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* Department of Physics and Astronomy, Institute of Optics, Department of Ophthalmology, and Department of Radiology, University of Rochester, Rochester, New York 14642
Correspondence: Address reprint requests to Thomas H. Foster, Dept. of Radiology, 601 Elmwood Ave., Box 648, Rochester, NY 14642. Tel.: 585-275-1347; E-mail: thomas.foster{at}rochester.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). In addition, relatively recent work has established that mitochondrial volume dysregulation may result from the opening of the permeability transition pore in response to a variety of insults, including oxidants. The opening of channels or swelling sufficient to rupture the outer mitochondrial membrane may cause the release of cytochrome c, which in turn leads to necrotic or apoptotic cell death (Green and Reed, 1998).
There has been significant renewed interest in the use of rigorous light scattering techniques to interrogate intracellular structure in cell systems and in tissue. With the exception of the extreme forward scattering direction where cell volume effects are important, Mourant et al. (1998) showed that scattering of visible light from cells is governed predominantly by the size and composition of intracellular organelles rather than by the volume of the cell as a whole. A study by Perelman et al. (1998) demonstrated that oscillations in the wavelength-dependent backscattering from cells and from tissue could be interpreted on the basis of nuclear size distributions. Backman et al. (1999) and Sokolov et al. (1999) extended the backscattering measurement to include polarization, which enabled the discrimination between singly and multiply scattered light in thick samples such as tissue. These reports and a related investigation using angle-resolved low coherence interferometry (Wax et al., 2002) shared with Perelman et al. (1998) an emphasis on the estimation of nuclear size in normal and malignant cell populations.
Mitochondria are also important scatterers of light in cells and tissue at visible wavelengths (Beauvoit et al., 1995). Optical property measurements of intact cells by Mourant et al. (1998) revealed a distribution of intracellular scatterers with volumes equivalent to spheres with diameters in the range 0.2–1.0 µm. Subsequent investigations by this group (Mourant et al., 2001, 2002) supported the idea that the dominant populations of scatterers in cells are smaller than nuclei. The 1.0–2.0-µm diameters at the high end of the range reported in their more recent articles are certainly compatible with mitochondria. Simple optical transmission measurements are sensitive to changes in mitochondrial morphology in preparations of isolated mitochondria, and such measurements have been used and reported for many years (for example, Packer, 1960). There are, however, very few reports of mitochondrial shape changes and swelling from optical measurements performed on intact cells. One such recent example is that of Boustany et al. (2002), who used an optical scatter imaging microscope to identify mitochondrial rounding in endothelial cell monolayers subjected to increased intracellular calcium.
In this article we describe the results of angle-resolved light scattering measurements performed on suspensions of intact murine mammary carcinoma cells, on these cells subjected to photodynamically induced oxidative stress, and on suspensions of mitochondria isolated from rabbit liver. The particular photosensitization strategy that we chose induces oxidative stress directly within the mitochondria, and this stress is known to lead to the demise of the cell through rapid apoptosis and/or necrosis, depending on the specific cell line and the details of the treatment (Kessel and Luo, 1999; Oleinick et al., 2002). Our analysis reveals that the changes to the scattering induced by photodynamic stress are consistent with an inhomogeneous mitochondrial swelling in which cytosol pools in the center of the organelle, displacing mitochondrial material to the periphery. Transmission electron microscopy images of the control and treated cells confirm these particular changes in mitochondrial morphology.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). After h-ALA incubation, the cells were again centrifuged and washed with serum free media and then centrifuged and washed in Hanks' balanced salt solution (HBSS). After a final centrifugation, the pellet was suspended in HBSS at a final volume of 2 ml. All handling of cells after incubation was done at very low light levels.
The cell suspension was then split into two groups. The control group was protected from any exposure to light. The treatment group was irradiated with 514 nm light at a fluence rate of 50 mW/cm2 for 200 s, conditions that were established by preliminary electron microscopy experiments. Greater than 90% of EMT6 cells subjected to this fluence stained with trypan blue immediately after treatment, indicating the onset of rapid necrosis (Lam et al., 2001). Less than 1 h elapsed between the time that the cells were taken out of incubation and the end of the light scattering experiments, and the time between irradiation and the end of the scattering measurements did not exceed 20 min.
Isolation of mitochondria
Mitochondria were isolated from fresh rabbit liver following the method of Senior et al. (1975). Immediately after the animal was sacrificed, the liver was harvested and kept on ice in a 10% saline solution. It was then sliced and washed in a buffer of 0.3 M sucrose, 1 mM EDTA, and 1 mM Tris HCl (hereafter Tris buffer) at a pH of 7.4. The tissue was homogenized in Tris buffer with 1 mg/ml bovine serum albumin (BSA) in a potter, and the slurry was centrifuged at 900 g for 5 min. The supernate was kept and centrifuged at 11,500 g for 5 min. The pellet was resuspended in buffer and BSA and centrifuged again. The fat layer was removed, and the pellet was suspended in Tris buffer and BSA.
The viability of the mitochondria was evaluated by measurements of oxygen consumption. A 3-ml covered cuvette was filled with 2 ml of a reaction buffer (24 mM glycylglycine, 10 mM MgCl2, 60 mM KH2PO4, and 87 mM sucrose) and 15 µl rotanone. The tip of an oxygen-sensitive electrode (model OM-4, Microelectrodes, Bedford, NH) was placed through a small hole in the top of the cuvette. Sufficient mitochondria were added to the cuvette to establish easily detectable oxygen consumption. Succinate (100 µl) was added to establish state 4 respiration, and then 20 µl ADP was added to establish state 3 respiration. After state 4 and state 3 respiration were verified with the oxygen electrode, the mitochondria were considered viable. The mitochondria were kept on ice, and all subsequent handling was done in the Tris buffer.
Angularly resolved light scattering
We constructed a goniometer to measure angularly resolved light scattering. A block diagram of the experimental setup is shown in Fig. 1. A scattering sample in aqueous suspension is placed in a cylindrical cuvette (model 540.115, Helma, Plainview, NY), which is positioned above the center of a 30.5-cm-diameter rotary stage (RT-12, Arrick Robotics, Tyler, TX). Red light (632.8 nm) from a 20-mW helium-neon laser is directed through the cuvette. In all of our measurements, the laser is linearly polarized perpendicular to the surface of the rotary stage. Light scattered from the sample is passed through a pinhole mounted midway to the edge of the rotary stage and colleted by an optical fiber (400-µm core diameter, 0.22 N.A.) that is mounted at the edge. Light exiting the fiber is measured by a photodiode (S2386-18L, Hamamatsu, Bridgewater, NJ) and digitized at 16 bits. A constant offset is introduced by light from a light-emitting-diode incident on the photodiode. A PC-controlled stepper motor rotates the stage continuously from 
3 to 110° with respect to the forward direction of the laser at an angular resolution of 0.2°. The angular position is read out from an optical encoder, and the stage position and photodiode voltage are simultaneously recorded. The data acquisition is automated and controlled by a homebuilt program written in LabView (National Instruments, Austin, TX). Each scan of the full angular range takes 2 min.
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) to account for the number of particles being sampled at a given angle (Bohren and Huffman, 1998).
Electron microscopy methods
For electron microscopy, cells were grown on glass chamber slides and fixed in 0.1 M phosphate-buffered 2.0% glutaraldehyde at room temperature for 2 h. After repeated rinsing in 0.1 M phosphate buffer, the cells were postfixed in 1.0% osmium tetroxide for 20 min. The slides were passed through a graded series of ethanol, infiltrated with Spurr epoxy resin, and fitted to inverted capsular molds containing fresh Spurr resin. After polymerization at 70°C, the hardened capsules containing the cells of interest were then "popped off" the surface of the glass slides by dipping the slides into liquid nitrogen (de Mesy Jensen and di Sant' Agnese, 1992). These blocks were trimmed and sectioned at 60–70-nm intervals with a diamond knife onto mesh copper grids. The grids were contrast enhanced with uranyl acetate for 15 min and lead citrate for 10 min and examined and photographed for quantitative analysis with a Hitachi 7100 transmission electron microscope at magnifications ranging from 3000x to 30,000x.
Mie theory fits
A Mie theory model was fit to the angularly resolved scattering data. In these fits, the indices of refraction for both the scattering center and the surrounding medium were held fixed at 1.40 and 1.38, respectively, to represent organelles in cytoplasm (Beuthan et al., 1996). We calculated Mie theory scattering spectra, 
for linearly polarized light scattered from particles with radius r ranging from 0.005 to 8.0 µm. The low and high ends of this range define rmin and rmax in Eq. 5. These values were chosen to span the radii of the scattering centers anticipated in the data; the diameter of a whole cell, for example, is 15 µm. Test functions, 
were built from Mie theory by integrating the scattering spectra against a particle size distribution, 
such that
 | (1) |
Here, 
is assumed to be a sum of log-normal distributions,
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and 
are related to the mean, µ, and standard deviation, 
, of the log-normal distribution as
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 | (4) |
We further constrained each distribution to be representative of physical scattering centers in cells by defining a parameter,
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Let 
be the measured angularly resolved scattering data. Because amplitudes of the scattered light intensity encountered in our experiments differ by four orders of magnitude, it was important to fit both and 
Mie-type scattering is highly forward directed, and as such many of the details of angularly resolved scattering at all but the smallest angles are best observed in Log (I()). Thus, fitting only I() strongly favors the most forward scattering angles at the expense of higher angle scattering. However, in the case of scattering from whole cells, we found that only fitting to Log I()) underestimated the extreme forward scattered data point, and we therefore included an I() term in the fit.
The fit itself was carried out by minimizing the function
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were chosen to weight each term approximately equally. Specifically, 
1 was set arbitrarily to 1.0, 2 to 7.0, and 3 to 1010. These relative amplitudes of 1 and 2 rendered the two integrals in Eq. 6 approximately equal near a minimum in 
2. This greatly improved the quality of the fits. With respect to the parameter Rj and its coefficient 3, we were guided by previous work of Mourant et al. (2002) in constraining the log-normal distributions so that their amplitudes were diminished at the tails relative to their peak value by a factor of 108. As an extension to that work, we increased this restriction to account for the relative cross sections for r = 8 µm versus 1-µm structures (
). Thus, 3 was chosen to be 1010. We note that relaxation of this parameter does not affect the mean of the distribution and more specifically does not affect the location of the strong peak of the distribution shown in Fig. 5. In particular, the choice of these parameters does not skew the results to discriminate against nuclear or whole-cell contributions. Fitting was done iteratively with the free parameters being the mean, µj, standard deviation, j, and the overall amplitude, aj, for each log-normal distribution 
The minimization was carried out using the simple downhill simplex of Nedler and Mead (Press et al., 1992).
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where r is the initial radius of the scattering sphere. This is equivalent to allowing the mean, µj, and standard deviation, j, of the distribution, 
to scale by the same constant 
. Mitochondria swelled by means of cytosol penetrating and forming a core in the center of the organelle, as shown in Fig. 9 C. The diameters of the core (
) and of the coating (
) were fixed for a particular by conservation of mitochondrial mass and are related by,
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. As in the initial Mie theory fits, we constructed test functions, T, from particle size distributions. The difference was that for one log-normal distribution, we used a coated sphere instead of a homogenous sphere model. The fit was again done with the simple downhill simplex by minimizing the function,
 | (8) |
Uncertainties in were estimated by determining the values on either side of the 2 minimum that increased 2 by 1 (Bevington, 1969).
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RESULTS |
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105 cells/ml in HBSS, which is in good agreement with Mourant et al. (2002).
The plots in Fig. 3 show the angularly resolved light scattering from intact control EMT6 cells and from cells subjected to ALA-sensitized photodynamic insult. The scattering from these two groups of cells is noticeably different for angles <30°. For larger angles, scattering from the two groups is virtually identical. It is apparent that the photodynamically treated cells scatter less total light than the control cells over this angular range and that the scattering distribution from the treated cells is more forward directed.
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2. Attempts to fit the data using three independent populations resulted in the fitting algorithm returning either zero amplitude for one of the populations or returning two identical populations. A typical fit using two log-normal distributions is shown in Fig. 4. The first population of scatterers had a mean diameter of 0.22 µm ± SD of 0.057 µm; the second had a mean diameter of 1.15 µm ± SD of 0.54 µm. The amplitudes of the two distributions extracted from the fits showed that there were 134 "small" particles for every "large" particle in an intact cell. Fig. 5 shows the relative contributions of these two populations to the observed light scattering. In this plot the normalized "relative scattered light intensity" is computed from the product of the particle size distribution obtained by the Mie theory fits and the scattering cross section. It is evident that most of the light is scattered by particles with diameters between 1 and 4 µm. More precisely, computing the area under the total light scattered curve in Fig. 5 for different particle sizes shows that 65% of the light scattered over this angular range is done by particles with diameters spanning 1–3 µm. Particles with diameters 0.5–4.0 µm scatter 85% of the light.
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We examined 70-nm-thick sections through the mitochondria, organelles with dimensions between 0.5 and 1.0 µm and 1.0–4.0 µm along the minor and major axes, respectively (Palade, 1953). These cross sections are ellipsoidal in nature. Our basis for comparison of the control versus treated cells was a measure of the major axis of this elliptical section for many mitochondria from both groups. Using the image processing package Image Pro (Media Cybernetics, San Diego, CA) we obtained 94 measurements of the major axes from EM sections of mitochondria that had been exposed to oxidative stress and 59 similar measurements from mitochondrial sections taken from the control group. A schematic of the sampling method is displayed as an inset of Fig. 8.
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Important features of the mitochondrial structure and the response to photodynamic insult are shown in the electron micrographs of Fig. 9, A and C. The distribution of the mitochondrial material becomes less homogeneous posttreatment, and the mitochondria take on the appearance of a coated sphere, as depicted schematically in Fig. 9, B and D. The original mitochondrial material appears to be concentrated in the outer region of the organelle, and a core of non-electron-dense material is present in most of the organelles.
Scattering results for photodynamically treated cells
The model for interpreting the light scattering from the ALA-sensitized, irradiated cells was created to be consistent with both the electron microscopy data as well as the Mie theory fits to the control cells as described above. The Mie theory fits to control cells and isolated mitochondria provide strong evidence that mitochondria dominate the forward angle scattering in intact cells. Electron microscopy shows that the mitochondria undergo swelling as a result of the photodynamically induced oxidative stress.
We considered several possible models based on various physical interpretations. A Mie theory model in which the mitochondria swelled in response to photodynamic insult and retained their original index of refraction could not represent the data, as this would cause the total scattering cross section to increase, and the data presented in Fig. 3 shows that the cross section in fact decreases. The possibility that, as a mitochondrion swelled, it took on a lower, homogeneous, volume-averaged index of refraction, 
was also examined. Fig. 10 shows how such a homogeneous swelling would affect the angularly resolved scattering from whole cells. It is clear that such a swelling effect is not consistent with the scattering angular distribution observed from the treated cells.
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65% (0.13/0.20) of the larger population are mitochondria.
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DISCUSSION |
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5 to 90°. Evidence for this comes first from the comparison of the size distributions extracted from fits to the whole cell and isolated mitochondria scattering data. Probably more convincing, however, is the fact that the analysis of the angle-resolved scattering from cells subjected to mitochondrial oxidative stress is remarkably compatible with the morphology changes observed under electron microscopy of identically treated cells.
The angle-resolved scattering plotted in Fig. 3 shows clear differences between the EMT6 cells subjected to photodynamic insult and the control, untreated cells. We emphasize that these measurements were made immediately after irradiation, and that the cells were intact; no significant lysis was observed at these early time points. Although some loss of cell volume regulation is probably also happening concurrently, our measurements would likely not be sensitive to this. Whole-cell volume is reported in the extremely forward-directed scattering (Salzman et al., 1990) that is not detected in our experiments, which begin sampling at 5° from the incident laser beam.
The fits to the angle-resolved scattering data in Fig. 4 reveal two populations of intracellular scatterers, which is generally consistent with the findings of Mourant et al. (1998) in two rat embryo fibroblast cell lines. In fact, the size estimates that we extract for the mean diameters of the two populations—0.22 and 1.15 µm—agree remarkably well with the range of intracellular scatterer dimensions reported by those authors. Although the mean of the larger size distribution is at 1.15 µm and this is indeed smaller than the diameter of a cell nucleus, this does not mean that the nucleus does not contribute to scattering in this angular range. The plots of Fig. 5 illustrate this clearly, where the size distributions extracted from the fits to the angle-resolved data of Fig. 4 are multiplied by the total scattering cross section as a function of particle diameter. These plots, which report the total light scattered as a function of particle size diameter, have several important features. First, although there are more than a 100-fold greater number of the small-diameter scatterers within a cell, these small-diameter structures scatter relatively little light in the angular range 5–90° as compared to the larger-diameter organelles. Second, although the mean of the larger-diameter size distribution was 1 µm, Fig. 5 shows that most of the light that is scattered is in fact scattered by particles with diameters of 2 µm, and the large-diameter tail of this distribution is certainly compatible with a contribution from the nucleus.
Fig. 6 presents angle-resolved data from a suspension of rabbit liver mitochondria. Here the Mie-theory fit is excellent, and the resulting size distribution, shown in Fig. 7, is significantly narrower than the large-diameter distribution that results from fits to the whole-cell data of Fig. 4. There are two explanations for these observations. First, as stated above the larger-diameter population of scatterers in the whole-cell case probably includes contributions from organelles other than mitochondria, and this will widen the size distribution. Second, with respect to the mitochondrial contribution itself, because the mitochondria in intact cells are ellipsoidal and randomly oriented whereas mitochondria in suspension tend to round, the isolated mitochondria will have a more tightly distributed range of effective diameters. Nevertheless, the peak and the mean of this distribution are in good agreement with those of the larger of the two scatterer populations from whole cells, as depicted in Fig. 7.
As noted above, this agreement is one piece of evidence that the larger size distribution extracted from fits to the whole-cell scattering data includes a significant contribution from mitochondria. The second, and perhaps more compelling evidence, comes from the fact that photodynamic insult changed the mitochondria in specific ways, and a coated sphere interpretation of the scattering measurements reported this change correctly. Figs. 8 and 9 summarize results of electron microscopy imaging performed on intact cells pre- and postphotodynamic stress. In particular, the representative EM images shown in Fig. 9, A and C, provided the rationale for the use of homogenous and coated sphere models in analyzing the scattering angular distributions from the control and treated EMT6 cells, respectively. Before leaving the images of Fig. 9, we note that the photodynamically treated mitochondria shown in Fig. 9 C bear a striking resemblance to mitochondria during state 4 to state 3 transition, as depicted for example in Fig. 14 of Hackenbrock (1966).
In Figs. 10, 11, and 12, we show the results of fitting the whole-cell scattering data from the treated cells using various candidate models that incorporated different assumptions about the morphology of the mitochondrial subjected to oxidative stress. The data are dramatically incompatible with models based on a homogeneously swollen sphere with a uniformly diluted refractive index (Fig. 10) and on a coated sphere in which the core has a refractive index equal to that of water (Fig. 11). The fact that the data are able to discriminate between these interpretations and one that incorporates a coated sphere with a cytosolic core is a measure of the excellent sensitivity of angularly resolved light scattering to very specific changes in the morphology of mitochondria, at least in this cell line. Interestingly, a coated sphere model was used previously by Brunsting and Mullaney (1974) to interpret scattering from Chinese hamster ovary (CHO) and from HeLa cells in terms of a nucleus surrounded by cytosol. Over part of the angular range studied in that work, the authors demonstrated in the CHO cells modestly better agreement with experiment using a coated versus homogeneous sphere model. Results with the HeLa cells were not shown but were apparently less encouraging. In a study of the angular dependence of visible light scattering from Escherichia coli cells, Cross and Latimer (1972) had good success by treating the bacteria as a coated ellipsoid, with the coat in this case representing the cell wall. As far as we are aware, ours is the first use of a coated sphere model to analyze the influence of morphological changes in mitochondria induced by any means on scattering angular distributions from whole cells, and it is the first direct experimental validation of a coated sphere model with EM. Although our coated sphere extension to a Mie theory analysis worked very well in this situation and seems to have captured the critical physical features of the problem, there does appear to be some structure in the angular distribution near 40° from the treated cells (see Fig. 12) that is not reproduced by the fitting function. It is possible that a rigorous comparison with other methods of treating these kinds of data, such as the finite-difference time-domain approach described by Drezek et al. (1999), would yield further insight. Finally, we emphasize that our measurements and analysis cannot strictly exclude the possibility that other organelles may also be responding to this form of oxidative stress in ways that may influence the scattering angular distributions.
There are several lines of investigation suggested by this study. It may be possible, for example, to use the methods described here to identify where changes in mitochondrial morphology occur in the apoptotic cascade and in the demise of cells subjected to a variety of insults (Green and Reed, 1998). This would enable a noninvasive assay capable of predicting the fate of cells without exogenous fluorescent reporters or other labeling. Encouraging work along these lines using optical scatter imaging microscopy has just been reported by Boustany et al. (2004). The sensitivity of the angle-resolved scattering to mitochondrial morphology and the similarity between the EM appearance of the mitochondria undergoing a state 4 to state 3 transition shown by Hackenbrock (1966) and those of our Fig. 9 C suggest that these measurements could be useful in fundamental studies of mitochondria in intact cells. Although studies of these kinds and others could be performed in suspension as we have done, the applicability of the light scattering technique would be greatly expanded if it could be implemented using adherent cells. In this regard, it is encouraging that Bartlett et al. (2004) recently suggested that mitochondrial signatures may be detected in a reflectance geometry using polarized light spectroscopy.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health grant CA68409 awarded by the National Cancer Institute.
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FOOTNOTES |
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Submitted on October 15, 2004; accepted for publication December 29, 2004.
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REFERENCES |
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) and a Mie theory best fit (solid line) to these data. The fit returned a diameter of 2 µm and a CV of 6%.
) and from EMT6 cells subjected to photodynamically induced oxidative stress (
). The treated cells scatter less light at small angles and the distribution is more forward peaked. Error bars are omitted for clarity.
