2.1.

Method for Modeling of Scattering with Water Loss and a Fixed Organelle Size Distribution

AVD invokes the loss of water from the cytoplasm, not accompanied by commensurate loss of ions or other dissolved/suspended matter. One consequence of this is an expected increase in the cytoplasmic index of refraction. To test the hypothesis that changing only the index of refraction of the cytoplasm could result in a change in scattering consistent with our early optical signal, a size distribution of scattering centers was extracted (using an inverse Mie theory formalism) from scattering measurements taken from a culture treated with staurosporine for 30 min and 4 h. As described in a previous publication, cells contain scatterers in a range of sizes that are distributed in a punctate manner, with large gaps between sizes.²² To represent this distribution, fitting was performed with a small number of sizes, each representing a specific class of organelle: Rayleigh scatterers (10 nm), lysosomes and peroxisomes (150, 200, and 300 nm), mitochondrial widths (600 nm), mitochondrial lengths (1.4 μm), and nuclei (8 μm). Due to the high frequency oscillations present in the scattered spectrum of large particles, any variation in size would have a marked effect on the spectrum included for fitting. To represent the biological variability in the size of cell nuclei, a Gaussian size distribution with a 20% C.V. was included in the Mie theory prediction of 8 μm particles. Distribution effects are not as pronounced for smaller sizes and were therefore not included in this simulation. An index of refraction of 1.41 was used for the organelles, while 1.39 was used for the refractive index of the cytoplasm after water loss, resulting in an index ratio of 1.01.

Further, while under normal conditions, the contribution of whole cell scattering is small due to the small index mismatch between intracellular and extracellular fluids, as water is lost from the cell, the index mismatch across the cell membrane increases. This increase in index ratio should result in an increased contribution of the whole cell to cellular scattering. Moreover, during apoptosis, adherent cells also partially detach from the substrate, causing them to “round up.” This effect is illustrated in Fig. 1. We postulate that these rounded-up cells may act more like scattering centers than those that remain attached to the plate, as relatively flat structures.^{14, 32}

Fig. 1

(a) Adherent cells normally have a relatively flat structure, though (b) shows a loss of adherence during apoptosis results in a more round shape.

To simulate the effect of whole-cell scattering consequent to the increased refractive index of the cytoplasm, Mie theory was used to predict the scattering from a Gaussian distribution of cell-sized spheres (11.5 μm mean diameter, 20% C.V.) after cytoplasm condensation, with n _{extracellular} = 1.35 and n _{intracellular} = 1.39, corresponding to an index ratio of 1.03. The resulting spectrum was included in the fitting matrix with the organelle spectra, for a total of eight possible sizes. For reference, the eight spectra used for fitting are presented in Fig. 2.

Fig. 2

Mie theory-predicted spectra present in the fitting matrix. The eight spectra correspond to single sizes of 10, 150, 200, 300, 600 nm, and 1.4 μm and two distributions of sizes of 8 and 11.5 μm mean diameter, both with 20% C.V.

2.2.

ESS Measurements

Wavelength-dependent measurements of scattering were made on cell cultures using instrumentation previously described elsewhere²² and depicted in Fig. 3. In brief, broadband white light from a 150 W xenon short-arc lamp was delivered to and collected from a plated cell sample using a fiber optic probe consisting of seven 100 μm fibers — six illumination fibers surrounding a single collection fiber. For clarity of the figure, only one pair of illumination and detection fibers are shown. The cells were grown in glass-bottomed 12 well plates (Mat-Tek Corporation, P12G-1.5-14-F), which were placed on the surface of a temperature-controlled water bath. The tip of the fiber probe was positioned above the plate, at a distance of ∼7 mm from the cell layer, such that the mean angle for collected light was 179.2 deg ± 0.6 deg. The unpolarized scattered light collected by the center fiber was transmitted to a spectrometer (Ocean Optics, USB4000) and analyzed. The probe's kinematic mounting system allows access to all 12 wells with precise repositioning.

Fig. 3

A schematic of the system used to measure wavelength-dependent backscattering. A fiber probe, containing 100 μm fibers in a standard six-around-one geometry (for clarity, only one pair is illustrated), collects light scattered at approximately 179 deg from plated cell cultures. The cultures are grown in glass-bottomed 12-well plates and are positioned at the surface of a temperature-controlled water bath. The probe translates to access all 12 wells on the plate.

Three separate spectral measurements, I(λ), are required for each collected spectrum: the raw spectrum from the sample (I _sample), a background measurement (I _background) to account for any scattering from components other than cells, and a spectral response (I _spec._resp.), acquired with a spectrally-flat diffuse reflector (Spectralon, Labsphere, Inc.), to account for spectral characteristics of the system response.²² The integration time for all sample and background measurements was 2 s, and the integration time for the spectral response was 10 ms, requiring each sample plate to be exposed to room air for no more than 3 min during measurements, with most of the time being used to maneuver the probe from well to well on the 12-well plate. Sample plates were returned to the incubator between measurements. The detector element of the spectrometer contains 2048 pixels, but to reduce pixilation noise, each measurement was smoothed with a eight-pixel boxcar corresponding to the resolution of the spectrometer (∼1.5 nm). The scattered light spectrum from each sample was calculated with the following expression: I _spectrum = (I _sample − I _background)/I _spec.resp., and then normalized to the area under the curve. Measurements were made for the spectral range 450 to 700 nm.

2.3.

Volume Decrease in the Absence of Apoptosis

To investigate cell volume decrease as the source of our early signal, cells were treated with differing concentrations of mannitol, a membrane-impermeable sugar alcohol, to increase the osmolarity of the medium and induce osmotic water loss. Three approaches were used to determine if the observed scattering spectral changes in apoptotic cells were consistent with the effects of volume decrease induced in a manner independent of apoptosis: caspase activity assay for apoptotic activity in cells treated with mannitol, quantification of the magnitude of volume loss by flow cytometry, and finally, scattering measurements made on the mannitol-treated cell cultures.

Osmotic volume loss was produced in CHO cultures by replacing the growth medium with fresh medium supplemented with one of the following: 50, 100, 200, 400, and 800 mm mannitol. The addition of these amounts of mannitol to isotonic growth medium (∼295 mOsm/L) resulted in solutions with the following hypertonic osmolarities: 345, 395, 495, 695, and 1095 mOsm/L, respectively. The effects of mannitol on apoptosis were determined using a luminescent assay for effector caspase activity (Caspase-Glo 3/7 Assay, Promega, Inc.). Full details of this procedure are presented in this work's companion paper. Two treatment times were assessed: 4 and 24 h.

To assess the degree of volume loss that resulted from each concentration of mannitol, the forward scattering from each population was assessed in a flow cytometer. To prepare samples for flow cytometry, cells were plated in individual 35-mm polystyrene dishes and grown to confluence. At 30 min and 4 h, the growth medium was replaced by medium alone or medium supplemented with mannitol at the following concentrations: 50, 100, 200, 400, and 800 mm. Cells were washed twice with phosphate buffered saline (PBS) supplemented with the same concentration of mannitol as the treatment, the washes were collected for analysis, and the cells were incubated with Accumax (Innovative Cell Technologies) for 20 min to obtain single cell suspensions for flow cytometry. Following incubation, a small volume of the collected washes was added back to the plates and cell aggregates were disrupted by agitation. One percent formaldehyde fixation was used to prevent volume changes after the end of the treatment period due to resuspension in the isotonic buffer required for flow cytometry. The samples were kept on ice until flow cytometry was performed.

Backscattering measurements were made with the system described in Sec. 2.2, on confluent samples of CHO cells grown under standard conditions. At the beginning of the experiment, the growth medium was aspirated and replaced with clear Dulbecco's Modified Eagle's Medium (DMEM) supplemented with one of five different concentrations of mannitol: 50, 100, 200, 400, and 800 mm or with fresh DMEM. The three scattering measurements described in Sec. 2.2 were made before treatment, then at 15 min post-treatment, 30 min, 1, 2, 4, and 6 h to capture the onset and progression of changes in the optical signal during early apoptosis.

2.4.

Inhibiting Early Features of Apoptosis

To determine whether the early optical signal is, at least partially, due to AVD, we investigated whether the signal is blocked when AVD induced by staurosporine is inhibited using the Cl⁻ channel blocker 4,4^′-diisothiocyanostilbene-2,2^′-disulfonic acid (DIDS), which inhibits early manifestations of staurosporine-induced apoptosis.⁴² Confluent cultures of CHO cells were treated with complete DMEM supplemented with one of the following: a control volume of 0.2% dimethyl sulfoxide (DMSO), 2 μm staurosporine in DMSO, 2 μm staurosporine in DMSO plus 200 μm DIDS, or 200 μm DIDS alone. The presence or absence of apoptosis in cultures treated with staurosporine or staurosporine plus DIDS was determined using the Caspase–Glo assay described in Sec. 2.3. At the beginning of the treatment period, the growth medium was replaced by growth medium supplemented with one of the following: 0.2% DMSO, 200 μm DIDS, 2 μm staurosporine, or 2 μm staurosporine plus 200 μm DIDS. Caspase activity was measured at 4 and 24 h following treatment.

To assess relative cell volume, cells were prepared for flow cytometry according to the protocol in Sec. 2.3. Each sample was treated with one of the following: 0.2% DMSO, 2 μm staurosporine in DMSO, or 2 μm staurosporine in DMSO plus 200 μm DIDS. Each sample was then washed and resuspended in isotonic PBS. Cell volumes were measured at 30 min and 4 h following treatment.

The three scattering measurements described in Sec. 2.2 were performed before treatment, then at 15 min post-treatment, 30 min, 1, 2, 4, and 6 h.

3.1.

Modeling Water Loss with Mie Theory

To help quantify the effects of water loss on cellular scattering, a size distribution of scattering centers was extracted from ESS measurements taken on cell cultures treated with staurosporine. Size extraction was performed through least-squares fitting of the experimentally measured ESS spectrum to the wavelength-dependent Mie theory predictions for single particle sizes and the distributions of sizes described in Sec. 2.1 (see Fig. 2). Thirty minutes following apoptosis induction, when organelle-based morphology changes are expected to be minimal, the best fit spectrum to scattering from apoptotic cells (Fig. 5, left) is comprised of 6% contribution from the whole cell, 7% contribution from cell nuclei, and the remaining 87% results from other organelles. Each of the resulting contributions are reasonable and consistent with those in a cell before treatment occurs (data not shown), with most of the scattering due to the smaller subcellular structures, and modest scattering from the nuclei and the whole cell which are expected to scatter predominantly in the forward direction. This model fit suggests that backscattering measurements are sensitive to AVD at early times, with the increased contribution of the whole cell accounting for much of the scattering difference. Interestingly, at 4 h post-treatment (Fig. 5, right), while the cell has not regained any volume, the spectrum is comprised entirely of scattering from the smaller organelles. Currently, the reason for this is unknown, but may be a result of organelle-based apoptotic changes, including the loss of Rayleigh scatterers due to cytoskeletal breakdown, the remodeling of individual mitochondria as well as fragmentation of the mitochondrial network, or subtle changes in nuclear micromorphology due to chromatin condensation.

Fig. 5

The best-fit spectra (thick dotted) to ESS measurements of cultures treated with staurosporine (thick solid) for 30 m (left) and 4 h (right) using the Mie predictions shown in Fig. 2.

3.2.

Inducing Volume Loss in the Absence of Apoptosis

To separate the effects of volume decrease itself from any associated apoptotic events, the level of apoptosis (caspase activity) was assayed at 4 and 24 h post-treatment for cells treated with each concentration of mannitol, used to induce apoptosis with minimal apoptosis. The average and range of values of three measurements are presented in Fig. 6.

Fig. 6

Caspase-3 and caspase-7 activity in CHO cells treated with increasing concentrations of mannitol. The height of each bar is the average of the three measurements while the error bars span the range of measurements. The caspase activity of CHO cells treated with staurosporine is included for comparison.

The two lowest concentrations of mannitol produce caspase activity that is similar to the low level in the untreated control, and is significantly less than the level produced by staurosporine. From this we conclude that concentrations of 50 mm mannitol and 100 mm mannitol induce minimal apoptosis in CHO cells, and can be used to separate the optical effects of volume decrease from those of apoptosis.

To determine whether the strength of the early optical response correlates with the amount of volume decrease produced by treatment with hypertonic solutions of mannitol, the volumes of these cell populations were measured with flow cytometry. The intensity of forward scattering was measured for 40,000 cells in each sample, and a histogram was generated. Histograms for cultures treated with five concentrations of mannitol and an untreated culture at each time point are overlaid in Fig. 7.

Fig. 7

Histograms of cell volume for CHO cells treated with increasing concentrations of mannitol as measured by flow cytometry.

At 30 min of treatment, each of the cell populations treated with mannitol exhibited a shift to the left, indicating a decrease in mean cell volume. This decrease in volume is progressive with increasing concentration of mannitol. At 4 h post-treatment, the cells treated with 50 and 100 mm mannitol recovered much, but not all, of their volume, whereas the cells treated with 200 mm mannitol, have recovered only some of their volume by 4 h. In contrast, cultures treated with 400 and 800 mm mannitol did not exhibit any volume recovery, consistent with the high degree of apoptosis measured by caspase activity.

Scattering measurements were made on four sets of samples treated with varying concentrations of mannitol. The average spectra of the four trials from cultures treated with 50 mm (dashed) and 100 mm mannitol (dotted) for 1 h are plotted with the average spectrum of the untreated control (solid), in Fig. 8.

Fig. 8

ESS data obtained from CHO cells treated with 50 mm (dashed line) and 100 mm (dotted line) mannitol for 1 h.

Before the samples were treated, they all produced nearly identical scattered spectra. Within 30 min of the addition of hyperosmotic medium, each sample showed changes in scattering consistent with the early optical signal produced by staurosporine treatment (similar to that shown in Fig. 4). As the concentration of mannitol increased, both the magnitude and the kinetics of the observed optical signal increased (not all data shown).

3.2.1.

Discussion: mannitol produces similar early effects as staurosporine

Given that caspase activity is minimal in cultures treated with 50 and 100 mm mannitol (Fig. 6), it is possible to separate the optical effects of apoptosis from the effects of volume decrease in these populations. At early times, cells treated with higher concentrations of mannitol produce larger changes in scattering, and this is consistent with a measured greater degree of volume loss.

After the initial shrinkage due to osmotic perturbation, viable cells can regulate their volume, to initial levels through regulatory volume increase (RVI). RVI is associated with NaCl influx with flow of osmotically obliged water.^{10, 33} Our volume measurements show that cells with small volume perturbations recover their initial volume after a transient period of shrinkage. A recovery to the untreated state is also evident in the scattering spectra. In the case of cells treated with 50 or 100 mm mannitol, the transient spectral change is reversed by 2 h and at some time between 4 and 6 h, respectively (data not shown). These timescales are consistent with volume recovery measured by flow cytometry. This suggests that the optical recovery seen in the cells not undergoing apoptosis is due to a recovery of volume, and that the early signal was due to volume loss.

Prolonged cell shrinkage by hypertonic stress that cannot be compensated for with RVI leads to apoptosis in many cell types.¹² One study found that experimental manipulations that reduce the cell volume to 70% of the original volume for periods of at least 3 h are sufficient to induce apoptosis.³⁴ In the case of CHO cells, cultures treated with both 400 and 800 mm mannitol exhibit significant volume loss without volume recovery (Fig. 6). The lack of volume recovery correlates with the persistence of the scattering changes as well as the presence of apoptosis in cells treated with mannitol at these concentrations.

Given that volume decrease in the absence of apoptosis produces scattering changes qualitatively similar to the early apoptotic signal, it is likely that the early optical signal observed upon apoptosis induction is due, in part, to volume loss.

We do note, however, that the magnitude of the optical response seen in the case of osmotically-induced water loss is not as large as that of the response produced by induction of apoptosis. Figure 9a compares the volume measurements of cells treated with 2 μm staurosporine and cells treated with 50, 100, and 800 mm mannitol. The ESS spectra from cultures incubated with mannitol are shown in Fig. 9b, along with a spectrum from a culture treated to undergo apoptosis with staurosporine. While the addition of 50 mm mannitol produces a volume loss similar to that of the 2 μm staurosporine, the optical response is small by comparison. A larger volume loss is required to produce a comparable optical response. Furthermore, the addition of 800 mm mannitol, which has been shown to induce apoptosis in CHO cells at similar levels to 2 μm staurosporine, also produces a strong optical response. These results suggest that, while the optical effects of mannitol-induced volume decrease are similar to early effects of apoptosis induction, volume loss alone cannot explain the early optical signal produced by apoptosis inducers. It is likely that additional early ultrastructural organelle morphology changes also contribute to the early optical signal, which will be addressed in future work.

Fig. 9

(a) A comparison of volume lost upon apoptosis induction due to 2 μm staurosporine and osmotic stress produced by the addition of 50, 100, and 800 mm mannitol. The ESS signals produced by treatment with 50, 100, 800 mm mannitol, and 2 μm staurosporine are shown in (b).

These findings are consistent with a recent study, where a weak correlation was found between changes in the ratio of wide-to-narrow angle scattering upon apoptosis induction and cell shrinkage,²³ and alterations in mitochondria were proposed to be responsible for early scattering changes (1 h post-treatment) in cells where apoptosis was induced using staurosporine. It is possible that mitochondrial alterations during apoptosis may also contribute to the early changes in wavelength-dependent backscattering observed here, though further experiments will be necessary to determine this.

Additionally, we note that the volume measurements were made on cells that had been removed from their substrates and resuspended. Due to the normally-adherent nature of CHO cells, their morphologies are flattened [as depicted in Fig. 1a] during optical measurements, but are spherical once resuspended. While the flow cytometry measurements give an indication of relative volume loss, it is unclear how this volume reduction affects the scattering properties of cells with a flattened geometry, making comparisons between absolute volume loss and absolute optical signal magnitude difficult.

Finally, during apoptosis, cells lose adherence to the substrate and rounding up of the cell is commonly observed [Fig. 1b]. In this case, phase contrast imaging of CHO cells treated with either 2 μm staurosporine or 400 to 800 mm mannitol show some rounding up when compared to a normally propagating culture and those treated with 50 to 200 mm mannitol (data not shown). While it is difficult to quantify the amount of rounding up that occurs in each sample, if examined qualitatively, a stronger early optical signal is observed in cultures that have rounded up. The optical properties of these rounded-up cells may differ from cells that have undergone an equivalent osmotic volume loss but retain adherence, and this may contribute to differences seen in the magnitude of the early optical signal.

3.3.

Impact of Chloride Channel Inhibitor DIDS

3.3.1.

ESS measurements

Figure 10 displays the average spectra of the control samples (thin solid) as well as those treated with staurosporine (thick solid) and staurosporine plus DIDS (thin dotted). Over the course of the experiment, the cells treated with staurosporine produce a scattering spectrum with all the features of a signal from an apoptotic population (see early and late scattering changes in Fig. 4). In the cells treated with staurosporine plus DIDS, however, the initial early signal is absent, resulting in a scattered spectrum indistinguishable from the culture treated with DMSO alone.

Fig. 10

Average ESS measurements of CHO cells (n = 9) treated with DMSO only (thin solid), staurosporine (thick solid), and staurosporine plus the chloride channel blocker DIDS (thin dotted). Control samples containing DIDS alone showed no change in scattering spectrum and are not shown.

3.3.2.

Caspase activity

Figure 11 shows that cells treated with staurosporine show a marked increase in caspase activity compared to the DMEM, DMSO and DIDS-only control. Staurosporine induced a clear increase in caspase activity at both 4 and 24 h, while DIDS largely blocked the increase in activity by staurosporine at 4 h and attenuated it at 24 h.

Fig. 11

Caspase-3 and caspase-7 activity (n = 3) as measured fluorometrically for an untreated culture and cells treated with DMEM only, 0.2% DMSO only, 50 μm z- 200 μm DIDS only, 2 μm staurosporine only, and 2 μm staurosporine plus 200 μm DIDS. Activity levels are shown at 4 h and 24 h.

3.3.3.

Loss of cell volume during apoptosis

To assess the effects of staurosporine plus DIDS on cell volume, a flow cytometry analysis was performed. Volume distribution measurements from populations of 40,000 cells treated with DMEM (thin solid), 2 μm staurosporine (thick solid), or 2 μm staurosporine plus 200 μm DIDS (thin dotted) are shown in Fig. 12.

Fig. 12

Volume measurements of cell populations treated with 2μm staurosporine (thick solid), 2 μm staurosporine plus 200 μm DIDS (thin dotted), or left untreated in DMEM alone (thin solid).

At 30 min and 4 h after staurosporine treatment, the plot of forward scattering shows the expected volume reduction and a broadening of the distribution when compared to the untreated sample. Cells treated with both staurosporine and DIDS do not show any significant loss of volume at 30 min after treatment. At 4 h, they do begin to lose some volume, though the loss is less than that of the cells treated with staurosporine alone.