Acoustic waves, diffraction gratings, and digital data acquisition systems bring about greater throughput, higher sensitivity, and higher resolution cytometry systems.

It’s more compact than a conventional flow cytometer, more powerful than traditional hydrodynamic focusing, and able to bypass fluidics systems in a single bound.

The world’s first portable acoustic cytometer (PAC) harnesses acoustic waves to focus cells into a tight, centered stream for analysis. The result is greater throughput and sensitivity than conventional flow cytometers without the need for large volumes of purified water and for thousands of dollars less.

The PAC, developed by researchers Gregory Kaduchak, Steven Graves, Gregory Goddard, John Martin, Robert Habbersett, Mark Naivar, and Michael Ward with the National Flow Cytometry Resource (NFCR) at Los Alamos National Laboratory (LANL), N.M., is the latest of six R&D 100 Awards won by the flow cytometry team at LANL.

The NFCR is funded by the National Center for Research Resources through the National Institutes of Health (NIH). According to James Freyer, director of the NFCR, it pursues the development of advanced flow cytometry instrumentation and applications and transfers these developments to the biomedical science community, continuing a 35-year tradition of flow cytometry technology development at LANL.

A combined acoustic particle focusing and optical cell recently developed by Acoustic Cytometry Systems acoustically focuses particles into a tight line for single particle optical interrogation within the optical cell. It is the particle delivery and optical interface for a portable flow cytometer. Image: Dixon Wolf

“Flow cytometry is a powerful biomedical technology that has been largely limited to use in first-world clinics and is the primary diagnostic for HIV progression analysis as well as many forms of cancer,” says Graves, team leader, Optical Spectroscopy and Instrumentation. “The PAC’s reduced cost and consumables requirements will make it possible to bring the power of flow cytometry to resource-limited areas of the world for medical diagnosis of diseases ranging from AIDS to cancer.”

Improving upon current technology

Conventional flow cytometers measure the physical and biochemical characteristics of cells or any particle. Among standard diagnostic equipment in clinical laboratories and medical centers, they produce blood cell counts and leukocyte subpopulation counts, and they monitor levels of lymphocytes, such as CD4+, as part of patients’ HIV/AIDS treatment plans.

A “sheath” fluid, usually a buffered saline solution, hydrodynamically focuses cells through a laser beam in conventional equipment, requiring both additional fluidic control systems and the use of large quantities of purified water. This hydrodynamic focusing results in extremely fast moving particles, which greatly increase other system requirements.

Conventional flow cytometers also require expensive light sources and detectors to adequately illuminate samples and ensure enough scattered and emitted light is collected for analysis. The complexity of the fluidics system and the need for high-quality lasers and detectors make most commercial flow cytometers bulky, expensive, and fragile. Consequently, their use is limited to laboratories with highly skilled workers and a fairly high level of infrastructure support.

The PAC uses a single ultrasonically vibrated capillary in place of a complex fluidics system. The capillary is comprised of glass with a piezoceramic device bonded to its outer surface, into the top of which sample cell suspension is injected. When energy is applied to the piezoceramic device, the device creates an ultrasonic field that drives the suspended cells to the center of the flow stream, aligning them in a narrow stream approximately 10 µm in diameter that passes through the laser beam. This allows for analysis at rates of up to 100,000 cells per second.

“Eliminating the sheath reduces instrument size and complexity, operating costs, use of consumables, and waste,” says Graves. “This is particularly important in the field or in less-developed areas of the world where clean water can be a scarce and valuable commodity.”

By using lower-cost lasers, such as those used in laser pointers, and simpler detection electronics, such as miniature photomultiplier tubes and single-chip digital signal processors, the PAC will make it possible for doctors or technicians to make diagnoses using a smaller, simpler, more rugged instrument that uses fewer consumables and generates minimal waste.

Any analyses currently made by using conventional flow cytometers may benefit from this technology, including screening potential new drugs, typing blood cancers, analyzing compatibilities for tissue transplants, screening for cancer markers or infectious agents, and monitoring cell populations and subpopulations to assess patients’ responses to anti-retroviral or chemotherapy drugs.

In an effort to bring new particle analysis capabilities such as the PAC to clinicians and researchers worldwide, Acoustic Cytometry Systems (ACS), LLC, a company spun off from LANL, was founded in 2006, in Los Alamos to commercialize acoustic focusing technology in flow cytometry and sample preparation.

In addition to the PAC, the NFCR has developed several other innovative particle analysis technologies recently including the high-sensitivity flow cytometer, a high-resolution spectral analysis flow cytometry system, and an open, reconfigurable cytometric acquisition system.

Analyzing molecules

DNA fragments flow through the flow channel from the bottom of the flow cell to the exit tubing at the top. The green (532 nm) laser beam passes through the focusing lens from right to left and then through the flow cell. Image: Los Alamos National Laboratory

Individual DNA molecules as small as 125 base pairs are now measurable in a simplified flow system with improved resolution and increased dynamic range using the high-sensitivity flow cytometer developed by Habbersett and James Jett, technical staff members at LANL.

According to Habbersett, this compact low-power system is sensitive enough to detect individual B-phycoerythrin molecules in solution, cleanly resolving them from the background. Originally developed for DNA fragment sizing, this benchtop instrument is also capable of the rapid flow analysis of subcellular organelles, bacteria, viruses, and engineered nanoparticles. Its most exciting application, however, may be extending the range of flow particle analysis to the molecular realm.

“Measuring the length of DNA fragments is arguably the most commonly performed biological measurement. Our low-cost system with this very high level of sensitivity could dramatically impact many types of biomedical assays,” says Habbersett. “This level of sensitivity should enable experiments that few other instruments can perform, such as detecting a few (<10) protein molecules interacting with a small DNA fragment.”

High-resolution spectral analysis

Although conventional multiparameter flow cytometers have proven successful, several types of analytical measurements would benefit from a more comprehensive and flexible approach to multi-color analysis.

The high-resolution spectral analysis flow cytometry system, developed by Goddard, a technical staff member at LANL, uses a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular CCD image sensor. This enables the user to obtain the complete emission spectrum of each particle as it passes through the laser.

The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to ensure modularity of the system. Evaluation of the prototype included wavelength characterization and calibration of the dispersive optics, explains Goddard, and benchmarking of this system demonstrated a single particle/cell detection sensitivity of 160 molecules of fluoroscein with a spectral resolution of approximately 1 nm.

“The strengths of the system are flexibility, relative simplicity, high spectral resolution, and excellent sensitivity,” says Goodard. “Minimal modifications are needed to add a diffraction grating and image sensor onto an existing flow cytometer, and the higher resolution gains the user flexibility in fluorophore selection. And, by replacing mirrors, filters, detectors, and detector electronics and with a grating, image sensor, and one set of control and acquisition electronics, the system becomes simpler, optically as well as electronically.”

Open, reconfigurable cytometric acquisition

The future of flow cytometry also requires advanced hardware and software that can be used to upgrade existing flow cytometers. The open, reconfigurable cytometric acquisition system (ORCAS) is a digital data acquisition system developed to support novel flow cytometry efforts. A commercial digital signal processing board processes the captured data and sends the results to the host computer that provides a user interface for acquisition, display, analysis, and storage. The system collects list mode data, correlated pulse shapes, and streaming data from a variety of detector types (e.g., photomultiplier tubes, solid-state detectors, photon counters, or CCDs) using Linux, Mac OS X, and Windows host computers.

“The system flexibility includes how it detects, captures, and processes event data. Custom data capture boards utilizing analog to digital converters and field programmable gate arrays (FPGAs) detect events and capture correlated event data,” says Naivar, technical staff member at LANL.

A recent report demonstrated that not only can this system support custom instrumentation, but ORCAS can be easily added to existing commercial instruments to provide improved data acquisition features.

“ORCAS extracts pulse features not found on commercial systems with excellent sensitivity and linearity over a wide dynamic range,” explains Naivar. “ORCAS is compact, scalable, flexible, and modular. Programmable feature extraction algorithms have exciting possibilities for both new and existing applications.”

—Mig Owens,
Communications specialist,
Los Alamos National Laboratory

Resources

• Acoustic Cytometry Systems (ACS), LLC, Los Alamos, N.M., 505-670-0232,
www.acousticyte.com

• Los Alamos National Laboratory (LANL), Los Alamos, N.M., 877-723-4101,
www.lanl.gov

• National Flow Cytometry Resource (NFCR), Los Alamos, N.M., 505-667-3912,
http://nfcr.lanl.gov