infrared imaging instrumentation, a number of enhancing features were continually made in the optics, in detector configurations, and in data collection paradigms. ? (B)Vibrational infrared spectroscopic imaging techniques involving cellular acetylation studies were conducted. Acetylation and deacetylation of nucleosome core histones is important for modulating gene expression. Increased protein acetylation leads to molecular and biological changes that affect the transcriptional activation of certain genes. Histone deacetylase inhibitors (HDACi) are emerging as a promising new class of anticancer drugs. The anticancer activity of HDACi is ascribed to hyperacetylation of both core histones and to a variety of non-histone proteins critical in maintenance of the malignant phenotype. We developed a spectroscopic imaging technique for assessing HDAC inhibitor activity. The method applies vibrational infrared spectroscopy and multivariate data analysis to examine spectral changes that are indicative of specific biochemical alterations associated with acetylation. Peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated with HDAC inhibitor SNDX-275, and spectral changes in the 2900 cm-1 methyl and methylene carbon-hydrogen stretching mode regions and in the 1650-1500 cm-1 amide I and II banding mode regions were assessed. Concentration-dependent increases in protein acetylation were detected and quantified in PBMCs exposed to HDACi SNDX-275 in vitro and in PBMCs from patients treated in vivo. The data demonstrate a new approach to the assessment of global acetylation that is sensitive and totally independent of antibodies, requires minimum cell processing, and is easily adapted to high thoughput screening. Peripheral blood cells were obtained also from patients enrolled in the NCI phase I study involving SNDX-275. The performance of our infrared spectroscopic method in monitoring drug effects in patients was very positive in detecting dosage-dependent protein acetylation in vivo.? ? (C) With respect to our visible reflectance imaging studies, a straightforward algorithm was developed for real time monitoring of oxygenation levels in blood cells and tissue based on the visible spectrum of hemoglobin. Absorbance images obtained from the visible reflection of white light through separate red and blue bandpass filters recorded by monochrome CCDs are combined to create enhanced images that suggest a quantitative correlation to the degree of oxygenated and deoxygenated hemoglobin in red blood cells. The filter bandpass regions are chosen specifically to mimic the color response of commercial 3-CCD cameras, representative of detectors with which the operating room laparoscopic tower systems are equipped. Adaptation of this filter approach is demonstrated for laparoscopic donor nephrectomies in which images are analyzed in terms of real time, in vivo monitoring of tissue oxygenation. ? (D) We developed our visible reflectance imaging methods for non- invasive monitoring of tissue oxygenation during nephrectomies. Standard methods for the assessment of organ viability during surgery are typically limited to visual cues and tactile feedback in open surgery. However, during laparoscopic surgery, these processes are impaired. This is of particular relevance during laparoscopic renal donation, where the condition of the kidney must be optimized despite considerable manipulation. However, there is no in vivo methodology to monitor renal parenchymal oxygenation during laparoscopic surgery. We have developed a method for the real time, in vivo, whole organ assessment of tissue oxygenation during laparoscopic nephrectomy to convey meaningful biological data to the surgeon during laparoscopic surgery. We apply the 3-CCD (charge coupled device) camera to monitor qualitatively renal parenchymal oxygenation with potential real-time video capability. We have validated this methodology in a porcine model across a range of hypoxic conditions and have then applied the method during clinical laparoscopic donor nephrectomies during clinically relevant pneumoperitoneum. 3-CCD image enhancement produces mean region of interest intensity values that can be directly correlated with blood oxygen saturation measurements (R2 > 0.96). Using the 3-CCD camera to qualitatively monitor tissue oxygenation provides a means of assessing intraoperative tissue oxygenation. This may be a useful method to avoid unintended ischemic injury during laparoscopic surgery. Preliminary results indicate that no significant changes in renal oxygenation occur as a result of pneumoperitoneum.? ? (E) An algorithm was developed for contrast enhancement for in vivo visible reflectance imaging of tissue oxygenation. An adaptation of a filter approach was demonstrated for use of existing 3-CCD cameras utilized in laparoscopic donor nephrectomies in which images are analyzed in terms of real time, in vivo monitoring of tissue oxygenation, an invaluable tool for use by surgeons.
| Crane, Nicole J; Schultz, Zachary D; Levin, Ira W (2007) Contrast enhancement for in vivo visible reflectance imaging of tissue oxygenation. Appl Spectrosc 61:797-803 |
| Bhargava, Rohit; Fernandez, Daniel C; Hewitt, Stephen M et al. (2006) High throughput assessment of cells and tissues: Bayesian classification of spectral metrics from infrared vibrational spectroscopic imaging data. Biochim Biophys Acta 1758:830-45 |
| Liang, Christine; Morris, Andrea; Schlucker, Sebastian et al. (2006) Structural and molecular hair abnormalities in trichothiodystrophy. J Invest Dermatol 126:2210-6 |
| Schlucker, S; Liang, C; Strehle, K R et al. (2006) Conformational differences in protein disulfide linkages between normal hair and hair from subjects with trichothiodystrophy: a quantitative analysis by Raman microspectroscopy. Biopolymers 82:615-22 |
