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Cytomation was selected by LLNL to bring MoFlo from prototype to commercial instrument, adding at the same time new features and functionality as well as a new level of engineering quality.
Sort and Analysis Speed
MoFlo is designed first and foremost as a very high speed sorter and analyzer. It offers the sort performance needed for the quantitation and/or isolation of rare cell populations with high recovery and purity, on time-scales that do not require operation for longer than an average working day and that do not place extreme demands on instrument stability. The design specification was for speeds at or above 25,000 cells per second, approximately 5 to 10 times as fast as existing instruments. Sorted cells must be viable, purity must be better than 95 percent at any speed, and yield must be solely as determined by Poisson statistics.
Modularity
Secondly, MoFlo is designed for maximum flexibility in the research laboratory. It is a requirement of LLNL that the specification of the electronics bus be in the public domain so that experimenters may, if desired, design and construct special-purpose interface electronics and use them in MoFlo. The system also must be expandable within reasonable physical bounds; that is, it must be easy to add extra sensors, extra electronics components, extra lasers and even to add sorting capability to a non-sorting MoFlo whenever desired. This specification contrasts dramatically with the mind-set of the established manufacturers, who have marketed increasingly inflexible instruments, incapable of expansion and essentially dedicated to either analysis or sorting.
Multiple Laser Capability
At the heart of MoFlo is a patent on an innovative electronics design which allows the system to precisely measure, analyze and make sort decisions on, any number of cells passing through the multiple laser intercept points. This capability eclipses competitors designs, which are able to handle just one cell at a time in the laser intercept region, and are thus performance-limited by the number of lasers. MoFlo suffers from no such limitation.
Optical
The foundation of MoFlo is a standard optical breadboard, with 1-inch center, quarter-20 holes for mounting of standard optical components. The size of the table is customized to any requirements, depending primarily on the number and type of lasers in your initial configuration.
Each laser beam is independently steered and focused for easy and stable alignment and re-alignment after any reconfiguration.
Each optical pathway preserves vertical polarization of laser light, for optimum illumination of the subject cells.
High-precision Newport-Klinger positioners are used on all optical stages, for maximum accuracy and stability and minimum hysteresis in alignment.
Two infrared cameras, one at the stream illumination point and one at the sort droplet breakoff, eliminate the need for tedious viewing microscopes.
Spatial, chromatic and temporal filtering , plus collimating lenses on each laser collection pathway, maximize signal quality and signal-to-noise ratio and minimize crosstalk.
Electronics
The MoFlo bus is public domain - you can design interfaces and special purpose cards to plug into it, for your own special needs. The electronics rack has a VME form factor, making compatible parts inexpensive, easily obtained and standardized.
The cycle time, regardless of the number of lasers in use, is 5.5 microseconds, and the sort module is capable of operation at clock rates (droplet formation rates) up to 200,000 per second.
Selected parameters may be displayed as pulses, stored dotplots and dynamic dotplots on three oscilloscopes.
Up to 5 ADC modules (10 channels) may be installed in the bus. Each ADC channel can generate peak, log or integral signals and each may be connected to any signal input, for maximum flexibility.
Sort decisions are executed by sets of 8 hardware lookup tables, each measuring 256 by 256 channels. Lookup table decisions may be logically combined to permit very complex bivariate decisions. Sorting is optimized for single drop operation, with two-drop, three-drop and phase-sensitive single drop options also selectable.
Drop delay is selectable with 1/16 drop precision, for superb purity and recovery even at the fastest rates.
The standard sorting configuration is 2-way, but a 4-way sorting option is available.
Optical crosstalk compensation between detectors collecting from a single laser beam is a standard facility in today's instruments. MoFlo adds inter-beam compensation for tomorrow's multi-color experiments.
Fluidics
All components of the fluid system are designed to operate at pressures of up to 100 psi. Where possible, all tubing, connectors and valves are of the type used in High Pressure Liquid Chromatography, for extremely low dead volumes, very stable physical characteristics and high resistance to accumulation of particles. Tubing is color-coded throughout for ease of use and safety.
The heart of the fluid system is the patented CytoNozzle flow cell, available now in 50, 70, 100 and 200 micron orifice diameters. The CytoNozzle is designed for operation at high pressures and flow rates, with high stability and minimal resonance effects, and is engineered with attention to minimizing cavities that can function as a source of contamination.
The MoFlo sample system minimizes contamination from sample to sample and is easily dismantled for autoclaving and sterilization.
Computer System and Software
Powering MoFlo is a Pentium computer with a 1 GByte hard disk, 16 MBytes of RAM and a 17-inch monitor. Larger disks, RAM and monitor are available as options.
The software is Cytomation's powerful CyCLOPS® flow cytometry software, modified for MoFlo. CyCLOPS is the only universal flow cytometry software interface in the industry, available for all manufacturers' instruments with Cytomation's CICEROreg. computer/electronics instrument upgrade. CyCLOPS controls the instrument, acquires listmode and histogram data in the standard FCS format, analyzes listmode and histogram files, and controls sorting by translating the operator's sort decisions into the hardware lookup tables, all in a modern, fast, windowed menu environment.
CyCLONETM Automated Cloning Device
For sorting on to slides or into 96-well or 384-well trays, the CyCLONE is the fastest, highest-precision, most flexible device available. Slides and trays with 24, 96 or 384 wells are standard, and non-standard devices of almost any dimension may be defined with the software for custom applications. CyCLONE also has a pre-programmed drop delay matrix mode for rapid automated drop delay determination.
Modularity
Every MoFlo configuration is assembled to the buyer's custom design, using the electronics, optical and fluidic basic building blocks. The minimum useful configuration is an analyzer consisting of a 2' by 2' optical breadboard with a single air-cooled laser, four detector channels (2 scatter and 2 fluorescence) and the standard computer system and software, without sorting. At the high end of the spectrum is a three laser, 25,000 events per second sorting instrument on a large optical table, with two or more detectors on each collection channel. Between is a range of configurations of varying power, varying number of detectors and with or without sorting capability.
Cytomation's goal is to produce a range of flow cytometry instrumentation with minimum obsolescence. In keeping with that goal, every MoFlo configuration is easily and inexpensively upgradeable to a bigger and better configuration. Even CICERO systems are upgradeable to MoFlo at minimal expense.
MoFlo is a revolutionary design today and will continue to set the standards in flow cytometers for research applications. Two areas of development are of primary importance in the near term:
The next major revision of MoFlo will come with extensive automation to enable alignment of all optics, fluidics and sorting without operator intervention and with unerring reproducibility.
Careful attention has been paid in the design of MoFlo to the necessity for future biohazard containment. Future MoFlo systems will feature an option for total isolation of the sort process from operator contact.
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