Dr. Gregory R. Goddard Profile

Dr. Gregory R. Goddard

Postdoctoral at Los Alamos National Lab

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Publications (3)

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Proceedings Article | 29 February 2008 Paper

Endogenous fluorescence lifetime of viable cells by flow cytometry

Jessica Houston, Mark Naivar, John Martin, Greg Goddard, Susan Carpenter, Judith Mourant, James Freyer

Proceedings Volume 6859, 68590T (2008) https://doi.org/10.1117/12.763816

KEYWORDS: Luminescence, Flow cytometry, Scattering, Calibration, Modulation, Signal detection, Tissues, Semiconductor lasers, Near infrared, Data acquisition

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Autofluorescence emission is commonly measured in flow cytometry and is used as a negative control in protocols that explore binding of exogenous fluorophores to cell receptors or other targets of interest. The presence of intrinsic fluorophores however may burden complex cytometry applications. For example it may be difficult to resolve fluorescence signals from multi-intensity and multi-color measurements when the de-convolved fluorescence in question falls close to the autofluorescence background. One possible solution to intensity and spectral overlap problems in flow cytometry is to acquire fluorescence decay kinetic measurements. To this end we focus on advancing time-dependent flow cytometry and conduct measurements of endogenous fluorescence lifetime. Instrument developments to a phase-sensitive flow cytometry (PSFC) system were coupled with lifetime measurements of intrinsic fluorophores from viable cell samples. The average lifetime of >300,000 individual rat fibroblast cells was measured at discrete wavelengths ranging from 457- to 785-nm using a 10-MHz intensity-modulated excitation beam. AC amplitude, DC, and phase-shift were resolved and the average lifetime from excitable endogenous species was measured. The lifetime results ranged from 1- to 6-ns over the broad spectral range. Cataloging lifetime values prefaces the use of phase-sensitive techniques in more complex systems and provides a priori measurements necessary for PSFC filtering known lifetime signals from Raman, or other emission and scattering events.

Proceedings Article | 28 February 2008 Paper

Cellular discrimination based on spectral analysis of instrinic fluorescence

G. Goddard, J. Houston, J. Martin, S. Graves, J. Freyer

Proceedings Volume 6859, 685908 (2008) https://doi.org/10.1117/12.763873

KEYWORDS: Luminescence, Particles, Sensors, Spectral resolution, Flow cytometry, Charge-coupled devices, Molecules, Calibration, Image sensors, Light scattering

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The increasing need for highly polychromatic approaches to flow cytometry, coupled with rapid technological advances, have driven the design and implementation of commercial instruments that measure up to 19 parameters using multiple lasers for excitation, an intricate optical filter/mirror arrangement, and analysis using fluorescence compensation approaches. Although such conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from higher density of spectral information and a more flexible approach to spectral analysis including, but certainly not limited to: spectral deconvolution of overlapping spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, cellular autofluorescence characterization, and flow based Raman spectroscopy. For these purposes, we have developed a high resolution spectral flow cytometer using an EMCCD camera with 1600 by 200 pixels, which is capable of detecting less than 200 fluorescein molecules with a spectral resolution of less than 3 nm. This instrument will enable high throughput characterization of single cell or particle emission spectra. For proof of principle instrument operation, we have begun characterization of intrinsic cellular autofluorescence, which is the major source of background for cell-based fluorescence assays. Specifically, we will describe recent work on the high resolution spectral characterization of autofluorescence for several commonly used cell types. Autofluorescence emission is known to cover over almost the entire spectrum from 300 to nearly 800 nm. These emissions are attributed to flavins, elastin, Indolamine dimers and trimers, NADH and collagen among other molecules. We will show that several unique autofluorescence spectra arise in the different cell lines thereby suggesting the possibility of discrimination of cell types based on intrinsic fluorescence.

Proceedings Article | 31 March 2006 Paper

Biomolecules as nanomaterials: interface characterization for sensor development

Gregory Goddard, Jennifer Whittier

Proceedings Volume 6172, 617206 (2006) https://doi.org/10.1117/12.658771

KEYWORDS: Proteins, Network security, Reflectivity, Calibration, Electrodes, Polymers, Polymerization, Molecules, Resistance, Sensors

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Extensive research is underway to understand and exploit the interface between biological materials and integrated systems Today, "nanotechnology" can be defined as a group of emerging technologies in which the structure of matter is controlled at the nanometer scale, the scale of small numbers of atoms, to produce novel materials and devices having useful and unique properties. An ideal biological candidate for use in nanoscale devices is the microtubule, an essential component of the eukaryotic cytoskeleton, which, unlike most proteins, has been shown to be electrically conductive. Due to the presence of an intrinsic dipole in the protein polymer, RF reflectance spectroscopy was chosen as an interrogation method. RF reflectance spectroscopy measures the electrical response of a sample in response to sinusoidally alternating currents as a function of frequency By interrogating the protein electrically, we are able to detect the polymerization state of the system, track any associated conductivity changes, and monitor binding of microtubule-associated proteins. We demonstrate manipulation of the microtubule system through the use of low-frequency electric fields, and discuss implications for sensor development.

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