Gregory Goddard

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

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.

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.

Single particle high resolution spectral analysis flow cytometry

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.

High-Resolution Spectral Analysis of Individual SERS-Active Nanoparticles in Flow

Nanoparticle spectroscopic tags based on surface enhanced Raman scattering (SERS) are playing an increasingly important role in bioassay and imaging applications. The ability to rapidly characterize large populations of such tags spectroscopically in a high-throughput flow-based platform will open new areas for their application and provide new tools for advancing their development. We demonstrate here a high-resolution spectral flow cytometer capable of acquiring Raman spectra of individual SERS-tags at flow rates of hundreds of particles per second, while maintaining the spectral resolution required to make full use of the detailed information encoded in the Raman signature for advanced multiplexing needs. The approach allows multiple optical parameters to be acquired simultaneously over thousands of individual nanoparticle tags. Characteristics such as tag size, brightness, and spectral uniformity are correlated on a per-particle basis. The tags evaluated here display highly uniform spectral signatures, but with greater variability in brightness. Subpopulations in the SERS response, not apparent in ensemble measurements, are also shown to exist. Relating tag variability to synthesis parameters makes flow-based spectral characterization a powerful tool for advancing particle development through its ability to provide rapid feedback on strategies aimed at constraining desired tag properties. Evidence for single-tag signal saturation at high excitation power densities is also shown, suggesting a role for high-throughput investigation of fundamental properties of the SERS tags as well.

Evaluation of a Green Laser Pointer for Flow Cytometry

Flow cytometers typically incorporate expensive lasers with high‐quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low‐cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A

Acoustic lysis of vegetative bacterial cells: Method and device development

160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom‐built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise ≤ 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow‐flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and ≤1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 μs (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of ∼75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times. Published 2007 Wiley‐Liss, Inc.

Acoustic focusing flow cytometry

A critical problem of many pathogen detection assays is the availability of intracellular protein and deoxyribonucleic acid (DNA). Acoustic lysis of suspended vegetative bacterial cells in a microfluidic system offers several advantages over conventional lysis techniques. The intracellular proteins and DNA are released and available for detection. A novel acoustic lysing alternative technique to the existing lysing methods for sample preparation and lysis step is proposed. We report here an efficient lysis device that uses acoustic excitation for performing lysis of Gram-positive and Gram-negative vegetative cells and has a high yield in a short amount of time. We also verified the condition of released protein since one of the major uses of vegetative cells lysis is for protein expression studies. Fluorimetry and flow cytometry were used to assess the degree of damage induced on the cells by the actual lysis method. The acoustic device allows the delivery of proteins in a non-denatured form, without adding chemicals, particles or other substances (e.g. enzymes) that could complicate the process or the detection procedure. The lysis device operates at low power (50–400 mW) and short time (3 min) and has high efficiency in comparison to current lysis standards (>85% vs. 12–50%).

Cellular discrimination based on spectral analysis of instrinic fluorescence

Acoustic cytometry is a new technology that replaces or partly replaces hydrodynamic focusing of cells or particles in flow cytometry with forces derived from acoustic radiation pressure. The ability to focus cells into a tight line without relying on hydrodynamic forces allows many possibilities outside the scope of conventional flow cytometry. Dilute samples can be processed quickly. Flow velocities can be varied allowing control of particle delivery parameters such as laser interrogation time and volumetric sample input rates. Recently, Life Technologies unveiled a flow cytometer that directs particles into the laser interrogation region using acoustic radiation pressure. In this talk, the application of acoustic cytometry in flow cytometry systems from fundamental principles to details of its implementation will be presented. Data will be shown for both the operational implementation of the acoustic focusing device as well as demonstrating its ability to perform for complex biological assays.

Analytical Performance of an Ultrasonic Particle Focusing Flow Cytometer

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.