A River Runs Through It

Flow cytometry instruments analyze suspensions of single cells by focusing them hydrodynamically in a columnar stream and causing them to flow past a point or points in the stream where lasers have been focused. As each cell passes through the point of excitation, it scatters the laser light, and if it is appropriately stained, it will also fluoresce. Sensors collect both the scattered light and fluorescence, and based on the timing of the pulses, the collected signals can be identified with an individual cell. When the stream is agitated, droplets form, some of which contain single cells. With some careful timing, droplets containing a desired cell can be either positively or negatively charged and then deflected away from the main stream and collected. Significant variations from this basic design include cells flowing in a quartz tube or across a microscope slide. Both of these configurations improve resolution but preclude physical sorting. The appeal of flow cytometry in the study of the cell cycle remains its capacity to eliminate the tedium of waiting for cells to grow up. If one accepts the idea that the cell cycle is adequately resolved by a one-dimensional DNA histogram and G1-S-G2 binning, then in a few minutes and for pennies in reagents, an age distribution can be had. If one has reservations about this view, it is still possible, for a fistful of dollars in reagents, to resolve the cycle into its cell cycle phase-specific populations using combinations of cyclins, proliferating cell nuclear antigen (PCNA), bromodeoxyuridine (BUDR), propidium iodide (PI), and sundry other surface and internal immunological markers. Compare this with staying up for 24 hours to collect synchronous cells and analyze them as they traverse the cell cycle, and you capture the instrument's appeal. Historically, three basic optical designs have been used, around which a variety of lasers or more conventional excitation sources, electronics, signal-collection devices, and software choices have been assembled: laser excitation of cells hydrodynamically focused in an open stream, a set-up that enables sorting but suffers somewhat in resolution due to instabilities in the stream the same basic instrument as above but with the cells focused in a stream flowing within a quartz cell from which sorting is not possible but resolution is improved compared to the open stream systems a microscope-based instrument in which cells flow across an optical surface under an epifluorescent objective. Currently, the only machine of this type available in this country is offered by Bio-Rad. The low price and high resolution of this machine should make it attractive to investigators with a variety of interests in addition to cell cycle studies. Flow machines can be purchased today with one, two, or three lasers (the greater the number of lasers, the more flexibility in fluor selection) and from 3 to 10 signal-collection devices (generally photomultiplier tubes or PMTs). Lasers are available with peak power wavelengths from 351 to 633 nm and in powers from 10 mW. Popular lasers for cell cycle applications include He/Ne at 633 nm, argon ion in wavelengths from 351 to 514 nm, and multi-line krypton ion. Electronics packages currently available make it possible to collect both the integrated fluorescence from each excited cell as well as the peak fluorescence, providing the ability, among other things, to detect those cells that are in the process of dividing and to distinguish them from adventitious doublets. The light-scattering properties, both forward angle and 90-degree scatter, of each cell are also collected, and this signal is most commonly used as a trigger to begin collection of fluorescence signals. With 10 physical collection devices and 3 lasers, it is possible to acquire more than 30 cellular measures, but most machines, limited by the number of analog-to-digital converters, can collect only 10 parameters from each cell. Most instruments will collect all signals in an uncorrupted, unbinned, list mode form, which gives flexibility in analyzing and displaying data. In practice, the primary limitation of flow cytometers lies in their inability to distinguish between fluors with overlapping excitation or emission spectra and the number of commercially available labeled fluors with compatible nonoverlapping signals. In practice, the primary limitation of flow cytometers lies in their inability to distinguish between fluors with overlapping excitation or emission spectra and the number of commercially available labeled fluors with compatible nonoverlapping signals. A way around fluor/reagent limitation has been employed successfully recently in some laboratories. Viable or fixed cells stained for markers of interest with 2 or 3 fluors of compatible excitation and emission spectra, can be sorted and then restained with a different set of fluors and re-analyzed or re-sorted. Clearly, to make this a feasible experiment, fast sort rates, multiple simultaneous sorts and a low abort rate (low loss of desired cells) due to machine electronics are necessary. Gone are the days when every flow cytometry laboratory had to have resident programming skills in order to display data adequately. While one may still have a quibble or two about the software packages sold with the instruments, they are vastly improved but may be platform specific. For many types of cell cycle analyses, the ability to collect data in list mode is essential. Several software houses offer after-market packages for specific applications and displays. The choice of such is very much a matter of taste, guided by one's underlying view of the cell cycle. In a few words, the more parameters the better. To many, the now classic one-dimensional flow cytometric DNA content histogram has come to be equated with the cell cycle. The new generation of machines can make it more certain that this definition is only approximately correct. A simple two-dimensional display of DNA content (integrated versus peak fluorescence) as determined by PI staining reveals that there are cell-cycle-specific populations as well as artifacts hiding behind the one-dimensional profile. In the figure (Mitotic Stages in Flow), a PI stain showing integrated DNA fluorescence on the y axis and PI peak fluorescence on the x axis shows that adventitious doublets, and possibly condensed premitotic (G2), mitotic, and newly divided cells may contaminate the "S-phase" cell populations. The danger, of course, is that these underlying cells will distort the estimation of cell cycle distributions. If these are minor contaminants in an analytical study, perhaps there is little need for concern. However, these contaminants are of greater concern if amplification techniques such as cloning of viable sorts or PCR are applied to sorted cell populations. In these instances, minor contaminants may significantly distort the results. Flow cytometry is on the move. Fast and well-designed electronic logic and multiple excitation and signal-collection devices make it possible to explore the cell cycle and growth regulation in ways that were not feasible a few years ago. These advances, which allow high sample throughput, incredible sort rates, and online or post-collection analyses, will move the field beyond simple one-dimensional views of the cell cycle. Finally, laser-scanning cytometry, though not strictly speaking a flow approach, promises to complement flow analysis. Following is a description of the flow cytometers currently in the market, with more detailed information contained in the table on the scientist web site (www.the-scientist.com). Bio-Rad BRYTE HS Flow Cytometer The BRYTE HS Flow cytometer from Bio-Rad is a new arc lamp flow cytometer for applications in microbiology, DNA analysis, and immunophenotyping. Use of a xenon or xenon-mercury arc lamp allows selection from a wide range of excitation wavelengths, from the UV through the visible spectrum. A variety of easily installed single and dual excitation and emission wavelength filter sets allows the use of any of the dye combinations used in flow cytometry. For projects involving very small particles such as bacteria, this instrument features particle counting capability, size detection down to <0.2 micron, forward and side scatter, and two- or three- color PMTs. Becton Dickinson FACSCalibur® Flow Cytometry System FACS Vantage Flow Cytometry System Becton Dickinson offers cytometers that meet both analysis as well as sorting needs. FACSCalibur Flow Cytometry System is a modular cytometer that offers up to four colors of fluorescence in addition to two scatter parameters. In addition to the blue laser line at 488 nm, FACSCalibur offers a second red diode laser at 635 nm for maximum sensitivity and flexibility in fluorochrome selection, while minimizing the need for spectral compensation for multicolor analysis. FACSCalibur can also be upgraded with FACS Loader for automated sample injection or with a unique catcher tube-sorting module, as well as many application-specific and general-purpose software packages. The FACS Vantage Flow Cytometer System is the most recent cell sorting instrumentation offered by B-D for the research lab. Like the FACSCalibur, FACS Vantage is fully modular for flexibility. It provides nonrectangular sort windows as a standard feature and enhanced fluidics for stability and reliability. The system features up to six colors of fluorescence and allows the use of up to three lasers. The new TurboSORT Plus upgrade streamlines high speed sorting, while CloneCytÔ Plus features single- or multi-cell deposition into a wide variety of devices. Also, the IndexSort feature of CloneCytPLUS maps the exact cell deposition location with the cellular signature found in the list mode file, allowing cellular function to be correlated with phenotype. Many other application-specific upgrades are available for this system. Coulter® EPICS® XL/XL-MCL Flow Cytometry System EPICS Elite ESP High Performance Cell Sorter The Coulter EPICS XL-MCL Flow Cytometer combines the analytical power of a research cytometer with a compact, clinical analyzer. The XL system features the capability to analyze up to four colors of fluorescence from a single air-cooled laser. The instrument is self-contained and biohazard safe. The user retains the flexibility to change filter elements for versatility in research settings. For high volume users, the XL MultiCarousel Loader (MCL) is capable of throughputs of up to 100 samples per hour. The fully featured XL-MCL incorporates positive bar-code identification and true vortex mixing prior to aspiration. The XL offers Digital Signal Processing (DSP) for linearity and drift-free amplification and compensation. The single laser design eliminates concerns regarding multibeam stability, signal delay, and alignment. Compensation is achieved by a digital compensation matrix. The new XL SYSTEM IITM Software fully automates instrument set-up and compensation for two-, three-, and four-color applications. Optional features in the software include tetraONE (the four-color, one-tube, automatic analysis algorithm) and reticONE (the automatic analysis algorithm for reticulocyte analysis). The ELITE ESP (Enhanced System Performance) High Performance Cell Sorter features the capability to analyze and sort up to six colors simultaneously while performing multiparameter applications, such as DNA quantification, functional studies, chromosome enumeration, or physiological measurements. The Elite ESP allows for rapid separation with sort rates up to 15,000 cells/sec, using four predefined sort modes or a fifth custom sort set-up. The Elite ESP offers a user-configurable optical platform. Laser preferences, from inexpensive, air-cooled lasers to top-of-the-line high-powered water-cooled lasers that fit on the optical bench, allow a variety of excitation wavelengths. Cytomation MoFlo High Speed Sorter and Analyzer The MoFlo High Speed Sorter and Analyzer combines the performance of a research sorter with the ease of use of more limited systems. The MoFlo line consists of a series of models, each upgradeable to 3-laser, 12-parameter fluorescence channels plus scattered light and 25,000 cell/sec performance. The modular flow cytometer systems have been designed to eliminate performance variability due to interdependent component adjustments. Independent optical paths allow simple adjustments for each beam. Maximum sort rates in excess of 25,000 cells/sec are achieved by a proprietary jet in air nozzle that minimizes divergence from laminar flow conditions. The fluidics system is designed to operate at pressures up to 100 psi. Transport tubing is terminated in Swage-type connectors to enable rapid dismantling for sterilization or mechanical cleaning. Sample injection is designed to eliminate contamination. The sample injector is equipped with a three-port inlet to facilitate kinetics experiments. The author can be reached at neumann@earthlink.net

A River Runs Through It

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