Cells
Researchers: Ragnhild Whitaker
We have developed a high-density platform for live single-cell sensing that provides accurate functional screening of biologically active compounds and cellular events in bacteria, yeast, or mammalian cells. In this array, coherent imaging fibers are selectively etched to form individual wells on the imaging array's distal end. Individual cells with fluorescent dyes and/or proteins were placed into each well.
Figure 1 Cells in wells
The fluorescent signals from each cell are spatially resolved and thousands of individual cell responses on a signal sensing substrate can be monitored simultaneously.
Figure 2 Cells fluoresce in wells
Because the wells can be sized according to the size of a specific cell type, this array provides a universal platform for a variety of living cell based assays. We have applied this living cell array platform to both fundamental and applied studies, including current studies on drug screening on G-protein coupled receptors and quorum sensing in Acyl-homoserine lactone responding bacteria. Previous works have included metal sensing, toxicity sensing, migration studies, gene transcription regulation mechanisms, and stochastic gene expression from individual cells.
Due to the extremely small dimensions, superior sensitivity, and easy and reproducible preparation, this universal living cell array platform provides a powerful tool for drug screening, toxicity testing, environmental analysis, and potentially for clinical diagnostic applications. A brief description of the two currently active projects can be found below.
Mammalian Drug Screening Application on Fiber-optic Platform:
G-protein coupled receptors (GCPRs) are the largest family of cell surface receptors, and an important target in drug development. Investigating ligand induced receptor activation by looking at intracellular calcium oscillations using calcium sensitive dyes is an established method for agonist detection and receptor function analysis, frequently used in High Throughput Screening (HTS). We used our fiber optic platform to follow calcium oscillations in single cells in real time. We used Chinese Hamster Ovarian (CHO) cells ectopically expressing GPCRs as our model system for the method development. The calcium oscillations were initiated by activating CCR5, ChemR23 or FPRL2 receptors expressed on CHO cells. Receptors were stimulated with several agonists at different concentrations. We were able to determine the percentage of live cells in a population responding to cell stimuli, the spread of response magnitude within a population of clonal cells, the number of outliers, and determine how calcium oscillations varied with agonist concentration or type of stimulating or non-stimulating agents. Using Principal Component Analysis (PCA) and K-nearest neighbor modeling, we were also able to unequivocally classify the time-resolved calcium traces from a single cell population as the type of stimuli or concentration. The classification of single cell traces was possible due to the large number of cells that can be simultaneously interrogated, returning data that are statistically significant. The same traces did not yield classification results when the fiber was analyzed as a bulk system. This method is powerful for identifying new potential drug targets or investigating the single cell behavior of an existing target or known receptor. Single cell mammalian drug screening employs fibers that are 22 µm in diameter, a size determined to be large enough to hold one cell only, without the cell covering more than one fiber.
Figure 3 : PCA analysis on CHO cell line expressing CCR5 GPCR receptor. The cell line was stimulated with three agonists as seen, and PCA was performed on data from one fiber (left) and data from three fibers pooled (right).
Classified as
Correctly Classified Instances: 1027 92 %
Incorrectly Classified Instances: 85 8 %
Table 1: Resulting matrix obtained when KNN analysis was performed on single cell data from cells expressing CCR5 GPCR. Ca2+ traces from single cells were analyzed, and data from two fibers pooled are demonstrated. The classification results enabled unmistakable identification of the agonist used to stimulate the cell population. Similar results were obtained when single cell data from several days were pooled.
Quorum sensing application on Fiber-optic Platform
Several bacterial species communicate with each other through signaling molecules released into the environment, a process termed quorum sensing. Gram negative bacteria communicate through Acyl Homoserine Lactones (AHL) of varying length. Quorum sensing is important for bacterial pathogenesis because it enables bacteria to sense when critical mass has been reached. When this critical mass of bacteria is reached, the AHL concentration surrounding the bacteria reaches what is called the quorum concentration. At this quorum concentration, genes that are under control of the quorum sensing system are expressed. Quorum controlled genes can be virulence factors, genes for bacteria–host interactions, and in most cases they include the expression of AHL molecules. The AHL expression therefore has a positive feedback loop. Studying the communication between bacteria has become important in understanding the mechanisms of quorum sensing, and as more and more bacteria grow resistant to conventional antibiotics, the development of drugs that can interrupt the communication, termed anti-pathogenic drugs, is of increasing interest.
We have used our fiber optic system to randomly deposit fluorescently labeled AHL producing strains and a GFP expressing AHL sensitive E.Coli strain to investigate how single bacteria express and respond to AHL. We are currently using a custom written image and data analysis program to extract time resolved single cell quorum sensing data from our fibers. Time resolved E.Coli GFP response profiles are analyzed with respect to number and placement of proximal AHL producers and response patterns. Responses from un-inhibited and inhibited communication between bacteria are compared, and by using this system, we hope to better characterize the way bacteria communicate by simultaneously detecting AHL communication in thousands of single bacteria responding to variety of environment conditions.
Figure 4: Example of configuration of AHL producing cells labeled in red and GFP expressing cells in green on a fiber. The image is produced by overlapping the fluorescence from the red Syto 64 dye and the fluorescence from GFP expressed by the E.Coli AHL sensor.
Some Select Publications
"Multi Analyte Single Cell Analysis with Multiple Cell Lines Using a Fiber Optic Array" Whitaker, R D.; Walt, D R.; Analytical. Chemistry, 2007, in press
“Fiber-Based Single Cell Analysis of Reporter Gene Expression in Yeast Two-Hybrid Systems” Whitaker, R D.; Walt, D R.; Analytical Biochemistry, 2007, 360(1), 63-74
“Detecting oxygen consumption in the proximity of Saccharomyces cerevisiae cells using self-assembled fluorescent nanosensors” Kuang Y.; Walt D R.; Biotechnology and bioengineering, 2007, 96(2), 318-325
“Monitoring "promiscuous" drug effects on single cells of multiple cell types” Kuang Y.; Walt D R.; Analytical Biochemistry, 2005, 345(2), 320-325
“Individual cell migration analysis using fiber-optic bundles” DiCesare, C.; Israel, B.; Walt D. R.; Analytical and Bioanalytical Chemistry, 2005, 382(1), 37-43.
“Simultaneously Monitoring Gene Expression Kinetics and Genetic Noise in Single Cells by Optical Well Arrays” Kuang Y.; Israel, B.; Walt D R.; Analytical Chemistry, 2004, 76(21), 6282-6286.
”Living Bacterial Cell Array for Genotoxin Monitoring” Kuang Y.; Israel, B.; Walt D R.; Analytical Chemistry, 2004, 76(10), 2902-2909.
“Application of high-density optical microwell arrays in a live-cell biosensing system” Taylor, L. C.; Walt, D. R.; Analytical Biochemistry, 2000, 278(2), 132-142
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