Optical bench configuration on the FACSVantage SE.
Our optical bench has eight photomultiplier tubes configured to detect fluorescent signals. Four of them (designated FL1, FL2, FL3 and FL6) are aligned with the primary laser positions (usually an argon laser emitting at 488 nm). Two of them (designated FL4-R and FL5-R) are aligned with the secondary laser (usually the helium-neon lasers emiting in the red), and two more (designated FL4-UV and FL5-UV) are aligned with the tertiary laser (usually the krypton-ion, emitting in the UV or violet). PMTs in FL3, 4-R, 5-R, 4-UV and 5-UV are Hamamatsu R3896-04 red-sensitive PMTs for maximum sensitivity of fluorochromes emitting above 600 nm.
(Below.) The FACSVantage SE optical bench (including the FL6, FL7 and FL8 upgrade). Primary laser (usually argon-ion) PMTs are in blue, secondary (usually HeNe) in red, tertiary (usually krypton-ion) in violet. Due to the recent PMT upgrade, the secondary (HeNe) PMTs are not shown in the photograph. Six fluorescent PMTs can be used simultaneously.
The optical bench can be configured with a variety of reflecting dichroics, long, short and narrow bandpass filters to allow simultaneous analysis of up to six fluorochromes simultaneously. We currently have the following dichroics and filter sets...
| Dichroic | Use |
| 380 LP | detection of side scatter during indo-1 analysis |
| 460 LP | indo-1 ratiometric analysis (extended reflection) |
| 480 LP | separation of Cascade Blue and Cascade Yellow or ELF-97 signals |
| 505 SP | standard indo-1 ratiometric analysis (behaves like 450 SP at 45 degree angle) |
| 560 SP | separation of FITC and PE signals |
| 610 SP | separation of FITC/PE and PE-Cy5 signals, Hoechst SP analysis |
| 640 LP | separation of APC and shorter wavelength signals |
| 690 LP | separation of APC and APC-Cy5.5 or APC-Cy7 signals |
| 710 LP | separation of PE and PE-Cy5.5 or PE-Cy7 signals |
| 748 LP | non-reflecting dichroic, detection or PE-Cy7 or APC-Cy7 signals |
| Filter | Use |
| 390/30 | indo-1 (bound Ca) enhanced sensitivity |
| 405/20 | indo-1 (bound Ca) standard sensitivity |
| 424/44 | DAPI, Hoechst 33342 and 32580, AMCA, Alexa Fluor 350, Marina Blue |
| 440/10 | Cascade Blue, Pacific Blue, Hoechst 34580 |
| 485/22 | indo-1 (free Ca) standard sensitivity, CFP |
| 495/20 | indo-1 (freeCa) enhanced sensitivity. CFP |
| 530/30 | fluorescein, Oregon Green 488, Alexa Fluor 488, Cy2, GFP, ELF-97, PKH2, PKH67 |
| 535/45 | fluorescein, Oregon Green 488, Alexa Fluor 488, Cy2, GFP, YFP, ELF-97, PKH2, PKH67 |
| 575/26 | PE, PI, Cy3, CF-3, CF-4, TRITC, PKH26 |
| 585/22 | PE, PI, Cy3, CF-3, CF-4, TRITC, PKH26 |
| 610/20 | lissamine rhodamine B, Rhodamine Red, Alexa Fluor 568 |
| 610/30 | lissamine rhodamine B, Rhodamine Red, Alexa Fluor 568 |
| 630/22 | PE-Texas Red, Texas Red, Alexa Fluor 594 |
| 660/20 | APC, Alexa Fluor 633, CF-1, CF-2, PBXL-1, PBXL-3 |
| 675/20 | Cy5, Alexa Fluor 647, TO-PRO-3 |
| 682/22 | Cy5, Alexa Fluor 647, Alexa Fluor 660, PE-Alexa Fluor 647, PE-Cy5, TO-PRO-3 |
| 710/20 | Cy5.5, Alexa Fluor 680, PE-Alexa Fluor 680, APC-Alexa Fluor 680 |
These tables in
Acrobat PDF format.
(Below). Examples of filters used on the FACSVantage SE.
Fluorochrome detection off the primary argon-ion intercept.
Up to four fluorochromes an be detected off the primary laser intercept. FITC (or other fluorescein substitutes) and PE are usually detected in FL1 and FL2. Fluorescent signals are passed through a 610 SP dichroic and a 560 SP dichroic, which splits the FITC and PE signals (530 and 575 nm respectively through their corresponding narrow bandpass filters (530/30 and 575/25) and PMTs. PE-Cy7 and PE-Cy5 can be detected through the FL3 and FL6 PMTs respectively. Fluorescent signals above 610 nm are deflected to the right, and subsequently split by a 690 LP dichroic to discriminate PE-Cy7 in FL3 (750 nm) from PE-Cy5 in FL6 (675 nm) using a 748 LP filter and a 675/20 narrow bandpass filter respectively.
(Below). Filter configuration for detecting FITC, PE, PE-Cy5 and PE-Cy7.
The argon-ion laser can also be tuned to several wavelengths in the UV, blue and green range. Click here for full laser information.
Fluorochrome detection off the secondary helium-neon laser.
The helium-neon laser (emitting at a fixed wavelength of 632 nm) can excite APC and the tandem conjugate APC-Cy7. These signals are separated using a 710 LP dichroic and detected through a 675/20 narrow bandpass filter and a 748 LP filter respectively. Click here to see more information on red-excited fluorochromes.
(Below). At left, the filter configuration for detecting APC and APC-Cy7 off the helium-neon laser. At right, EL4 cells were labeled with biotin-anti-Thy1.2 followed by either APC-avidin or APC-Cy7-avidin, followed by mixing of the labeled cells and analysis using the laser and filter configuration shown.
Fluorochrome detection off the tertiary krypton-ion laser.
FL4-UV and FL5-UV are aligned with the secondary laser intercept and are used to detect fluorchromes excited off of the krypton and/or HeNe lasers. The krypton laser can be tuned to two UV (351 and 357 nm) and two violet wavelengths (407 nm and 415 nm), allowing excitation of the DNA dyes Hoechst 33342, Hoechst 33258 or DAPI, and phenotyping fluorochromes Cascade Blue and Cascade Yellow. Simultaneous detection of Cascade Blue and Cascade Yellow are shown below. Click here for sample data using Cascade Blue and Cascade Yellow.
(Below). At left, the filter configuration for detecting Cascade Blue and Cascade Yellow with the krypton laser tuned to 407 nm. At right, EL4 cells were labeled with biotin-anti-Thy1.2 followed by either Cascade Blue-avidin or Cascade Yellow-avidin, followed by mixing of the labeled cells and analysis using the laser and filter configuration shown.
The krypton-ion laser can also be tuned to several wavelengths in the blue, green and yellow range. Click here for full laser information, and here for information on yellow-excited (568 nm) probes.
Multiple fluorochrome detection off the primary argon-ion and secondary HeNe lasers.
Multiple fluorochromes can be analyzed using two or more lasers simultaneously.
(Below). Filter configuration for detecting FITC, PE, PE-Cy5, APC and APC-Cy7 simultaneously, and data from a pilot 5-color experiment. Mouse EL4 cells were labeled biotin-anti-Thy1.2 followed by FITC, PE, PE-Cy5, APC and APC-Cy7-conjugated avidin. Cells were then mixed and analyzed using the illustrated filter configuration.
Multiple fluorochrome detection off the primary argon-ion, secondary HeNe and tertiary krypton-ion lasers.
(Below). Above, filter configuration for detecting FITC, PE, PE-Cy5, APC, APC-Cy7 and Cascade Blue in a pilot 6-color experiment using three laser excitation. Below, EL4 cells were labeled with biotin-anti-Thy1.2 followed by either FITC, PE, PE-Cy5-APC, APC-Cy7 or Cascade Blue-avidin, followed by mixing of the labeled cells and analysis using the laser and filter configuration shown below..
