We measured the optical properties of freshly excised kidneys with renal parenchymal tumors to assess the feasibility of photodynamic therapy (PDT) in these patients. Kidneys were collected from 16 patients during surgical nephrectomies. Spatially resolved, white light, steady-state diffuse reflectance measurements were performed on normal and neoplastic tissue identified by a pathologist. Reflectance data were fit using a radiative transport model to obtain absorption (μ a ) and transport scattering coefficients (μ ′ s ), which define a characteristic light propagation distance, δ . Monte Carlo (MC) simulations of light propagation from cylindrical diffusing fibers were run using the optical properties extracted from each of the kidneys. Interpretable spectra were obtained from 14 kidneys. Optical properties of human renal cancers exhibit significant inter-lesion heterogeneity. For all diagnoses, however, there is a trend toward increased light penetration at longer wavelengths. For renal cell carcinomas (RCC), mean values of δ increase from 1.28 to 2.78 mm as the PDT treatment wavelength is increased from 630 to 780 nm. MC simulations of light propagation from interstitial optical fibers show that fluence distribution in tumors is significantly improved at 780 versus 630 nm. Our results support the feasibility of PDT in selected renal cancer patients, especially with photosensitizers activated at longer wavelengths.
Angularly resolved light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology, with particular sensitivity to organelles the size of mitochondria and lysosomes. We have recently reported on a lysosome-staining-based method that provides scattering contrast between stained and unstained cells, and through the use of appropriate models, we extracted a size distribution and contribution to cellular light scattering that we attributed to lysosomes. We provide an independent measurement of the lysosomal size distribution and contribution to cellular light scattering by exploiting photodynamic ablation of lysosomes and observing its effect on angularly resolved light scattering measurements. From these measurements, we conclude that lysosomes scatter approximately 14% of the light from EMT6 cells at 633 nm and that their size distribution has a mean and standard deviation of 0.8 and 0.4 µm, respectively.
Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly
resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6
cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic
therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated
sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of
Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More
recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of
lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an
absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering
measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as
quantified by uptake of acridine orange, and can induce downstream effects including release of
mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell
Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the
induction of lysosomal morphology changes requires a fluence significantly higher than that reported for
cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial
swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes
remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of
scattering measurements at different wavelengths to different organelle populations.
Angularly resolved light scattering and wavelength-resolved darkfield scattering spectroscopy measurements were performed on intact, control EMT6 cells and cells stained with high-extinction lysosomal- or mitochondrial-localizing dyes. In the presence of the lysosomal-localizing dye NPe6, we observe changes in the details of light scattering from stained and unstained cells, which have both wavelength- and angular-dependent features. Analysis of measurements performed at several wavelengths reveals a reduced scattering cross section near the absorption maximum of the lysosomal-localizing dye. When identical measurements are made with cells loaded with a similar mitochondrial-localizing dye, HPPH, we find no evidence that staining mitochondria had any effect on the light scattering. Changes in the scattering properties of candidate populations of organelles induced by the addition of an absorber are modeled with Mie theory, and we find that any absorber-induced scattering response is very sensitive to the inherent refractive index of the organelle population. Our measurements and modeling are consistent with EMT6-cell-mitochondria having refractive indices close to those reported in the literature for organelles, approximately 1.4. The reduction in scattering cross section induced by NPe6 constrains the refractive index of lysosomes to be significantly higher. We estimate the refractive index of lysosomes in EMT6 cells to be approximately 1.6.
We have previously described changes in angle-resolved light scattering measured from intact cells in suspension subjected to photodynamic therapy using photosensitizers that localize primarily to mitochondria. These changes were analyzed with a Mie theory-based model. For the sensitizers Pc 4 and ALA-induced protoporphyrin IX, the scattering data from PDT-treated cells was consistent with a coated sphere model, in which mitochondrial morphology changes were the predominant mechanism governing the scattering changes. This interpretation was supported by electron microscopy. Here we describe quite different changes in angle-resolved light scattering from cells sensitized with the lysosomal-localizing photosensitizer LS11. Unlike the case of the mitochondrial-localizing photosensitizers, analysis of these post-treatment scattering data reveals a shift toward a larger mean organelle diameter in the larger of the two particle size distributions identified from Mie-theory analysis of scattering from control cells. Further, the post-treatment scattering angular distributions are well interpreted in terms of homogeneous rather than coated spheres. On the basis of these results and results of fluorescence microscopy of LS11-PDT treated monolayers, we propose that the initial, pre-treatment scatterer population is comprised of lysosomes and mitochondria. LS11 PDT ablates a significant fraction of the lysosomes, leaving a relatively unperturbed population of mitochondria to dominate the scattering. These findings suggest that scattering measurements are capable of reporting a variety of PDT-induced changes to cell organelles. They further suggest that photodynamic action is a useful biophysical tool for understanding basic mechanisms of light scattering from intact cells.
Angularly-resolved light scattering is an established method of particle sizing. Scattering from intact cells provides information about the size distributions of intracellular scatterers. Mitochondria are important light scatterers, especially at forward angles. Nuclei play an important part in scattering light at extreme forward angles and in backscattering geometries. Because changes in mitochondrial morphology are among the early responses to photodynamic therapy (PDT) using mitochondrial-localizing sensitizers and because these changes may be important in determining the fate of the cell, it is interesting to consider light scattering as a means of assessing the response of cells and tissue to PDT. Simple transmission measurements in an absorption spectrophotometer report a rapid reduction in scattering in cells subjected to aminolevulinic acid (ALA)-PDT. ALA-PDT with a fluence of 5 J cm-2 induces a change in the angularly-resolved light scattering from EMT6 cells in suspension within approximately 45 minutes of irradiation. At earlier times following this fluence, the scattering differs only slightly from that observed with control cells. Analysis of the post-treatment scattering data at forward angles is consistent with mitochondrial swelling. Qualitatively similar changes in scattering are observed immediately after a fluence of 10 J cm-2 in cells sensitized with Pc 4.
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