The BD FACSAria III sorter is built on the solid foundation of patented technologies, superior multicolor performance, and legendary ease-of-use that opened the complex world of cell sorting to a broader audience of researchers and wider range of applications. The fluidics and optical systems are precisely integrated to maximize signal detection. A patented flow cell with gel-coupled cuvette and patented octagon and trigon detection system allow the system to achieve unrivaled sensitivity and resolution.
Sensitivity for Multicolor and Sorting Applications
The fluidics system in the BD FACSAria III cell sorter is pressure driven. Positive air pressure forces sample cells through an optically gel-coupled cuvette flow cell. Hydrodynamic focusing guides particles in a single-file stream through the cuvette, where laser light intercepts the stream at the sample interrogation point
Gel-coupled cuvette flow cell
At the heart of the BD FACSAria III is a quartz cuvette flow cell in true fixed alignment with the laser and gel coupled to the collection optics. For greater sensitivity, the BD FACSAria III incorporates a next-generation cuvette in the flow cell. Its patented design helps ensure that lasers are precisely focused on the sample stream, that they generate the greatest signal, and that the maximum amount of emitted light is collected.
Fixed alignment minimizes startup time, improves experiment-to-experiment reproducibility, and enables automated daily quality control. Most importantly, it also improves collection efficiency and optimizes resolution needed for multicolor applications, even at high-speed sorting settings.
In addition to other benefits, the next generation flow cell in the BD FACSAria III is designed to improve resolution for side population applications and DNA cell cycle analyses.
High-performance analysis, high-performance sorting
The BD FACSAria III analysis performance is comparable to state-of-the-art highly sensitive analyzers. This is accomplished by using a similar gel-coupled flow cell design and the fixed optical architecture of the BD FACSCanto™ II and BD LSRFortessa systems.
This design architecture achieves high numerical aperture light collection. The flow cell and nozzle design enable low particle speeds in the analysis zone for maximum light collection, and then accelerate the particle through the nozzle at stream speeds to achieve the drop rates required for high-performance sorting.
Through the precise coordination of the optical and fluidics systems, the BD FACSAria III delivers exceptional optical detection sensitivity compared to traditional stream-in-air systems, in which particle speeds are the same for both analysis and sorting.
Nozzles for a range of particles
A choice of nozzles lets users sort a wide range of particle sizes. Nozzles are available in four sizes: 70, 85, 100, and 130 microns. Nozzles are readily accessible and easy to change, with a design offering tight registration for a secure fit. This means a reproducible drop profile after every nozzle exchange, resulting in reproducible instrument setup and alignment. The software sort setup matches pressure and sort settings to the nozzle being used.
Easy aseptic setup and cleaning
Innovations in the fluidics system such as easy-to-insert nozzles, automated sort setup, and easy-to-change filters make setup fast and simple. The fluidics design features integrated valve manifolds and a streamlined fluidics path. Software wizards make aseptic sort setup easy and effective. In addition, after a sample tube is run, both the inside and outside of the sample injection tubing are flushed to minimize carryover.
BD FACS Accudrop technology simplifies drop-delay determination
Patented BD FACS Accudrop technology assists the user to see the best drop-delay value. Software automation simplifies drop-delay determination. Once the drop-delay is calculated, the system automatically adjusts to maintain a constant break-off, called the Sweet Spot. Automatic clog detection stops the sort and protects the collection tubes if a clog is detected.
After passing through the cuvette, the stream accelerates through the nozzle, and droplets are formed for sorting. Since particle interrogation occurs above the nozzle, insertion and removal of the nozzle can occur without realigning the optics or the fluid stream.
Engineered into the System
A self-contained fluidics cart supplies sheath and cleaning fluids and collects waste from the cytometer. The cart also provides the air pressure and vacuum needed to achieve pressure from 5 to 75 psi, to accommodate a variety of cell sorting applications. BD FACSDiva™ software adjusts the air pressure. The fluidics cart is typically positioned directly under or to the left of the cytometer.
The fluidics cart holds a 10-L stainless steel sheath tank, a 5-L stainless steel tank used for shutting down the instrument with ethanol, and 10-L waste container. The sheath tank can be autoclaved. In addition, the cart holds three 5-L auxiliary cleaning fluid containers used in conjunction with the automated Prepare for Aseptic Sort mode.
Sample injection chamber
During acquisition, the sample injection chamber is pressurized, forcing the sample to the cuvette flow cell. To simplify acquisition, the chamber temperature and agitation settings are controlled using BD FACSDiva software. A variety of tube holders are provided, from 15-mL centrifuge tube to 1.0-mL microtube size. To minimize clogging, 35- and 50-micron sample line filters are available.
From the sort block to the collection chamber
After leaving the nozzle, particles pass through the sort block that houses the deflection plates. The novel design fixes the plates in position for more efficient and reproducible deflection into a collection device in the sort collection chamber.
The sort block also houses an aspirator drawer that keeps the sort collection tubes covered until sorting begins and automatically closes to protect the tubes when the Sweet Spot is on and a clog is detected.
The sort collection chamber’s universal mount design makes inserting the tube holders easier. The holders are designed to help maintain aseptic conditions when removing sort tubes. Temperature control for sort collection tubes, slides, and plates is available as an option.
Engineered with aerosol management in mind, the BD FACSAria™ III features an enclosed pathway from the sample injection chamber to the sort collection tubes. For an added level of aerosol management, the BD™ Aerosol Management Option (AMO) evacuates the sort collection chamber and traps aerosolized particles during sorting.
Increased efficiency for multicolor detection
Innovations in the optical system, pioneered by BD, efficiently maximize signal detection and greatly increase sensitivity and resolution for each color in a multicolor assay. Enhanced sensitivity and resolution mean that even dim populations can be readily identified and sorted.
The optics system allows optimizing multicolor assays and panel design for superior results. The design provides researchers a choice of laser excitation wavelength(s) that illuminate cells in the sample. Collection optics direct light scatter and fluorescence signals through spectral filters to detectors. Innovative designs for both the excitation and collection optics reduce excitation losses and dramatically improve collection efficiency, yielding better information from each sample.
The excitation optics consist of multiple fiber-launched fixed-wavelength lasers, beam-shaping optics, and achromatic focusing lenses that produce beam spots that are spatially separated and concentrated (9 μm x 65 μm). The more concentrated the beam spot, the higher the signal produced as each fluorescent-labeled particle passes through the laser spot. Laser light is focused into the gel-coupled cuvette flow cell. Optical gel coupling to the fluorescence objective lens enables transmission of the greatest amount of emitted light from the interrogation point to the collection optics. Since the optical pathway and the sample core stream are fixed, alignment is constant from day to day and from experiment to experiment.
Fixed alignment also ensures that there is no variability in experiment results introduced by manual optical adjustments.
Fiber optics deliver emitted light from the gel-coupled cuvette to the detector arrays. The collection optics are set up in patented octagon pathways that maximize signal detection from each laser illuminated beam spot. This is accomplished by transmitting the highest wavelengths (which have the fewest photons of light) to the first photomultiplier tube (PMT), and reflecting lower wavelengths to the next PMT through a series of longpass dichroic mirrors.
This design is based on the principle that light reflection is more efficient than light transmission. Emitted light travels to each PMT via reflection and is transmitted through only two pieces of glass to reach each detector. Therefore, colors can be detected with minimum light loss.
Bandpass filters in front of each PMT allow spectral selection of the collected wavelengths. Importantly, this arrangement simplifies filter and mirror changes within the optical array and requires no further alignment, for maximum signal strength.
Precision optical design
The many innovations in the BD FACSAria Fusion’s optical system, such as the patented gel-coupled cuvette and octagon detection system, and the 9-μm x 65-μm beam spot, are designed to work together to maximize sensitivity and resolution. This precision design delivers a more efficient optical system enabling the use of lower powered lasers, which in turn reduces the total cost of instrument operation.
Flexibility to Expand, Upgrade to Protect Investments
For many users, the capability of an advanced cell sorter is defined by its flexibility, which in turn is defined by the number of parameters that can be detected simultaneously. With flexibility built in, the BD FACSAria™ III can support six lasers and four spatially separated beam spots. Choose up to six laser wavelengths—633 nm, 561 nm, 488 nm, 445 nm, 405 nm, and 375 nm—and up to 20 detector positions, to measure up to 18 colors simultaneously.
Users can design a configuration that meets their lab’s budget and site requirements today and have a growth path for the future. They can expand the BD FACSAria III system with additional lasers via a field upgrade.
Lower cost of ownership
The unique, efficient design of the optical system delivers a lower cost of operation than stream-in-air sorters. The BD FACSAria III delivers maximum sensitivity and resolution using fiber launched solid state lasers. No special power or cooling is needed for these lower powered air cooled lasers.
Control and Analysis
Compatible with Other BD Analyzers and Sorters
BD FACSDiva™ software efficiently controls the setup, acquisition, and analysis of flow cytometry data from the BD FACSAria™ III workstation. BD FACSDiva software is common across many BD cell analyzers and cell sorters, including BD FACSCanto™ and BD™ LSR systems. Researchers gain application flexibility because it is easier to move the assay design and optimization to another platform, for example, from analysis to sorting.
The Cytometer Setup and Tracking (CS&T) feature of BD FACSDiva software establishes baseline settings and optimizes instrument sensitivity and fluorescent resolution. The software reduces the chances of operator error, and ensures consistency of results. It allows for the creation of application-specific settings for rapid performance of routine experiments in a more consistent manner. Tracking capabilities in the software measure a number of instrument settings and report on performance, simplifying daily quality control. Levey-Jennings plots help users understand instrument performance and identify maintenance issues.
Acquisition and analysis
BD FACSDiva software enables researchers to preview and record data from multiple samples with an automated acquisition process. The software manages acquisition templates, experiment layouts, and compensation procedures to further facilitate data acquisition.
For efficient and convenient analysis, the software provides automated hierarchical snap-to gating, user selectable plot configurations, and batch analysis function. Recorded data can be analyzed by creating plots, gates, population hierarchies, and statistical views on a BD FACSDiva global worksheet. Once the global worksheet is saved, it can be used to analyze multiple sample tubes from an experiment, thereby saving time. Other productivity benefits come from features such as user-definable batch analysis and automated gate resizing, pausing between data files, exporting statistics, and printing before proceeding to the next data file.
The gel-coupled cuvette and electronics operate together to deliver the maximum amount of signal information about each particle. The electronic sampling rate is precisely matched to the speed of the particles flowing through the cuvette. The BD FACSAria III electronic design has demonstrated linear and accurate event data acquisition at up to 70,000 events per second (shown in graph below).