Chapter 2: Mass Cytometry and CyTOF XT
CyTOF XT System Components Overview
36
CyTOF XT User Guide
bands regardless of initial position or energy. The relationship between time of ion flight to
the detector and their mass-to-charge ratio (m/z) is:
where t
0
and A are derived from the mass calibration procedure. ICP ionizes metal atoms
predominantly into a single positive-charge state. Each packet of ions resolves into a series
of bands, with the lightest probes reaching the detector first and each successively heavier
mass reaching the detector later. Each time-resolved band of ions of mass m is separated
from its m ±1 neighbor by 20
–
25 ns.
After the first packet of ions is pushed out and detected, a second pulse pushes out the
next packet of ions for detection and the cycle repeats until data acquisition is complete.
Event Detection and Acquisition Data File Format
Data for each 13 µs push is digitized sequentially and integrated to obtain ion counts for the
channels selected for analysis. The data is processed according to cell event selection
criteria set by the user. These criteria include a minimum signal threshold and a range for
event duration consistent with single-cell events and bead event detection. As a result, the
data acquired contains the integrated number of total ion counts for each selected analyte
on a per-event basis. The raw data is saved in LMD format and processed to generate the
FCS 3.1 file before the next sample acquisition starts. The FCS file can be used for further
analysis in compatible software programs.
Detector
The ions separated in the TOF chamber are detected using a discrete dynode electron
multiplier. When an ion strikes the first dynode of the detector, secondary electrons are
liberated. These electrons strike the next dynode, where they generate more electrons.
This process is repeated, resulting in an electron pulse that is captured by the anode of the
detector. The output analog signal is amplified and converted by a dual 12-bit digitizer to
digital values at 1 ns sampling intervals. The digitizer trigger delay dictates the first mass
channel (the lowest mass registered) to be recorded per push, while the segment length
dictates the mass range to be recorded per push. Instruments are set to collect data from
up to 135 mass channels (each corresponding to 1 amu), starting at mass 75 for CyTOF XT.
Summary of Contents for CyTOF XT
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