Light scattering spectroscopy for pre-cancer diagnosis

Light scattering spectroscopy (LSS) is a promising non-invasive technique for studying epithelial tissue properties with sub-micron resolution. The technique is sensitive to refractive index variations and can therefore be used to extract optical properties, and particle size distributions, in cellular and sub-cellular matrices. An important area of research at the Spectroscopy Laboratory is applying LSS for pre-cancer (dysplasia) diagnosis. LSS is well-suited to this task, given that the majority of human cancers are epithelial in origin [1], and that pre-cancerous progression is often correlated to significant changes in cellular morphology (e.g., nuclear enlargement and polymorphism) [2-6] .

Here, we present an animal cancer study utilizing the LSS technique, to determine its potential as a pre-cancer diagnosis method. The work relies on a well-established rat esophagus cancer model, using carcinogen-treated Fisher 344 rats [7-9]. The rats were sacrificed 20 weeks after being dosed with the caricnogen nitrosomethylbenzylamine (NMBA), and their esophagi extracted and studied by LSS within 1 hour after the rats’ deaths. Two locations (10mm2 each) were studied for each rat tissue, and there were 5 rats sacrificed for each of the following study groups: (i) Normal; (ii) curcumin-treated; (iii) NMBA-treated, and; (iv) NMBA- and curcumin-treated rats. Curcumin was included in this study as a possible chemopreventive agent. Immediately after analysis by LSS, the rat tissues were fixed and stained for histopathological analysis (Figure 1).

Figure1. Rat histology, at 20x.magnification. (a) Normal tissue; (b) moderate dsyplasia; (c) severe dysplasia. B-basal cell layer, K-keratin layer. All of the images are on the same scale. The scale bar is 100 um.

Our LSS technique is based on illuminating a tissue sample with a polarized, collimated beam of white light from a Xe arc lamp source (Figure 2). The backscattered light from the tissue is collected over a range of polar backscattering angles ?=0-5o, and dispersed in a CCD imaging spectrograph for spectral analysis in the range ?=450-750nm [6]. Two polarization components of the backscattered light, parallel and perpendicular to the incident beam polarization, are collected for discrimination against the large diffuse scattering signal inherent in turbid biological tissue samples. The polarized residual signal has been shown to provide scattering information from the topmost layer of tissue (optical depth <2), and thereby provides scattering properties specific to the tissue epithelium.

Figure 2. Schematic of the LSS instrument. S - source of unpolarized white light, L1, L2 – lenses, P1, P2 - polarizers, M - mirror, BS - beam splitter, SPEC - spectrograph. The CCD records the scattered intensity distribution with respect to scattering angle and wavelength.

The LSS spectra shown on Figure 3(a) are for exact backscattering (?=0o), and are representative of LSS spectra obtained at all scattering angles. Spectra for individual LSS polarizations were normalized relative to a diffuse reflectance standard spectrum, as in [5,6]. The normalized perpendicular LSS spectra were linearly weighted and subtracted from the parallel component, in such a manner as to eliminate the absorptive contribution of hemoglobin to the LSS spectra, and thereby minimize the diffusely backscattered LSS component. All residual spectra thus obtained were systematically well fit by an inverse power law, I(?)??-? (Figure 2(b)).

Figure 3. (a) Parallel and perpendicular polarization signals normalized to diffuse reflectance standard. (b) Polarized normalized residual, with perpendicular component weighted (a = constant) to remove hemoglobin absorption band.

Good correlation was found between the histopathology of the excised rat esophagi and the exponent of the inverse power law fit in their LSS residual spectra, ? (Figure 4). Rat esophagus tissue labeled as either moderately or severely dysplastic showed systematically lower values of ? than normal tissue. This result is significant as a potential marker of pre-cancerous evolution in epithelial tissue, and shows that LSS can be pursued as a quantitative and non-invasive approach to cancer diagnosis.

Figure 4. Exponent vs. rat group. The symbols represent histopathological diagnosis.

Work is also underway to explore the biophysical significance of the optical parameter, gamma. Mie theory simulations indicate that our inverse power law LSS spectra can result from an inverse power law in scattering particle size distribution, N(d) is proportional to d^B?, with particle diameters in the range 25nm < d < 1um [10]. We note that the lower diameter limit thus obtained is significantly below the Rayleigh optical resolution limit. In addition, the parameter ? can be associated with fractal behavior of the scattering medium [11] and may thus shed light on the bulk organization and optical properties in normal and diseased biological tissue. Future research at the Spectroscopy Laboratory will explore these issues in greater depth.

Recent Publications

1. R.S. Cotran, S.L. Robbins, V. Kumar, Pathological Basis of Disease (W.B. Saunders Company, Philadelphia, 1999).
2. J. Mourant, T. Johnson, S. Carpenter, A. Guerra, T. Aida, J. Freyer, J Biomed Opt.,7, 3 (2002).
3. K. Sokolov, R. Drezek, K. Gossage, and R. Richards-Kortum, Optics Express 5, 13 (1999).
4. M. Bartlett, H. Jiang, Phys. Rev. E 65, 031906 (2002).
5. V. Backman, R. Gurjar, K. Badizadegan, I. Itzkan, R. R. Dasari, L. T. Perelman and M. S. Feld, IEEE J Sel Top Quant. Elec. 5, 1019 (1999).
6. V. Backman, V Gopal, M. Kalashnikov, K. Badizadegan, R. Gurjar, A. Wax, I. Georgakoudi, M. Mueller, C.W. Boone, R. R. Dasari, and M. S. Feld, IEEE J Sel Top Quant. Elec. 7, 6 (2001).
7. K.M. Pozhariski, Tumors of the Esophagus, In: VS Turusov (ed) Pathology of Tumors in Laboratory Animals, Vol 1, Part 1, IARC Scientific Publications No. 5, pp 87-100, Lyons, France, IARC, 1973).
8. R.M. Hodgson, F. Schweinsberg , M. Wiessler, P. Kleihues, Cancer Res. 42, 7 (1982)
9. C.W. Boone, G.D. Stoner, J.V. Bacus, V. Kagan, M.A. Morse, G.J. Kelloff, J.W. Bacus, Cancer Epidemiol Biomarkers Prev., 9, 1149 (2000).
10. V. Backman, G. Popescu, M. Hunter, M. Kalashnikov, C.W. Boone, A. Wax, V. Gopal, K. Badizadegan, G.D. Stoner and M.S. Feld, in preparation (2004).
11. A. Wax, C. Yang, V. Backman, K. Badizadegan, C.W. Boone, R.R. Dasari and M.S. Feld, Biophys. J. 82, 2256 (2002).