Sharon L. Thomsen

PATHOLOGIC ANALYSIS OF PHOTOTHERMAL AND PHOTOMECHANICAL EFFECTS OF LASER–TISSUE INTERACTIONS

Abstract— —Pathologic analysis of the biologic effects and mechanisms of laser–tissue interactions requires correlation of the irradiation parameters with the biologic status and response of the target tissues over time. The photobiologic mechanisms of laser‐induced tissue injury can be separated into three categories, photochemical, photothermal and photomechanical. Anatomic pathologic analysis of laser‐induced lesions reveals alterations that represent either specific markers of the photobiologic mechanism or non‐specific reactions to tissue injury. Repair, regeneration and wound healing of laser induced lesions appear to be non‐specific responses to the type of tissue damage rather than the photobiologic mechanism producing the lesion.

Near-infrared Raman spectroscopy for in vitro detection of cervical precancers

Abstract— In this study, we investigate the potential of near‐infrared Raman spectroscopy to differentiate cervical precancers from normal tissues, inflammation and metaplasia and to differentially diagnose low‐grade and high‐grade precancers. Near infrared Raman spectra were measured from 36 biopsies from 18 patients in vitro. Detection algorithms were developed and evaluated relative to histopathologic examination. Algorithms based on empirically selected peak intensities, ratios of peak intensities and a combination of principal component analysis for data reduction and Fisher discriminant analysis for classification were investigated. Spectral peaks were tentatively identified from measured spectra of potential chromophores. Empirically selected normalized intensities can differentiate precancers from other tissues with an average sensitivity and specificity of 88 ± 4% and 92 ± 4%. Ratios of un‐normalized intensities can differentiate precancers from other tissues with a sensitivity and specificity of 82% and 88% and high‐grade from low‐grade lesions with a sensitivity and specificity of 100%. Using multivariate methods, intensities at eight frequencies can be used to differentiate precancers from all other tissues with a sensitivity and specificity of 82% and 92% in an unbiased test. Raman algorithms can potentially separate benign abnormalities such as inflammation and metaplasia from precancers. Comparison of tissue spectra to published and measured chromophore spectra indicate that the most likely primary contributors to the tissue spectra are collagen, nucleic acids, phospholipids and glucose 1‐phos‐phate. These results suggest that near‐infrared Raman spectroscopy can be used for cervical precancer diagnosis and may be able to accurately separate samples with inflammation and metaplasia from precancer.

Rate Process Analysis of Thermal Damage

Kinetic models of thermal damage in tissues can be used to describe pathologic end points obtained with laser irradiation. Many treatment end-point goals involve relatively low temperature coagulation or desiccation of tissue, and these end points can be conveniently described by rate process models. Thermal damage is exponentially dependent on temperature and linearly dependent on time of exposure. Damage processes can be modeled as first-order rate processes for which two experimentally derived coefficients are sufficient. The rate process models apply well to the prediction of damage thresholds and less well as the damage becomes complete, since several of the fundamental assumptions are violated. In order to be useful in evaluating laser dosimetry, the kinetic model must be coupled to quantitative pathological analysis. This chapter describes quantitative markers of thermal damage and experimental methods for estimating relevant kinetic coefficients in both constant-temperature and transient thermal history experiments. As expected, transient in vivo thermal history data yield a noisy kinetic plot; however, estimates of the appropriate rate coefficients often can be made.

CERVICAL PRECANCER DETECTION USING A MULTIVARIATE STATISTICAL ALGORITHM BASED ON LASER-INDUCED FLUORESCENCE SPECTRA AT MULTIPLE EXCITATION WAVELENGTHS

Abstract— A portable fluorimeter was developed and utilized to acquire fluorescence spectra from 381 cervical sites in 95 patients at 337, 380 and 460 nm excitation immediately prior to colposcopy. A multivariate statistical algorithm was used to extract clinically useful information from tissue spectra acquired in vivo. Two full‐parameter algorithms were developed using tissue fluorescence emission spectra at all three excitation wavelengths (161 excitation‐emission wavelength pairs) for cervical precancer (squamous intraepithelial lesion [SIL]) detection: a screening algorithm that discriminates between SIL and non‐SIL with a sensitivity of 82 ± 1.4% and specificity of 68 ± 0.0%, and a diagnostic algorithm that differentiates high‐grade SIL from non‐high‐grade SIL with a sensitivity and specificity of 79 ± 2% and 78 ± 6%, respectively. Multivariate statistical analysis was also employed to reduce the number of fluorescence excitation‐emission wavelength pairs needed to redevelop algorithms that demonstrate a minimum decrease in classification accuracy. Two reduced‐parameter algorithms that employ fluorescence intensities at only 15 excitation‐emission wavelength pairs were developed: the screening algorithm differentiates SIL from non‐SIL with a sensitivity of 84 ± 1.5% and specificity of 65 ± 2% and the diagnostic algorithm discriminates high‐grade SIL from non‐high‐grade SIL with a sensitivity and specificity of 78 ± 0.7% and 74 ± 2%, respectively. Both the full‐parameter and reduced‐parameter screening algorithms discriminate between SIL and non‐SIL with a similar specificity (±5%) and a substantially improved sensitivity relative to Pap smear screening. A comparison of the full‐parameter and reduced‐parameter diagnostic algorithms to colposcopy in expert hands indicates that all three have a very similar sensitivity and specificity for differentiating high‐grade SIL from non‐high‐grade SIL.

PHOTODYNAMIC THERAPY WITH PHOTOFRIN II INDUCES PROGRAMMED CELL DEATH IN CARCINOMA CELL LINES

Abstract The mode of cell death following photodynamic therapy was investigated from the perspective of programmed cell death or apoptosis. Human prostate carcinoma cells (PC3), human non‐small cell lung carcinoma (H322a) and rat mammary carcinoma (MTF7) were treated by photodynamic therapy. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder indicative of internucleosomal cleavage of DNA during apoptosis. The magnitude of the response and the photodynamic therapy dosage required to induce DNA fragmentation were different in PC3 and MTF7. The MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal response at the LD50 but had a marked response at the LD85. Thus, apoptosis did not ensue as quickly in PC3 as in MTF7. The H322a cells were killed by photodynamic therapy but failed to exhibit any apoptotic response. The results also suggested that apoptosis in these cell lines has a minor requirement for de novo protein synthesis and no requirement for de novo RNA synthesis. This study indicates that although apoptosis can occur during photodynamic therapy‐induced cell death, this response is not universal for all cancer cell lines.

Anisotropy in the absorption and scattering spectra of chicken breast tissue.

Oblique incidence reflectometry is a simple and accurate method for measuring the absorption and the reduced-scattering coefficients of turbid media. We used this technique to deduce absorption and reduced-scattering spectra from wavelength-resolved measurements of the relative diffuse reflectance profile of white light as a function of source-detector distance. In this study, we measured the absorption and the reduced-scattering coefficients of chicken breast tissue in the visible range (400-800 nm) with the oblique incidence probe oriented at 0 degrees and 90 degrees relative to the muscle fibers. We found that the deduced optical properties varied with the probe orientation. Measurements on homogenized chicken breast tissue yielded an absorption spectrum comparable with the average of the absorption spectra for 0 degrees and 90 degrees probe orientations measured on the unhomogenized tissue. The reduced-scattering spectrum for homogeneous tissue was greater than that acquired for unhomogenized tissue taken at either probe orientation. This experiment demonstrated the application of oblique-incidence, fiber-optic reflectometry to measurements on biological tissues and the effect of tissue structural anisotropy on optical properties.

Spectroscopic diagnosis of cervical intraepithelial neoplasia (CIN) in vivo using laser-induced fluorescence spectra at multiple excitation wavelengths

The diagnostic contribution of cervical tissue fluorescence spectra acquired in vivo at 380 and 460 nm excitation were analyzed using a general multivariate statistical algorithm.

Development of a multivariate statistical algorithm to analyze human cervical tissue fluorescence spectra acquired in vivo

A general multivariate statistical algorithm has been developed to analyze the diagnostic content of cervical tissue fluorescence spectra acquired in vivo.

Changes in birefringence as markers of thermal damage in tissues

Light microscopy using polarized transmission illumination or routinely stained histologic sections shows changes of the native birefringence of certain tissues constituents when they are heated by laser irradiation or electrosurgical current. The naturally occurring birefringence of cardiac muscle disappears permanently when the muscle is frozen, thawed, and heated to temperatures in excess of 42 degrees C in vitro. Partial loss of the native birefringence of collagen occurs in canine urinary bladder coagulated by laser irradiation and pericardium heated with electrodes. In addition, thermally coagulated collagens have variable birefringence color shifts when compared to the adjacent unaffected collagens in stained histologic sections.<<ETX>>

Primate retina imaging with polarization-sensitive optical coherence tomography

Polarization-sensitive optical coherence tomography (PSOCT) is applied to determine the depth-resolved polarization state of light backreflected from the eye. The birefringence of the retinal nerve fiber layer (RNFL) was observed and measured from PSOCT images recorded postmortem in a Rhesus monkey. An image-processing algorithm was developed to identify birefringent regions in acquired PSOCT retinal images and automatically determine the thickness of the RNFL. Values of the RNFL thickness determined from histology and PSOCT were compared. PSOCT may provide a new method to determine RNFL thickness and birefringence for glaucoma diagnostics.