Jeremy D. Wilson

Mie theory interpretations of light scattering from intact cells

We present a Mie theory-based analysis of angularly resolved light scattering from intact cells that involves the product of a particle size distribution rho and the scattering cross section sigma. The analysis shows that the ability to size intracellular organelles depends strongly upon the assumed functional form of the particle size distribution; however, the product sigma rho extracts the dominant light scatterers regardless of this choice. We examine goniometer measurements from our own and another laboratory and find that this analysis reconciles seemingly conflicting results and confirms that mitochondrial-sized organelles dominate scattering from intact cells in the angular range 50 degrees - 90 degrees.

Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes

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.

Lysosome-damage-induced scattering changes coincide with release of cytochrome c

Light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology. Apoptosis, or programmed cell death, is associated with distinct morphological signatures such as mitochondrial swelling and nuclear condensation as well as characteristic biochemical signaling pathways, many of which are initiated by the release of cytochrome c from the mitochondria into the cytosol. In this Letter, we examine the time course of mitochondrial morphology changes as reported by light scattering and the subcellular location of cytochrome c measured by immunofluorescence microscopy in response to intracellular cell death signaling induced by photodynamic damage to lysosomes. We report that within this system, release of cytochrome c from the mitochondria into the cytosol occurs approximately simultaneously with mitochondrial-morphology-induced light scattering changes, providing further evidence that light scattering has the potential to play an important role in future studies of cell death biology.

Irradiation‐Induced Enhancement of Pc 4 Fluorescence and Changes in Light Scattering are Potential Dosimeters for Pc 4‐PDT†

Phthalocyanine 4 (Pc 4) is a promising photosensitizer currently in clinical trials. Photobiological responses to Pc 4 photodynamic therapy (Pc 4‐PDT) have been characterized extensively, but relatively little has been done to evaluate dose metrics for this sensitizer. We describe an irradiation‐induced increase in fluorescence in tumor cell monolayers. This increase is due solely to enhanced fluorescence from Pc 4, as confirmed by confocal spectroscopy. In EMT6 cells incubated with 250 nM Pc 4 for 24 h, the maximum increase in fluorescence is approximately 3.7‐fold above baseline levels. This increase occurs over a range of fluences, 0.05 – 0.6 J cm−2, where clonogenic survival decreases by 3 orders of magnitude. Light scattering measurements performed on similarly treated EMT6 cells in suspension suggested a Pc 4‐PDT‐mediated mitochondrial swelling of approximately 13% at 0.6 J cm−2, where fluorescence enhancement saturates under these treatment conditions. Fluorescence imaging and light scattering experiments performed at a five‐fold lower Pc 4 incubation concentration revealed a reduced fluorescence enhancement at a five‐fold higher fluence, which produced comparable mitochondrial swelling. Taken together, these data suggest that Pc 4 is initially aggregated at high local concentration in mitochondria and that irradiation relaxes the quenching of Pc 4 fluorescence through a mechanism that may involve mitochondrial swelling.

Microscope enabling multimodality imaging, angle-resolved scattering, and scattering spectroscopy.

We present the design, construction, and initial characterization of a multifunctional imaging/scattering spectroscopy system built around a commercial inverted microscope platform. The system enables co-registered brightfield, Fourier-filtered darkfield, and fluorescence imaging; monochromatic angle-resolved scattering measurements; and white-light wavelength-resolved scattering spectroscopy from the same field of view. A fiber-based illumination system provides illumination-wavelength flexibility and a good approximation to a point source. The performance of the system in its various data acquisition modes is experimentally verified using fluorescent microspheres. This multifunctional instrument provides a platform for studies on adherent cells from which the biophysical implications of subcellular light scattering can be studied in conjunction with sensitive fluorescence-based techniques.

Characterization of lysosomal contribution to whole-cell light scattering by organelle ablation

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 microm, respectively.

Optical property measurements establish the feasibility of photodynamic therapy as a minimally invasive intervention for tumors of the kidney

Abstract. 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 (μa) and transport scattering coefficients (μs′), 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.

Light scattering reports early mitochondrial responses to photodynamic therapy (Invited paper)

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.

Fluence- and time-dependant lysosomal and mitochondrial damage induced by LS11 PDT characterized with light scattering

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.

PDT-induced changes in light scattering from cells using lysosomal vs. mitochondrial-localizing photosensitizers

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.