流式细胞仪器参数设置与追踪(cst)
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23-13536-00 Rev. 01 Cytometer Performance—CS&T 19
CS&T
CS&T is a fully automated software and research reagent system, unique to BD digital cytometers, providing: • Characterization • Setup • Tracking
23-13536-00 Rev. 01
Cytometer Performance—CS&T
4
Factors Affecting Detector Efficiency
• Laser power and alignment
• Optical efficiency • Optics cleanliness or damage • PMT red spectrum sensitivity • Poor PMT performance (age)
• Dirty flow cell
• Dirty or degraded filter
23-13536-00 Rev. 01
Cytometer Performance—CS&T
5
Background Signal—Br
Br is a measure of optical background.
Unbound antibody or fluorochrome Scatter from the flow cell and ambient light Raman scatter
• Violet laser parameters exhibit high Br due to autofluorescence.
23-13536-00 Rev. 01
Cytometer Performance—CS&T
8
Qr and Br: A Look at Some Real Data
• Qr and Br characterizations are for both FITC and PerCP-Cy5.5 parameters. • Plots gated on CD3+ lymphocytes.
1 Br
.
23-13536-00 Rev. 01
Cytometer Performance—CS&T
7
Factors Affecting Br
• Dirty flow cell. • Damaged optical component.
• Blue laser parameters FITC and PE exhibit high Br due to Raman scatter.
time
23-13536-00 Rev. 01
Cytometer Performance—CS&T
16
Factors Affecting SDEN
• PMT connectors • PMT noise • Cables near power sources • Analog to digital conversion error • Window extension
23-13536-00 Rev. 01
Cytometer Performance—CS&T
17
Window Extension
intensity processed event electronic abort below threshold
threshold
window gate
time
window extensions
Spectral overlap from another marker on a cell
Autofluorescence
Br is the number of fluorochrome molecules that would produce a signal equivalent to the background signal.
Small deviations in linearity in the last decade can result in significant compensation errors.
Linear MFI Measured MFI Spillover Compensation Error 142000 140000 20% 2000 X 20% = 400
Cytometer 2 APC detector photoelectrons
Cytometer 1 APC detector photoelectrons
Which cytometer has higher Qr for APC detection?
23-13536-00 Rev. 01
High Qr—Low Br High Qr—High Br Low Qr—High Br
23-13536-00 Rev. 01
Cytometer Performance—CS&T
9
Resolution
• Resolution is the ability to distinguish between populations based on fluorescense. • Resolution is a key factor in sensitivity.
• Standard Deviation of Electronic Noise—SDEN
23-13536-00 Rev. 01
Cytometer Performance—CS&T
2
Detection Efficiency—Qr Qr
is proportional to
Q=
# photoelectrons # fluorochrome molecules
Cytometer Performance—CS&T
23-13536-00 Rev. 01
Know Your Cytometer
Cytometer Characterization
For each fluorescence parameter:
• Detector Efficiency—Qr • Background Signal—Br • Resolution (high signal)—%rCV for a bright particle • Linear Range—Linearity Min and Max Limits
Cytometer Performance—CS&T
3
Why is Qr Important?
• Higher Qr → more sensitivity. • When other factors are held constant, sensitivity (as quantified by stain index) is proportional to Qr . Low Qr High Qr
• Low SDEN → higher sensitivity and larger dynamic range.
intensity
• Most critical for parameters with low cellular autofluorescence:
− − − − APC-H7 PE-Cy7 PerCP-Cy5.5 APC
23-13536-00 Rev. 01
Cytometer Performance—CS&T
11
What is Linearity?
• Linearity is the proportionality of output to input for fluorescence measurements.
X Ab 2XBiblioteka BaiduAbs
2X Abs 2000 = X Abs 1000
0
1000
2000
3000
23-13536-00 Rev. 01
Cytometer Performance—CS&T
12
Why is Linearity Important?
• Accurate fluorescence compensation
intensity
• Caution: smaller window extensions can amplify other measurement errors. − Laser delay − Linearity
threshold
time
window extensions
Sensitivity Exercise
Cytometer Factors Affecting Linearity
• Quality of PMT tube and its socket
• Preamp and electronics • Laser beam shape and alignment
23-13536-00 Rev. 01
Good Resolution
Poor Resolution
Fluorescence Intensity
• A practical measure of resolution is the %rCV of a reference particle in the mid to upper end of the scale.
window extensions overlap
23-13536-00 Rev. 01
Cytometer Performance—CS&T
18
Window Extension and SDEN
• Smaller window extensions can lower the SDEN contribution to variation relative to the integrated pulse signal.
A B C D
FITC MFI
PE MFI
68
80
1796
75
5921
80
73000
365
• Samples are BD™ CompBead particles stained with varying levels of FITC-Ab.
• Compensation is calculated using samples A and C.
23-13536-00 Rev. 01
Cytometer Performance—CS&T
10
Cytometer Factors Affecting Resolution
• Uniform excitation of particles − Laser power − Laser beam shape and alignment − Flow rate • Window extension
• Quantitative measurement studies
–DNA –Antigen-antibody binding
23-13536-00 Rev. 01
Cytometer Performance—CS&T
13
Effect of Non-Linearity on Compensation
23-13536-00 Rev. 01
Cytometer Performance—CS&T
6
Why is Br Important?
• Higher Br → less sensitivity.
• When other factors are held constant, sensitivity (as quantified by stain index) is proportional to Low Br High Br
• This cytometer has a poor linear range, with >2% deviation from linearity above the Max Linearity Channel at 50,000.
23-13536-00 Rev. 01 Cytometer Performance—CS&T 14
Cytometer Performance—CS&T
15
Standard Deviation of Electronic Noise—SDEN
• SDEN is the signal variation due to low level background noise contributed by the electronics system.
CS&T
CS&T is a fully automated software and research reagent system, unique to BD digital cytometers, providing: • Characterization • Setup • Tracking
23-13536-00 Rev. 01
Cytometer Performance—CS&T
4
Factors Affecting Detector Efficiency
• Laser power and alignment
• Optical efficiency • Optics cleanliness or damage • PMT red spectrum sensitivity • Poor PMT performance (age)
• Dirty flow cell
• Dirty or degraded filter
23-13536-00 Rev. 01
Cytometer Performance—CS&T
5
Background Signal—Br
Br is a measure of optical background.
Unbound antibody or fluorochrome Scatter from the flow cell and ambient light Raman scatter
• Violet laser parameters exhibit high Br due to autofluorescence.
23-13536-00 Rev. 01
Cytometer Performance—CS&T
8
Qr and Br: A Look at Some Real Data
• Qr and Br characterizations are for both FITC and PerCP-Cy5.5 parameters. • Plots gated on CD3+ lymphocytes.
1 Br
.
23-13536-00 Rev. 01
Cytometer Performance—CS&T
7
Factors Affecting Br
• Dirty flow cell. • Damaged optical component.
• Blue laser parameters FITC and PE exhibit high Br due to Raman scatter.
time
23-13536-00 Rev. 01
Cytometer Performance—CS&T
16
Factors Affecting SDEN
• PMT connectors • PMT noise • Cables near power sources • Analog to digital conversion error • Window extension
23-13536-00 Rev. 01
Cytometer Performance—CS&T
17
Window Extension
intensity processed event electronic abort below threshold
threshold
window gate
time
window extensions
Spectral overlap from another marker on a cell
Autofluorescence
Br is the number of fluorochrome molecules that would produce a signal equivalent to the background signal.
Small deviations in linearity in the last decade can result in significant compensation errors.
Linear MFI Measured MFI Spillover Compensation Error 142000 140000 20% 2000 X 20% = 400
Cytometer 2 APC detector photoelectrons
Cytometer 1 APC detector photoelectrons
Which cytometer has higher Qr for APC detection?
23-13536-00 Rev. 01
High Qr—Low Br High Qr—High Br Low Qr—High Br
23-13536-00 Rev. 01
Cytometer Performance—CS&T
9
Resolution
• Resolution is the ability to distinguish between populations based on fluorescense. • Resolution is a key factor in sensitivity.
• Standard Deviation of Electronic Noise—SDEN
23-13536-00 Rev. 01
Cytometer Performance—CS&T
2
Detection Efficiency—Qr Qr
is proportional to
Q=
# photoelectrons # fluorochrome molecules
Cytometer Performance—CS&T
23-13536-00 Rev. 01
Know Your Cytometer
Cytometer Characterization
For each fluorescence parameter:
• Detector Efficiency—Qr • Background Signal—Br • Resolution (high signal)—%rCV for a bright particle • Linear Range—Linearity Min and Max Limits
Cytometer Performance—CS&T
3
Why is Qr Important?
• Higher Qr → more sensitivity. • When other factors are held constant, sensitivity (as quantified by stain index) is proportional to Qr . Low Qr High Qr
• Low SDEN → higher sensitivity and larger dynamic range.
intensity
• Most critical for parameters with low cellular autofluorescence:
− − − − APC-H7 PE-Cy7 PerCP-Cy5.5 APC
23-13536-00 Rev. 01
Cytometer Performance—CS&T
11
What is Linearity?
• Linearity is the proportionality of output to input for fluorescence measurements.
X Ab 2XBiblioteka BaiduAbs
2X Abs 2000 = X Abs 1000
0
1000
2000
3000
23-13536-00 Rev. 01
Cytometer Performance—CS&T
12
Why is Linearity Important?
• Accurate fluorescence compensation
intensity
• Caution: smaller window extensions can amplify other measurement errors. − Laser delay − Linearity
threshold
time
window extensions
Sensitivity Exercise
Cytometer Factors Affecting Linearity
• Quality of PMT tube and its socket
• Preamp and electronics • Laser beam shape and alignment
23-13536-00 Rev. 01
Good Resolution
Poor Resolution
Fluorescence Intensity
• A practical measure of resolution is the %rCV of a reference particle in the mid to upper end of the scale.
window extensions overlap
23-13536-00 Rev. 01
Cytometer Performance—CS&T
18
Window Extension and SDEN
• Smaller window extensions can lower the SDEN contribution to variation relative to the integrated pulse signal.
A B C D
FITC MFI
PE MFI
68
80
1796
75
5921
80
73000
365
• Samples are BD™ CompBead particles stained with varying levels of FITC-Ab.
• Compensation is calculated using samples A and C.
23-13536-00 Rev. 01
Cytometer Performance—CS&T
10
Cytometer Factors Affecting Resolution
• Uniform excitation of particles − Laser power − Laser beam shape and alignment − Flow rate • Window extension
• Quantitative measurement studies
–DNA –Antigen-antibody binding
23-13536-00 Rev. 01
Cytometer Performance—CS&T
13
Effect of Non-Linearity on Compensation
23-13536-00 Rev. 01
Cytometer Performance—CS&T
6
Why is Br Important?
• Higher Br → less sensitivity.
• When other factors are held constant, sensitivity (as quantified by stain index) is proportional to Low Br High Br
• This cytometer has a poor linear range, with >2% deviation from linearity above the Max Linearity Channel at 50,000.
23-13536-00 Rev. 01 Cytometer Performance—CS&T 14
Cytometer Performance—CS&T
15
Standard Deviation of Electronic Noise—SDEN
• SDEN is the signal variation due to low level background noise contributed by the electronics system.