斯坦福大学流式细胞仪补偿原理课件 (很详细)
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FACS compensation
James Tung, David Parks, Wayne Moore, Herzenberg Laboratory, and Stanford Shared FACS Facility
Fluorescence compensation
FITC spectral overlap detected in the PE channel must be subtracted from the PE signal
Cy5PE-A, FSC-A subset 10900.fcs Event Count: 7804
SSC-A, FSC-A subset 10814.fcs Event Count: 9253
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
10000
10 3
10 3
10 3
FITC fluorescence in the PE channel
1000
1000
100
100
10
10
10
100
1000
10000
10
100
1000
10000
FITC (B220) fluorescence in the FITC channel
Properly compensated values distribute symmetrically around the median of the negative population, but log display only shows values above zero.
SSC-A, FSC-A subset 1-35 Specimen_001 unstained FL580 Event Count: 7920
10 5
10 5
10 5
PE
10 4 PE-A
10 4 PE-A
10 4 PE-A
8000
10 3
10 3
10 3
10 2
10 2 0 10 2 10 3 FL-A 10 4 10 5 10 2 10 3 FL-A 10 4 10 5
0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0
0
0
Cy5PE-A, FSC-A subset 10894.fcs Event Count: 5908
300 400
12-color FACS
440/40 540/80
Krypton Laser 407 nm
Spectra of dyes
525/50
575/25
630/22
665/30
Argon Laser 488 nm
Excitation spectrum Emission spectrum Collection Filter
0 FL-A SSC-A, FSC-A subset 1-34 Specimen_001 unstained FL610 Event Count: 7877
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
12000
10 3
10 3
10 3
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-3 Specimen_001 beads FL670 Event Count: 9139 10 4 10 5
0
0
SSC-A, FSC-A subset 1-41 Specimen_001 stained FL670 Event Count: 7069
10 2 0 10 2 10 3 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-2 Specimen_001 beads FL610 Event Count: 9065 10 4 10 5
0 FL-A SSC-A, FSC-A subset 1-39 Specimen_001 stained FL610 Event Count: 7488
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
PE-A
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
0
0
0
Cy5PE-A, FSC-A subset 10896.fcs Event Count: 6577
Cy5PE-A, FSC-A subset 10908.fcs Event Count: 6792
SSC-A, FSC-A subset 1-33 Specimen_001 unstained FL643 Event Count: 7885
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE PE-A
18000
10 3
10 3
10 3
10 2 0 10 2 10 3 10 4 10 5
Cy5PE-A, FSC-A subset 10902.fcs Event Count: 7829
SSC-A, FSC-A subset 10816.fcs Event Count: 9243
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
10 3
10 3
10 3
7800
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-4 Specimen_001 beads FL580 Event Count: 9080 10 4 10 5
0
0
SSC-A, FSC-A subset 1-38 Specimen_001 stained FL580 Event Count: 6914
Excitation spectrum Emission spectrum
Fluorescein (FITC) PE
525/50
Collection Filter
(wavelength/bandpass)
575/25
300
400
500
600
700
800
Argon Laser 488 nm
Cascade Blue Alexa 430 Fluorescein PE TRPE Cy5PE Cy5.5PE Cy7PE Alexa 594 APC Cy5.5APC Cy7APC
SSC-A, FSC-A subset 1-36 Specimen_001 unstained FL550 Event Count: 7987
Cy7PE Calibration Aria
10 5 10 4 PE-A 10 3
Stained cells
unstained cells
10 5 10 5 10 4 PE-A 10 4 PE-A
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
Cy7PE IgD
0
0
0
0
Cy5PE-A, FSC-A subset 10898.fcs Event Count: 6927
Cy5PE-A, FSC-A subset 10912.fcs Event Count: 6772
SSC-A, FSC-A subset 10822.fcs Event Count: 9232
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-5 Specimen_001 beads FL550 Event Count: 9153 10 4 10 5
FL B220
0
0
0
SSC-A, FSC-A subset 1-37 Specimen_001 stained FL550 Event Count: 6966
Cy5PE-A, FSC-A subset 10904.fcs Event Count: 7638
SSC-A, FSC-A subset 10818.fcs Event Count: 9233
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4
10 3
10 3
10 3
5900
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10
3
10
2
10
2
0 0 10 10 10 PECY55-A 10
10
1
10
1
10 10 Cy55PE: CD24 biotin
2
3
10
4
Fluorescence compensation
Compensated Uncompensated
10000 10000
(FITC spectral overlap subtracted from PE)
FL Calibration Aria
10 5 10 4 PE-A 10 3
Stained cells
10 5 10 4 PE-A
unstained cells
10 5 10 4 PE-A
beads Bead MFI 36000
10 3
10 3
10 2 0 10 2 10 3 FL-A 10 4 10 5
SSC-A, FSC-A subset 1-32 Specimen_001 unstained FL670 Event Count: 7962
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
26000
10 3
10 3
10 3
10 2 0 10 2 10 3 FL-A 10 4 10 5
SSC-A, FSC-A subset 10820.fcs Event Count: 9203
10 5
10 5
10 5
PEБайду номын сангаас
10 4 PE-A
10 4 PE-A
10 4 PE-A
4500
10 3
10 3
10 3
10 2
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5 10 2 10 3 10 4 Cy7PE-A 10 5
Digital electronics vs. Analog electronics
Diva
FlasherII
5 10 4 10
10
5
10
4
10
4
10
3
PECY5-A
PECY5-A
Cy5PE
2 3 4 5
3 10 2 10 1 10 1 10 2 3 4 10 10 10 PECY55-A 5 10
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-1 Specimen_001 beads FL2600 Event Count: 9084 10 4 10 5
0
0
SSC-A, FSC-A subset 1-40 Specimen_001 stained FL643 Event Count: 7486
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
0
0
0
Cy5PE-A, FSC-A subset 10892.fcs Event Count: 7032
beads
Bead MFI
14000
10 3
10 3
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
0
0
0
Cy5PE-A, FSC-A subset 10888.fcs Event Count: 7531
(wavelength/bandpass)
720/45
785/50
625/40
660/40
705/50
Dye Laser 595 nm
750LP
500
600
700
800
Wavelength (nm)
Factors that affect compensation
• Machine Standardization -- Calibration • Digital-linear vs. Analog-LogAmp Electronics • Analog hardware compensation • Log display for samples • Different lots of tandom dyes
James Tung, David Parks, Wayne Moore, Herzenberg Laboratory, and Stanford Shared FACS Facility
Fluorescence compensation
FITC spectral overlap detected in the PE channel must be subtracted from the PE signal
Cy5PE-A, FSC-A subset 10900.fcs Event Count: 7804
SSC-A, FSC-A subset 10814.fcs Event Count: 9253
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
10000
10 3
10 3
10 3
FITC fluorescence in the PE channel
1000
1000
100
100
10
10
10
100
1000
10000
10
100
1000
10000
FITC (B220) fluorescence in the FITC channel
Properly compensated values distribute symmetrically around the median of the negative population, but log display only shows values above zero.
SSC-A, FSC-A subset 1-35 Specimen_001 unstained FL580 Event Count: 7920
10 5
10 5
10 5
PE
10 4 PE-A
10 4 PE-A
10 4 PE-A
8000
10 3
10 3
10 3
10 2
10 2 0 10 2 10 3 FL-A 10 4 10 5 10 2 10 3 FL-A 10 4 10 5
0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0
0
0
Cy5PE-A, FSC-A subset 10894.fcs Event Count: 5908
300 400
12-color FACS
440/40 540/80
Krypton Laser 407 nm
Spectra of dyes
525/50
575/25
630/22
665/30
Argon Laser 488 nm
Excitation spectrum Emission spectrum Collection Filter
0 FL-A SSC-A, FSC-A subset 1-34 Specimen_001 unstained FL610 Event Count: 7877
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
12000
10 3
10 3
10 3
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-3 Specimen_001 beads FL670 Event Count: 9139 10 4 10 5
0
0
SSC-A, FSC-A subset 1-41 Specimen_001 stained FL670 Event Count: 7069
10 2 0 10 2 10 3 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-2 Specimen_001 beads FL610 Event Count: 9065 10 4 10 5
0 FL-A SSC-A, FSC-A subset 1-39 Specimen_001 stained FL610 Event Count: 7488
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
PE-A
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
0
0
0
Cy5PE-A, FSC-A subset 10896.fcs Event Count: 6577
Cy5PE-A, FSC-A subset 10908.fcs Event Count: 6792
SSC-A, FSC-A subset 1-33 Specimen_001 unstained FL643 Event Count: 7885
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE PE-A
18000
10 3
10 3
10 3
10 2 0 10 2 10 3 10 4 10 5
Cy5PE-A, FSC-A subset 10902.fcs Event Count: 7829
SSC-A, FSC-A subset 10816.fcs Event Count: 9243
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
10 3
10 3
10 3
7800
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-4 Specimen_001 beads FL580 Event Count: 9080 10 4 10 5
0
0
SSC-A, FSC-A subset 1-38 Specimen_001 stained FL580 Event Count: 6914
Excitation spectrum Emission spectrum
Fluorescein (FITC) PE
525/50
Collection Filter
(wavelength/bandpass)
575/25
300
400
500
600
700
800
Argon Laser 488 nm
Cascade Blue Alexa 430 Fluorescein PE TRPE Cy5PE Cy5.5PE Cy7PE Alexa 594 APC Cy5.5APC Cy7APC
SSC-A, FSC-A subset 1-36 Specimen_001 unstained FL550 Event Count: 7987
Cy7PE Calibration Aria
10 5 10 4 PE-A 10 3
Stained cells
unstained cells
10 5 10 5 10 4 PE-A 10 4 PE-A
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
Cy7PE IgD
0
0
0
0
Cy5PE-A, FSC-A subset 10898.fcs Event Count: 6927
Cy5PE-A, FSC-A subset 10912.fcs Event Count: 6772
SSC-A, FSC-A subset 10822.fcs Event Count: 9232
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-5 Specimen_001 beads FL550 Event Count: 9153 10 4 10 5
FL B220
0
0
0
SSC-A, FSC-A subset 1-37 Specimen_001 stained FL550 Event Count: 6966
Cy5PE-A, FSC-A subset 10904.fcs Event Count: 7638
SSC-A, FSC-A subset 10818.fcs Event Count: 9233
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4
10 3
10 3
10 3
5900
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10
3
10
2
10
2
0 0 10 10 10 PECY55-A 10
10
1
10
1
10 10 Cy55PE: CD24 biotin
2
3
10
4
Fluorescence compensation
Compensated Uncompensated
10000 10000
(FITC spectral overlap subtracted from PE)
FL Calibration Aria
10 5 10 4 PE-A 10 3
Stained cells
10 5 10 4 PE-A
unstained cells
10 5 10 4 PE-A
beads Bead MFI 36000
10 3
10 3
10 2 0 10 2 10 3 FL-A 10 4 10 5
SSC-A, FSC-A subset 1-32 Specimen_001 unstained FL670 Event Count: 7962
10 5
10 5
10 5
10 4 PE-A
10 4 PE-A
10 4 PE-A
26000
10 3
10 3
10 3
10 2 0 10 2 10 3 FL-A 10 4 10 5
SSC-A, FSC-A subset 10820.fcs Event Count: 9203
10 5
10 5
10 5
PEБайду номын сангаас
10 4 PE-A
10 4 PE-A
10 4 PE-A
4500
10 3
10 3
10 3
10 2
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5 10 2 10 3 10 4 Cy7PE-A 10 5
Digital electronics vs. Analog electronics
Diva
FlasherII
5 10 4 10
10
5
10
4
10
4
10
3
PECY5-A
PECY5-A
Cy5PE
2 3 4 5
3 10 2 10 1 10 1 10 2 3 4 10 10 10 PECY55-A 5 10
10 2 0 10 2 10 3 FL-A 10 4 10 5
10 2 0 0 10 2 10 3 FL-A SSC-A, FSC-A subset 1-1 Specimen_001 beads FL2600 Event Count: 9084 10 4 10 5
0
0
SSC-A, FSC-A subset 1-40 Specimen_001 stained FL643 Event Count: 7486
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
0
0
0
Cy5PE-A, FSC-A subset 10892.fcs Event Count: 7032
beads
Bead MFI
14000
10 3
10 3
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
10 2 0 10 2 10 3 10 4 Cy7PE-A 10 5
0
0
0
Cy5PE-A, FSC-A subset 10888.fcs Event Count: 7531
(wavelength/bandpass)
720/45
785/50
625/40
660/40
705/50
Dye Laser 595 nm
750LP
500
600
700
800
Wavelength (nm)
Factors that affect compensation
• Machine Standardization -- Calibration • Digital-linear vs. Analog-LogAmp Electronics • Analog hardware compensation • Log display for samples • Different lots of tandom dyes