Nada N. Boustany

Microscopic Imaging and Spectroscopy with Scattered Light

Optical contrast based on elastic scattering interactions between light and matter can be used to probe cellular structure, cellular dynamics, and image tissue architecture. The quantitative nature and high sensitivity of light scattering signals to subtle alterations in tissue morphology, as well as the ability to visualize unstained tissue in vivo, has recently generated significant interest in optical-scatter-based biosensing and imaging. Here we review the fundamental methodologies used to acquire and interpret optical scatter data. We report on recent findings in this field and present current advances in optical scatter techniques and computational methods. Cellular and tissue data enabled by current advances in optical scatter spectroscopy and imaging stand to impact a variety of biomedical applications including clinical tissue diagnosis, in vivo imaging, drug discovery, and basic cell biology.

Optical scatter imaging: subcellular morphometry in situ with Fourier filtering

We demonstrate a quantitative optical scatter imaging (OSI) technique, based on Fourier filtering, for detecting alterations in the size of particles with wavelength-scale dimensions. We generate our scatter image by taking the ratio of images collected at high and low numerical aperture in central dark-field microscopy. Such an image spatially encodes the ratio of wide to narrow angle scatter and hence provides a measure of local particle size. We validated OSI on sphere suspensions and live cells. In live cells, OSI revealed biochemically induced morphological changes that were not apparent in unprocessed differential interference contrast images. Unlike high-resolution imaging methods, OSI can provide size information for particles smaller than the cameras spatial resolution.

A ZnO Nanostructure-Based Quartz Crystal Microbalance Device for Biochemical Sensing

We report a ZnO-nanostructure-based quartz crystal microbalance (nano-QCM) device for biosensing applications. ZnO nanotips are directly grown on the sensing area of a conventional QCM by metalorganic chemical vapor deposition (MOCVD). Scanning electron microscopy (SEM) shows that the ZnO nanotips are dense and uniformly aligned along the normal to the substrate surface. By using superhydrophilic nano-ZnO surface, more than tenfold increase in mass loading sensitivity of the nano-QCM device is achieved over the conventional QCM. The ZnO nanotip arrays on the nano-QCM are functionalized. The selective immobilization and hybridization of DNA oligonucleotide molecules are confirmed by fluorescence microscopy of the nano-QCM sensing areas.

Ultraviolet Resonance Raman Spectroscopy of Bulk and Microscopic Human Colon Tissue

A sensitive ultraviolet resonance Raman (UVRR) system, which integrates bulk and microscopic capabilities, is described for use with human tissue. The system incorporates a quasi-continuous tunable laser excitation source in the range 200–300 nm, a liquid nitrogen-cooled charge-coupled device (CCD), a single grating spectrometer, dielectric longpass laser line rejection filters, and cooled specimen holders to minimize bio- and photo-degradation. The system was used on normal human colon tissue, and was compared to our previously used Nd:YAG-based system. Spectra with higher signal-to-noise ratio (S/N) than has previously been achieved, and with less than 5% photobleaching, are presented. The present system outperforms our previous system by improving the S/N in bulk normal colon spectra by a factor of greater than 10. We show for the first time UVRR spectra collected from the various microscopic regions of human colon in a thin section preparation, and the line shapes from the different microscopic morphological components are related to the bulk colon spectra. At 251 nm the epithelial cells and the lamina propria are the major contributors to the UVRR spectrum of bulk colon tissue. Our data suggest that combined bulk and microscopic UVRR spectroscopy of human tissue could provide a valuable tool for bioassay and histochemistry.

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.

Raman spectroscopy for cancer detection: instrument development and tissue diagnosis

Raman spectroscopy can provide quantitative molecular information about the biochemical composition of human tissues exhibiting various stages of disease. Fluorescence interference is ubiquitous in Raman spectra of biological samples excited with visible light. However, it can be avoided by using near-infrared (NIR) or ultraviolet (UV) excitation. We are exploring the potential of these methods for detecting precancerous/cancerous changes in human tissues. The NIR studies use 830 nm excitation from a Ti:sapphire laser. Raman signals are collected by an imaging spectrograph/deep-depletion CCD detection system. High quality tissue spectra can be obtained in a few seconds or less. The UV resonance Raman studies employ wavelengths below 300 nm for selective excitation of nucleic acids, proteins and lipids. Excitation is provided by a frequency tripled/quadrupled mode-locked Ti:sapphire laser, and Raman light is collected by a one meter spectrograph/UV-enhanced CCD detector. The two systems can be coupled to appropriate microscopes for extracting morphological and biochemical information at the cellular level, which is important for understanding the origin of the Raman spectra of bulk tissue. The results of the initial studies for cancer detection in various human tissues are reported here.

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.