Immune Monitoring Laboratory at Dartmouth-Hitchcock Medical Center


CMS - 1.6.8 - Pouebo

4-Color FACSCalibur (342W Borwell)


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

FACSCalibur blue laserFACSCalibur red laser

Technical Specifications




Fluorescence sensitivity

750 molecules of equivalent soluble fluorescein

Fluorescence resolution

< 3% for PI labeled chick erythrocyte nuclei

Forward and side scatter sensitivity

Sensitivity enables the separation of fixed platelets from noise

Forward and side scatter resolution

Optimized scatter performance for resolving lymphocytes, monocytes, and

Excitation optics

Optical platform

Fixed optical assembly


15mW Coherent 488nm, air cooled, argon ion laser
635nm red laser

Emission optics

Optical Coupling

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

Four fluorescence detectors

High performance, high dynamic range photomultipliers (PMTs) with bandpass filters: 530 nm (FITC), 585 nm (PE/PI), and >670 nm (PerCP, PE-Cy5, PE-Cy7) and 661 nm (APC)

Sample concentration

Single-cell suspension of 10e05 to 2 x 10e07cells/ml recommended range.

Sample flow rates

Three selectable flow rates of 60 μl/min, 35 μl/min, and 12 μl/min;
Particle velocity in flow cell: approximately 6 meters/second.

Signal processing

Dynamic range

12-bit data acquisition
1,024 linear channels displayed in a 4-decade logarithmic display

Pulse processing

Width and area measurements for discriminating doublets; available for all
fluorescence parameters.


Time available correlated to any parameter

Analogue Compensation

Electronics provides analogue correction of spectral overlap

Data management

Central processing

Intel processor

CellQuest v3.3

Apple Mac G4 computer with OS 9.2.2 operating system


19-inch Dell LCD monitor

Data File Structure

Flow Cytometry Standard (FCS) 2.0/3.0

Adapted from