Figure: CytoFLEX S multicolor flow cytometers lasers

Multicolor flow cytometer / cell sorter BD FACSAria Fusion Cell Sorter (BD Life Sciences)

BD Life Sciences

Optics

Excitation Optics

Optical Platform

Fixed optical alignment of all Class IIIb lasers with the cuvette flow cell. All lasers are solid state.

Beam height: 9 ±3 μm

Beam width: 65 ±7 μm

Beam shaping: elliptical

Power Out of the Laser Head

405 nm: >85 mW

488 nm: >50 mW

640 nm: >100 mW

Steering Optics

Fiber optics steer the fixed-alignment laser beams onto the expansion prisms to focus them on the cuvette flow cell.

Emission Optics

Optical Coupling

The quartz cuvette flow cell is gel-coupled by refractive index-matching optical gel to the fluorescence objective lens for optimal collection efficiency. Numerical aperture: 1.2

Forward Scatter Detector and Filters

Photodiode with 488/10 bandpass filter for the 488-nm laser.

Side Scatter Detector

Photomultiplier with a 488/10 bandpass filter for the 488-nm laser.

Fluorescence Detectors and Filters

An octagon technology (BD Octagon™) detector array for the 488-nm and 405-nm lasers.

A trigon technology (BD Trigon™) detector array for the 640-nm laser.

Detector arrays enable user-defined detection configurations, filters and mirrors are user changeable.

Wavelengths Detected from the 488-nm Laser without the 561-nm Laser Installed

530/30-nm FITC, Alexa Fluor® 488

585/42-nm PE

616/23-nm PE-Texas Red®, propidium iodide (PI)

695/40-nm PerCP-Cy™5.5 or PI, or 675/20-nm PerCP

760/60-nm PE-Cy™7

Wavelengths Detected from the 640-nm Laser

670/30-nm APC, Alexa Fluor® 647

780/60-nm APC-Cy7 or APC-H7

Wavelengths Detected from the 405-nm Laser

450/40-nm BD Horizon Brilliant™ Violet 421, V450, VPD450, Pacific Blue™, DAPI

525/50-nm BD Horizon Brilliant™ Violet 510 or V500, AmCyan

610/20-nm BD Horizon Brilliant™ Violet 605

Fluidics

General Operation

Sheath and cleaning fluid tanks, as well as waste collection, are located in an easy-to-reach drawer at the base of the system. An auxiliary air input is designed to connect the cytometer to a lab’s air filtration and drying system. If needed, a third-party compressor is available as an option. Sheath pressure is adjustable from 5 to 75 psi.

Fluidic Reservoirs

Autoclavable 10-L sheath and waste containers and 5-L cleaning reservoirs are provided.

Sample Flow Rates

Adjustable dynamic range of sample flow rates

Nozzles

70, 85, 100 and 130-μm nozzles are removable and can be sonicated.A registered key-fit position at the bottom of the cuvette provides fixed stream alignment.

Sample Collection Cooling and Heating

Refrigerator/heater option is available to provide cooling or heating for sort collection into tube holders, multiwell plates and slides.

Automatic Cell Deposition Unit

Sorts into slides and 6, 24, 48, 96 and 384-well plates. Index sorting can be enabled when sorting single cells. This capability indexes the cell surface phenotype to the well containing that cell.

Sort Performance

Drop Drive Frequency

Range: 1–100,000 Hz

Purity and Yield

At 70 psi and 87 kHz with an average threshold rate of 25,000 events persecond, a four-way sort achieved a purity of >98% and a yield >80% of Poisson’s expected yield. Higher threshold rates up to 70,000 events per second can be achieved without affecting purity. However, yield will decrease based on Poisson’s statistics.

Sort Collection Devices

Two-way sorting: 12 x 75-mm and 15-mL tubes

Four-way sorting: 1.5-mL microtubes and 12 x 75-mm tubes

Sort Collection Cooling

Water recirculator for refrigeration or heating (optional)

BD FACS™ Accudrop

Red diode laser provided forfully automated drop-delay determination

Automated drop breakoff monitoring

Automated clog detection and sort tube protection system using Sweet Spot technology

Signal Processing

Converter

10-MHz analog-to-digital converter. Pulse sampling is precisely matched to the particle flow rate in the cuvette. Particles travel slower compared to conventional stream-in-air sorters. This increases the light collected, resulting in better sensitivity. High-speed sorting is achieved by accelerating the stream through the nozzle, achieving drop rates comparable to stream-in-air sorters. The flow cell design and electronics are matched to maximize signal while maintaining maximum sort speed, purity and yield.

Workstation Resolution

262,144 channels

Data Acquisition Channels

20 parameters (18 fluorescence and 2 scatter)

Fluorescence Compensation

No limit to inter- and intra-beam compensation

Pulse Processing

Height, area and width measurements available for any parameter. Ratio measurements are also available.

Time

Time can be correlated to any parameter for kinetic experiments or other applications.

Channel Threshold

Available for any parameter from any laser, with the ability to use multiple thresholds from different lasers simultaneously.

Loading

Sample Input Sizes

Microtubes, 12 x 75-mm and 15-mL tubes

Polystyrene or polypropylene tubes can be used.

Sample Input Agitation

Adjustable through the software to keep the sample constantly suspended

Temperature Control

Sample input: 4ºC, 20ºC, 37ºC and 42ºC (adjusted in the software)

Sample output: water recirculation unit (optional)