Digital and multicolor flow cytometry

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Digital and multicolor flow cytometry

• Holden Maecker

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An LSRII is not a Calibur with more colors

• Optics fiber optics and octagons/trigons
• Fluidics optimized for high flow rates
• Electronics digital signal processing

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PMT Octagon and Trigon
PMT
Bandpass filter
Longpass filter
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Filter Nomenclature Conventions

• Longpass filter allows light above a certain
  wavelength to pass, reflects shorter wavelengths
• Example 505LP 505 nm longpass
• Bandpass filter allows light within a certain
  range of wavelengths to pass (above and below a
  specified midpoint)
• Example 530/30BP 515-545 nm bandpass

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Effect of flow rate

• Higher flow rates mean a broader sample stream
  (less precise focusing)
• Less precise focusing means less accurate
  fluorescence measurement of dim populations
  (population spreading)
• Higher flow rates also increase the frequency of
  coincident events (can be gated out based on FSC
  area vs. height)
• In practice, flow rates up to 8000-12,000 events
  per second are acceptable on the LSRII (vs.
  2000-3000 events per second on a FACS Calibur)

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Digital Signal Processing

• Generates high resolution fluorescence values
  that can include negative numbers
• No compression of populations at the low end of
  the fluorescence scale
• More accurate representation of dim populations
• Allows compensation to be performed in the
  software at any time
• Uncompensated data and any associated
  compensation matrix are both stored
• Compensation can be changed at any time
• Peak area and peak height can both be recorded
  for all parameters

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Biexponential display of digital data
Antibody capture beads stained with 3 levels
of an APC reagent The transformed display shows
aligned populations In the APC-Cy7 dimension
APC-Cy7 Area
All populations align correctly
APC Area
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Spillover affects resolution senstivity
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Conclusions

• LSRII optics (using octagons and trigons) mean
  more efficient light collection and flexibility
  in use of detectors and filters
• LSRII fluidics allow running at higher flow rates
  with minimal compromise to the data
• Digital signal processing provides more accurate
  representation of dim populations, and more
  accurate and flexible compensationbut
  logarithmic data display may not be appropriate
• More colors mean more spillover issues, with loss
  of resolution sensitivity in affected detectors