Live Cell Biomass Tracking for Basic, Translational, and Clinical Research

Single cell mass is tightly regulated throughout generations and the cell cycle, making it an important marker of cell health. Abnormal changes in cell size can be the first indication of dysfunction in response to environmental stimuli such as cytotoxic drugs. Described here is the further development of high-speed live cell interferometry (HSLCI) to concurrently measure the changes in single cell mass of thousands of cells over time. Critically, the high-throughput nature of HSLCI provides realistic pictures of tumor heterogeneity. This throughput enabled HSLCI to correctly predict in vivo carboplatin sensitivity of three triple negative breast cancer patient derived xenografts, while also characterizing the spectrum of drug response from apoptosis to senescence to drug resistance. HSLCI quantified previous qualitative observations of increases in cell size and losses in cell density in senescent cells, and importantly observed proliferative recovery in cells demonstrating thee senescent characteristics. Furthermore, the addition of a micropipette system has enabled the isolation of rare (~1%) drug resistant cells for further study with molecular biology methods. Together, this work highlights HSLCI’s versatility and potential for clinical, translational, and basic research

Live Cell Interferometry: A Novel Quantitative Phase Imaging Technique for Rapid Characterizations of Tumor Heterogeneity, Drug Resistance, Cell Fates and Biophysical Properties

Cell mass is an important biophysical property that provides a crucial link between external physical measurements and internal cellular processes such as cell cycle progression, division, differentiation and cell death. Advancements in quantitative phase imaging (QPI) techniques have enabled many in-depth studies of cell mass in the context of basic and translational research. This thesis describes the development and implementation of live cell interferometry (LCI) as a novel QPI technique that is high speed, high throughput, precise and label-free. It was validated as a powerful tool in dissecting tumor heterogeneity and drug resistance in patient derived melanoma cell lines. LCI analysis of cell fate in response to mitotic inhibitors provided valuable insights in cancer drug development and dose selection. Furthermore, it was utilized to characterize many other biophysical responses in fundamental research such as cardiomyocyte hypertrophy in cardiac wound healing responses. These studies showcased the unique capabilities and advantages of LCI in applications from bench to bedside

Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM)

© JoVE 2006-2014. All Rights Reserved. Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide

Diffusive and directional intracellular dynamics measured by field-based dynamic light scattering

Quantitative measurement of diffusive and directional processes of intracellular structures is not only critical in understanding cell mechanics and functions, but also has many applications, such as investigation of cellular responses to therapeutic agents. We introduce a label-free optical technique that allows non-perturbative characterization of localized intracellular dynamics. The method combines a field-based dynamic light scattering analysis with a confocal interferometric microscope to provide a statistical measure of the diffusive and directional motion of scattering structures inside a microscopic probe volume. To demonstrate the potential of this technique, we examined the localized intracellular dynamics in human epithelial ovarian cancer cells. We observed the distinctive temporal regimes of intracellular dynamics, which transitions from random to directional processes on a timescale of ∼0.01 sec. In addition, we observed disrupted directional processes on the timescale of 1∼5 sec by the application of a microtubule polymerization inhibitor, Colchicine, and ATP depletion. © 2010 Optical Society of America

Single-cell analysis of cell competition using quantitative microscopy and machine learning

Cell competition is a widely conserved, fundamental biological quality control mechanism. The cell competition assay of MDCK wild-type versus mutant MDCK Scribble-knockdown (ScribKD) relies on a mechanical mechanism of competition, which posits that the emergence of compressing stresses within the tissue at high confluency drive the competitive outcome. According to this mechanism, proliferating wild-type cells out-compete mutant ScribKD cells, resulting in their apoptosis and apical extrusion. Previous studies show that there is an increased division rate of wild-type cells in neighbourhoods with high numbers of ScribKD cells, but what still remains a mystery is whether this is a cause or consequence of increased apoptosis in the “loser” cell population. This project also interrogated the competitive assay of wild-type versus RasV12 , which is hypothesized to operate on a biochemical mechanism and results in the apical extrusion (but not apoptosis) of the loser RasV12 population. For both these mechanisms of competition it is still unknown which population of cells are driving the winner/loser outcome. Is the winner cell proliferation prompting the loser cell demise? Or is an autonomous loser elimination prompting a subsequent winner cell proliferation? In my research, I have employed multi-modal, time-lapse microscopy to image competition assays continuously for several days. These data were then segmented into wild-type or mutant instances using a Convolutional Neural Network (CNN) that can differentiate between the cell types, after which they were tracked across cellular generations using a Bayesian multi-object tracker. A conjugate analysis of fluorescent cell-cycle indicator probes was then utilised to automatically identify key time points of cellular fate commitment using deep-learning image classification. A spatio-temporal analysis was then conducted in order to quantify any correlation between wild-type proliferation and mutant cell demise. For the case of wild-type versus ScribKD , there was no clear evidence for the wild-type cells mitoses directly impacting upon the ScribKD cell apoptotic elimination. Instead, a subsequent analysis found that a more subtle mechanism of pre-emptive, local density increases around the apoptosis site appeared to be determining the eventual ScribKD fate. On the other hand, there was clear evidence of a direct impact of wild-type mitoses on the subsequent apical extrusion and competitive elimination of RasV12 cells. Both of these conclusions agree with the prevailing classification of cell competition types: mechanical interactions are more diffuse and occur over a larger spatio-temporal domain, whereas biochemical interactions are constrained to nearest neighbour cells. The hypothesized density-dependency of ScribKD elimination was further quantified on a single-cell scale by these analyses, as well as a potential new understanding of RasV12 extrusion. Most interestingly, it appears that there is a clear biophysical mechanism to the elimination in the biochemical RasV12 cell competition. This suggests that perhaps a new semantic approach is needed in the field of cell competition in order to accurately classify different mechanisms of elimination

An investigation into the effects of substrate properties on the mechanics of corneal epithelial cells

Cells respond to mechanical changes in their extracellular environment, as reflected by various cell behaviours and observed through changes in the tissue biomechanics. Types of cell behaviour that are regulated by mechanical cues in the microenvironment of the cell are cell spreading, migration, proliferation and differentiation. Cell migration is a key part of many biological processes including corneal wound repair. Changes in the biomechanical properties of the cornea can be induced by refractive and therapeutic treatments and also by diseases of the eye or other illnesses. A Rabbit Corneal Epithelial (RCE) cell line was used to study cell mechanics and cell migration. Polydimethylsiloxane (PDMS), a biocompatible silicone elastomer, was used as a substrate to culture RCE cells. In order to promote cell attachment and growth, the hydrophilicity of the PDMS surface was increased by treating it with oxygen-rich cold atmospheric pressure plasma, which was confirmed by surface characterisation techniques. Cell attachment and growth studies over time comparing plasma and non-plasma treated PDMS showed an increase in RCE cell growth and area coverage on plasma treated PDMS. [Continues.

Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy

Biology would not be where it is today without fluorescence microscopy. It is arguably one of the most commonly used tools in the biologists toolbox and it has helped scientists study the localization of cellular proteins and other small things for decades, but it is not without its limitations. Due to the diffraction limit, conventional fluorescence microscopy is limited to micrometer-range structures. Science has long relied upon electron microscopy and X-ray crystallography to study phenomena that occur below this limit. However, many of lifes processes occur between these two spatial domains. Super-resolution microscopy, the next stage of evolution of fluorescence microscopy, has the potential to bridge this gap between micro and nano. It combines superior resolutions of down to a few nanometers with the ability to view objects in their natural environments. It is the ideal tool for studying the large, multi-protein complexes that carry out most of lifes functions, but are too complex and fragile to put on an electron microscope or into a synchrotron. A form of super-resolution microscopy called SMLM Microscopy shows especially high promise in this regard. With its ability to detect individual molecules, it combines the high resolution needed for structural studies with the quantitative readout required for obtaining data on the stoichiometry of multi-protein complexes. This thesis describes new tools which expand the toolbox of SMLM with the specific aim of studying multi-protein complexes. First, the development of a novel fluorescent tagging system that is a mix of genetic tagging and immuno-staining. The system, termed BC2, consists of a short, genetically encodable peptide that is targeted by a nanobody (BC2 nanobody). The system brings several advantages. The small tag is not disruptive to the protein it is attached to and the small nanobody can get into tight spaces, making it an excellent tag for dense multi-protein structures. Next, several new variants of some commonly used green-to-red fluorescent proteins. The novel variants, which can be converted with a combination of blue and infrared light are especially useful for live-cell imaging. The developed fluorescent proteins can also be combined with photo-activatable fluorescent proteins to enable imaging of several targets with the same color protein. Finally, an application of the latter technique to study the multi-protein kinetochore complex and gain first glimpses into its spatial organization and the stoichiometry of its subunits

The NASA SBIR product catalog

The purpose of this catalog is to assist small business firms in making the community aware of products emerging from their efforts in the Small Business Innovation Research (SBIR) program. It contains descriptions of some products that have advanced into Phase 3 and others that are identified as prospective products. Both lists of products in this catalog are based on information supplied by NASA SBIR contractors in responding to an invitation to be represented in this document. Generally, all products suggested by the small firms were included in order to meet the goals of information exchange for SBIR results. Of the 444 SBIR contractors NASA queried, 137 provided information on 219 products. The catalog presents the product information in the technology areas listed in the table of contents. Within each area, the products are listed in alphabetical order by product name and are given identifying numbers. Also included is an alphabetical listing of the companies that have products described. This listing cross-references the product list and provides information on the business activity of each firm. In addition, there are three indexes: one a list of firms by states, one that lists the products according to NASA Centers that managed the SBIR projects, and one that lists the products by the relevant Technical Topics utilized in NASA's annual program solicitation under which each SBIR project was selected

Superresolved Three-Dimensional Analysis of the Spatial Arrangement of the Human Immunodeficiency Virus Type-1 (HIV-1) Envelope Glycoprotein at Sites of Viral Assembly

Human Immunodeficiency Virus type 1 (HIV-1) replicates by forcing infected host cells to produce new virus particles, which assemble form protein components on the inner leaflet of the host cell\u27s plasma membrane. This involves incorporation of the essential viral envelope glycoprotein (Env) into a structural lattice of viral Gag proteins. The mechanism of Env recruitment and incorporation is not well understood. To better define this process, we seek to describe the timing of Env-Gag encounters during particle assembly by measuring angular positions of Env proteins about the surfaces of budding particles. Using three-dimensional superresolution microscopy, we show that Env distributions are biased toward the necks of budding particles, indicating incorporation of Env late in the assembly of the lattice. We show that this behavior is dependent on the host cell type and on the long cytoplasmic tail of Env. We propose a model wherein Env incorporation is regulated by opposing mechanisms: Gag lattice trapping of Env cytoplasmic tails, and intracellular sequestering of Env during lattice assembly