Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.
Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers.
The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an ‘epigenetic’ drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat’s differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.
The literature review examines three topics: (1) ethnic identity and craft; (2) northern Thailand and hill tribes; and (3) design thinking, vulnerability, and resilience.
Empirical research was conducted with hill tribe craftspeople in northern Thailand. Seven types of capital—human, social, natural, physical, financial, cultural, and emotional capital—were identified through interviews and observation. Those types of capital indicated what the craftspeople wanted and needed.
The key findings were as follows: First, social capital has a close relationship with both human capital and emotional capital, indicating that for craftspeople, networks and membership ensure knowledge and increase connections with friends and family. Secondly, emotional capital is affected by financial capital. Financial capital refers to the monetary resources used to achieve craftspeople’s livelihood objectives. The craftspeople required high order volumes to earn to more money and thus improve their economic condition; they experienced more stress when order volumes were low. Third, financial capital is not related to social and cultural capital. Graphs implied certain relationship among them, with the reasons varying depending on the individual craftsperson’s environment. A high level of social and cultural capital does not affect low financial capital, and vice versa. Finally, cultural capital directly influences emotional capital because the happiness of hill tribe craftspeople is related to their identity and dignity as craftspeople.
Hydrophobic platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) was physically incorporated into micelles formed from poly(ε-caprolactone)-block-poly(ethylene glycol) to enable the application of PtTFPP in aqueous solution. Micelles were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM) to show an average diameter of about 140 nm. PtTFPP showed higher quantum efficiency in micellar solution than in tetrahydrofuran (THF) and dichloromethane (CH2Cl2). PtTFPP in micelles also exhibited higher photostability than that of PtTFPP suspended in water. PtTFPP in micelles exhibited good oxygen sensitivity and response time. This study provided an efficient approach to enable the application of hydrophobic oxygen sensors in a biological environment.
Single-cell studies of phenotypic heterogeneity reveal more information about pathogenic processes than conventional bulk-cell analysis methods. By enabling high-resolution structural and functional imaging, a single-cell three-dimensional (3D) imaging system can be used to study basic biological processes and to diagnose diseases such as cancer at an early stage. One mechanism that such systems apply to accomplish 3D imaging is rotation of a single cell about a fixed axis. However, many cell rotation mechanisms require intricate and tedious microfabrication, or fail to provide a suitable environment for living cells. To address these and related challenges, we applied numerical simulation methods to design new microfluidic chambers capable of generating fluidic microvortices to rotate suspended cells. We then compared several microfluidic chip designs experimentally in terms of: (1) their ability to rotate biological cells in a stable and precise manner; and (2) their suitability, from a geometric standpoint, for microscopic cell imaging. We selected a design that incorporates a trapezoidal side chamber connected to a main flow channel because it provided well-controlled circulation and met imaging requirements. Micro particle-image velocimetry (micro-PIV) was used to provide a detailed characterization of flows in the new design. Simulated and experimental results demonstrate that a trapezoidal side chamber represents a viable option for accomplishing controlled single cell rotation. Further, agreement between experimental and simulated results confirms that numerical simulation is an effective method for chamber design.
Background:
Environmental heat exposure is a public health concern. The impacts of environmental heat on mortality and morbidity at the population scale are well documented, but little is known about specific exposures that individuals experience.
Objectives:
The first objective of this work was to catalyze discussion of the role of personal heat exposure information in research and risk assessment. The second objective was to provide guidance regarding the operationalization of personal heat exposure research methods.
Discussion:
We define personal heat exposure as realized contact between a person and an indoor or outdoor environment that poses a risk of increases in body core temperature and/or perceived discomfort. Personal heat exposure can be measured directly with wearable monitors or estimated indirectly through the combination of time–activity and meteorological data sets. Complementary information to understand individual-scale drivers of behavior, susceptibility, and health and comfort outcomes can be collected from additional monitors, surveys, interviews, ethnographic approaches, and additional social and health data sets. Personal exposure research can help reveal the extent of exposure misclassification that occurs when individual exposure to heat is estimated using ambient temperature measured at fixed sites and can provide insights for epidemiological risk assessment concerning extreme heat.
Conclusions:
Personal heat exposure research provides more valid and precise insights into how often people encounter heat conditions and when, where, to whom, and why these encounters occur. Published literature on personal heat exposure is limited to date, but existing studies point to opportunities to inform public health practice regarding extreme heat, particularly where fine-scale precision is needed to reduce health consequences of heat exposure.
This report summarizes the proceedings of the workshop focusing primarily on two sessions: the first related to social vulnerability mapping and the second related to the identification and prioritization of interventions necessary to address the impacts of climate-sensitive hazards.