2.1.

Homogeneous Aqueous Suspensions of Spheres

Monodisperse polystyrene spheres (Polysciences, Warrington, Pennsylvania) with diameters of 0.356, 0.548, 0.771, and $1.053\mu \mathrm{m}$ were suspended in water and adjusted to have scattering coefficient ${\mu }_{\mathrm{s}}\sim 0.01\mu {\mathrm{m}}^{-1}$ . The refractive index of polystyrene spheres is 1.6 and results in the index ratio $m=1.2$ $(=n_{\text{polystyrene}}∕n_{\text{water}}=1.6∕1.33)$ . The aqueous sphere suspensions were sandwiched between a microscope slide and a $22\times 22\text{-}\mathrm{mm}$ coverslip separated by $75\text{-}\mu \mathrm{m}$ -thick double-sided tape (Scotch^®, 3M), and then sealed with a 1:1:1v/v/v mixture of vaseline, lanolin, and paraffin (VALAP).

2.2.

Molecular Biology and Generation of Stable Cell Lines

Immortalized baby mouse kidney (iBMK) cells, W2 cells, and iBMK cells lacking Bax and Bak (D3 cells) were generously provided by the laboratory of Dr. E. White at Rutgers University. iBMK cells were maintained at $38°\mathrm{C}$ in a 5% $\mathrm{C}{\mathrm{O}}_2$ in-air atmosphere in high glucose Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (V:V), $100\text{-units}∕\mathrm{ml}$ penicillin, and $100\text{-}\mu \mathrm{g}∕\mathrm{ml}$ streptomycin. For microscopy, cells were cultured on glass cover slips, to which they readily attach. The preparation of iBMK cells that stably expressed YFP and YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ was described previously.¹⁹ Expression of YFP constructs was confirmed by fluorescence microscopy of the live cells. YFP fluorescence was detected with a YFP filter cube (Carl Zeiss Filter 46), excitation bandpass: $500\pm 10\mathrm{nm}$ , emission filters: $>515\mathrm{nm}$ long-pass dichroic filter, followed by a $535\pm 15\mathrm{nm}$ bandpass filter. Since the overexpressed Bcl- ${\mathrm{x}}_{\mathrm{L}}$ was linked to YFP, we used the YFP-transfected cells to control the effects of YFP transfection.

2.3.

Optical Scatter Image Acquisition and Analysis

The optical scatter imaging setup used here was described previously.¹⁹ The wavelength of the incident light on the sample was $630\mathrm{nm}$ $\pm 5\mathrm{nm}$ . The images were collected using an oil immersion objective, $\mathrm{NA}=1.4$ . In a Fourier plane conjugate with the objective’s back focal plane, a variable iris diameter with a center beam stop was switched between high numerical aperture (NA) and low NA positions, collecting light within a solid angle bound at the oil immersion objective by $3\mathrm{deg}<{\theta }_{\mathrm{HNA}}<90\mathrm{deg}$ for high NA, and $3\mathrm{deg}<{\theta }_{\mathrm{LNA}}<10\mathrm{deg}$ for low NA. The angles 3, 10, and $90\mathrm{deg}$ are set by the radius of the beam block in the center of the variable diameter iris, the edge of the iris used for the low NA acquisition, and the numerical aperture of the objective, respectively. These angles were measured using the diffraction pattern of a graticule with spacing $b=10\mu \mathrm{m}$ in the object plane. These scattering angle ranges, which were reported previously,¹⁹ are corrected here to account for the index mismatch between the aqueous culture medium $(\mathrm{n}=1.33)$ and the $63\times$ objective’s immersion oil $(\mathrm{n}=1.5)$ . When corrected for the index mismatch between water and oil, these angular ranges result in collecting light scattered by the sample within a solid angle bound by $3.38\mathrm{deg}<{\theta }_{\mathrm{HNA}}<90\mathrm{deg}$ for high NA, and $3.38\mathrm{deg}<{\theta }_{\mathrm{LNA}}<11.29\mathrm{deg}$ for low NA. As before, the ratio of high-to-low NA background-subtracted signal was measured at each pixel in the image and defined as the OSIR. The OSIR was subsequently converted to particle diameter, as described in the next section.

Each coverslip with attached live cells was mounted by means of a steel plate onto the stage of the inverted microscope. Just before mounting onto the microscope’s stage, the DMEM growth medium was replaced with Leibovitz L15 medium (Invitrogen, Carlsbad, California) supplemented with 10% fetal bovine serum, $100\text{-units}∕\mathrm{ml}$ penicillin, and $100\text{-}\mathrm{mg}∕\mathrm{ml}$ streptomycin. Leibovitz’s L15 was used to maintain the cells in a non- $\mathrm{C}{\mathrm{O}}_2$ -equilibrated environment and did not contain phenol red, whose color could cause light absorption. The cells were monitored by DIC, fluorescence, and optical scatter microscopy at room temperature and room air.

Optical scatter images were acquired in IPlab (BD Biosciences Bio-imaging, Rockville, Maryland) and processed in MatLab (The MathWorks, Natick, Massachusetts). In each experiment, a segment was manually defined around every cell in the differential interference contrast (DIC) images. These segments were overlaid onto the optical scatter images such that data analysis was limited to regions containing a cell. Only positively fluorescent cells were analyzed in the transfected cell variants. An OSIR threshold, 1.15 to 40, was applied and only pixels within this range were analyzed. The upper and lower thresholds were used to exclude values of OSIR that fell outside of the theoretical expected range. An upper value of 40 was conservatively chosen to account for noisy data. These threshold values exclude $\sim 20\mathrm{\%}$ of the pixels within a typical cell.

2.4.

Conversion of Optical Scatter Image Ratio Pixel Histograms to Particle Size Distributions

The optical scatter images directly encode at each pixel the measured value of the OSIR, corresponding to the intensity ratio of high-to-low NA scatter. For spheres, this intensity ratio was inferred from the Mie theory²¹ as:

Fig. 1

Numerical simulations of light scattering by spheres with index ratio $m=1.02$ (dot), $m=1.04$ (line), $m=1.1$ (dash), and $m=1.2$ (dash dot). The simulations were done with an $x$ -parameter increment of 0.1 for the range $x=0.1$ to 1, and an $x$ parameter increment of 1 for $x>1$ . $x=\pi Dn∕\lambda$ , $\lambda =0.630\mu \mathrm{m}$ , $n=1.33$ .

We used the $m=1.04$ data in Fig. 1 to convert the cell OSIR to diameter, and the $m=1.2$ curve to convert the sphere suspension OSIR data. Moreover, in the case of the polystyrene spheres with $m=1.2$ , the largest diameter tested was $1.053\mu \mathrm{m}$ to remain in the monotonically decreasing region of the curve. With refractive index ratio $m=1.04$ , the calculated OSIR based on the Mie theory decreases nonlinearly and monotonically from 34.65 to 1.15 as a function of sphere diameter for diameter values going from $0.01\text{to}2.38\mu \mathrm{m}$ . Beyond diameters of $2.38\mu \mathrm{m}$ , the OSIR value does not decrease monotonically but remains under 1.15. Below diameters of $0.01\mu \mathrm{m}$ , the calculated OSIR changes negligibly but remains above 34.65. Thus, cell pixels with OSIR less than 1.15 or OSIR greater than 34.65 were not considered in this histogram analysis. In the OSIR range 1.15 to 40, less than 0.05% of the total pixels exist between 34.65 and 40.

The histograms of pixel distribution as a function of diameter were further analyzed by converting the number of pixels at each diameter to number of particles $N$ to yield a particle size distribution. At each diameter $D$ the number of particles $N$ was calculated as $N=(\text{number}\text{of}\text{pixels}\times a)∕\pi (D^2∕4)$ , where $a$ is the area of one image pixel in the object plane, and making the simple assumption that the particles are on average sectioned across their midline in the plane of the image. The pixel area $a$ corresponded to a $0.49\mu \mathrm{m}\times 0.49\mu \mathrm{m}$ area in the object based on the image of a calibrated graticule. For the cell data, the number of particles at each diameter was normalized to the number of cells tested to yield a 2-D particle size distribution per cell. For all sphere samples, the tested sample area was $250\times 250\mu \mathrm{m}$ .

2.5.

Apoptosis Experiments

With the cells on the microscope stage, apoptosis was induced by replacing the L15 growth medium with the same supplemented with $0.4\mu \mathrm{M}$ staurosporine (STS) (Sigma Chemical, Saint Louis, Missouri) prepared from a $4\mathrm{mM}$ stock solution of STS in DMSO. Treatment with the same volume of DMSO in L15 growth medium, but without STS, was used as a negative control.

2.6.

Cell Death Assay

Cell death resistance was assessed by measuring the percentage of dead cells in response to STS treatment. Cells were cultured in 12-well plates, and treated with $0.4\mu \mathrm{M}$ STS at 90% confluence. After $24\mathrm{h}$ , $40$ -mM propidium iodide (Invitrogen) was added to the incubated cultures for $15\min$ . The cells were collected from the plates by pipetting and trituration. Microscopic observation of the plates insured that all cells were collected by this process. The cell suspension was concentrated to $\sim 5\times {10}^5\text{cells}∕\mathrm{ml}$ by centrifugation and partial removal of the supernatant. The number of cells with positive propidium iodide fluorescence in the final cell suspension was counted in a hemacytometer, and was taken as the number of dead cells that had lost membrane integrity. Propidium iodide fluorescence was visualized with a rhodamine filter cube (filter number 20, Carl Zeiss). The results from at least three different culture plates were analyzed under each condition.

3.1.

Effect of Bcl- ${\mathrm{x}}_{\mathrm{L}}$ and Bax/Bak on Staurosporine-Induced Cell Death

Figure 2 shows images of the transfected Bax/Bak expressing (W2) and Bax/Bak null (D3) iBMK cells. YFP expression resulted in diffuse fluorescence throughout the cells, while expression of YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ showed a filamentous pattern consistent with mitochondrial localization. To confirm the antiapoptotic effect of Bcl- ${\mathrm{x}}_{\mathrm{L}}$ , we challenged the cells with STS to induce apoptosis and measured the percentage of dead cells. After $24\mathrm{h}$ of treatment with $0.4\mu \mathrm{M}$ STS, the percentage of dead cells was $72.30\pm 4.09\mathrm{\%}$ and $75.01\pm 11.76\mathrm{\%}$ for W2 cells and W2 cells expressing YFP, respectively [Fig 2]. W2 cells overexpressing YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ were resistant to cell death with only $4.81\pm 2.18\mathrm{\%}$ dead cells. iBMK D3 cells, which lack the proapoptotic proteins Bax and Bak, and YFP expressing D3 cells showed only $14.59\pm 0.46\mathrm{\%}$ and $12.24\pm 2.21\mathrm{\%}$ cell death, respectively. Overexpression of YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ in the D3 cells further decreased cell death to $7.04\pm 1.53\mathrm{\%}$ . The inhibition of cell death by Bcl- ${\mathrm{x}}_{\mathrm{L}}$ overexpression and by knockout of Bax/Bak also supports the fact that apoptosis is the cell death process being induced by STS.

Fig. 2

(a) Fluorescence images of the transfected iBMK cell variants. W2 express Bax/Bak and D3 are Bax/Bak null. The transfectants stably expressed YFP or YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ . (b) Percentage of dead iBMK cells assessed by propidium iodide exclusion after $24\mathrm{h}$ of treatment with $0.4\text{-}\mu \mathrm{M}$ STS. Error bars represent the standard deviation of the mean of at least three different culture plates.

3.2.

Alterations in Light Scattering at Onset of Apoptosis

In a previous publication,² STS treatment caused a decrease in OSIR by $\sim 25\mathrm{\%}$ from baseline in nontransfected CSM 14.1 and YFP expressing CSM14.1 cells. In contrast, the OSIR decreased by less than 10% after STS treatment in the CSM cells expressing YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ . To investigate the reproducibility of these results, we performed the same experiment on the Bax/Bak expressing iBMK W2 cells and their transfected variants (Figs. 3 and 4 ). The OSIR per cell was first calculated by averaging the OSIR pixel values within each cell segment. In Fig. 3, the average OSIR per cell was also normalized to the initial OSIR value immediately after STS addition $(t=0)$ . The OSIR averages reported in Fig. 3 (red lines) and Fig. 4 are subsequently calculated by averaging the OSIR values per cell over the cell population under a given condition. The nontransfected W2 cells and YFP-W2 variants showed an average $\sim 32\mathrm{\%}$ and $\sim 20\mathrm{\%}$ OSIR decrease, respectively, and the YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ W2 variant showed only $\sim 8\mathrm{\%}$ decrease in the average OSIR per cell after treatment with STS for $60\min$ . Changes in OSIR were 3% after $60\min$ of DMSO treatment in the absence of STS. To further investigate the relationship between the initial OSIR decrease and apoptotic cell death, we measured the OSIR response to STS challenge in the apoptosis-resistant Bax/Bak null iBMK D3 cells. Nontransfected D3, YFP-D3, and YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ -D3 variants all showed a monotonic decrease in OSIR. The decrease in OSIR after $60\min$ of STS treatment was 26% for D3, 29% for YFP-D3, and 16% for YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ D3 cells. The slight increase in OSIR after $40\min$ for D3 and D3-YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ was not statistically significant. Individual OSIR cell traces (gray lines) are displayed in Fig. 3, along with the average (red lines in Fig. 3) for each cell variant treated with STS. Although the average responses of the nontransfected W2 and D3 cells are similar, the W2 cell responses are more heterogeneous. In both variants, the OSIR responses of some cells also increased first and then decreased. The nonnormalized initial OSIR values at $t=0$ just prior to STS treatment and final values after $60\min$ of STS treatment are shown in Fig. 4 for all the cell variants. The decrease in OSIR in the W2 cells and its inhibition by overexpression of Bcl- ${\mathrm{x}}_{\mathrm{L}}$ reproduce the trends observed previously in the CSM14.1 cells.² However, unlike the previously tested CSM 14.1 cells, the YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ expressing W2 cells had a similar initial OSIR compared with their parental W2 counterparts.

Fig. 3

Relative OSIR changes in individual cells (gray lines) treated with STS. The red lines are the average responses of all the cells tested (the number of cells tested in each case is reported on each panel). For each cell (each individual gray trace), the OSIR value at a given time point corresponds to the average OSI pixel value within that cell’s segment normalized to the initial average OSI pixel value at $\text{time}=0\min$ . just before STS treatment. The red traces show the mean ± the standard error of the mean of these normalized average OSIR values calculated on a cell-by cell basis. (Color online only.)

Fig. 4

Average OSIR values per cell before treatment (black bars, $0\min$ ) and $60\min$ after treatment with either DMSO or STS (gray bars). Data are the mean OSIR per cell ± the standard deviation of the mean. The star indicates a $p$ value $<0.001$ based on a Student $t$ -test (comparing values at $0\min$ with those at $60\min$ in each case).

3.3.

Alterations in Light Scattering versus Cell Morphology

STS is a general kinase inhibitor^{22, 23} and causes cell shrinkage. We first investigated cell shrinkage by observing cellular morphology with differential interference contrast (DIC) and compared these images with the optical scatter images of the different cell variants before and after $60\min$ of STS treatment (Fig. 5 ). All variants, except the W2 and D3 cells expressing YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ , appeared shrunken after STS challenge. The fact that the D3 YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ cells did not shrink but still showed an OSIR decrease in response to STS suggests that the OSIR decrease is not due to the shrinkage of the cells but is more likely due to the morphological alteration of subcellular organelles. To further confirm that the OSIR decrease is not correlated with cell shrinkage, we plotted the percentage OSIR decrease versus percentage cell shrinkage between 0 and $60\min$ for all the cells tested (Fig. 6 ). The decrease in cell area was estimated by counting the number of pixels with “active” OSIR values between 1.15 and 40 within each cell. While this excludes some background regions within the cells, this number of pixels was linearly correlated (correlation value $>0.9$ ) with the area of the cell measured by segmentation of the DIC images. Thus, we used the percent decrease in the number of active OSIR pixels as a measure for cell shrinkage. The plot in Fig. 6 shows little correlation between the decrease in OSIR and cell shrinkage with low correlation values $\left(R^2\right)$ , whether a linear or polynomial fit to the data is used ( $R^2=0.15$ and $R^2=0.22$ , respectively). Moreover, STS-induced cell shrinkage is not related to sensitivity or resistance to apoptosis, since both the W2 and D3 cells shrunk in the first $60\min$ of STS treatment.

Fig. 5

DIC and OSI images of nontransfected, YFP-expressing, and YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ -expressing W2 and D3 cells before $(t=0\min )$ and after $(t=60\min )$ treatment with $0.4\mu \mathrm{M}$ STS.

Fig. 6

OSIR decrease versus cell shrinkage after $60\min$ of STS treatment. The red dashed line is a linear fit to the data, and the blue dashed line is a third order polynomial fit. Each black dot represents a single cell (data include all W2, D3, and variants). (Color online only.)

After $24\mathrm{h}$ of treatment, all cells, including the propidium iodide negative or apoptosis resistant cells, were rounded up (data not shown) and could be collected easily by pipetting up and down. Thus, Bcl- ${\mathrm{x}}_{\mathrm{L}}$ only delayed the cell shrinkage induced by STS, and this effect does not depend on Bax/Bak, as the cell shrinkage was delayed in both W2 and D3 cell variants.

3.4.

Estimated Particle Size Distribution of Spheres in Aqueous Suspensions

The OSIR pixel histograms were converted to particle size distributions as described in Sec. 2.4 to get an estimate of subcellular particle size. We first applied the method to aqueous polystyrene spheres as model particles of known size. Figure 7 shows the pixel histograms obtained from the optical scatter images of the spheres [Fig. 7]. Figure 7 shows the corresponding particle diameter distributions obtained from the conversion of the pixel histograms. The mean $\text{diameter}\pm \text{standard}$ deviation of 0.356, 0.548, 0.771, and $1.053\text{-}\mu \mathrm{m}$ spheres are $0.332\pm 0.044$ , $0.521\pm 0.072$ , $0.638\pm 0.151$ , and $0.800\pm 0.212\mu \mathrm{m}$ , respectively. The peak histogram values were 0.357, 0.529, 0.669, and $0.83\mu \mathrm{m}$ . Fitting the distributions to a Gaussian, we retrieved means and standard deviations of $0.361\pm 0.096$ , $0.538\pm 0.121$ , $0.701\pm 0.211$ , and $0.929\pm 0.309\mu \mathrm{m}$ $(\mu \pm \sigma )$ , respectively. These values were more representative of the actual sphere diameters, especially for larger diameters where the measured particle distribution is skewed to the right due to the inability to interpret the lower OSIR values outside the dynamic range. The histograms show good discrimination between the different particle sizes. However, the variance in the estimated diameter increases from $\sim 18$ to 30% as particle diameter increases and is significantly larger than the expected variance of 2 to 3% given by the sphere manufacturer. The measured variance in OSIR pixel value across the image of a given sphere suspension [Fig. 7] is the initial source of these errors. While the mean OSIR value measured is representative of the sphere size, image pixel values can vary significantly due to movement of the spheres and diffraction in cases where the pixel size is smaller than the particle’s geometric cross section. The increase in variance for larger particles is likely due to the “flattening” of the OSIR decrease at high diameters (Fig. 1). Thus, small changes in OSIR at diameters greater than $0.8\mu \mathrm{m}$ correspond to large relative changes in size, where as large OSIR changes for diameters less than $0.5\mu \mathrm{m}$ correspond to small changes in size.

Fig. 7

(a) OSI images of aqueous polystyrene sphere suspensions with diameters 0.356, 0.548, 0.771, and $1.053\mu \mathrm{m}$ . (b) OSI pixel histograms corresponding to the images shown in (a). (c) Particle diameter distributions obtained from the OSI pixel histograms. The distributions are shown normalized to the maximum number of particles.

3.5.

Estimated Subcellular Particle Size Distributions

Figures 8 and 8 show the pixel histograms of the W2 and D3 variants, respectively. The solid lines are the histograms before STS treatment $(t=0\min )$ and the dotted lines are the histograms after $60\min$ of treatment with STS. The OSIR pixel histograms are shown after normalization to the number of cells. The histograms were converted to particle size distributions [Figs. 8 and 8]. Figures 9 and 9 show the means and standard deviations of the particle size distributions as a function of time after treatment. The subcellular particle size distributions are broad with standard deviations on the order of 40%. Consistent with the average OSIR decrease measured on the cell-by-cell basis (Figs. 3 and 4), the mean of the histogram, which gives an estimate of mean subcellular particle diameter, increases for the W2 $\left(0.95\text{to}1.40\mu \mathrm{m}\right)$ and D3 $\left(1.08\text{to}1.53\mu \mathrm{m}\right)$ cells and their YFP transfected counterparts ( $1.12\text{to}1.32\mu \mathrm{m}$ for W2-YFP and $1.08\text{to}1.42\mu \mathrm{m}$ for D3_YFP) after treatment with STS for $60\min$ . The mean diameter remains largely unchanged for the DMSO treated cells $\left(1.04\text{to}1.08\mu \mathrm{m}\right)$ and the W2 cells transfected with YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ $\left(1.05\text{to}1.14\mu \mathrm{m}\right)$ . In contrast, the histogram mean increased for the YFP-Bcl- ${\mathrm{x}}_{\mathrm{L}}$ -D3 cells $\left(0.99\text{to}1.15\mu \mathrm{m}\right)$ , but this increase was less pronounced than that observed in the untransfected D3 or YFP-D3 cells. Analysis of the histograms also retrieved a population of particles with sizes smaller than $100\mathrm{nm}$ , corresponding to pixels with OSIR values above $\sim 34.65$ . Due to the sparse number of particles in this size range, it was difficult to discern differences between the cell variants. However, the data suggest that there may be an overall lower number of particles in this nanoscale size range in the Bax/Bak null D3 cells compared with the W2 cells.

Fig. 8

OSI pixel histograms of (a) W2 and (b) D3 cell variants shown before (solid lines) and after (dotted lines) $60\min$ of treatment with STS or DMSO. Particle size distribution of W2 (c) and D3 (d) cell variants before (solid lines) and after (dotted lines) $60\min$ of treatment with STS or DMSO. The distributions were obtained by converting the corresponding histograms in (a) and (b). In (c) and (d) we included an inset to magnify the histograms for diameters above $0.3\mu \mathrm{m}$ .

Fig. 9

(a) Means and (b) standard deviations of the subcellular particle size distributions as a function of time.