Scattering Cross Section Measurements

To accurately measure the total amount of light scattered by the cell suspensions, the scattering cross sections were measured with the setup shown in Figure 5.21.

  

Figure 5.21: Experimental setup for scattering cross section measurements.

The beam from the laser passed through a pinhole aperture and through a 2 mm pathlength cuvette. The total distance from the sample cuvette to the detector was sufficiently long $(~6\: \text{m})$ to ensure that only collimated light ( $\theta<0.03^\circ$) was measured. The unscattered portion of the beam was directed to a photodetector (Newport 835) which was shielded from the scattered light by enclosing it in a black container.

Measurements were first taken with a water-filled cuvette to account for losses due to the cuvette walls. The collimated transmission, T_c, was computed from

$$T_c = \frac{P_{H_2O}}{P_s}$$ (5.8)

where P_H2O and P_s are the detected powers of the blank and sample cuvettes, respectively. If T_c, was greater than 0.9 the sample was considered dilute and the attenuation is described by Beer's law,

$$T_c = e^{-\mu_t d}$$ (5.9)

where d is the pathlength. Since the sample has little absorption, the total attenuation is dominated by scattering

$$\mu_t \approx \mu_s = C_{sca}N_v ,$$ (5.10)

where C_sca is the scattering cross section and N_v is the number of scatterers per volume. Therefore,

T_c = e^{-C_scaN_v d}, (5.11)

and the scattering cross section is

$$C_{sca} = \frac{-\ln T_c}{N_v d}.$$ (5.12)

To test the experimental arrangement, collimated transmission measurements of a dilute suspension of $1\: \mu\text{m}$ spheres at $\lambda=633 \:\text{nm}$ were used to compute the scattering cross section with Equation 5.12 and compare with Mie theory. The measured cross section was $2.8 \pm 0.5 \mu m^2$ and Mie theory predicts a cross section of $2.7 \mu m^2$.

To measure the effect of acetic acid on scattering cross section, a procedure similar to that of the goniometer measurements was followed. The transmitted intensity from a water filled cuvette was recorded prior to each measurement with the cells. The cell suspension with acetic acid contained 50% PBS and 50% acetic acid (6%) and measurements were taken 1-2 minutes after application of acetic acid.

The measured scattering cross sections of tumor cells are plotted in Figure 5.22 at wavelengths of 543, 633, and 790 nm. At all wavelengths the cross sections increased when the cells were mixed with acetic acid, with the greatest increase occurring at $\lambda=543 \: \text{nm}$. The uncertainty indicated in the graph results from the variations in the measured power levels which were significant since the measured transmission values were greater than 0.95 in most cases.

  

Figure 5.22: Measured scattering cross sections of tumor cells at three wavelengths, demonstrating the effect of acetic acid.

The effect of acetic acid on a suspension of normal cells is shown in Figure 5.23 at wavelengths of 543 and 633 nm.

  

Figure 5.23: Measured scattering cross sections of normal cells at 543 and 633 nm.

As in the tumor cells, the cross sections increase with acetic acid, although the increase appears to be less dramatic. Note however, that the cross sections for the normal cells in PBS are greater than the cross sections measured for the tumor cells, and the uncertainty in the measurements of the normal cells is greater than the tumor cells.

The increased cross sections of the normal cells compared to the tumor cells results from the fact that they are larger in size. The two types of cells are from different cell lines and therefore, the absolute value of the cross sections cannot be directly compared. However Figures 5.22 and 5.23 illustrate that the relative increase between the two types of cells is larger for the tumor cells than the normal cells.

The larger uncertainty in the measurements of the normal cells could be due to the strong tendency of the normal cells to adhere to each other and form large clusters, as shown in Figure 5.24, whereas the tumor cells remained separate.

  

Figure 5.24: Low magnification images of tumor (top) and normal (bottom) cell suspensions demonstrating the tendency of the normal cells to adhere to each other and form clusters.

Before each measurement the normal cells were pipetted several times to disperse the cells, but all of the clusters were not broken up.

The scattering cross section measurements for the normal and tumor cells indicate that acetic acid causes an increase in total scattering for both cell types. While these results cannot be directly compared to the acetowhite change in the cervix since the cells are from a breast cell line, they support the hypothesis that scattering is increased due to an alteration of the nuclear proteins. The scattering increase may be enhanced in the tumor cells because the amount of nuclear protein is greater and the nuclei are larger [78]. In tissue, this increase in scattering could be further enhanced in diseased areas because the epithelium contains a larger number of cells and is thicker than in normal areas.