Measurement of Single Cell Refractive Index, Dry Mass, Volume, and Density Using a Transillumination Microscope

Phillips, Kevin G.; Jacques, Steven L.; McCarty, Owen J. T.

doi:10.1103/physrevlett.109.118105

Cited by 145 publications

(115 citation statements)

References 15 publications

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“…The principle of NIQPM and its application to quantifying the spatial cellular density map in a cell has been established and described elsewhere. 15,17,[26][27][28][29][30] Briefly, NIQPM consists of an image acquisition step and a postprocessing procedure to determine the phase profile of optical waves transmitted through the specimen. Through-focus intensity measurements [ Fig.…”

Section: Noninterferometric Quantitative Phase Microscopymentioning

confidence: 99%

Network signatures of nuclear and cytoplasmic density alterations in a model of pre and postmetastatic colorectal cancer

et al. 2014

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Abstract. Cells contributing to the pathogenesis of cancer possess cytoplasmic and nuclear structural alterations that accompany their aberrant genetic, epigenetic, and molecular perturbations. Although it is known that architectural changes in primary and metastatic tumor cells can be quantified through variations in cellular density at the nanometer and micrometer spatial scales, the interdependent relationships among nuclear and cytoplasmic density as a function of tumorigenic potential has not been thoroughly investigated. We present a combined optical approach utilizing quantitative phase microscopy and partial wave spectroscopic microscopy to perform parallel structural characterizations of cellular architecture. Using the isogenic SW480 and SW620 cell lines as a model of pre and postmetastatic transition in colorectal cancer, we demonstrate that nuclear and cytoplasmic nanoscale disorder, micron-scale dry mass content, mean dry mass density, and shape metrics of the dry mass density histogram are uniquely correlated within and across different cellular compartments for a given cell type. The correlations of these physical parameters can be interpreted as networks whose nodal importance and level of connection independence differ according to disease stage. This work demonstrates how optically derived biophysical parameters are linked within and across different cellular compartments during the architectural orchestration of the metastatic phenotype.

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Section: Noninterferometric Quantitative Phase Microscopymentioning

confidence: 99%

Network signatures of nuclear and cytoplasmic density alterations in a model of pre and postmetastatic colorectal cancer

et al. 2014

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“…However, the DM measurement is limited by knowledge of α which is often taken as a constant, assumed value [13][14][15]. Several analyses of cell properties have been presented based on evaluating dry mass using quantitative phase imaging [16][17][18]. These studies of dry mass are ultimately limited by knowledge of α and in some scenarios, it may be preferable to leave the analysis in terms of optical volume.…”

Section: Introductionmentioning

confidence: 99%

Influence of defocus on quantitative analysis of microscopic objects and individual cells with digital holography

Rinehart

Park

Wax

2015

Biomed. Opt. Express

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Digital holography offers a unique method for studying microscopic objects using quantitative measurements of the optical phase delays of transmitted light. The optical phase may be integrated across the object to produce an optical volume measurement, a parameter related to dry mass by a simple scaling factor. While digital holography is useful for comparing the properties of microscopic objects, especially cells, we show here that quantitative comparisons of optical phase can be influenced by the focal plane of the measurement. Although holographic images can be refocused digitally using Fresnel propagation, ambiguity can result if this aspect is not carefully controlled. We demonstrate that microscopic objects can be accurately profiled by employing a digital refocusing method to analyze phase profiles of polystyrene microspheres and red blood cells.

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“…DIC was commercialized by Zeiss in 1965in close collaboration with Nomarski 6 . In 1981, two laboratories demonstrated the first recorded live cell DIC imagery with the incorporation of video cameras into the optics train of the DIC microscope 7,8 , and bright field [18][19][20][21][22] to determine optical path-length, phase, mass density, refractive index, and cellular volume.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

Phillips

Baker‐Groberg

McCarty

2014

JoVE

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We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using "off-the-shelf" microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods. Video LinkThe video component of this article can be found at

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Measurement of Single Cell Refractive Index, Dry Mass, Volume, and Density Using a Transillumination Microscope

Cited by 145 publications

References 15 publications

Network signatures of nuclear and cytoplasmic density alterations in a model of pre and postmetastatic colorectal cancer

Network signatures of nuclear and cytoplasmic density alterations in a model of pre and postmetastatic colorectal cancer

Influence of defocus on quantitative analysis of microscopic objects and individual cells with digital holography

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

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