FACSAria cell sorter

 

 

 

Optical Bench

Before you decide on a fluorochrome for a flow cytometric assay, you need to know:
  • the wavelengths of the lasers in the cytometer
  • the wavelengths of the filters in front of the photomultiplier tubes (PMTs) in the cytometer
  • the absorption and fluorescence wavelengths of fluorochromes to be used

These can be simulated using the BD fluorescence spectrum viewer:

 

There are 2 possible filter configurations for the violet laser PMTs:

 

Configuration #1

450/40 PMT:

  • brilliant violet 421
  • VioBlue (Miltenyi Biotec)
  • live/dead violet fixable amine dye (Invitrogen)
  • pacific blue
  • BD Horizon V450
  • Alexa fluor 405
  • eFluor 430
  • DAPI
  • Hoechst

575LP dichroic then 605/20 PMT:

  • brilliant violet 605
  • eFluor 605NC

625LP dichroic then 655/20 PMT:

  • brilliant violet 650
  • eFluor 650NC

 

Configuration #2

450/40 PMT:

  • brilliant violet 421
  • VioBlue (Miltenyi Biotec)
  • pacific blue
  • live/dead violet fixable amine dye (Invitrogen)
  • BD Horizon V450
  • Alexa fluor 405
  • eFluor 430
  • DAPI
  • Hoechst

502LP dichroic then 510/50 PMT:

  • brilliant violet 510
  • live/dead aqua fixable amine dye (Invitrogen)
  • BD Horizon V500

 

530/30 PMT:

  • FITC
  • alexa fluor 488
  • GFP
  • YFP
  • live/dead green fixable amine dye
  • CFSE

575/26 PMT

  • PE

610/20 PMT

  • PE-CF594 (BD)
  • PE-Texas red
  • PE-alexa fluor 610
  • live/dead red fixable amine dye

695/40 PMT

  • PerCP
  • PerCP-Cy5.5
  • eFluor 610

780/60

  • PE-Cy7
  • PE-vio770

660/20 PMT

  • APC
  • alexa fluor 647
  • eFluor 660
  • live/dead far red fixable amine dye

685LP then 720/40 PMT

  • alexa fluor 700

760LP PMT

  • APC-Cy7
  • APC-H7 (BD)
  • APC-alexa fluor 750
  • APC-eFluor 780
  • APC-vio770 (Miltenyi Biotec)
  • live/dead near IR fixable amine dye

 

 

 

 

 

Technical Specifications

 

Specification

Comments

Performance

Fluorescence sensitivity

FITC 125 molecules of equivalent soluble fluorochrome

PE 125 molecules of equivalent soluble fluorochrome

Fluorescence resolution

CV < 3% for PI labelled chick erythrocyte nuclei

CV < 3% for Hoechst labelled chick erythrocyte nuclei

Forward and side scatter sensitivity

Sensitivity enables the separation of fixed platelets from noise

Identification of bacteria and 0.5 micron beads

Forward and side scatter resolution

70,000 events/sec

Excitation optics

Optical platform

Fixed optical assembly, upon cuvette flow cell

Lasers

20mW Coherent Sapphire 488nm, air cooled, argon ion laser
25 mW 405 Coherent VioFlame
20 mW 635 JDS Uniphase HeNe

Emission optics

Optical Coupling

Quartz cuvette is coupled to emission lens by refractive index-matching optical gel for optimum collection efficiency

Fluorescence Detectors

Three fixed fibre apertures

Twelve fluorescence detectors

High performance, high dynamic range photomultipliers with filters

Steering Optics

Fibre optics steer the three laser beams onto the beam expansion prisms, and then are focused upon the cuvette flow cell

Signal processing

Workstation resolution

Five decades for peak detection, offset less than 1/10,000 full scale

Data acquisition channels

14 parameters, 12 fluorescent and two scatter

Dynamic range

18-bit data acquisition
262,144 linear channels displayed in a 5-decade logarithmic display

Pulse processing

Width and area measurements available for all fluorescence parameters

Ratio measurements for intra-laser parameters

Time

Time available correlated to any parameter

Channel triggering

Available on any laser signal

Data management

Central processing

Dual Intel Xeon processors

Data storage

Nine and 80-GB internal hard drive, internal drive with 250-MG removable disk storage space capacity, 3.5-inch floopy drive, DVD/CD-RW combo drive, Iomega Zip 750 MB

Data File Structure

Flow Cytometry Standard 3.0 or 2.0

Software

BD FACSDiva software v3.2

Fluidics

General

Fluidics cart provides sheath and cleaning fluid to instrument and receives waste
No air or vacuum required
Adjustable sheath pressure

Sample flow rate

Adjustable

Sample injection chamber

Sample input agitation

Adjustable through software

Temperature control

Software adjustable, 4, 20, 37 and 42 o C

Aerosol management

Standard feature

Sample injection chamber and sample collection tube area provide a sealed area to minimise aerosols

Sort performance

Sort collection devices

Two way sorting and four way sorting in to sample tubes

70um, 100um and 130um diameter nozzle sizes available for sorting at 20,000, 10,000 and 5,000 cells per second at 70, 20 and 12 psi.

Automatic cell deposition unit allows for sorting onto slides or plates

Water re-circulator for refrigeration/heating collected sample

http://www.icms.qmul.ac.uk/flowcytometry/instruments/facsaria/index.html - top