Field-based angle-resolved light-scattering study of single live cells

G. R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139, USA.
Optics Letters (Impact Factor: 3.29). 07/2008; 33(14):1596-8. DOI: 10.1364/OL.33.001596
Source: PubMed


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.

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    • "This approximation is often applied to biological cells [32] [57] [39] [40], interstellar dusts [35] [58] [59] and atmospheric aerosols [60] [61] [62]. In the case of biological cells [32] [33] [34], as in many other situations [35], it mainly produces errors on the energy scattered at large angles (i.e. a very small proportion of the total energy scattered by microorganisms), which explains the successful use of this approximation for the study of microorganisms' cross-sections in oceanography. In the following, we therefore consider that microorganisms are homogeneous and we define a single complex refractive index m "
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    ABSTRACT: A generic methodological chain for the predictive calculation of the light-scattering and absorption properties of photosynthetic microorganisms within the visible spectrum is presented here. This methodology has been developed in order to provide the radiative properties needed for the analysis of radiative transfer within photobioreactor processes, with a view to enable their optimization for large-scale sustainable production of chemicals for energy and chemistry. It gathers an electromagnetic model of light-particle interaction along with detailed and validated protocols for the determination of input parameters: morphological and structural characteristics of the studied microorganisms as well as their photosynthetic-pigment content. The microorganisms are described as homogeneous equivalent-particles whose shape and size distribution is characterized by image analysis. The imaginary part of their refractive index is obtained thanks to a new and quite extended database of the in vivo absorption spectra of photosynthetic pigments (that is made available to the reader). The real part of the refractive index is then calculated by using the singly subtractive Kramers–Krönig approximation, for which the anchor point is determined with the Bruggeman mixing rule, based on the volume fraction of the microorganism internal-structures and their refractive indices (extracted from a database). Afterwards, the radiative properties are estimated using the Schiff approximation for spheroidal or cylindrical particles, as a first step toward the description of the complexity and diversity of the shapes encountered within the microbial world. Finally, these predictive results are confronted to experimental normal-hemispherical transmittance spectra for validation. This entire procedure is implemented for Rhodospirillum rubrum, Arthrospira platensis and Chlamydomonas reinhardtii, each representative of the main three kinds of photosynthetic microorganisms, i.e. respectively photosynthetic bacteria, cyanobacteria and eukaryotic microalgae. The obtained results are in very good agreement with the experimental measurements when the shape of the microorganisms is well described (in comparison to the standard volume-equivalent sphere approximation). As a main perspective, the consideration of the helical shape of Arthrospira platensis appears to be a key to an accurate estimation of its radiative properties. On the whole, the presented methodological chain also appears of great interest for other scientific communities such as atmospheric science, oceanography, astrophysics and engineering.
    Journal of Quantitative Spectroscopy and Radiative Transfer 08/2015; 161. DOI:10.1016/j.jqsrt.2015.03.025 · 2.65 Impact Factor
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    • "Furthermore, it may account for differences caused by the time-dependent effect of acetic acid on tissue scattering and absorption. The vasoconstrictive and light scattering effects of acetic acid [35,36] may affect the extracted hemoglobin concentration, effective blood vessel radius, and also the scattering parameters. Recent work in our laboratory, which uses the same data set described in this paper, showed that without parameter normalization, HSIL sites could be differentiated from non-HSIL with AUC, sensitivity, and specificity of only 0.68, 78% and 67%, respectively [29]. "
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    ABSTRACT: This study develops a spectroscopic algorithm for detection of cervical high grade squamous intraepithelial lesions (HSILs). We collected reflectance and fluorescence spectra with the quantitative spectroscopy probe to measure nine spectroscopic parameters from 43 patients undergoing standard colposcopy with directed biopsy. We found that there is improved accuracy for distinguishing HSIL from non-HSIL (low grade SIL and normal tissue) when we "normalized" spectroscopy parameters by dividing the values extracted from each clinically determined suspicious site by the corresponding value extracted from a clinically normal squamous site from the same patient. The "normalized" scattering parameter (A) at 700nm, best distinguished HSIL from non-HSIL with sensitivity and specificity of 89% and 79% suggesting that a simple, monochromatic instrument measuring only A may accurately detect HSIL.
    Biomedical Optics Express 10/2011; 2(10):2917-25. DOI:10.1364/BOE.2.002917 · 3.65 Impact Factor
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    ABSTRACT: Conventional interferometric microscopy techniques such as digital holographic microscopy and quantitative phase microscopy are often classified as 3D imaging techniques because a recorded complex field image can be numerically propagated to a different depth. In a strict sense, however, a single complex field image contains only 2D information on a specimen. The measured 2D image is only a subset of the 3D structure. For the 3D mapping of an object, multiple independent 2D images are to be taken, for example at multiple incident angles or wavelengths, and then combined by the so-called optical diffraction tomography (ODT). In this Letter, tomographic phase microscopy (TPM) is reviewed that experimentally realizes the concept of the ODT for the 3D mapping of biological cells in their native state, and some of its interesting biological and biomedical applications are introduced.
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