Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels (original) (raw)
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Confocal light absorption and scattering spectroscopic microscopy
Applied Optics, 2007
We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with lightscattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.
Noninvasive sizing of subcellular organelles with light scattering spectroscopy
IEEE Journal of Selected Topics in Quantum Electronics, 2003
A long-standing impediment for applications of optical techniques in cellular biology is the inability to characterize subcellular structures whose dimensions are much less than about 1 m. In this paper, we describe a method based on light scattering spectroscopy that can find the size distribution of subcellular organelles as small as 100 nm with an accuracy of 20 nm. We report experiments using aqueous suspensions of subcellular organelles enriched in mitochondria, zymogen granules, and microsomes. From the observed light scattering spectra, we extract size distributions that are in excellent agreement with the results of electron microscopy. Further studies are underway to extract the shapes of organelles in addition to their sizes.
2002
Light scattering spectroscopy (LSS) is a promising optical technique developed for quantitative characterization of tissue morphology as well as in vivo detection and diagnosis of disease such as early cancer. LSS employs a wavelength dependent component of light scattered by epithelial cells and other tissues to obtain information about subcellular structure. We present two novel modalities of LSS, LSS imaging and scattering angle sensitive LSS (a/LSS). LSS imaging provides quantitative information about the epithelial cell nuclei, such as nuclear size, degree of pleomorphism, hyperchromasia, and amount of chromatin. It allows mapping these histological properties over wide areas of epithelial lining. We show that LSS imaging can be used to detect precancerous lesions in optically accessible organs. Using a/LSS, which enables characterization of tissue components with sizes smaller than the wavelength of light, we show that the number of subcellular components with the sizes between 30 nm and few microns scales with the size according to an inverse power-law. We show that the size distribution exponent is an important parameter characterizing tissue organization, for example the balance between stochasticity and order, and has a potential to be applicable for early cancer diagnosis and characterization.
Assessing light scattering of intracellular organelles in single intact living cells
Optics Express, 2009
We report a method of assessing the contribution of whole cell body and its nucleus to the clinically most relevant backward light scattering. We first construct an experimental system that can measure forward scattering and use the system to precisely extract the optical properties of a specimen such as the refractive index contrast, size distribution, and their density. A system that can simultaneously detect the backscattered light is installed to collect the backscattering for the same specimen. By comparing the measured backscattering spectrum with that estimated from the parameters determined by the forward scattering experiment, the contribution of cell body and nucleus to the backward light scattering is quantitatively assessed. For the HeLa cells in suspension, we found that the cell body contributes less than 10% and cell nucleus on the order of 0.1% to the total backscattering signal. Quantitative determination of the origin of backscattered light may help design a system that aims for detecting particular structure of biological tissues.
Optical Biopsy IV, 2002
Light scattering spectroscopy (LSS) is a promising optical technique developed for quantitative characterization of tissue morphology as well as in vivo detection and diagnosis of disease such as early cancer. LSS employs a wavelength dependent component of light scattered by epithelial cells and other tissues to obtain information about subcellular structure. We present two novel modalities of LSS, LSS imaging and scattering angle sensitive LSS (a/LSS). LSS imaging provides quantitative information about the epithelial cell nuclei, such as nuclear size, degree of pleomorphism, hyperchromasia, and amount of chromatin. It allows mapping these histological properties over wide areas of epithelial lining. We show that LSS imaging can be used to detect precancerous lesions in optically accessible organs. Using a/LSS, which enables characterization of tissue components with sizes smaller than the wavelength of light, we show that the number of subcellular components with the sizes between 30 nm and few microns scales with the size according to an inverse power-law. We show that the size distribution exponent is an important parameter characterizing tissue organization, for example the balance between stochasticity and order, and has a potential to be applicable for early cancer diagnosis and characterization.
Microscope laser light scattering spectroscopy of single biological cells
Cell biophysics, 1985
A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 #J,m) 3. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method. Index Entries: Microscope laser light scattering spectroscopy, of cells; laser light scattering spectroscopy, of cells; light scattering spectroscopy, of cells by laser microscopy; spectroscopy of cells, microscope laser light scattering; cells, laser light scattering spectroscopy of.
Field-based angle-resolved light-scattering study of single live cells
2008
We perform field-based angle-resolved light-scattering measurements from single live cells. We use a laser interferometer to acquire phase and amplitude images of cells at the image plane. The angular scattering spectrum is calculated from the Fourier transform of the field transmitted through the cells. A concurrent 3D refractive index distribution of the same cells is measured using tomographic phase microscopy. By measuring transient increases in light scattering by single cells during exposure to acetic acid, we correlate the scattering properties of single cells with their refractive index distributions and show that results are in good agreement with a model based on the Born approximation.
Optical scatter imaging: subcellular morphometry in situ with Fourier filtering
Optics Letters, 2001
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 camera's spatial resolution.
Light scattering spectrophotometric device for cellular structure characterization
Light scattering spectrophotometry is a new optical-probe technique suited for accurately measuring of in situ cellular structure features. This paper presents an original experimental optical device (design and construction) witch is based on the Mie light scattering theory and microscopically control of the investigated field.
arXiv (Cornell University), 2017
Here we report a method for visualization of volumetric structural information of live biological samples with no exogenous contrast agents. The process is made possible through a technique that involves generation, synthesis and analysis of three-dimensional (3D) Fourier components of light diffracted by the sample. This leads to the direct recovery of quantitative cellular morphology with no iterative procedures for reduced computational complexity. Combing with the fact that the technique is easily adaptive to any imaging platform and requires minimum sample preparation, our proposed method is particularly promising for observing fast, volumetric and dynamic events previously only accessible through staining methods. Main Text: Imaging and visualization of the three-dimensional (3D) structural information in cellular and sub-cellular environments will profoundly influence the way we perceive the underlying mechanisms of the complex biological world. At present, 3D imaging techniques usually involve exogenous fluorescent dyes. A problem with using dyes, however, is that it is sometimes undesirable to stain cells for certain important biomedical research topics, including stem cell and fertility studies, and the process can become difficult when multiple fluorophores are involved. On the other hand, there has been considerable recent interest in developing highresolution, 3D, label-free imaging techniques. Indeed, while fluorescent dye has the capacity to target specific cellular components, with a high degree of reliability, rapidly obtaining highresolution volumetric data with intrinsic contrast is particularly desirable in providing complementary information about the background biological context (1). For label-free techniques, the image contrast is intrinsic, arising from spatial variations in the optical refractive index (RI denoted as n) of the object. Although label-free imaging techniques span various electromagnetic (EM) wavelengths including infrared (IR) and/or near-infrared (NIR), excitation using non-linear optics (2) and x-rays (3,4) as well as electrons (5), visible light (VIS) is more readily adaptable to existing biological laboratory infrastructure and is relatively non-destructive to biological samples. In this case, however, the effects of light diffraction need to be taken into account to obtain a more accurate representation of the propagation of light through such objects. Here, we present a new approach to optical tomographic imaging where unique quantitative spatial mapping of subcellular structures is demonstrated. This enables localized functional interpretations in relative biomedical studies, hence bridging the significant gap in traditional imaging between function and structure. The proposed modality provides structural information in full 3D, with no iterative procedures nor any staining.