Robert M. Pasternack

Attachment of 3-(Aminopropyl)triethoxysilane on silicon oxide surfaces: dependence on solution temperature.

Parameters important to the self-assembly of 3-(aminopropyl)triethoxysilane (APTES) on chemically grown silicon oxide (SiO 2) to form an aminopropyl silane (APS) film have been investigated using in situ infrared (IR) absorption spectroscopy. Preannealing to approximately 70 degrees C produces significant improvements in the quality of the film: the APS film is denser, and the Si-O-Si bonds between the molecules and the SiO 2 surface are more structured and ordered with only a limited number of remaining unreacted ethoxy groups. In contrast, post-annealing the functionalized SiO 2 samples after room temperature reaction with APTES (i.e., ex situ annealing) does not lead to any spectral change, suggesting that post-annealing has no strong effect on the horizontal polymerization as suggested earlier. Both IR and ellipsometry data show that the higher the solution temperature, the denser and thinner the APS layer is for a given immersion time. Finally, the APS layer obtained by preannealing the solution at 70 degrees C exhibits a better stability in deionized water than the APS layer prepared at room temperature.

Optical Scatter Imaging with a digital micromirror device

We had developed Optical Scatter Imaging (OSI) as a method which combines light scattering spectroscopy with microscopic imaging to probe local particle size in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter at each pixel in the full field of view. In this paper, we replace the variable diameter Fourier filter with a digital micromirror device (DMD) to extend our assessment of morphology to the characterization of particle shape and orientation. We describe our setup in detail and demonstrate how to eliminate aberrations associated with the placement of the DMD in a conjugate Fourier plane of our microscopic imaging system. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This improved OSI system could be further developed to automate morphological quantification and sorting of non-spherical particles in situ.

Optical scatter changes at the onset of apoptosis are spatially associated with mitochondria

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.

Highly sensitive size discrimination of sub-micron objects using optical Fourier processing based on two-dimensional Gabor filters

We use optical Gabor-like filtering implemented with a digital micromirror device to achieve nanoscale sensitivity to changes in the size of finite and periodic objects imaged at low resolution. The method consists of applying an optical Fourier filter bank consisting of Gabor-like filters of varying periods and extracting the optimum filter period that maximizes the filtered object signal. Using this optimum filter period as a measure of object size, we show sensitivity to a 7.5 nm change in the period of a chirped phase mask with period around 1 microm. We also show 30 nm sensitivity to change in the size of polystyrene spheres with diameters around 500 nm. Unlike digital post-processing our optical processing method retains its sensitivity when implemented at low magnification in undersampled images. Furthermore, the optimum Gabor filter period found experimentally is linearly related to sphere diameter over the range 0.46 microm-1 microm and does not rely on a predictive scatter model such as Mie theory. The technique may have applications in high throughput optical analysis of subcellular morphology to study organelle function in living cells.

Measurement of subcellular texture by optical Gabor-like filtering with a digital micromirror device

We demonstrate an optical Fourier processing method to quantify object texture arising from subcellular feature orientation within unstained living cells. Using a digital micromirror device as a Fourier spatial filter, we measured cellular responses to two-dimensional optical Gabor-like filters optimized to sense orientation of nonspherical particles, such as mitochondria, with a width around 0.45 microm. Our method showed significantly rounder structures within apoptosis-defective cells lacking the proapoptotic mitochondrial effectors Bax and Bak, when compared with Bax/Bak expressing cells functional for apoptosis, consistent with reported differences in mitochondrial shape in these cells. By decoupling spatial frequency resolution from image resolution, this method enables rapid analysis of nonspherical submicrometer scatterers in an undersampled large field of view and yields spatially localized morphometric parameters that improve the quantitative assessment of biological function.

Detection of mitochondrial fission with orientation-dependent optical Fourier filters

We utilize a recently developed optical imaging method based on Fourier processing with Gabor‐like filters to detect changes in light scattering resulting from alterations in mitochondrial structure in endothelial cells undergoing apoptosis. Imaging based on Gabor filters shows a significant decrease in the orientation of subcellular organelles at 60 to 100 minutes following apoptosis induction and concomitant with mitochondrial fragmentation observed by fluorescence. The optical scatter changes can be detected at low resolution at the whole cell level. At high resolution, we combine fluorescence imaging of the mitochondria with optical Fourier‐based imaging to demonstrate that the dynamic decrease in organelle orientation measured by optical Gabor filtering is spatially associated with fluorescent mitochondria and remains largely absent from nonfluorescent subcellular regions. These results provide strong evidence that the optical Gabor responses track mitochondrial fission during apoptosis and can be used to provide label‐free, rapid monitoring of this morphological process within single cells.

Quantifying subcellular dynamics in apoptotic cells with two-dimensional Gabor filters

We demonstrate an optical Fourier filtering method which can be used to characterize subcellular morphology during dynamic cellular function. In this paper, our Fourier filters were based on two-dimensional Gabor elementary functions, which can be tuned to sense directly object size and orientation. We utilize this method to quantify changes in mitochondrial and nuclear structure during the first three hours of apoptosis. We find that the technique is sensitive to a decrease in particle orientation consistent with apoptosis-induced mitochondrial fragmentation. The scattering signal changes were less pronounced in the nucleus and the remainder of the cytoplasm. Particles in these regions were less oriented than mitochondria and did not change orientation significantly.

Direct and highly sensitive measurement of the spatial arrangement of microstructures within biological samples

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 particles or mediums 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.

Optimizing Filter Design for Optical Scatter Microscopy

We show a method to quantify the morphology of biological structures by observing the sample through two-dimensional optical Fourier filters implemented on a liquid crystal device. We present filter configurations with sensitivity to sample texture.

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access. The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10s of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the objects field transform rather than the discretized objects intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view. In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.