Tamara M. Johnson

Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics

We have studied the optical properties of mammalian cell suspensions to provide a mechanistic basis for interpreting the optical properties of tissues in vivo. Measurements of the wavelength dependence of the reduced scattering coefficient and measurements of the phase function demonstrated that there is a distribution of scatterer sizes. The volumes of the scatterers are equivalent to those of spheres with diameters in the range between ~0.4 and 2.0 mum. Measurements of isolated organelles indicate that mitochondria and other similarly sized organelles are responsible for scattering at large angles, whereas nuclei are responsible for small-angle scattering. Therefore optical diagnostics are expected to be sensitive to organelle morphology but not directly to the size and shape of the cells.

Predictions and measurements of scattering and absorption over broad wavelength ranges in tissue phantoms

Predictions from Mie theory regarding the wavelength dependence of scattering in tissue from the near UV to the near IR are discussed and compared with experiments on tissue phantoms. For large fiber separations it is shown that rapid, simultaneous measurements of the elastic scatter signal for several fiber separations can yield the absorption coefficient and reduced scattering coefficient. With this information, the size of the scattering particles can be estimated, and this is done for Intralipid. Measurements made at smaller source detector separations support Mie theory calculations, demonstrating that the sensitivity of elastic scatter measurements to morphological features, such as scatterer size, is enhanced when the distance between the source and detector fibers is small.

Light scattering from cells: the contribution of the nucleus and the effects of proliferative status.

As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei. The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells. Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures. An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined. Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells. Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus. This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells. Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria.

Polarized angular-dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.

Measuring absorption coefficients in small volumes of highly scattering media: source-detector separations for which path lengths do not depend on scattering properties.

The noninvasive measurement of variations in absorption that are due to changes in concentrations of biochemically relevant compounds in tissue is important in many clinical settings. One problem with such measurements is that the path length traveled by the collected light through the tissue depends on the scattering properties of the tissue. We demonstrate, using both Monte Carlo simulations and experimental measurements, that for an appropriate separation between light-delivery and light-collection fibers the path length of the collected photons does not depend on scattering parameters for the range of parameters typically found in tissue. This is important for developing rapid, noninvasive, and inexpensive methods for measuring absorption changes in tissue.

Non-invasive measurement of chemotherapy drug concentrations in tissue: preliminary demonstrations of in vivo measurements

Measurements of the tissue concentrations of two chemotherapy agents have been made in vivo on an animal tumour model. The method used is based on elastic scattering spectroscopy (ESS) and utilizes a fibre-optic probe spectroscopic system. A broadband light source is used to acquire data over a broad range of wavelengths and, therefore, to facilitate the separation of absorptions from various chromophores. The results of the work include measurements of the time course of the drug concentrations as well as a comparison of the optical measurements with high performance liquid chromatography (HPLC) analysis of the drug concentrations at the time of sacrifice. It is found that the optical measurements correlate linearly with HPLC measurements, but give lower absolute values.

Elastic scattering spectroscopy as a diagnostic tool for differentiating pathologies in the gastrointestinal tract: preliminary testing

We report preliminary clinical testing of elastic-scattering spectroscopy for the detection of pathologies of the gastrointestinal tract. Tissue pathologies are detected and diagnosed using spectral measurements of elastically scattered light in an optical geometry that results in sensitivity to both the absorption and scattering properties of the tissue, over a wide range of wavelengths (300 to 750 nm). The system employs a small fiber optic probe, which is amenable to use with most endoscopes or catheters, or to direct surface examination, as well as interstitial needles. In this paper we report the results of preliminary clinical measurements on various organ sites of the gastrointestinal tract. In several instances the data indicate promise for this diagnostic method to distinguish malignant and dysplastic conditions from normal or other diagnoses.

Characterizing Mammalian cells and cell phantoms by polarized backscattering fiber-optic measurements.

Fiber-optic, polarized elastic-scattering spectroscopy techniques are implemented and demonstrated as a method for determining both scatterer size and concentration in highly scattering media. Measurements of polystyrene spheres are presented to validate the technique. Measurements of biological cells provide an estimate of the average effective scatterer radius of 0.5-1.0 mum. This average effective scatterer size is significantly smaller than the nucleus. In addition, to facilitate use of polarization techniques on biological cells, polarized angular dependent scattering from cell suspensions was measured. The light scattering from cells has properties similar to those of small spheres.

Methods for measuring the infrared spectra of biological cells

Infrared (IR) spectroscopy of biological cells is a growing area of research, with many papers focusing on differences between the spectra of cancerous and noncancerous cells. Much of this research has been performed using a monolayer of dehydrated cells. We posit that the use of monolayers can introduce artefacts that lead to an apparent but inaccurate measurement of differences between cancerous and noncancerous cells. Additionally, the use of dried cells complicates the extraction of biochemical information from the IR spectra. We demonstrate that using suspensions of viable cells in aqueous suspension reduces measurement artefacts and facilitates determining the concentration of the major biochemical components via a linear least-squares fit of the component spectra to the spectrum of the cells.

Angular dependent light scattering from multicellular spheroids

We demonstrate that the effects of cell-cell contact and of changes in cell shape have only a minor effect on the angular distribution of light scattering from mammalian fibroblast cells. This result is important for the development of light scattering as a noninvasive tool for tissue diagnostics such as cancer detection. Changes in cell organization that are not accompanied by changes in internal cellular structure may not be measurable. On the other hand, changes in internal cellular structure should be measurable without interference from changes in overall cellular organization. The second major result of this work is that there are small but significant differences between light scattering of tumorigenic and nontumorigenic cells grown in a three-dimensional culture system. The cause of the differences in light scattering are likely due to the nontumorigenic cells arresting in the G1 phase of the cell cycle, while the tumorigenic cells continue to proliferate.