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Flow Cytometers |
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The Flow Cytometry Core Facility is equipped with 6 flow cytometers that are categorized into 2 types:
The flow cytometers in the facility are equipped with an amazing variety of laser excitation wavelengths, which are shown in the following table:
Core Facility Bench Top Analyzer Flow Cytometers: There are 3 bench top analyzer flow cytometers in the core that are manufactured by Becton Dickinson Immunocytometry Systems, San Jose, California, and consist of 2 upgraded, dual-laser FACScans and a four-laser LSRII. Know that FACS™ is an acronym for Fluorescence-Activated Cell Sorting and is Becton Dickinson's registered trademark.
The core is equipped with 2 upgraded FACScan flow cytometers that are each equipped with a low-power (15mW) argon laser that emits blue light at 488 nanometers as well as a low-power (30mW) helium-neon diode laser that emits red light at 633 nanometers. The core's FACScans are upgraded from the factory's original configuration that has only 1 laser and 3 fluorescent detectors. Both FACScans have two detectors for laser-light scatter and 4, not 3, detectors for fluorescence in the green, orange and red/dark red regions of the color spectrum. The detector array permits the use of a great number of fluorophores, allowing for up to 7 parameters for every particle interrogated (time is also a parameter). The optical filters on the FACScans are not interchangeable (each fluorescence detector "sees" on a single fluorescence color) so the choices of fluorophores are similarly constrained, however, a tremendous number of fluorophores can be detected with it's configuration. Both FACScans are equipped with pulse processing for cell doublet discrimination and have an optional 20-liter cubitainer system that has 5 times the buffer and waste capacity of the factory system.
The LSRII flow cytometer is the only digital bench top analyzer flow cytometer in the facility. The LSRII has optical detectors and removable optical filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the FACScans. The core's LSRII is upgraded from the standard factory configuration that has only 3 lasers and 10 fluorescent detectors. The core's LSRII has 4, not 3, lasers with 2 detectors for laser-light scatter and 12, not 10, detectors for fluorescence in the following configuration:
LSRII optical configuration:
The core's LSRII is also equipped with pulse processing for cell doublet discrimination and provides investigators with up to 15 parameters for every particle interrogated. Core Facility High-Speed Cell Sorter Flow Cytometers: There are 3 high-speed cell flow cytometers in the core facility that consist of 3 different models by 2 manufacturers. The facility is equipped with a DakoCytomation MoFlo and a Dako (formerly DakoCytomation) MoFlo XDP. Dako is based in Fort Collins, Colorado. The 3rd sorter in the facility is a Becton Dickinson FACSAria. The primary function of the high-speed cell sorter flow cytometers are for physically sorting cells or particles of interest and for analysis requirements that cannot be met on the bench top analysis flow cytometers in the facility. For example, the sorter flow cytometers are the only flow cytometers in the facility that are equipped with cyan, green, yellow, orange or far red laser lines, consequently, investigators requiring access to these excitation lines are granted access to the sorter flow cytometers regardless if their goal is to physically sort cells (particles) of interest, otherwise, as stated previously, the primary function of the sorter flow cytometers is for physically sorting cells or particles of interest. Unlike the bench top analyzer flow cytometers, which are operated by the investigators themselves, sorter operation is provided as a service. This is because the instruments are much more complex than the bench top analyzers and consequently require much more experience and training to achieve proper function. Also, because of the open design of the MoFlos, which permits great flexibility in experimental design, their components are more easily damaged and they are expensive to replace. Because the sorters use a stream-in-air sorting method, they aerosolize the samples. Consequently, the sorters cannot be used for sorting hazardous samples. Non-hazardous living cells can be sorted and recovered in sterile form for subsequent culture or for in vitro functional studies. Accommodations can be made for AECOM investigators requiring sorting of hazardous specimens off site - please inquire in advance.
The MoFlo has three high-powered tunable-wavelength lasers which consist of an argon, a krypton and an argon-krypton mixed-gas laser. The MoFlo also has detectors and removable filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the other cytometers, i.e., mCherry, mRFP, Texas Red (yellow laser excitation), among many others. There are other important differences between this cytometer and the others in the facility. Among them are:
The diminished fluorescence detection of the MoFlo, like all jet-in-air sorters, over the bench top analyzers is a property of the fluidic and optical system that is needed for sorting. The three bench top analyzers have a closed cuvette that is optically coupled to the fluorescence collector lens which is analogous to an oil immersion objective on a microscope whereas the MoFlo has a jet-in-air system with a "dry" fluorescence collector lens. Cells or particles and enveloping sheath are pushed out of a small ceramic nozzle as a fine stream that crosses the laser beam(s) and fluorescence is collected by a dry microscope objective that is focused on the laser/stream intersection. It follows that, as with dry microscope objectives, less light is collected, the sensitivity will always be less. The magnitude of the difference in the fluorescence varies with the fluorochrome but a general rule is for the jet-in-air sorters to have approximately a ½-decade lower fluorescence than the bench top analyzers. . It is a common misconception that a sorter is able to do everything that a bench top analyzer is able to do plus a whole lot more simply because a sorter costs more than they do. As mentioned previously, the MoFlo is equipped with three high-powered tunable-wavelength lasers, which are detailed in the following table. The typical configuration for immunophenotyping using the MoFlo is detailed below:
A strength of the open architecture of the MoFlo design is that any of its lasers can be placed into any of its optical pathways. Persons may be interested to know that the MoFlo was conceived at Lawrence Livermore National Laboratories (home of the world's fastest computer!) as a tool for the rapid isolation of chromosome-specific DNA libraries for the Human Genome Project. The MoFlo was the first commercially available high-speed cell sorter flow cytometer when it was introduced in 1994. Dako MoFlo XDP high-speed cell sorter flow cytometer [12-color, 4-laser system with optional 4-way sorting, aerosol containment, cloning option and sample station/sort receptacle heater/chiller]; available as a service with online booking. The MoFlo XDP - AECOM's was the first commercial installation of this model flow cytometer in the world - is almost identical in 'architecture' to the core's 'original' (existing) MoFlo. The 2 MoFlos have the identical optical pathways and fluidic systems though the MoFlo XDP is equipped with 12, not 8, fluorescent detectors. The primary difference between AECOM's 2 MoFlos is in the electronics where the MoFlo XDP uses completely digital processing whereas the original MoFlo uses analog-to-digital processing though there is no difference in their sorting speed. The fluorescent detectors are a newer generation than those that exist in the existing MoFlo and the company claims that the XDP is more sensitive than the original MoFlo. Perhaps the most notable difference between the 2 MoFlos is that the MoFlo XDP is equipped with 4, not 3, lasers and, as mentioned previously, there are 12, not 8, fluorescent detectors. The MoFlo XDP has 3 high-powered tunable-wavelength lasers, which consist of an argon, a krypton and an argon-krypton mixed-gas laser (virtually identical as the original MoFlo) though, unlike the 'original' MoFlo, the MoFlo XDP is also equipped with a high-powered 592nm (orange) fiber laser, which shares the mixed-gas laser pathway. Investigators that are interested to utilize the orange fiber laser must forgo concurrent use of the mixed-gas laser. The MoFlo XDP's lasers are detailed in the following table. Like the original MoFlo, the MoFlo XDP has detectors and removable optical filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the bench top analyzer cytometers, such as mCherry and Texas Red (yellow laser excitation) as well as the newly published far-red fluorescent proteins mKate and Katushka (orange laser excitation), among many other fluorophores. As with the original MoFlo, there are other important differences between this cytometer and the bench top analyzer flow cytometers in the facility. Among them are:
As mentioned previously, the MoFlo XDP has the same architecture and the 'original' MoFlo in the facility though the MoFlo XDP has 12, not 8, fluorescent detectors in the following configuration for immunophenotyping:
Becton Dickinson FACSAria high-speed cell sorter flow cytometer [12 color, 4 laser system with optional 4-way sorting, aerosol containment, cloning option and sample station/sort receptacle heater/chiller]; available as a service with online booking. The core's FACSAria is an upgraded, 'Special Order' system that differs from the standard factory configuration that is equipped with 10 fluorescent detectors and 3 lasers. The core's FACSAria is equipped with 12, not 10, fluorescent detectors and 4, not 3, solid-state lasers as detailed in the following table. The core's FACSAria is equipped with an optional forward scatter photomultiplier tube (PMT) which is in consideration of investigators interested to sort particles <1 micron in size. The forward scatter PMT is in addition to the standard forward scatter diode, which exists in all the cytometers in the facility.
The FACSAria is equipped with the following lasers in the detector configuration as described:
A 14-color, 5-laser FACSAria is available to investigators that is not physically located in the core facility though it is under the auspices and oversight of the core's administration and staff. The 5-laser FACSAria is equipped with the almost the identical lasers and forward scatter PMT that the 4-laser FACSAria has but the 5-laser FACSAria is also equipped with a green 532 nanometer laser. The lasers are and associated optics are:
Differences in sorter model performance There are notable differences in performance between the disparate sorters in the facility that investigators that are sorting must be aware of. Known differences are detailed below. The take-home message should be that each model sorter in the facility has strengths and weaknesses that investigators should take into consideration when planning experiments that involve sorting. FACSAria advantages:
FACSAria disadvantages:
As stated previously, the take-home message is that each model sorter in the facility has strengths and weaknesses that investigators must take into consideration when planning experiments that involve sorting. |
Jinghang Zhang is solely responsible for the content of this site. Comments, concerns and questions regarding it should be addressed to her. |
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Becton Dickinson FACScan analog bench top analyzer flow cytometers [4-color, 2-laser systems]; available to trained users with online booking.
Becton Dickinson LSRII digital bench top flow cytometer [12-color, 4-laser system], available to trained users with online booking.
DakoCytomation MoFlo high-speed cell sorter flow cytometer [8-color, 3-laser system with optional 4-way sorting, cloning option and sample station/sort receptacle heater/chiller]; available as a service with online booking.
