Inertial flow focusing: a case study in optimizing cellular trajectory through a microfluidic MEMS device for timing-critical applications.

Although microfluidic micro-electromechanical systems (MEMS) are well suited to investigate the effects of mechanical force on large populations of cells, their high-throughput capabilities cannot be fully leveraged without optimizing the experimental conditions of the fluid and particles flowing through them. Parameters such as flow velocity and particle size are known to affect the trajectories of particles in microfluidic systems and have been studied extensively, but the effects of temperature and buffer viscosity are not as well understood. In this paper, we explored the effects of these parameters on the timing of our own cell-impact device, the µHammer, by first tracking the velocity of polystyrene beads through the device and then visualizing the impact of these beads. Through these assays, we find that the timing of our device is sensitive to changes in the ratio of inertial forces to viscous forces that particles experience while traveling through the device. This sensitivity provides a set of parameters that can serve as a robust framework for optimizing device performance under various experimental conditions, without requiring extensive geometric redesigns. Using these tools, we were able to achieve an effective throughput over 360 beads/s with our device, demonstrating the potential of this framework to improve the consistency of microfluidic systems that rely on precise particle trajectories and timing.

Toward the measurement of multiple fluorescence lifetimes in flow cytometry: maximizing multi-harmonic content from cells and microspheres.

Flow cytometry is a powerful means for in vitro cellular analyses where multi-fluorescence and multi-angle light scattering can indicate unique biochemical or morphological features of single cells. Yet, to date, flow cytometry systems have lacked the ability to capture complex fluorescence dynamics due to the transient nature of flowing cells. In this contribution we introduce a simple approach for measuring multiple fluorescence lifetimes from a single cytometric event. We leverage square wave modulation, Fourier analysis, and high frequency digitization and show the ability to resolve more than one fluorescence lifetime from fluorescently-labelled cells and microspheres. Illustration of a flow cytometer capable of capturing multiple fluorescence lifetime measurements; creating potential for multi-parametric, time-resolved signals to be captured for every color channel.

Digital data acquisition and processing.

A flow cytometer is made up of many different subsystems that work together to measure the optical properties of individual cells within a sample. The data acquisition system (also called the data system) is one of these subsystems, and it is responsible for converting the electrical signals from the optical detectors into list-mode data. This unit describes the inner workings of the data system, and provides insight into how the instrument functions as a whole. Some of the information provided in this unit is applicable to everyday use of these instruments, and, at minimum, should make it easier for the reader to assemble a specific data system. With the considerable advancement of electronics technology, it becomes possible to build an entirely functional data system using inexpensive hobbyist-level electronics. This unit covers both analog and digital data systems, but the primary focus is on the more prevalent digital data systems of modern flow cytometric instrumentation.

Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts.

Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells.

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.

Capture of Fluorescence Decay Times by Flow Cytometry.

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction.

Digital analysis and sorting of fluorescence lifetime by flow cytometry.

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 micros, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142 degrees to 1.6 degrees. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 micros and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to approximately 98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will allow both better dissemination of this technology and better exploitation of the traditionally underutilized parameter of fluorescence lifetime.

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach.

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.

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 $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 < or = 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 < or =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 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 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.

Open, reconfigurable cytometric acquisition system: ORCAS.

A digital signal processing (DSP)-based digital data acquisition system has been developed to support novel flow cytometry efforts. The system flexibility includes how it detects, captures, and processes event data. Custom data capture boards utilizing analog to digital converters (ADCs) and field programmable gate arrays (FPGA) detect events and capture correlated event data. A commercial DSP board processes the captured data and sends the results over the IEEE 1394 bus to the host computer that provides a user interface for acquisition, display, analysis, and storage. The system collects list mode data, correlated pulse shapes, or streaming data from a variety of detector types using Linux, Mac OS X, and Windows host computers. It extracts pulse features not found on commercial systems with excellent sensitivity and linearity over a wide dynamic range. List mode data are saved in FCS 3.0 formatted files while streaming or correlated waveform data are saved in custom format files for postprocessing. Open, reconfigurable cytometric acquisition system is compact, scaleable, flexible, and modular. Programmable feature extraction algorithms have exciting possibilities for both new and existing applications. The recent availability of a commercial data capture board will enable general availability of similar systems.