Biomedical Engineering Reference
In-Depth Information
markers with the least amount of expression on a given population should be detected
with a reagent that is labeled with the brightest fluorochrome. However, this weakly
expressed antigen should be stained with a fluorochrome-tagged reagent that does not
have spillover issues with another fluorochrome reagent recognizing a cell marker that
is highly expressed [64]. A final word of caution is to take into account that a
fluorochrome as a single reagent may give different results when employed in a
multicolor reagent cocktail and this is a fidelity issue. To determine if this is a problem,
one can compare the antibody-fluorochrome conjugate by itself and compare the
results with the results obtained for this reagent when it is in the multicolor reagent
cocktail. One should try and use reagent combinations that have good fidelity when
used in a multicolor reagent cocktail.
1.6 COMPENSATION
Due to overlap in the emission spectra of different dyes, it is often not possible to
choose emission filters that uniquely measure only one of the dyes in a multicolor
experiment. Due to this spectral overlap, one fluorochrome can contribute a signal to
several detectors; therefore, the contribution in detectors not assigned to that
fluorochrome must be removed from the total signal in those detectors. Compensation
is an artificial means of eliminating spectral overlap between two different fluor-
ochromes by mathematical means and is not just a subtraction process [65, 66].
Compensation between detectors can be performed either by hardware, after signal
detection but before logarithmic conversion and/or digitization, or by uncompensated
data that are analyzed post-collection by software (Figure 1.5).
1.7 THRESHOLD AND GATES
An electronic threshold is defined as a gate for acquiring signals. Only events with
intensity greater than the threshold will be processed and analyzed. Thresholding is
most often used to eliminate debris. A gate is a boundary that can be used to identify
subpopulations and this limits the number of events that are analyzed (note that these
events are acquired in the listmode data file but not analyzed). Gates are often used to
identify the lymphoid cells for analysis.
1.8 ANALYSIS
The electric pulses that are detected by the PMTs are amplified (log amplification is
most often used to measure fluorescence). These amplified signals are converted from
analog to digital. Data can be stored as a listmode file, which consists of a complete
listing of all events and parameters that were measured [67]. One can take a listmode
file and subject the data to analysis such as regions and gating but one cannot adjust
amplification or fluorescence [68].
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