Chapter 2: Mass Cytometry and CyTOF XT
CyTOF XT System Components Overview
CyTOF XT User Guide
37
Data Acquisition and Analysis
This section describes the process whereby the ions separated by mass in the TOF
chamber generate signals that are converted into digital values and analyzed.
Dual Count Scale
CyTOF XT resolves multi-element samples using TOF, with ions from each isotope arriving at
the detector centered in discrete 20
–
25 ns time windows (within each 13 µs push), depending
on their mass-to-charge ratio. At very low ion concentrations, the probability of pulse signal
overlap is negligible, and ion count is most precisely determined by simply counting the
number of pulses as the intensity per ion varies. As ion concentration increases, ion pulses
begin to arrive at the detector at the same time. In this situation, pulse count underestimates
the true ion count, but at this signal level there are enough ions to use an average intensity
per ion to scale the integrated intensity and determine the number of ions.
The range of data that CyTOF XT collects requires collection of dual data, which means that
pulse count and intensity values are collected for every channel. CyTOF Software plots the
entire data range on a single dual signal scale, the units of which are actual counts of
particles that hit the detector. To achieve this, two things are done. First, a dual count
coefficient is applied to convert analog intensity into actual counts according to the
following formula:
Counts = intensity x dual count coefficient
Second, a dual switchover threshold is applied, below which pulse count is used, and above
which counts from coefficient-converted analog intensity are used. Using the dual count scale,
CyTOF XT quantifies bound antibody labels per cell across a wide range of signal input.
Figure 28. Impact of analyte concentration on signal measurement. At low analyte concentration (left),
pulses do not overlap. Because each pulse delivers a different number of electrons to the anode and
therefore a different intensity value, it is more precise to count pulses when ion concentration is very low.
Here the pulse count is 1. At higher analyte concentrations (right), pulses overlap, and counting pulses
underestimates the true number of ions that hit the detector. Here the pulse count is 8 (if we count
discernible peaks) even though 16 ions hit the detector. Thus, at high analyte concentration, it is more
accurate to use integrated intensity and convert this intensity value to counts using a calibration coefficient.
Summary of Contents for CyTOF XT
Page 1: ...FLDM 00254 Rev 03...
