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Core R&D:

The NFCR's current NIH grant focuses on three main areas for its core R&D activities:

Field-Based Manipulations in Acoustically-Focused Sample Streams
The particles in traditional flow cytometers are focused hydrodynamically. While this method forms a tight core for consistent sample analysis, it limits the analysis to short interrogation times and unidirectional flow.   In contrast, samples that are acoustically focused to the center of a stream can be controlled to flow at varying rates, including reverse flow for reanalysis of interesting events, as well as being regularly spaced along the flow axis.  Additional advantages include orientation of the particle in the sample stream which creates more uniform scatter signatures as well as the ability to concentrate particles of a determined size to the center of the stream.
(Greg Kaduchak, PI)

Next Generation Optically Activated Large Particle Sorting
With few exceptions, cell sorters rely on droplet based sorting of particles.  While this works well for smaller particles, because of fluid dynamic effects the size of particles that can be sorted is limited to ~100 µm with rates of less than 1000 events/sec.  Using a combination of synchronous particle delivery, droplet on demand, parallel flow cells, low cost excitation and emission detection components and miniaturized data acquisition we will develop high-thoughput large particle sorting systems.  Our target is to be able to sort particles up to 1 mm at rates of more than 10,000 events/sec.
(Steve Graves, PI)

An Integrated Phase-Spectral Flow Cytometer
Flow cytometers traditionally collect light in discrete wavelength increments defined by physical filters in the collection optics: this configuration limits the number of discrete signals that can be captured.  We have shown that by using dispersive optics and a CCD array for collection, a wide range of fluorescence wavelengths can be captured in small increments, resulting in complete spectral resolution of fluorescence on a single cell basis.  Another commonly encountered challenge in flow cytometry is that of fluorophores with overlapping spectra.  When dyes are excited, they remain in the excited state for a defined period of time, dependant on their inherent properties and environment.  We have demonstrated that by using phase resolved measurements the fluorescence lifetime can be measured in a flow cytometer, resolving dyes with similar spectra and discriminating autofluorescence.  We are combining these two capabilities in a single instrument that will be able to distinguish an increased number of fluorescence parameters, allowing more sensitive resolution for complex samples and increased potential for multiplexing.
(Jim Freyer, PI)

NFCR-Associated Projects:

Many NFCR personnel have independent funding that involve flow cytometric instrumentation and methods development:

Magnetization Measurements in Flow
In collaboration with colleagues in the Biophysics Group at LANL, we are developing a flow cytometer with magnetic detection capabilities. By integrating a superconducting quantum interference device (SQUID) for detection of magnetization with conventional fluorescence detection approaches, the new instrument will enable new approaches to multiplexed particle analysis and multiparameter cellular analysis. (Michelle Espy, PI)
Protease Analysis
Proteases are involved in a host of biological processes, from bacterial pathogenesis to coagulation. Taking advantage of our experience in the mechanistic analysis of molecular interactions, we are investigating the recognition and cleavage mechanisms of bacterial proteases with the aim of developing new diagnostics and therapeutics. (Steve Graves, PI)
Ultrasensitive Nucleic Acid Detection
We are using fluorescence correlation spectroscopy of single molecules combined with novel labeling strategies to detect nucleic acid targets at the zeptomole level. This approach is being developed for RNA detection and molecular haplotyping. (Hong Cai, PI)
Bacterial Bioforensics
As part of a consortium of National Laboratories, we are applying state-of-the-art molecular analysis methods to the forensic analysis of bacterial pathogens. We are using the DNA Fragment Sizing Flow Cytometer to perform restriction fragment analysis of whole genomes to fingerprint organisms without the need for DNA sequence information. For analysis of DNA sequence-based signatures, we are using SNP analysis on microsphere arrays to interrogate functionally- and phylogenetically-important sequences. (Babs Marrone, PI)

Collaborative Projects:

Influenza Biosurveillance
Working with collaborators in the Theoretical Biology Group at LANL, we are developing new approaches to infectious disease surveillance that take advantage of rapid nucleic acid sequence analysis.
Leukemia Detection
We have developed an approach for the rapid detection and identification of genetic abnormalities associated with leukemia. We are working with a number of clinical collaborators to validate and disseminate this technology.

Specialized Instrument Capabilities:

High Sensitivity Flow Cytometer
The High Sensitivity Flow Cytometer uses slow sheath flow (ul/min) and photon counting APDs to perform sensitive fluorescence measurements, capable of detecting single molecules of phycoerythrin.
Rapid Kinetic Flow Cytometer
Uses computer controlled stepper motor driven syringes to perform rapid mixing and sample delivery. Mounted on a commercial optical bench, the RKFC provides multilaser, multicolor detection capability with a measurement deadtime of less than 100 msec.
Thermal Cycling Flow Cytometer
Peltier-based temperature control units accurately and dynamically control the sample temperature. Capable of ramping the temperature for ambient to 95°C and back in less than one minute.
Phase Sensitive Flow Cytometer
Uses modulated laser beams and custom electronics to perform phase resolved measurements and fluorescence lifetime analysis of single cells.
DNA Fragment Sizing Flow Cytometer
Based on the High Sensitivity Flow Cytometer, this instrument is optimized for the analysis of intercalating dyes in double stranded DNA. A suite of software tools has been developed to support DNA fragment sizing applications.

In addition, the NFCR houses several commercial benchtop analyzers, sorters, and other fluorescence instrumentation.



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