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

Phillips, Kevin G.; Baker‐Groberg, Sandra M.; McCarty, Owen J. T.

doi:10.3791/50988

Cited by 9 publications

(12 citation statements)

References 31 publications

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“…18 Briefly, cellular density of neutrophils was quantified using NIQPM on an upright optical microscope (Axio Imager; Carl Zeiss, Gottingen, Germany) equipped with a ×63/1.4 numerical aperture (NA) oil immersion objective and an air-coupled condenser lens providing Köhler illumination at an NA of 0.1. Through-focus bright field imagery of the cells was acquired using an illumination wavelength of 540 ± 20 nm.…”

Section: Methodsmentioning

confidence: 99%

“…Knowledge of the phase of transmitted waves through the sample enables the enumeration of the projected mass density, defined as the sum of the three-dimensional density along the optical axis of the microscope, through the following relationship, 17,18 …”

Section: Methodsmentioning

confidence: 99%

“…The measurement techniques in this study are readily accessible to other researchers as the NIQPM approach and DIC rely only on a standard optical microscope. 18 …”

Section: Introductionmentioning

confidence: 99%

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Critical Behavior of Subcellular Density Organization During Neutrophil Activation and Migration

et al. 2015

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Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Critical Behavior of Subcellular Density Organization During Neutrophil Activation and Migration

et al. 2015

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“…Modern optical engineering approaches, including the transport of intensity equation (TIE) method, have enabled the quantification of the phase, denoted [radian], of transmitted waves through cellular specimens (26). This is crucial, as knowledge of the phase enables the enumeration of the projected mass density, defined as the sum of the three dimensional density along the optical axis of the microscope, through the following relationship,…”

Section: Noninterferometric Quantitative Phase Microscopy (Niqpm): Trmentioning

confidence: 99%

A physical sciences network characterization of circulating tumor cell aggregate transport

King

Phillips

Mitrugno

et al. 2015

American Journal of Physiology-Cell Physiology

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Circulating tumor cells (CTC) have been implicated in the hematogenous spread of cancer. To investigate the fluid phase of cancer from a physical sciences perspective, the multi-institutional Physical Sciences-Oncology Center (PS-OC) Network performed multidisciplinary biophysical studies of single CTC and CTC aggregates from a patient with breast cancer. CTCs, ranging from single cells to aggregates comprised of 2-5 cells, were isolated using the high-definition CTC assay and biophysically profiled using quantitative phase microscopy. Single CTCs and aggregates were then modeled in an in vitro system comprised of multiple breast cancer cell lines and microfluidic devices used to model E-selectin mediated rolling in the vasculature. Using a numerical model coupling elastic collisions between red blood cells and CTCs, the dependence of CTC vascular margination on single CTCs and CTC aggregate morphology and stiffness was interrogated. These results provide a multifaceted characterization of single CTC and CTC aggregate dynamics in the vasculature and illustrate a framework to integrate clinical, biophysical, and mathematical approaches to enhance our understanding of the fluid phase of cancer.circulating tumor cell; metastasis; breast cancer; cell lines; fluid dynamics; physics of cancer; hemodynamics; quantitative phase microscopy; microfluidics; immersed finite element method WHILE THE PRESENCE of circulating tumor cells (CTCs) in the vasculature has been implicated in the metastatic cascade of epithelial carcinomas, new evidence has revealed a putative role of CTC aggregates as a potential form of stromal-assisted metastasis across a variety of epithelial tumor types (6). In concert, the presence of homotypic interactions among CTCs leading to aggregation occurring at sites of endothelial attachment suggest the involvement of CTC aggregates in the hematogenous dissemination of cancer. Despite compelling evidence suggesting a role for CTC aggregates in metastatic progression (28), the physics underlying CTC vascular transport including the influence of blood flow, coagulation, intercellular adhesion, and collisions with cells of the vasculature, such as red blood cells (RBCs) and endothelial cells (ECs), remains poorly understood. Better physical understanding of CTC transport and mechanobiology could enable, for example, new strategies to monitor patient response to chemotherapy (22).Here, we demonstrate a multidisciplinary approach (29) that utilizes clinical measurements on single CTCs and CTC aggregates from a patient with breast cancer as a quantitative guide in the rational design of in vitro and in silico models to investigate the dynamics of CTC transport in the vasculature. Using the high-definition (HD) CTC assay to identify circulating tumor cells in the blood, coupled with quantitative phase microscopy (QPM) to quantify the subcellular density organization of CTCs, we profiled the biophysical properties of CTCs in a patient with breast cancer. These metrics, including geometric and density features,...

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“…17 We have previously utilized the non-interferometric quantitative phase microscopy (NIQPM) technique to measure dry mass and density of formed platelet aggregates and thrombi, circulating tumor cells isolated from ovarian cancer patients, and isogenic primary and metastasized colon adenocarcinoma cell cytoplasm and nuclei. 2,9,27,28 Here we performed NIQPM and high numerical aperture (NA) 2D and 3D image segmentation in conjunction with DAPI and fluorescent γ-H2AX antibody staining to elucidate HNSCC cell body, cytoplasm, nuclei, and γ-H2AX foci volume, height, area, and mass density after exposure to 8 Gy of IR. 8 Gy of IR is a standard robust single dose treatment for cancer patients and for in vitro studies.…”

Section: Introductionmentioning

confidence: 99%

Effect of Ionizing Radiation on the Physical Biology of Head and Neck Squamous Cell Carcinoma Cells

et al. 2015

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Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer worldwide. Although there are numerous treatment options for HNSCC, such as surgery, cytotoxic chemotherapy, molecularly targeted systemic therapeutics, and radiotherapy, overall survival has not significantly improved in the last 50 years. This suggests a need for a better understanding of how these cancer cells respond to current treatments in order to improve treatment paradigms. Ionizing radiation (IR) promotes cancer cell death through the creation of cytotoxic DNA lesions, including single strand breaks, base damage, crosslinks, and double strand breaks (DSBs). As unrepaired DSBs are the most cytotoxic DNA lesion, defining the downstream cellular responses to DSBs are critical for understanding the mechanisms of tumor cell responses to IR. The effects of experimental IR on HNSCC cells beyond DNA damage in vitro are ill-defined. Here we combined label-free, quantitative phase and fluorescent microscopy to define the effects of IR on the dry mass and volume of the HNSCC cell line, UM-SCC-22A. We quantified nuclear and cytoplasmic subcellular density alterations resulting from 8 Gy X-ray IR and correlated these signatures with DNA and γ-H2AX expression patterns. This study utilizes a synergistic imaging approach to study both biophysical and biochemical alterations in cells following radiation damage and will aid in future understanding of cellular responses to radiation therapy.

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

Cited by 9 publications

References 31 publications

Critical Behavior of Subcellular Density Organization During Neutrophil Activation and Migration

Critical Behavior of Subcellular Density Organization During Neutrophil Activation and Migration

A physical sciences network characterization of circulating tumor cell aggregate transport

Effect of Ionizing Radiation on the Physical Biology of Head and Neck Squamous Cell Carcinoma Cells

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