Biomedical subjects

Gregory Goddard

Publications and source records attributed to Gregory Goddard.

4 recordsLinked to original sources

PubMed2006

Single particle high resolution spectral analysis flow cytometry.

BACKGROUND: While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. METHODS: Our system utilizes a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular charge-coupled-device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system. RESULTS: Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements. Coefficients of variation of integrated spectral fluorescence intensity of several sets of standard fluorescent microspheres ranged from 1.4 to 4.8% on the spectral system. Spectral discrimination of free versus PI bound to cells is also demonstrated. CONCLUSIONS: It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi-fluorophore labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, and allow more precise resolution of the relative contribution of individual fluorophores in multiply-tagged samples, thereby enabling a range of new applications involving the spectral analysis of single cells and particles.

Calibration↗

PubMed2006

Ultrasonic particle-concentration for sheathless focusing of particles for analysis in a flow cytometer.

BACKGROUND: The development of inexpensive small flow cytometers is recognized as an important goal for many applications ranging from medical uses in developing countries for disease diagnosis to use as an analytical platform in support of homeland defense. Although hydrodynamic focusing is highly effective at particle positioning, the use of sheath fluid increases assay cost and reduces instrument utility for field and autonomous remote operations. METHODS: This work presents the creation of a novel flow cell that uses ultrasonic acoustic energy to focus small particles to the center of a flowing stream for analysis by flow cytometry. Experiments using this flow cell are described wherein its efficacy is evaluated under flow cytometric conditions with fluorescent microspheres. RESULTS: Preliminary laboratory experiments demonstrate acoustic focusing of flowing 10-microm latex particles into a tight sample stream that is approximately 40 microm in diameter. Prototype flow cytometer measurements using an acoustic-focusing flow chamber demonstrated focusing of a microsphere sample to a central stream approximately 40 microm in diameter, yielding a definite fluorescence peak for the microspheres as compared with a broad distribution for unfocused microspheres. CONCLUSIONS: The flow cell developed here uses acoustic focusing, which inherently concentrates the sample particles to the center of the sample stream. This method could eliminate the need for sheath fluid, and will enable increased interrogation times for enhanced sensitivity, while maintaining high particle-analysis rates. The concentration effect will also enable the analysis of extremely dilute samples on the order of several particles per liter, at analysis rates of a few particles per second. Such features offer the possibility of a truly versatile low-cost portable flow cytometer for field applications.

Acoustics↗

PubMed2005

Ultrasonic particle concentration in a line-driven cylindrical tube.

Acoustic particle manipulation has many potential uses in flow cytometry and microfluidic array applications. Currently, most ultrasonic particle positioning devices utilize a quasi-one-dimensional geometry to set up the positioning field. A transducer fit with a quarter-wave matching layer, locally drives a cavity of width one-half wavelength. Particles within the cavity experience a time-averaged drift force that transports them to a nodal position. Present research investigates an acoustic particle-positioning device where the acoustic excitation is generated by the entire structure, as opposed to a localized transducer. The lowest-order structural modes of a long cylindrical glass tube driven by a piezoceramic with a line contact are tuned, via material properties and aspect ratio, to match resonant modes of the fluid-filled cavity. The cylindrical geometry eliminates the need for accurate alignment of a transducer/reflector system, in contrast to the case of planar or confocal fields. Experiments show that the lower energy density in the cavity, brought about through excitation of the whole cylindrical tube, results in reduced cavitation, convection, and thermal gradients. The effects of excitation and material parameters on concentration quality are theoretically evaluated, using two-dimensional elastodynamic equations describing the fluid-filled cylindrical shell with a line excitation.

Journal Article↗

PubMedDate not supplied

Conformation and structure of polymeric contrast agents for medical imaging.

The structure of Gd-DTPA-polylysine, Gd-DOTA-polylysine, Gd-SCN-Bz-DOTA-polylysine, and Gd-DTPA-poly(glu:lys) was investigated with circular dichroism, gel permeation chromatography, low angle light scattering, and proton longitudinal relaxivity. Molecular modeling calculations were performed and predicted helical secondary structure for charged Gd-chelator residues, i.e., Gd-DTPA, when the DTPA conjugation levels reached 90% and higher. This helical secondary structure was observed with circular dichroism. The conformational transition from coiled to extended linear was observed also by gel permeation chromatography and by proton relaxivity measurements. The helical secondary structure was not observed when the chelator was changed to DOTA. The residue charge interactions were eliminated in this case since the Gd-DOTA complex had no net charge. For this construct, the gel permeation and relaxivity measurements indicated a coiled conformation. An extended linear conformation was regained when the chelator complex was changed to Gd-SCN-Bz-DOTA, which had a net negative charge. The functional aspects of these structures were investigated by MR imaging of an animal tumor model. The linear extended polymer constructs gave 10-fold higher tumor signals then the coiled-collapsed constructs, indicating a much higher degree of trans-endothelial transport in the tumors.

Animals↗