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Digital and multicolor flow cytometry. Holden Maecker. 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. PMT Octagon and Trigon. PMT. Bandpass filter. Longpass filter.
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Digital and multicolor flow cytometry Holden Maecker
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
PMT Octagon and Trigon PMT Bandpass filter Longpass filter
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
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)
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
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
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 compensation—but logarithmic data display may not be appropriate • More colors mean more spillover issues, with loss of resolution sensitivity in affected detectors