Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 µM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 µm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.
We combine optical scatter imaging (OSI) with fluorescence imaging of mitochondria to investigate the spatial relationship between the optical scatter signal and the location and structure of mitochondria within endothelial cells undergoing apoptosis. The OSI data corroborate our previous results showing a decrease in the intensity ratio of wide-to-narrow angle scatter [optical scatter image ratio (OSIR)] during the first 60 min of apoptosis. In addition, we find here that this is followed by an increase in OSIR concurrent with mitochondrial fragmentation. We demonstrate that the dynamic change in light scattering is spatially associated with subcellular regions containing fluorescently labeled mitochondria, and remains absent from adjacent nonfluorescent regions dominated by other organelles. These results lend strong support to the hypothesis that mitochondria act as the source of the optical scatter changes measured at the onset of apoptosis.
Light scattering methods for assessing structural properties of cells and tissues quantitatively measure
morphometric parameters directly without the need for staining. We demonstrate an optical scattering filtering method
used in a biological setting that is sensitive to quantifying object orientation and aspect ratio. These parameters are
measured in cells both sensitive to and resistant to mitochondrial-mediated apoptosis, the latter having been
demonstrated to have shorter mitochondria than apoptosis competent cells. The implementation of the digital
micromirror device (DMD) allows for robust filtering of the scatter data, which we implement with Gabor-like filters
chosen for their ability to intelligently confine the filter response both in the image and in the scatter regimes. By
strategically applying Gabor-like filters to the specific frequencies and orientations in the scatter data, relative changes in
object size, orientation and aspect ratio may be derived. Furthermore, using a DMD and filtering the optical scatter data
in analog allows us to decouple image resolution from frequency resolution and measure these parameters with high
sensitivity for objects within the resolution of the optical system despite an undersampled, lower resolution digital
image. As a result, this measurement may be made at lower magnifications with higher throughput and ultimately on a
larger population of living and unstained cells imaged simultaneously.
Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron
sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the
intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically
with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect
mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be
observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology
assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the
DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical
filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been
widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model
system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles
on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to
characterize the morphology of subcellular organelles to help understand fundamental biological processes.
Intrinsic optical properties of biological tissue can be modulated with specific genetic alterations, and used as a
phenotypic response to probe specific signaling pathways. Utilizing this approach, we used optical scatter imaging to
probe the effect of BCL-xL on subcellular particle size distribution within monolayers of living CSM14.1 and iBMK
cells. Expression of YFP-Bcl-xL shifted the center of the subcellular particle diameter distribution from 1.5μm to 2μm
in CSM 14.1 cells and from 1.5 to 1.8 μm in iBMK cells. This shift was also observed in cells expressing YFP-TM, in
which YFP is directly fused to the C-terminal transmembrane (TM) domain of BCL-xL, but not in cells expressing YFP
or YFP-BCL-xL-ΔTM, which lack the TM domain. YFP and YFP-BCL-xL-ΔTM were diffusely distributed in the
cytoplasm, while YFP-TM and YFP-BCL-xL were localized on the mitochondria. The measured particle sizes,
combined with the localization of the TM domain to the mitochondria, suggest that morphological disturbances of the
mitochondrial membrane effected by the TM domain of Bcl-xL, underlie the measured optical scatter changes. We have
also found that expression of BCL-2, another anti-apoptotic protein, in iBMK cells, results in a subcellular particle
diameter increase similar to that induced by BCL-xL. However, BCl-xL-ΔTM induced as much apoptosis resistance as
BCL-xL. Thus, mitochondrial morphology changes are not required for apoptosis resistance. Nonetheless, expression of
YFP-TM also conferred a moderate level of apoptosis resistance, while apoptosis resistant iBMK cells lacking Bax and
Bak, showed an increase in the light scattering contribution of particles less than 1.5 μm in diameter. Our results suggest
a possible secondary role of the BCL-xL TM domain in apoptosis resistance. However, the functional relationship
between mitochondrial morphology and apoptosis resistance remains to be fully elucidated.
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