Flow Cytometry

The Technology of ViraCor Laboratories’ Immunology Program
ViraCor Laboratories’ Immune Monitoring Program capabilities were recently expanded within our Clinical Immunology Laboratory by acquiring a custom-configured BD Biosciences LSR II Flow Cytometer that is capable of detecting up to 12 parameters (2 scatter and 10 fluorescent) per cell. This flow cytometer is equipped with 3 fixed-angle, solid state laser sources: Coherent Sapphire 20 mW 488 nm Sapphire Blue Laser, JDS Uniphase 18 mW 633 nm HeNe Red Laser, and the Coherent VioFlame PLUS 50 mW 405 nm Violet Laser.

By distributing a large number of fluorochromes over 3 lasers, spectral overlap is reduced and sensitivity is significantly improved. The beam from each of the three lasers is independently steered to the flow cell using Newport Instruments gimbaled optical mounts and laser dichroics. After light from each of the three spatially separated laser beams has interrogated the sample within the flow cell, the emitted fluorescent signals are delivered by fiber optics to the collection optics including both octagon and trigon detector arrays. The optical arrays in the LSR II transmit the highest wavelengths (lower energy) to the first photomultiplier tube (PMT) and reflect the lower wavelengths (higher energy) to the next PMT through a series of longpass dichroic mirrors, which allows for more colors to be detected with minimal loss in signal intensity. Photons that pass through the longpass dichroic mirror either travel through the respective bandpass filter and are detected by the PMT or are reflected by the bandpass filter. The optical configuration of the LSR II can be quickly altered by changing the longpass and bandpass filters required for the detection of a specific fluorochrome.

Data acquisition/analysis on the LSR II is fully digital, allowing for the measurement of total fluorescence, thus providing more information per cell. The LSR II in our immunology laboratory has been customized to detect up to 12 parameters for any given clinical sample:

• 2 scatter parameters (forward scatter and side scatter)
• 5 fluorochromes on the 488 nm octagon array
• 2 fluorochromes with the 633 nm red laser on the 633 nm trigon array
• 3 fluorochromes on the 405 nm octagon array

The experimental design, instrument control, and data analysis on the LSR II are driven by the BD FACSDiva 6.0 Software. The LSR II is an extremely sensitive and scalable flow cytometer, which provides our clinical researchers the capability to gather maximum information from each clinical specimen.

Figure 1(a)

Figure 1(b)

Figure 1.  The BD LSR II with the cover closed (a) and the cover open (b).  The 3 lasers are basically centered on the instrument deck with the flow cell, collection optics, and fiber optics splitter in the right forward position.  The octagon and trigon detector arrays are located on the rear of the instrument deck. 

Figure 2

Figure 2.  Lasers - the Coherent Sapphire 20 mW 488 nm Sapphire Blue Laser (far right), JDS Uniphase 18 mW 633 nm HeNe Red Laser (center rear), and the Coherent VioFlame PLUS 50 mW 405 nm Violet Laser (far left). 

Figure 3(a)

Figure 3(b)

Figure 3.  Laser Beam Steering Optics and Flow Cell.  (a) Collection optics and flow cell.  (b) Flow cell; to improve the numerical aperature of the system, the BD LSR II utilizes an improved signal focusing objective mated to the flow cell, which contains an optical gel layer. 

Figure 4(a)

Figure 4(b)

Figure 4.  Optical Detector Arrays. (a) 488 nm octagon array with 5 fluorochromes and SSC detectors.  The 405 nm octagon array (not pictured) has a similar appearance but with only 2 fluorochrome detectors.  (b) 633 nm trigon array with 3 fluorochrome detectors. 

Table 1.  LSR II Laser and Detector Configuration.