Quantitative morphological assessment of biological cells and their subcellular environment is important to characterize
cellular state in normal and diseased tissue and cellular response to various experimental treatments. Recently, we
showed that optical Gabor-like filtering of light scattered by spheres yields an optical measurement which varies linearly
with diameter. In addition, the sensitivity to changes in size was superior to post-processing of digital images. Here, we
extend our previous results by showing that the linear relationship between Gabor filter period and particle size holds
over a size range from 100nm to 2000nm. We also show that this relationship is independent of the particle's or
medium's refractive index. Using simulations, we provide a theoretical basis for our findings. Unlike previous methods,
this technique does not require the presence of single isolated particles and thus may be used to directly extract the
characteristic size associated with the local texture of heterogeneous objects. We therefore discuss this applicability of
our method in heterogeneous samples consisting of collagen and living cells.
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.
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