BD Biosciences LSR II.

Our core recently acquired a LSR II, the latest benchtop flow cytometer developed by BD Biosciences and a significant redesign over the original LSR.  This instrument is equipped with four laser sources:  a Coherent Sapphire 20 mW 488 nm solid state laser (replacing the originally utilized air-cooled argon-ion laser), a JDS Uniphase helium-neon 17 mW 633 nm laser for red excitation, and a Coherent Vioflame 25 mW 408 nm violet laser diode.  A Lightwave Electronics 355 nm frequency-tripled Nd-YAG laser will be added at the end of 2002 and will provide UV excitation, replacing the 325 nm HeCad laser used on earlier instruments.  The instrument is currently set up to detect four fluorochromes using 488 nm excitation, two with the 633 nm HeNe, two with the 408 nm violet diode and two off the UV, giving us up to 10 fluorescent parameter detection simultaneously.  The LSR II has full digital data acquisition/analysis capability using software similar to that used for the FACSVantage DiVa; unlike the DiVa, which still can acquire data under the old analog system, the LSR II is a fully digital system.

(Below).  The LSR II.   Upper photo, covers open; bottom photo, internal instrument configuration.   The lasers are in the forward portion of the instrument; the flow cell, collection optics and fiber optic splitter are on the right forward portion of the instrument; the PMT "octagons" and "trigons" are located to the rear of the instrument.

Lasers.   The LSR II uses the same basic instrument "bench" as the old LSR.  The lasers are mounted in roughly the same positions of the LSR, although the solid-state 488 nm is far smaller and has virtually no cooling requirements beyond a heat sink, saving considerable space.  The beams are independently steered using Newport Instruments gimbaled optical mounts and laser dichroics accessible from above the beams paths, making multiple beam alignments far simpler than on the old LSR.

(Below).  The LSR II lasers.  From the back, the Coherent Sapphire 488 nm 20 mW solid state; the Cyonics 633 nm 20 mW HeNe gas laser; and the Coherent VioFlame 408 nm 15 mW violet laser diode.  The Lightwave 355 nm UV laser will be added in the forward-most position later.

(Below).  Laser beam steering optics. 

(Below).  Schematic of laser beam steering optics.  Beam order is arbitrary.

Collection optics and fiber optics. Like the LSR, the LSR II uses a standard FACSCalibur quartz flow cell.  However, the LSR II utilizes an improved signal focusing objective, mated to the flow cell with an optical gel layer.    This change in collection optics improves the numerical aperture of the system (BD claims) to 1.2, a major improvement over earlier systems.  The resulting four sets of signals (each from one laser) are focused on four pinholes, behind which are four fiber optics light collection points.  Each fiber optic channels signals to one of four PMT clusters.  The pinhole system simplifies the laser delay settings, which remain relatively fixed after initial calibration and require no daily delay adjustment.

(Below).  Flow cell, collection optics and fiber optic junction.

PMTs and filters/dichroics.  The LSR II uses "clusters" of PMTs, dichroics and filters mounted in light-tight molded plastic units, referred to as "octagons" or "trigons" based on the number of detectors.  The emission signals are transmitted into the PMT clusters via fiber optics, where the signals are appropriately passed or reflected into the multiple detectors.  Isolating each group of PMTs into a rigid, free-floating unit solves one of the problems with the old LSR, namely the mounting of the detectors directly on the optical bench, where they were very prone to mechanical disruption and loss of alignment.   The instrument uses standard 25 mm narrow bandpass filters in removable mounts, allowing easy alteration of the optical configuration as well as the use of our existing filters.  The longpass dichroics are a non-standard diameter and are spec'd to 13 degrees incidence of reflection to accomodate the design of the optical path; although we cannot use our own dichroics in this system, the existing ones are interchangeable.

(Below).  Default configuration for the primary 488 nm detectors. Left, the primary laser "octagon". Right, the current default configuration for the primary PMT "octagon".   This configuration allows four-color analysis of FITC, PE, PerCP-Cy5.5 and PE-Cy7 off the primary 488 nm solid laser.  The two "distal" detector slots in the signal path have no PMTs or dichroics.

(Below).  Modified onfiguration for the detection of PE-Cy5. Although the default configuration can be used for PE-Cy5 analysis (instead of PerCP-Cy5.5), it is not optimal.  For a more optimal configuration, the PerCP-Cy5.5 bandpass filter can be changed to the PE-Cy5 670/14, and the dichroic in front of the detector changed to the 635 LP (custom fabricated by Omega Optical).

(Below).  Proposed configuration for six-color analysis with the primary 488 nm detector octagon.  Two additional PMTs can be added to the primary 488 nm octagon for six-color analysis.  Filter and dichroics for PE-Texas Red and PE-Cy5.5 would be added to the configuration, and the side scatter, FITC and PE detector positions would be shifted to accommodate the additional detectors.

(Below).  Default configuration for the secondary violet diode detectors.  The violet laser diode beam occupies the secondary beam/signal position.  Left, the secondary PMT "trigon". Right, the current default configuration for the secondary PMT trigon.   This configuration allows simultaneous detection of two blue and green violet-excited fluorochromes, such as Cascade Blue and ELF-97 (illustrated).    The "distal" detector slot in the signal path has no PMT or dichroic.

(Below).  Default configuration for the third position UV detectors.  The UV 355 nm solid state laser beam will occupy the third beam/signal position.  Left, the third PMT "trigon". Right, the current default configuration for the third PMT trigon.  This configuration allows simulaneous detection of two UV-excited fluorochromes, such as the ratiometric calcium probe indo-1 (illustrated).   The "distal" detector slot in the signal path has no PMT or dichroic.

(Below).  Default configuration for the fourth position red HeNe detectors.  The 633 nm HeNe laser beam will occupies the fourth beam/signal position.  Left, the fourth PMT "trigon". Right, the current default configuration for the fourth PMT trigon.  This configuration allows simulaneous detection of two red-excited fluorochromes, such as APC and APC-Cy7.   The "distal" detector slot in the signal path has no PMT or dichroic in the default configuration.

(Below).  Addition of a third detector to the fourth position 633 nm detector trigon.  A third PMT can be added to this trigon, allowing simultaneous detection of APC, APC-Cy5.5 and APC.Cy7.  Go here to see sample data.

(Below).  LSR II  dichroics.  The indicated custom fabricated dichroics were prepared for specific applications and have a 13 degree angle of incidence.

(Below).  FACSVantage DiVa filters.  Although these filters are supplied with the instrument, any standard 25 mm bandpass filters can be substituted.

  Click here to see data from the 488 solid state blue-green, 633 nm HeNe red or 408 nm violet diode lasers.

Go here to download the LSR II Overview.

Go here to download the most recent version of the LSR II User Handbook.

Download Adobe Acrobat Reader here .

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