Digital and multicolor flow cytometry Holden Maecker An

Digital and multicolor flow cytometry Holden Maecker

An LSRII is not a Calibur with more colors n Optics: fiber optics and octagons/trigons n Fluidics: optimized for high flow rates n Electronics: digital signal processing

PMT Octagon and Trigon PMT Bandpass filter Longpass filter

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

Effect of flow rate n n 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 n Generates high resolution fluorescence values that can include negative numbers n n n Allows compensation to be performed in the software at any time n n n No compression of populations at the low end of the fluorescence scale More accurate representation of dim populations 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 APC-Cy 7 Area Antibody capture beads stained with 3 levels of an APC reagent The transformed display shows aligned populations In the APC-Cy 7 dimension All populations align correctly APC Area

Spillover affects resolution senstivity

Conclusions n n 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