Lectura Desconectados

Cytotechnology (2012) 64:109–130
DOI 10.1007/s10616-011-9415-0
REVIEW
Flow cytometry: retrospective, fundamentals and recent

instrumentation
Julien Picot • Coralie L. Guerin •
Caroline Le Van Kim • Chantal M. Boulanger
Received: 10 October 2011 / Accepted: 28 November 2011 / Published online: 21 January 2012
Springer Science+Business Media B.V. 2012
Abstract Flow cytometry is a complete technology come from the common endeavor of physicists,
given to biologists to study cellular populations with biophysicists, biologists and computer engineers,
high precision. This technology elegantly combines who succeeded, by providing new concepts, to bring
sample dimension, data acquisition speed, precision flow cytometry to current maturity. The aim of this
and measurement multiplicity. Beyond the statistical paper is to present a complete retrospective of the
aspect, flow cytometry offers the possibility to phys- technique and remind flow cytometry fundamentals
ically separate sub-populations. These performances before focusing on recent commercial instrumentation.
Keywords Flow cytometry Retrospective

Julien Picot and Coralie L. Guerin contributed equally to this
work. Fundamentals Instrumentation Analyzer
Cell-sorter
J. Picot (&) C. Le Van Kim
Institut National de la Transfusion Sanguine, 75739 Paris
Cedex 15, France
e-mail: [email protected] Seventy seven years of technological innovation
J. Picot C. Le Van Kim As flow cytometry is a technology that combines

INSERM, U665, 75739 Paris, France several concepts (Shapiro 2003), it is difficult to
J. Picot C. Le Van Kim estimate a single starting point. However, the main
University Paris Diderot, Sorbonne Paris Cité, Paris, originality residing in observation of aligned cells one
France behind another into a fluid sheath was described for the
first time by Moldavan (1934) for cell counting on the
C. L. Guerin C. M. Boulanger
University Paris Descartes, Sorbonne Paris Cité, 24th August 1934 in Montreal.
75006 Paris, France During World War II, the US Army was interested
in developing a system that rapidly detects bacterial
C. L. Guerin C. M. Boulanger biowarfare agents in aerosols. Gucker and O’Konski
PARCC INSERM UMR970, 75737 Paris, France
(1947) set up and developed a photoelectronic counter
C. L. Guerin which injected an air stream containing particles into a
Hematology Department and INSERM U765, Paris sheath stream on a dark-field microscope focal point.
Descartes University, Paris, France This first air-fluxes cytometer detected 0.5 micron
C. L. Guerin events by scattered light. In 1942, Coons et al. used,
European Georges Pompidou Hospital Paris, Paris, France for the first time, an anthracene-associated antibody
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110 Cytotechnology (2012) 64:109–130
(UV-excitable fluorochrome) to detect Streptococcus green and red fluorescence emission (530 and 640 nm
pneumoniae. One decade later, Coons and Kaplan respectively). In 1972, Bonner et al. developed a new
(1950) described the first fluorescein isothiocyanate system named fluorescence activated cell sorting
(FITC) antibody association. In 1945, Coulter devel- (FACS) which purified sub-populations (Bonner et al.
oped a process that permits counting events and 1972) and was equipped with a water-cooled argon
measuring cell size by conductance variation. In this laser to analyze FITC- and Rhodamin-coupled antibody
primary system where cells pass one by one into a very fluorescence. To develop cell sorting, Fulwyler (1965)
thin capillary and cross a light beam, extinction number and Kamentsky and Melamed (1967) used a droplet
corresponded to the cell number. However this tech- deflection system developed by Sweet (1965) at
nique led to many clogs. In 1949, Coulter precisely Stanford University (Stanford, CA, USA) for ink jet
measured cell volume in a saline suspension based on printers in 1965. With this droplet deflection technol-
electrical impedance variation proportionality due to the ogy, the saline sample stream was broken into droplets
fact that sheath fluid and cell suspension are ionic liquids containing cells. Those of interest, with selected
and cells surrounded by a lipid membrane are poor measurement values, were electrically charged at the
conductors compared to saline fluid. Coulter’s cell droplet break-off point and droplets were then deflected
counters evolved into cell analyzers and were rapidly by the means of an electric field into a collection tube.
adopted by clinical laboratories for blood cell counting Their larger cell sorter rapidly measured scattered lights
(Brecher et al. 1956; Mattern et al. 1957). at several angles (Mullaney et al. 1969; Salzman et al.
In 1953, Crosland-Taylor used the laminar coaxial 1975a, 1975b; Steinkamp et al. 1973), fluorescence and
flux properties described by Reynolds in 1883, to measured cell volume by electrical impedance varia-
develop a device counting red blood cells suspended in tion. In 1973, Hulett et al. developed a rapid cell sorter
a sheath fluid through a capillary (Crosland-Taylor (Hulett et al. 1973). In 1974, the first commercial flow
1953). Simultaneously, photomultiplier tubes cytometric differential counter was the Hemalog-
appeared, increasing optic signal precision. In the D (Technicon, Tarrytown, NY, USA), which led to
middle of the 1960s, Fulwyler described the electronic the establishment of a white blood cell classification
separation of cells by volume (Fulwyler 1965). (Mansberg et al. 1974; Ornstein and Ansley 1974). The
Concurrently, Katmentsky et al. (1965) described a same year, Becton–Dickinson (now BD Biosciences,
rapid cell spectrophotometer, which made rapid San Jose, CA, USA) commercialized a second cell
absorption measures. In 1965, he also studied optical sorter version named FACS II, which measured FSC
properties of cells passing across a laser beam, such as and 530 nm emission fluorescence. Simultaneously, the
scattered light intensity (forward scatter) and UV-light Max Planck Institute (Göttingen, Germany) built a
absorption (DNA quantity) after staining. In 1967, he multiparameter cytometer cell sorter (Arndt-Jovin and
developed a spectrophotometric cell sorter (Kament- Jovin 1974), and the Partec/Phywe society (Göttingen,
sky and Melamed 1967). In the late 60s, Göhde’s Germany) commercialized the Impulsecytophotometer
Partec (Münster, Germany) developed an analyzer ICP 22. In 1975, Partec commercialized the two-
(Dittrich and Gohde 1969) built around a Zeiss parameter Particle Analyzing System PASTM 8000. In
fluorescent microscope for DNA analysis with ethi- the middle of the decade, Coulter Electronics (now
dium bromide (LePecq and Paoletti 1967). One year Beckman Coulter, Fullerton, CA, USA), known for
later, Phywe (Göttingen, Germany) commercialized it hematologic counters, made their first cell sorter named
for the first time under the name impulsecytophotom- two-parameter-sorter-1 (TPS-1) with an air-cooled
eter ICP11. 35 mW argon ion laser, measuring FSC and one
In 1970, Kamentsky founded Bio/Physics Systems fluorescence. Several excitation wavelengths were
(Mahopac, NY, USA) and in 1971 sold the Cytograph introduced to flow cytometry: the first analyzer
and Cytofluorograph systems. For the first time, Van (Curbelo et al. 1976) used five illuminating beams
Dilla et al. (1969) equipped these analyzers with from a single arc lamp; the second named Cytomat-R
respectively 633 nm He–Ne laser and 488 nm argon (Shapiro et al. 1977) with three laser beams was built by
ion lasers. Excitation by a laser instead of a lamp allows Shapiro in order to analyze FSC, SSC and several
optimal beam focusing on cells. Cytofluorograph fluorescence colors (Shapiro et al. 1976) allowing
allowed the measurement of forward scatter light, morphological gating (Shapiro 1977). Both could detect
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Cytotechnology (2012) 64:109–130 111
up to 30,000 events/s. In 1977/78, Coulter Electronics Partec commercialized the two Particle Analyzing
developed the EPICS (Electronically Programmable Systems PAS-III and PAS-IV. In 1994, Cytomation
Individual Cell Sorter) system with a 5 watts argon laser (Dako/Cytomation and now Beckman Coulter) com-
and data multiparametric analysis. In 1978, at the mercialized the MoFlo MLS flow cytometer developed
Conference of the American Engineering Foundation in by van den Engh, the first high-performance cell sorter
Pensacola, Florida, the cytophotometry technique was for high-speed sorting applications. From the mid-90s,
renamed flow cytometry, a term that quickly became smaller diode and solid-state lasers were more and more
popular. The same year, the Society for Analytical often incorporated into flow cytometers. Because many
Cytology (later renamed International Society for objects are too large and too fragile for conventional
Analytical Cytology and then International Society flow cytometry, in 1998, the first Complex Object
for Advancement of Cytometry) that edits the journal Parametric Analyzer Sorter (COPASTM) was intro-
Cytometry was created. In 1978, Schlossman started the duced. Over the next decade, Union Biometrica
production of monoclonal antibodies directed against expanded the COPAS platform into a family of fully
blood lymphoid antigens. New fluorochromes for automated systems for high throughput analysis, sorting
multicolor flow cytometry were also developed and dispensing of large objects ranging from 20 to 1,500
(Reinherz et al. 1979). In 1979, Partec commercialized microns.
the PAS-II instrument. At the same time, NIH scientists In 2000, Apogee Flow Systems Ltd sold systems
added a 568 nm krypton laser to the flow cytometer and dedicated to environmental bacteria detection. The US
after a developmental phase, FACS IV was the first Army chose the A40 military flow cytometer (Apogee
dual-laser cytometer created, commercialized by Flow Systems) for its high sensitivity to small particles.
Becton–Dickinson (Steinkamp et al. 1979). Coulter In 2001, a violet diode laser was used for the first time
and Ortho (that bought Bio/Physics Systems) manu- on a flow cytometer (Shapiro and Perlmutter 2001).
factured flow cytometers that measured FSC, SSC and The same year, the BD LSRTM II flow cytometer
fluorescence, and analyzed several thousand events/s. proposed detection of fourteen fluorescences, a pow-
After successfully performing a dual-color immunoflu- erful tool for elucidation of the complex immune
orescence experiment, Loken et al. (1977) introduced system (De Rosa and Roederer 2001; De Rosa et al.
fluorescence compensation in the process. 2001). One year later, in 2002, the FACSAriaTM (BD
In the early 1980s, optical emission systems Biosciences) became the first cell sorter with a fixed
appeared. In the mid-1980s, Lawrence Livermore optical emission system. Partec inserted a flow cytom-
National Laboratory (LLNL) created the first high etry laboratory into a french car: CyLabTM was the first
speed cell sorter prototype (Peters et al. 1985) for mobile flow cytometry lab. Partec later proposed the
human chromosome separation (Gray et al. 1987). This CyLabTM Plus, an advanced mobile HIV monitoring
high-speed cell sorter could sort 20,000 cells/s at laboratory based on a 4-wheeldrive transporter vehicle
200 psi (pound per square inch) pressure, three times using CyFlow technology which can also run on a car
faster than conventional sorters (8,000 cells/s at 12 psi). battery (12 V DC power). In 2004, a Partec’s CyFlow
In 1985, cell analyzers allowed up to three color integrated the International Space Station (ISS). The
analysis, such as EPICS C analyzer (Leif et al. 1985) same year, new nanocrystal semi-conductor fluoro-
(Coulter) with an arc lamp source or FACScanTM chromes named Qdots appeared. All five different
(Becton–Dickinson) with a 15 mW air-cooled argon nanocrystals are excited with the same long-wave-
laser source. Competition started between the different length UV lamp and their size determines their color.
flow cytometer manufacturers concerning the simulta- With Qdot technology, Perfetto et al. (2004) realized
neous analysis capacity of fluorescence. In 1987, Partec seventeen different fluorescences per cell experiment.
introduced Cell Analyzer CA-II. In 1989, LLNL In 2007, van den Engh developed a new high-speed cell
produced a second generation of high speed cell sorters sorter named InfluxTM (Cytopeia) and sold his society
allowing 200,000 events/s sorting at less than 100 psi to BD Biosciences while Beckman Coulter acquired
pressure, with parallel organization of the data analysis Dako/Cytomation.
process (van den Engh and Stokdijk 1989). In 2010, Sony, Merck Millipore and Danaher
In 1990, flow cytometer capacity increased to respectively acquired iCyt, Guava Technologies and
measure seven fluorescences simultaneously. In 1991, Beckman Coulter. New ergonomic and compact
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112 Cytotechnology (2012) 64:109–130
analyzers appeared: Cube 8 (Partec), easyCyteTM 8HT built-in sample tray for a 96-well microplate and/or
(Merck Millipore) and the first acoustic focusing tubes. Some systems are temperature-controlled,
(Ward et al. 2009) cytometer Attune (Applied whereas others offer the possibility to add reagents
BiosystemsTM by Life TechnologiesTM). Furthermore, or drugs during analysis.
new analyzers appeared LSRFortessaTM (BD Biosci- Cells pass one by one across laser(s) beam(s) for
ences), GalliosTM/NaviosTM (Beckman Coulter) and a individual analysis. This hydrodynamic focusing
high-speed cell sorter AstriosTM MoFlo (Beckman (Fig. 1 part 1) is based on coaxial laminar flow
Coulter). This year, in 2011, BD Biosciences acquired dynamic properties described in 1883 by Reynolds. To
Accuri Cytometers and commercialized a new bench- ensure a good quality of hydrodynamic focusing, the
top analyzer, the FACSVerseTM. Two new bench-top fluidic system must be very stable. The sample
analyzers appeared MACSQuant VYB (Miltenyi suspension is first pressurized and then injected into
Biotec) and Auto40 (Apogee Flow systems). Partec a sheath core flow before passing thought a nozzle.
produces the CyFlow Cube Sorter, a new bench-top The cellular suspension speed is dependent on the
mechanical cell sorter. sheath fluid pressure that is fixed for an analyzer and
Today, approximately seventeen societies manu- adjustable for a cell-sorter. The higher the sample
facture analyzers and cell sorters. Some flow cytom- pressure the more cells have an opportunity to move
eters simultaneously analyze up to 32 parameters at laterally in the stream, causing a decrease in precision
200,000 events/s, and sort up to 100,000 cells/s into of hydrodynamic focusing, and consequently a drop in
6-way sorting. quality of the sample analysis, due to increases in the
Coefficient of Variation of the peak values. The nozzle
design is essential for obtaining laminar fluxes (Ful-
Flow cytometry fundamentals wyler 1977) in order to transport cells to the center of
the stream: nozzle aperture should be narrow and
Flow cytometry is a powerful tool for the analysis of perfectly calibrated (between 50 and 2,000 microns,
multiple individual cell parameters from heteroge- depending on applications). This hydrodynamic
neous populations. Flow cytometry is used in several focusing results from several stability developments
multicolor applications for biology or functional (Steen 1990), and is used in most flow cytometers.
studies as well as a broad range of research applica- However, other focus concepts exist, such as micro-
tions including: immuno-phenotyping, multi-paramet- capillary technology or acoustic focusing (Ward et al.
ric DNA analysis, proliferation, fluorescent protein, 2009) which allows alignment of cells by sound waves
cell counting… Thousands of cells per second pass (Goddard et al. 2006, 2007; Goddard and Kaduchak
one by one through one or more laser beams in a flow 2005) without damage to cells (as ultrasound for fetus
cytometer, which measure scattered lights at several in utero medical exam).
angles and fluorescence emissions. Some of them are Flow cell could be qualified as the heart of the
also capable of analyzing cell volume by electrical system because it is the place where laser beam and
impedance variation. Three parts constitute a flow cells interact (Fig. 1 part 2). That is the starting point
cytometer: first, the fluidic system permits hydrody- of scattered lights (forward scatter and side scatter)
namic focusing; second the excitation source and and fluorescence signal emissions detected with an
optical emission systems from the wavelength filters optical collection system before amplification and
to the detectors constitute the optical part and finally electronic digitalization. This intersection between
the electronic system digitalizes the signal to be excitation light source and cells is occurring in a
analyzed with specific computer software. quartz chamber or in the air at the nozzle exit. Due to
For flow cytometry acquisition, cells need to be in the important variation of refraction indices between
suspension in a tube or a plate. Some flow cytometers air and sheath fluid, the reflection phenomenon implies
are equipped with a carousel or a rack loader (16, 30, lack of light signal so the amount of emission
32 or 40 tubes capacity) containing a bar-code wavelength collected in the air is less important than
identification option and a vortex mixing system prior in quartz chambers; numerical aperture of the objec-
to sample aspiration. Some analyzers are equipped tive and sheath fluid pressure are important in this
with a high throughput 96 or 384-well plate loader or a case. In contrast to quartz chambers, optical systems in
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Cytotechnology (2012) 64:109–130 113
Fig. 1 Flow cytometry

principle
the air have to be aligned daily (Watson 1999) to analyzer which uses mercury arc-lamp for UV-excita-
ensure sensitivity and quality. tion. Since 2001, violet laser diodes are used as light
Cell illumination with an excitation light source sources for cytometry (Shapiro and Perlmutter 2001).
provides scattered (forward scatter and side scatter) Use of several excitation sources increases the number
and fluorescent lights. The first flow cytometers were of fluorochromes detectable simultaneously and
equipped with a mercury arc lamp, but light focaliza- directly impacts on the number of cellular parameters
tion on a small analysis area and light emissions correlated (Crissman and Steinkamp 1982; Perfetto
collection were very poor, unlike those delivered by a et al. 2004). Some flow cytometers were equipped with
coherent and monochromatic laser beam, direct or four different excitation sources (Greimers et al. 1996)
fibered through prisms and optical lens (Van Dilla et al. or more, today cell sorters can be equipped with up to
1969) also used to control illumination point geometry. ten lasers. Laser co-linearity (several excitation
Indeed, contrary to circle geometry, the elliptic and sources in the same plan) implies alignment simplicity
now the rectangular geometry laser beam homoge- and stability but involves more fluorescence splitting
neously illuminate cells whatever the lateral cell than in a laser non-co-linearity system (one plan by
position in sheath fluid. The variable power intensity excitation sources), which allows effective fluores-
laser (15–200 mW) is function of the flow cell (quartz cence splitting, but creates a spatial gap, electronically
or jet-in-air). Emission fluorescence is quantitatively converted to a temporal gap (time laser delay) in order
proportional to excitation intensity of the fluoro- to synchronize several light signals from one cell.
chrome. At the present time, excitation light sources When cell pass through the excitation source, the
are systematically diode lasers or lasers, except for one laser beam is refracted in all directions. Light diffusion
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114 Cytotechnology (2012) 64:109–130
at small angles (forward scatter light, FSC, FALS) is (threshold), often FSC, below which events are not
collected in the axis of the laser beam by a photodiode considered by the electronic system.
or a photomultiplier tube; the magnitude of the The flow cytometer electronics (Fig. 1 part 4)
forward scatter light is correlated to the relative-size measures several thousand events per second, limited
of the cells. A shutter bar masks the signal due to the however by electronic dead time (Snow 2004), which
laser beam on the detector. Some analyzers permit a is today less than 5 ls. Most efficient cell sorters have
choice between 1–8 and 1–19 collection of FSC no dead time, and thus no hard aborts. Analyzers
signal (field stop) to detect, respectively, only large digitalize signal with analogic to digital converter
events or total events. Light diffusion at large angles (ADC) by converting voltage value to digital value
(side scatter light, SSC, WALS) is collected at 90 of and determining the channel number: 256 (8 bits),
the laser beam like fluorescence emission lights 1,024 (10 bits), etc. on logarithmic or linear scale
(Fig. 1 part 3). Orthogonal side scatter light is a (Fig. 1 part 5). Some analogical and all new digital
combination of diffusion, reflection and refraction flow cytometers allow retreatment of the data and
caused by structural complexity into the cell. In flow modifications of fluorescence compensation matrix
cytometry, diffusion phenomena are very complex post-acquisition. Flow cytometers simultaneously
(Salzman et al. 1979). Side scatter and fluorescent analyze pulse height, width and area for each cell
lights are filtered by dichroic mirrors and adequate allowing doublet-exclusion. Actually, the highest
emission filters (band pass, short pass or long pass) resolution is 32 bits (232 channels), divided on a five
(Steinkamp et al. 1974) into an emission optical decades logarithm scale (up to seven decades for
system which directs lights at different wavelengths some). Most recent cytometers offer a biexponential
towards appropriate detectors. The role of the dichroic scale under the axis. Events visualization is charac-
mirror is fundamental because it selects, reflects and terized by exhibiting pseudo-linear like behavior for
transmits light signal band pass towards specific values near zero, and transitioning it to a pseudo-
detectors collecting specific fluorescence. Light logarithmic behavior values distant from zero (Novo
reflected by dichroic mirror is better than transmitted and Wood 2008; Parks et al. 2006). Thus, events with
light, which originates in a multicolor staining, which lower fluorescence intensities are grouped and cell
signal passes through several filters and loses a populations appear homogeneous.
significant part of its initial intensity. Thus, architec- Cell sorters offer the possibility of isolating
ture of the emission optical system is essential to subpopulations of cells of interest with high recovery
minimize losses and usually the user has the possibil- and high degree of purity from heterogeneous cell
ity of changing optical filters for each fluorescent mixtures based on light scattering and fluorescent
measurement. characteristics.
Detectors (photodiode and photomultiplier tubes) Two kinds of sorting mechanisms can be described
convert and amplify light signals (photons) from cell (Ashcroft and Lopez 2000).
passage into a laser beam to an electric signal. This First, the mechanical sorter employs a mechanical
pulse is then digitalized, recorded and treated by catcher tube to sort cells of interest. The catcher tube is
specific software. Photodiodes are used for high located in the upper portion of the flow cell and moves
intensity signals and photomultiplier tubes (PMT) into the stream to collect the cells. If the cell is
for low intensity signals that need to be amplified with identified as a cell of interest, it is captured by the
dynodes succession into PMT. Some analyzers also catcher tube and collected into a tube or a concentration
allow increasing gain, generating significant unspe- module; otherwise it is dispatched to the waste tank.
cific signals (background noise) so that is preferable to Ideal for pathogenic samples, this method produces no
first increase PMT voltage before gain for low biohazardous aerosols, but is considerably slower in
intensity signals. sorting rate (300 cells/s with BD FACSCaliburTM and
An impulsion’s characteristics are pulse height or CyFlow Sorter module by Partec). The mechanical
peak value, pulse width and pulse area or integral system permits sorting of only one population. These
value. Impulsion length is also named flight time or flow cytometers are designed with a closed fluid
collection integration time. Trigger is a parameter system, thereby making them ideally suited for work-
chosen by the user based on a discrimination value ing with potentially biohazardous samples.
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Cytotechnology (2012) 64:109–130 115
Second, most high-speed cell sorters use electro- the MoFlo BTA and the MoFlo MLS (Cytomation)
static deflection of droplets (‘‘jet-in-air’’ method) were previously described (Chapman 2000; Nunez
(Chapman 2000). The advantage of this system is 2001).
the possibility to sort from one to six sub-populations
of cells (depending on cell sorters) simultaneously at Small bench-top analyzers
low- or high-speed. As sorting generates aerosols in
the chamber (Schmid and Dean 1997; Schmid et al. In the last few years, new bench-top flow cytometers
1997) working with potentially biohazardous samples have appeared, combining analytical power of
(Perfetto et al. 2003) will require implementation of research dedicated cytometers into robust, ergonomic,
appropriate precautions. ultra-compact and easy-to-use analyzers. These new
The cell suspension is directed into a stream, which systems are easy to handle and less demanding
emerges from a vibrating nozzle and breaks up into regarding location space, maintenance and service.
individual droplets (Fig. 1 part 6). The nozzle vibra-
tion conditioned by the drop drive frequency (ddf) is A50-Micro, A50-Universal, Auto40 (Apogee Flow
the number of drops generated per second proportion- systems)
ally to amplitude level. The fluidic system stability
allows a good evaluation of the cell localization inside A50-Micro, for extremely small particles applications,
the droplets (Petersen and van den Engh 2003) if all with three lasers (375, 405, 488, 532 or 635 nm)
conditions are accomplished. A droplet containing a detects up to 3 light scatters (3 detectors) and four
cell of interest is positively or negatively charged and fluorescence colors. The A50-Universal analyzer is
goes through an electric field between two deflection equipped with up to three lasers (375, 405, 488, 532 or
plates before being deflected into collection tubes 635 nm) and detects FSC, SSC and four fluorescence
(Fig. 1 part 7). colors simultaneously. The newest Auto40 is equipped
Several parameters should be considered for cell with a laser interchangeable without realignment (488
sorting optimization. According to type of cells, it is or 532 nm) and permits analysis of FSC, SSC and
necessary to adjust sheath fluid pressure linked to the three fluorescence colors simultaneously. Optical filter
orifice diameter of the nozzle tip; the number of drops blocks are interchangeable without realignment. All
formed per second depends upon nozzle vibration these analyzers determine volumetric sample and give
(frequency and amplitude). The design specification absolute counts. Analysis rate is up to 20,000 events/s.
for a cell sorting instrument also includes set up of Sample acquisition (16 bits—4.8 logarithmic decades)
break-off point (point where the stream breaks into and data analyses are performed by a software in an
droplets), drop delay (distance between the laser beam integrated computer. Post-acquisition compensation
interception of the cell and the break-off point), and isn’t allowed (FCS 2.0).
purity level (sort mode’s choice). These calculations
can be automatic. Accuri C6, CSampler (BD Biosciences)
Thus it is possible to sort from 1 to 6 ways
simultaneously into several containers (0.6, 1.5, 2.0, After purchase this year of Accuri Cytometers, BD
5.0, 15 or 50 mL tubes, multi-well plates until 1,536, Biosciences commercializes two new bench top
slides). analyzers: BD Accuri C6 and CSampler. Equipped
There are many possible applications resulting with 488 and 640 nm solid-state lasers, these flow
from the use of high-speed cell sorters (Ibrahim and cytometers analyze up to six parameters (FSC, SSC
van den Engh 2003). and four fluorescences). For high throughtput acqui-
sition, Accuri C6 can be associated with HyperCyt
(IntelliCytTM Corporation). The CSampler is
Recent commercial instrumentation equipped with a 48 or 96-well plate or 24 tubes
capacity loader. Analysis rate is up to 10,000 events/s.
The EPICSAltraTM (Beckman Coulter), the FAC- The user can change bandpass and dichroic filters.
ScanTM and FACSVantageTM SE (Becton–Dickin- Sample acquisition (24 bits—7 decades digitalization)
son), the PASIII (Partec), the Galaxy Pro (Dako), and and data analysis are performed using CFlow
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116 Cytotechnology (2012) 64:109–130
software (PC), with CFlow zoom function. Post- car battery (12 V DC power). All cytometers deter-
acquisition compensations are allowed (FCS 3.1). mine true volumetric absolute counting. Optical filters
are interchangeable. Optional RobbyWellTM permits
AttuneTM (Applied BiosystemsTM by Life analysis of 96-well plates. Sample acquisition (16
TechnologiesTM) bits—4 decades) and data analysis with CyFlow
Cube 6 and Cube 8 are performed by CyViewTM
Since 2010, Life technologiesTM Corporation proposes software (PC). Sample acquisition (16 bits) and data
an acoustic focusing analyzer that uses sound waves to analysis with CyFlow ML and CyFlow SL are
precisely control cell movement (Goddard and Kadu- performed by FloMax software (PC). Post-acquisi-
chak 2005; Goddard et al. 2006, 2007). AttuneTM tion compensations are possible (FCS 3.0).
equipped with a 50 mW 405 nm and 20 mW 488 nm
solid-state lasers, analyzes FSC, SSC and six colors. The CytoSense, CytoSub, CytoBuoy (CytoBuoy b.v.)
user can change bandpass and dichroic filters. Sample
rate is adjustable from 25 to 1,000 lL/min and electron- CytoBuoy b.v. is a dutch company which manufac-
ics have the capability to run up to 20,000 events/s. tures particle analysis instruments and software for
Sample acquisition (six decades) and data analyses are mainly aquatic and marine science. Since 2001, the
performed by AttuneTM Cytometric Software (PC). Post- bench top scanning flow cytometer CytoSense is
acquisition compensations are allowed (FCS 3.0). designed for pico- , nano- and micro-plankton studies.
It combines classical flow cytometry data with silico-
CyAnTM ADP (Beckman Coulter) images of the measured particles and targeted video
imaging. This instrument is equipped with 2 lasers
After the acquisition of the Dako Cytomation society, (460 or 488 or 532 or 561 nm and 445 or 635 or 640 or
Beckman Coulter commercializes CyAnTM ADP, a 660 nm) and up to 10 detectors. The CytoSense works
high-speed analyzer (70,000 events/s) equipped with with a hydrodynamic sheath fluid injection system
three solid-state lasers (405, 488 and 642 nm) to with external and recirculating mode, and an auto-
analyze nine fluorescence colors simultaneously. For adaptive speed controlled from 0.2 to 20 lL/s. The
high throughtput screening, CyAnTM ADP integrated CytoSense may be extended with a video imaging-in-
to the HyperCyt plate loader (IntelliCytTM Corpora- flow at rates of up to 1,000 scans per second. The
tion) enables 384-well plates. Fast sample acquisition CytoSub module ‘‘Shallow’’ allows a submerged use
(12 bits—4 decades) and high capacity data analyses in shallow waters (depth of 20 meter max.) and
(up to one hundred million event data files) are CytoSub module ‘‘Deep’’ transforms the CytoSense
performed by Summit software (PC). Post-acquisi- flow cytometer into a CytoSub instrument for
tion compensations are allowed (FCS 3.0). submerged operation down to 200 meters. The Buoy
module transforms the CytoSense into a CytoBuoy
CyFlow Cube 6/Cube 8, CyFlow ML, CyFlow SL analyzer for moored operation, with 8 solar panels
(Partec) systems with rechargeable batteries and flashlight,
telemetry and Argos transponder. CytoUSB software
Pioneer in flow cytometry since 1968, the German is used for instrument operation and storage of
society Partec has since 2000 developed flexible and measured data files (8 bits—3.5 decades). CytoClus
modular CyFlowcytometers. The newest CyFlow software is used for data analysis.
Cube 6/Cube 8 are, respectively, equipped with two or
three lasers (large choice available), analyze FSC, EclipseTM (Sony)
SSC and up to four or six fluorescence colors
simultaneously. Since acquisition of iCyt in February 2010, Sony
With five light sources, CyFlow ML detects up to commercializes the EclipseTM flow cytometer.
sixteen optical parameters: 2 9 FSC, SSC and thir- Equipped with four fiber coupled diode solid state or
teen colors simultaneously. With a 50 mW 488 nm DPPS lasers (30 mW 405, 25 mW 488, 30 mW 561
blue solid-state laser, CyFlow SL analyzes FSC, SSC and 30 mW 642 nm) the EclipseTM simultaneously
and three fluorescent colors. CyFlow SL can run on a analyzes FSC, SSC and five fluorescence colors up to
123
Cytotechnology (2012) 64:109–130 117
an analysis rate of 5,000 events/s. An autoloader and data analysis are performed using InCyte
permits the analysis of 96- to 384-well plate and tubes acquisition and analysis software (PC) (equivalent
(40-tube rack). Plate or rack shake at three intensity 18 bits—4 decades). Post-acquisition compensations
levels (5–30 s) or aspirate/dispense at three different are allowed (FCS 3.0). In Guava easyCyteTM 5HT,
speeds (1–12 cycles) are possible. Sample rate is 6HT, 6HT/2L and 8HT flow cytometers, built-in
adjustable from 5 to 200 lL/min. The EclipseTM sample tray provides walk-away automation for a
analyzer measures accurate sizing by electronic vol- 96-well microplate and ten microtubes.
ume measurement and accurate absolute cell count
with high precision syringe delivery mechanism. MACSQuant, MACSQuant VYB (Miltenyi Biotec)
Sample acquisition (24 bits—6 logarithmic decades)
and data analysis are performed by iFlow software With a tactile-screen and fluid level light warnings,
(PC). Post-acquisition compensations are possible MACSQuant is an analyzer equipped with three air-
(FCS 3.0). cooled lasers (405, 488 and 635 nm) to detect FSC,
SSC and eight colors simultaneously. MACSQuant
FACSVerseTM (BD Biosciences) VYB is the newest analyzer (2011) equipped with three
lasers (Violet, Yellow, Blue), respectively, 405, 561
FACSVerseTM is the latest machine from BD Biosci- and 488 nm to detect eight colors. Analysis rates are up
ences, commercialized since June 2011. This analyzer to 10,000 events/s. 5, 15 and 50 mL tubes or 96-well
combines a fibered three lasers excitation system, 405, plate can be maintained at cooling temperature. These
488 and 640 nm to a ten parameters collection optic analyzers determine volumetric or absolute cell count-
(FSC, SSC and eight fluorescences). A new optical ing. These systems incorporate a barcode reader to
geometry design in heptagon equips this analyzer with identify MACS reagents and their associated staining
automatic filter detection. It proposes a universal protocols. Emission filters are unchangeable. MACS-
loader option that can load 96- or 384-well plates or QuantifyTM Software (PC) performs sample acquisition
various kinds of tubes with possibility of 30-tube rack (16 bits—5 decades digitalization) and data analysis.
with 1D or 2D bar-code reader, or 40-tube rack Post-acquisition compensations are allowed (FCS 3.0).
without bar-code reader. Plate or rack shakes before
analysis. Sample rate is adjustable at 12, 45-55, 60 and Cell analyzers
120 lL/min. BD FACSuiteTM Software (PC) per-
forms sample acquisition (18 bits—5 decades) and CELL LAB QUANTATM SC/SC MPL (Beckman
data analysis. Post-acquisition compensations are Coulter)
allowed (FCS 3.0). This cytometer offers bi-exponen-
tial scales. The CELL LAB QUANTATM SC or SC Multi-
Platform Loader (MPL) flow cytometer is equipped
Guava easyCyteTM 5-5HT/6-6HT/6/2L-6HT/2L/8- with a 20 mW 488 nm air-cooled and mercury arc
8HT (Merck MilliporeTM) lamp (100 W) for UV-illumination sources (366, 405
and 435 nm). This flow cytometer simultaneously
After the acquisition of Guava Technologies society, measures electronic volume by impedance variation,
Merck MilliporeTM commercializes microcapillary SSC and three fluorescence colors. The MPL version
bench top analyzers. Guava easyCyteTM 5-5HT and permits use of microtubes and 24- , 96- and 384-well
6-6HT permits, with one blue laser, to analyze FSC, plates. Emission filters are unchangeable. Cell Lab
SSC and, respectively, up to three and four fluores- Quanta Collection Software (PC) performs sample
cence colors. Guava easyCyteTM 6/2L-6HT/2L and acquisition (4 decades) and data analysis. Post-acqui-
Guava easyCyteTM 8-8HT flow cytometers are sition compensations are not allowed (FCS 2.0).
equipped with dual 75 mW blue and 40 mW red
lasers (488 and 640 nm) and permit to analysis of EPICS XLTM/XL-MCLTM (Beckman Coulter)
simultaneous FSC, SSC and, respectively, four (3/1)
and six (4/2) fluorescence colors. Sample is mixed Beckman Coulter manufactures since 1993 the
using an automated paddle mixer. Sample acquisition COULTER EPICS XL/XL-MCL flow cytometer.
123
118 Cytotechnology (2012) 64:109–130
The XL system features the capability to analyze FSC, analysis by CXP Analysis software (PC). Post-acqui-
SSC and four fluorescence colors with a single 15 mW sition compensations are allowed (FCS 3.0) for both.
488 nm air-cooled laser. The XL-MCL system offers
walk-away sample handling with the Multi Carousel GalliosTM/NaviosTM (Beckman Coulter)
Loader (MCL) (32 tubes capacity). This device
incorporates positive bar-code identification and vor- Recently, Beckman coulter proposed new cytometers,
tex mixing prior to sample aspiration. The optical GalliosTM and NaviosTM, respectively, developed for
emission filters are fixed. Sample acquisition (10 clinical and research applications. These cytometers
bits—4 decades) and data analysis are performed by feature three solid-state fibered lasers (405, 488 and
XL SYSTEM IITM software or EXPO32TM ADC 638 nm) for FSC, SSC and ten fluorescence colors
software (PC). Post-acquisition compensation is not analysis. These systems offer walk-away sample
allowed (FCS 2.0). handling with the Multi Carousel Loader (MCL) (32
tubes capacity), positive bar-code identification and
incorporated vortex mixing prior to sample aspiration.
FACSCantoTM, FACSCantoTM II (BD Biosciences)
The user can change bandpass as well as dichroic
filters. NaviosTM Software (PC) performs sample
FACSCantoTM evolution permits obtention of an
acquisition (20 bits—5 decades) at 25,000 events/s
analyzer equipped with a 30 mW 405 nm solid-state
and data analysis. Post-acquisition compensations are
laser, a 20 mW 488 nm air-cooled laser and a 17 mW
allowed (FCS 3.0).
633 nm laser delivered by optic fibers to analyze eight
fluorescence colors simultaneously. An automatic
LSRTM, LSRTM II, LSRFortessaTM, LSRFortessaTM
fluidic pump maintains fluidic stability. The optical
SORP (BD Biosciences)
system geometry is composed of two trigons (violet
and red lasers) and one octagon (blue laser). The user
Since 1999, the first version of LSRTM was profoundly
can change both bandpass and dichroic filters.
changed to obtain LSRFortessaTM and LSRFortessaTM
FACSCantoTM II offers walk-away sample handling
SORP in 2010. These new versions are equipped with
with the carousel loader (40 tubes capacity) or high
five lasers (upgraded to seven with a large choice from
throughput sampler 96- or 384-well plate loader.
16 different wavelengths and a wide range of powers)
Sample rate is adjustable at 12, 60 and 120 lL/min.
delivered by fiber optics to detect FSC, SSC and up to
BD FACSDivaTM Software (PC) performs sample
eighteen colors simultaneously (20 PMT max.). User
acquisition (18 bits—5 decades) and data analysis.
chooses laser wavelengths in the SORP version. The
Post-acquisition compensations are allowed (FCS
optical system has transmission pathways in trigon and
3.0). These cytometers offer bi-exponential scales.
octagon geometry. In addition, user can change both
bandpass and dichroic filters. For screening assays,
FC500 MCL/MPL (Beckman Coulter) LSRTM II and LSRFortessaTM offer walk-away sam-
ple handling with high throughput sampler 96/384-
FC500 MCL flow cytometer features the capability to well microtiter plate. Sample rate is adjustable at 12,
analyze FSC (flied stop 1–8 or 1–19), SSC and five 60 and 120 lL/min. BD FACSDivaTM Software (PC)
fluorescence colors with 20 mW 488 nm and 20 mW performs sample acquisition (18 bits—5 decades) and
635 nm air-cooled lasers. FC500 MCL system offers data analysis. Post-acquisition compensations are
walk-away sample handling with the Multi Carousel allowed (FCS 3.0). These cytometers offer bi-expo-
Loader (MCL) (32 tubes capacity), positive bar-code nential scales.
identification and incorporated vortex mixing prior to
sample aspiration. Sample rate is adjustable at 15, 30 S1000, S1000Ex, SE500 (Stratedigm)
and 60 lL/min. Sample acquisition (20 bits—4
decades) is performed by CXP acquisition Software. The S1000 and S1000Ex analyzers with four lasers
FC500 MPL version offers Multi-well Plate Loader analyze FSC (optional PMT detector), SSC and eight
(MPL). Sample acquisition (20 bits—4 decades) is or fourteen fluorescence colors respectively. S1000
performed by MXP acquisition software and data and S1000Ex permit a large choice of solid-state lasers
123
Cytotechnology (2012) 64:109–130 119
(372, 405, 488, 532, 561 and 640 nm). SE500 flow from 0.2 to 20 lL/s. The CytoSense Sorter is equipped
cytometer, with dual 488 and 640 nm solid-state lasers with a piezo operated fluid switch sorter working at
(40 mW), analyzes FSC (optional PMT detector), SSC rates of up to 100 sorts per second. CytoUSB software
and four fluorescence colors. Analysis rate is 10,000 is used for instrument operation and storage of
events/s for all cytometers. The CellCapTure Software measured data files (8 bits—3.5 decades). CytoClus
(PC) performs sample acquisition and data analysis. software is used for data analysis.
Post-acquisition compensations are allowed (FCS 3.0)
(Table 1). FACSCaliburTM (BD Biosciences)
Mechanical cell sorters Manufactured since 1994, FACSCaliburTM uses a

488 nm air-cooled argon gas laser (15 mW) and a
CyFlow Cube Sorter (Partec) smaller 635 nm diode laser for the six parameters
detection (FSC, SSC, 3/1 fluorescences). An auto-
With two lasers, the latest CyFlow Cube Sorter matic fluidic pump maintains fluidic stability. Emis-
analyzes FSC, SSC and up to three fluorescence colors sion filters are unchangeable. FACSCaliburTM offers
simultaneously and determines true volumetric abso- walk-away sample handling with the carrousel loader
lute counting. Optical filters are interchangeable. (40 tubes capacity) or high throughput sampler 96- and
Optional RobbyWellTM permits the analysis of 384-well plate loader. FACSCaliburTM can analyze
96-well plates. Sample acquisition (16 bits—4 dec- cell suspensions at the rate of over 1,000 events per
ades) and data analysis are performed by CyViewTM second at three different speeds (12, 35 or 60 lL/min).
software (PC). Post-acquisition compensations are Unlike the traditional cell analyzer, FACSCaliburTM is
possible (FCS 3.0). equipped (in option) with a sorting module. It can sort
cell suspensions at a rate of 300 cells per second and
CyFlow space (Partec) collect them in a 50 mL tube. Sample acquisition (10
bits digitalization—4 decades) and data analysis are
CyFlow space analyzer with an open optical system performed by CellQuestTM Pro Software (Macintosh).
and 3 lasers (375, 407, 488, 561 and 638 nm Post-acquisition compensation is not allowed (FCS
available), allows simultaneous analyzing of FSC, 2.0).
SSC and seven fluorescence colors. Built-in CyFlow
Sorter modules for diamond piezo-based, closed, non- Deflection cell sorters
destructive and non-hazardous cell and particle sorting
are optionally available. Optional RobbyWellTM BioSorterTM, CopasTMBIOSORT, CopasTMSELECT,
allows analysis of 96-well plates. Sample acquisition CopasTMPLUS, CopasTMXL (Union Biometrica Inc)
(16 bits—4 decades) and data analysis are performed
by FloMax software (PC). Post-acquisition compen- The BioSorter is a continuous flow system capable of
sation is possible (FCS 3.0). analyzing, sorting objects ranging in size from 10 to
1,500 lm via different interchangeable fluidic and
CytoSense Sorter (CytoBuoy b.v.) optic core assemblies (FOCA) optimized for a partic-
ular size range (250, 500, 1,000, 2,000 lm). An axial
The CytoSense Sorter is a bench top scanning light-loss detector measures relative axial size and
mechanical sorter designed for pico-, nano-, and optical density is determined by the total integrated
micro-plankton studies, combining classical flow signal of the light blocked. Four excitations lasers
cytometry data with silico-images of the measured max. (405 or 445 or 460 and 488, 561, 640 or 660 nm
particles and targeted video imaging. This instrument are available); the fluorescence intensity can be
is equipped with 2 lasers (460 or 488 or 532 or 561 nm simultaneously measured at three different wave-
and 445 or 635 or 640 or 660 nm) and up to 10 lengths. Samples are introduced via a 50 mL conical
detectors. The CytoSense works with a hydrodynamic tube (one and two liter sample cups are optional) with
sheath fluid injection system with external and recir- suspended stirrer. An X–Y–Z stage allows dispensing
culating mode. Auto-adaptative speed is controlled into 24- , 48- or 96-well microtiter plates, tubes and
123
Table 1 Analyzers comparison
120
Holder Fluidic
123
Carousel Plate loader Tubes (mL) Agitation (A)/ Focusing type Flow
Temperature cell type
control (T)/Barcode
reader (B)
Benchtop A50 Micro/Universal No No 0.5, 1, 1.5, 2, 5 No Hydrodynamic Quartz

Accuri C6 No No 0.5, 1, 1.5, 2, 5 No Hydrodynamic Quartz
AccuriCSampler 24 48–96 0.5, 1, 1.5, 2, 5 No Hydrodynamic Quartz
Attune No No 5 No Hydrodynamic ? acoustic Quartz
Auto40 No No 5 No Hydrodynamic Quartz
CyAn ADP No No 5 No Hydrodynamic Quartz
CyFlow Cube 8 Yes 96 5 No Hydrodynamic Quartz
CyFlow ML No 96 5 No Hydrodynamic Quartz
CyFlow SL No No 5 No Hydrodynamic Quartz
CytoSense, CytoSub, CytoBuoy No No NI No Hydrodynamic NI
Eclipse 40 96–384 5 A Hydrodynamic Quartz
FACSVerse 30 or 40 96–384 0.5, 1.5, 5, 15, 50 A/B Hydrodynamic Acier
Guava easyCyte 8HT 10 96 1.2, 1.5 A Microcapillary Quartz
MACSQuant/MACSQuant VYB 24 96 5, 15, 50 A/T/B Hydrodynamic Quartz
Analyzers Cell Lab Quanta SC/SC MPL No 24–384 5 No Hydrodynamic Quartz
EPICS XL/XL-MCL 32 No 5 A/B Hydrodynamic Quartz
FACSCanto II 40 96–384 5 No Hydrodynamic Quartz
FC500 MCL/MPL 32 96 5 A/B Hydrodynamic Quartz
Gallios/Navios 32 No 5 A/B Hydrodynamic Quartz
LSRFortessa, LSRFortessa SORP No 96–384 5 No Hydrodynamic Quartz
S1000, S1000Ex No No NI No Hydrodynamic Quartz
SE500 No No NI No Hydrodynamic Quartz
Table 1 continued
Optic Electronic
Total Excitation Total PMT FSC Alignment fixed Excitation Number Number Acquisition Flow
laser lasers fluorescence improvment (F) or manuel light of bits of rate - event/s cytometry
available (M) decades (according to standart
manufacturers) (FCS)
Benchtop A50 Micro/ 4 375, 405, 488, 532, 4 Diode F Direct 16 4.5 20.000 2.0
Universal 635
Accuri C6 2 488, 640 4 Diode F Direct 24 7 10.000 3.1
AccuriCSampler 2 488, 640 4 Diode F Direct 24 7 10.000 3.1

Attune 2 405, 488 6 Diode F Direct NI 6 20.000 3.0
Auto40 1 488 or 532 3 Diode F Direct 16 4.5 20.000 2.0
CyAn ADP 3 405, 488, 642 9 Diode F Direct 12 4 70.000 3.0
CyFlow Cube 8 4 355, 375, 407, 488, 6 Diode F NI 16 4 10.000 3.0
532, 561, 594, 638,
785, 365-UV-LED
CyFlow ML 5 407, 488, 532, 561, 13 Diode F Direct 16 4 10.000 3.0
638, 660
CyFlow SL 1 488 3 Diode F Direct 16 4 10.000 3.0
CytoSense, 2 460, 488, 532, 561, 8 Diode F NI 8 3.5 1.000 NI
CytoSub, 445, 635, 640, 660
CytoBuoy
Eclipse 4 405, 488, 561, 642 5 Diode F Optic 24 6 5.000 3.0
fiber
FACSVerse 3 405, 488, 640 8 Diode F Optic 18 5 10.000 3.0
fiber
Guava easyCyte 2 488, 640 6 Diode F Direct 18 4 1.000 3.0
8HT
MACSQuant/ 3 405, 488, 561, 635 8 Diode F Direct 16 5 10.000 3.0
MACSQuant
VYB
121
123
Table 1 continued
122
Optic Electronic
123
Total Excitation Total PMT FSC Alignment fixed Excitation Number Number Acquisition Flow
laser lasers fluorescence improvment (F) or manuel light of bits of rate - event/s cytometry
available (M) decades (according to standart
manufacturers) (FCS)
Analyzers Cell Lab 1 ? arc 488 ? mercury arc 3 Diode F Direct NI 4 NI 2.0
Quanta SC/ lamp lamp (366, 405,
SC MPL 435)
EPICS XL/ 1 488 4 Diode F Direct 10 4 10.000 2.0
XL-MCL
FACSCanto II 3 405, 488, 633 8 Diode F Optic fiber 18 5 10.000 3.0
FC500 MCL/ 2 488, 635 5 Diode/ F Direct 20 4 10.000 3.0
MPL 1–18
Gallios/Navios 3 405, 488, 561, 638 10 W2 F Optic fiber 20 5 25.000 3.0
LSRFortessa, 7 355, 375, 407, 488, 18 PMT F Optic fiber 18 5 40.000 3.0
LSRFortessa 532, 561, 594, (UV
SORP 638, 785 excepted)
S1000, 4 372, 405, 488, 532, 14 PMT F Direct NI NI 10.000 3.0
S1000Ex 561, 640
SE500 2 488, 640 4 PMT F Direct NI NI 10.000 3.0
NI no indication
Cytotechnology (2012) 64:109–130 123
bulk receptacles. The z-direction allows adjustment Management Option (evacuates the sort collection
for different height plates and tubes. The BioSorterTM chamber and traps aerosolized particles during sort-
instrument is controlled by FlowPilot-ProTM software ing), Cytometer Setup and Tracking (this feature
(PC) with real-time data acquisition (16 bits) via on- establishes baseline settings), Sweet Spot Technology
board customized electronics. (Automated clog detection and sort tube protection
CopasTMBIOSORT, CopasTMSELECT, CopasTM system) or Easy aseptic setup and cleaning.
PLUS, CopasTMXL are flow systems capable of BD FACSDivaTM Software (PC) performs sample
analyzing, sorting and dispensing objects ranging in acquisition (18 bits—5 decades) and data analysis.
size from 20 to 1,500 lm via different engineered Post-acquisition compensations are allowed (FCS
fluidic paths and optimized quartz flow cell sizes 3.0). This cell sorter offers bi-exponential scales.
(FOCA) for a specific object size range to achieve the
highest accuracy and sensitivity possible. The BIO-
SORT, SELECT, PLUS and XL version are respec- InfluxTM (BD Biosciences)
tively equipped with 250, 500, 1,000 and 2,000 lm
flow cell. Multi-laser excitations (3 lasers max.) This cell sorter was created by Cytopeia in 2007, and
among 325, 375, 405, 488/514, 561, 635, 640 or take over by BD Biosciences in 2008. This high-speed
670 nm are available and permit to detect three sorter (200,000 events/s) can include a ten laser paths
fluorescence colors. All systems are equipped with and seven pinholes optical wavelength lasers (355,
an X–Y stage, which allows dispensing into 24- , 48- 405, 445, 457, 488, 515, 532, 561, 594, 640 and
and 96-well microtiter plates. Only the CopasTM 785 nm available) and requires a daily optical align-
BIOSORT permits sorting into 384-well plate. All ment. A pinhole camera view ensures that the
CopasTM systems are controlled by COPASTM soft- fluorescence is in alignment with the detectors. It
ware (PC) for operation and analysis (16 bits). can measure 24 parameters simultaneously and cal-
culates 24 9 24 compensations. In order to create
droplets for sorting, the nozzle assembly is coupling to
FACSAriaTM III (BD Biosciences) an acoustical system. A bubble detector in the sample
line detects air bubbles from the sample tube. Any
The first BD FACSAriaTM has been available since parameter can be used as the threshold but only from
2003. The third version was commercialized in 2010. the primary laser; lasers and detectors can be switched
This high-speed cell sorter (70,000 events/s) can to change laser sequence. Peak heights are measured
support six fixed alignment air cooled solid state by default; area and width measurements are available
lasers, with a 375, 405, 445, 488, 561 and 633 nm for a maximum of 8 parameters. There is a 16-bits
wavelength, and four spatially separated beam spots in analog-to-digital conversion. An exchangeable detec-
a quartz-cuvette. Emitted light is delivered by fiber tor module allowing for the measurement of small
optics to twenty detectors simultaneously in a collec- particles can be equipped with polarization sensitive
tion optics set up in patented octagon- and trigon- detector. Pressure can be adjusted from 1 to 90 psi and
shaped pathways. Threshold and height, area, and nozzles are available in five sizes: 70, 86, 100, 140 and
width measurements are available for any parameter. 200 microns. A six-way sorting is possible; tube
Pressure can be adjusted from 5 to 75 psi and holders include sizes from micro tubes to 50 mL tubes
nozzles are available in four sizes: 70, 85, 100 and 130 and collection is also possible on 6- , 24- , 48- , 96- and
microns. 35- and 50-micron sample line filters can be 384-well plates or on slide. Sample and sort collection
added. Four-way sorting is possible; tube holders tubes can be cooled or heated by an optional circu-
include sizes from micro tubes to 15 mL tubes and lating water bath. There are several automatic menus,
collection is also possible on a 384-well plate or on such as BD FACSTM Accudrop (to assist the user in
slide. Sample and sort collection tubes can be the best drop delay value determination) and BDTM
maintained at a cooling or heating temperature. There Aerosol Management Option (to evacuate the sort
are several automatic menus such as BD FACSTM collection chamber and trap aerosolized particles
Accudrop (this technology assists the user in deter- during sorting). Post-acquisition compensations are
mining the best drop delay value), BDTM Aerosol allowed (FCS 3.0).
123
124 Cytotechnology (2012) 64:109–130
JSANTM (Bay Bioscience Co.) custom Biosafety Level II cabinet option. Summit
software (PC) performs sample acquisition (32 bits—5
Optical detection fibers of the Japan-made Sorter decades) and data analysis. It can calculate 18 9 18
ANalyzer (JSAN) corresponding to each excitation digital compensation matrix. Post-acquisition com-
laser are closely fixed at the flow cell. Four lasers (375 pensations are allowed (FCS 3.0).
or 405, 488, 534 or 561 and 638 nm) on the grade level
with flow cell can be mounted with the spatially
MoFlo XDPTM (Beckman Coulter)
separated beams system. It can acquire any ten
parameters simultaneously with eight fluorescences
This high-speed cell sorter (70,000 events/s) is com-
and generate 8 9 8 digital matrix fluorescence com-
patible with an array of laser options (water cooled,
pensations. Pressure can be adjusted to 30 psi. The
free-space, fiber coupled). It can mount up to 6 lasers
OptiDrop function permits monitoring the droplet
(3 pinholes) and 12 fluorescences. It is a 32-bits high-
parameters and stop sort in case of abnormality; the
resolution 5-decades multi-channel digital system
OptiDelay automatically calculates drop delay. The
under the control of the Summit software, which
sorter provides a 2-way sorting at 20,000 events/s
allows an 18 9 18 auto-compensation matrix. Sheath
speed. CloneMate option device enables direct sorting
pressure range goes from 4 to 100 psi and there are
of a single cell or more into each well in a multiwell
eight nozzle sizes (50, 70, 80, 90, 100, 120, 150 and
plate. In option, user can choose the JSAN body color
200 lm). The IntelliSort II function is a beadless drop
among black (default), blue, green and red color.
delay determination and monitoring function. The
AppSAN software (PC) performs sample acquisition
sorter provides a 4-way sorting for multiple popula-
(20 bits—6 decades) and data analysis. Post-acquisi-
tions, with independent sort mode capability for each
tion compensation is not allowed (FCS 2.0).
tube. There are a variety of collection devices with
temperature control for all sorts of outputs: 0.5, 1.0,
MoFlo AstriosTM (Beckman Coulter)
1.5, 5.0, 7.0, 15 and 50 mL tubes are accepted. The
Cyclone module could collect into multi-well plates
This is a high-speed cell sorter (70,000 events/s)
up to 1,536 wells and on slides. The aerosol evacuation
commercialized in 2010. It is controlled by a touch
system (AES) removes aerosols from the sort cham-
screen control panel for instrument set up and sort
ber. A Biosafety cabinet option exists.
monitoring. It can combine seven lasers (355, 405,
488, 532, 561, 592 and 642 nm) for seven pinholes.
Emission light is transmitted by optical fiber to thirty Reflection (Sony)
PMT dispatched in 49 positions. Sheath pressure range
goes from 4 to 100 psi and there are eight nozzle sizes After the acquisition of iCyt in February 2010, Sony
(50, 70, 80, 90, 100, 120, 150 and 200 lm). The commercializes the Reflection cell-sorter. This high-
IntelliSort function permits to automatically calculate speed parallel sorting (70,000 events/s) contains one to
optimal droplet break off and to monitor it. The sort four Highly Automated Parallel Sort (HAPS) modules
rescue function protects sample and sorted cells in per instrument that may be operated remotely by
case of stop sorting. A six-way sorting can be done multiple users. It can mount up to 7 lasers (355, 405,
with mixed mode sorting: each sort stream is capable 488, 532, 561, 592 and 640 nm) and count up to 24
of having its own sort mode (Enrich, Purify and fluorescent detectors. Pressure can be adjusted from 5
Single) programmed. There is a variety of collection to 100 psi and 50 to 200 lm nozzles are available.
devices with temperature control for all sort outputs— Each module can divided sort into 4 streams and cells
1.5, 5, 15 and 50 mL tubes are accepted. The of interest are collected into 0.6–50 mL tubes. These
Cyclone II module can collect into multi-well plates modules can be mounted inside a biological safety
(from 6- to 1,536-wells) and disposes up to 1,536 spots cabinet to protect samples, products, and operators
on a slide. The sorter is equipped with an Aerosol from biohazard contamination. Wide forward scatter
Evacuation System (AES) filters particles from all collection angle allows FSC detection increasing.
chambers where aerosols have the potential of being Sample acquisition (14 bits) and data analysis are
generated. Additionally, the MoFlo AstriosTM has a performed by WinList software.
123
Cytotechnology (2012) 64:109–130 125
SynergyTM BTS/BSC (Sony) High-throughput acquisition is achieved by using a

F-theta lens with high-speed galvanometer mirrors
The SynergyTM instrument platform supports two high that scan a large field (3.59/NA 0.25 magnification)
performance cell sorter modules, two automated cell on 6–1,536-well microplates, T-25 and T-75 flasks.
analyzer modules, or a sorter/analyzer combination on a The CCD detector (2,052 9 2,052 pixels) has a
single bench-top frame. Configured with up to eight resolution of 1 lm/pixel. The CeligoTM software
lasers (among 375, 405, 445, 488, 491, 532, 561, 594 performs both image acquisition and analysis.
and 642 nm) focused on five spatially separated inter- The LEAPTM (Laser-Enabled Analysis and Pro-
rogation points, the SynergyTM can acquire up to 24 cessing System) is a microplate-based cytometry
simultaneous parameters at up to 100,000 events/s. This system for non-invasive in situ process, allowing the
instrument is configured with up to 32 detectors per use of various adherent and non-adherent cell types,
module: up to 26 user-configurable fluorescent detectors which is capable of sorting by killing unexpected cells
(PMT) and 6 scatter diodes. With the optional Sort (1,000 targets/s) with a UV-laser (355 nm). Diameter
deposition System (SDS), this high-speed cell sorter of the laser spot is include between 10 and 50 lm.
(70,000 events/s) supports sorting into popular plate Multiple magnifications (39, 59, 109 and 209/0.25
formats and 4-way sorting into most popular collection NA) are possible by using a F-theta lens on specific
tube formats. Nozzle sizes include 70, 85, 100 and 130 plate formats C-LectTM 6- , 12- , 96- and 384-well
microns. Sample acquisition and data analysis are plates. The LEAPTM uses cooled emCCD detector
performed by WinList acquisition and analysis software (1,024 9 1,024 pixels). The LEAPTM software per-
respectively. The iCyt SynergyTM BSC can be seam- forms both image acquisition and analysis.
lessly integrated with a Baker SterilGARD III
Advance Biological Safety Cabinet (BSC) (Table 2). Imaging flow cytometry (Fluid imaging technologies;
Amnis Corporation)
Perspectives
Manufactured since 1999, the FlowCAM (Fluid
New technologies in the cytometry field have been Imaging Technologies) was the first bench top digital
developed to extend possibilities. imaging analyzer for particle or cell measurements in
solution, originally developed for the oceanographic
Microfluidic cytometry (On-chip Biotechnologies) research community and now used for other applica-
(Cho et al. 2010a) tions. The FlowCAM acquires high resolution
microscopic color or monochrome images of cells
FISHMAN-R is a Japanese analyzer that enables flow (29, 49, 109 and 209 magnification) with a size
cytometry on a microfluidics chip. With dual lasers range from 2 lm to 2 mm at a rate of up to 10,000
(473 and 640 nm) it analyzes FSC, SSC and up to four images/min and determines two fluorescences (488 or
colors and could detect particles size from 0.5 to 532 nm laser) and up to 23 morphology parameters for
20 lm (bacteria and cells). Acquisition rate is 3,000 each particle. Portable and Submersible FlowCAM
events/s. The sample is not diluted with sheath fluid versions exist. The FlowCAM V-1000 is a small
after analysis, so that it is possible to re-use it for other analyzer with two magnifications (29–49 or 49–109
experiments afterwards. Sample acquisition and data or 10–209). VisualSpreadsheet software performs
analysis are performed using the same software (PC). acquisition and analysis for all analyzers.
The ImageStreamX (Amnis Corporation) is a mul-
Adherent cell cytometry (Cyntellect, Inc.) tispectral imaging flow cytometer that combines the
strength of flow cytometry and fluorescence micros-
The CeligoTM is the first adherent cell cytometer, copy in a single platform; it can digitally image
which analyzes cells in their environment with min- millions of cells directly in flow. This technology
imal sample manipulation. With three combinations of enables the identification of single cell based on
excitation LED (377, 483 and 531 nm) and block distribution of fluorescence markers and morphology
filters, the CeligoTM analyzes adherent or non-adher- of the cell with brightfield and darkfield (SSC) at a rate
ent cells in brightfield and three fluorescence colors. of above 1,000 cells per second. The system combines
123
Table 2 Cell sorters comparison
126
Holder Fluidic Optic
Carousel Plate Tubes Agitation Pressure Flow Nozzle Total Excitation lasers Total PMT FSC Alignment Excitation
123
loader (mL) (A)/ (psi) cell diameter laser available fluorescence improvment fixed light
Temperature type (lm) (F) or
control (T)/ manuel
Barcode (M)
reader (B)
Mechanical CyFlow Yes 96 5 No No Quartz No 2 355, 375, 407, 3 Diode F NI

cell Cube Sorter 488, 532, 561,
sorters 594, 638, 785,
365-UV-LED
CyFlowspace No 96 5 NI No Quartz No 5 405, 488, 532, 7 NI F Direct
561, 642, 660
CytoSense No No NI No No NI No 2 460, 488, 532, 8 Diode F NI
Sorter 561, 445, 635,
640, 660
FACSCalibur 40 96–384 5 B No Quartz No 2 488, 635 4 Diode F Direct
Deflection BioSorter No No 50, A NI Quartz 250, 4 405 or 445 or 3 Diode NI NI
cell 1,000, 500, 460, 488, 561,
sorters 2,000 1,000, 640 or 660
2,000
Copas No 2 9 96 50 A NI Quartz 250 or 3 325, 375, 405, 3 Diode NI NI
BIOSORT/ 500 or 488/514, 561,
SELECT/ 1,000 635, 640 or
PLUS/XL or 670
2,000
FACSAria III No No 0.5, 2, 5, A/T 5–75 Quartz 70, 85, 6 375, 405, 445, 18 Diode F Optic
15 100, 488, 561, 633 fiber
130
Influx No No 5 A/T 1–90 Jet in 70, 86, 7 (\ 10) 355, 405, 445, 22 Diode M Direct
air 100, 457, 488, 515,
140, 532, 561, 594,
200 640, 785
JSAN No No 5 NI 30 Jet in NI 4 375 or 405, 488, 8 Diode NI Direct
air 534 or 561,
638
Table 2 continued
Holder Fluidic Optic
Carousel Plate Tubes Agitation Pressure Flow Nozzle Total Excitation lasers Total PMT FSC Alignment Excitation
loader (mL) (A)/ (psi) cell diameter laser available fluorescence improvment fixed light
Temperature type (lm) (F) or
control (T)/ manuel
Barcode (M)
reader (B)
MoFlo No No 0.5, 1.0, A/T 4–100 Jet 50, 70, 100, 7 355, 405, 488, 30 Diode F Optic fiber
Astrios 1.5, 5, in 200 532, 561, 592, (UV
7, 15, air 642 excepted)
50
MoFlo No No 0.5, 1.0, A/T 4–100 Jet 50, 70, 80, 6 355, 405, 488, 12 Diode M Direct and
XDP 1.5, 5, in 90, 100, 532, 561, 592, optic fiber
7, 15, air 120, 150, 640 (UV
50 200 excepted)
Reflection No No NI NI 5–100 Jet 50–200 7 355, 405, 488, 24 Diode NI NI
in 532, 561, 592,
air 640
Synergy No No NI NI NI Jet 70, 85, 100, 8 375, 405, 445, 24 Diode NI NI
BTS/ in 130 488, 491, 532,
BSC air 561, 594, 642
Electronic Collect Biosafety
Number Number Acquisition Sort Flow Automatic Number Plate/ Tubes Temperature Aerosol Under
of bits of decades rate - event/s rate - event/s cytometry drop of sort slide (mL) control (T) containment hood
(according to (according to standart delay (D)/clog ways (AC) (UH)
manufacturers) manufacturers) (FCS) detection (C)
Mechanical CyFlow 16 4 10.000 300 3.0 No 1 No NI NI NI NI

cell sorters Cube Sorter
CyFlowspace 16 4 10.000 300 3.0 No 1 No NI NI NI NI
CytoSense 8 3.5 1.000 100 NI No 1 No 50 No No No
Sorter
FACSCalibur 10 4 10.000 300 2.0 No 1 No 50 No No No
Deflection BioSorter 16 NI NI NI NI NI NI P24–96 50 NI NI NI
cell sorters Copas 16 NI NI 30 2.0 NI NI P24–384 50 NI NI NI
BIOSORT/
SELECT/
PLUS/XL
FACSAria III 18 5 100.000 70.000 3.0 D/C 4 P 6–384/S 1.5, 5, 15 T AC NI
Influx 16 5 200.000 200.000 3.0 D/C 6 P 6–384/S 0.5, 1,5, 5, T AC UH
15, 50
JSAN 20 6 NI 20.000 2.0 D/C 2 P96 5, 15 T NI NI
127
123
128 Cytotechnology (2012) 64:109–130
a brightfield lamp, five excitation lasers (405, 488,

Under
560, 592 and 658 nm) and twelve channels of
(UH)
hood
UH
UH
UH
UH
detection. The full Color Brightfield option provides
a full spectrum brightfield light source that allows the
containment
Biosafety
ImageStreamx system to replicate the RGB brightfield

Aerosol
(AC)
imagery of a microscope. The MultiMag option

AC
AC
AC
AC
provides 209, 409 and 609 magnification. The
Temperature
extended depth of field technology uses a combination

control (T)
of specialized optics and unique image processing

algorithms to project all structures within the cell into
NI
NI
T
one crisp plane of focus.

0.6, 1.5, 5, 15,
0.6, 1.5, 5, 15,

The new FlowSight (May 2011), based on the same
5.0, 7.0, 15,
1,5, 5, 15,50
0.5, 1.0, 1.5,
technology, combines a brightfield lamp, four excita-

tion lasers (80 mW 405, 50 mW 488, 50 mW 561 and
Tubes
(mL)
50
50
50
100 mW 642 nm) and twelve channels of detection to
analyze simultaneously brightfield, darkfield and ten
P 6–1,536/
P 6–1,536/
fluorescence colors per cell at a rate of 2,000 cells/s.

Plate/
slide
P96
P96
S
The FlowSight operates with a pixel size of one

micron (209/0.6NA magnification).
Number
For high throughput analysis, the ImageStreamX

of sort
494
ways
and the FlowSight are equipped with a 96-well plate

6
autosampler. These systems use the IDEAS software

Automatic
delay (D)/
detection
Collect
to quantify.
drop
clog
D/C
D/C
(C)
NI
NI
Mass cytometry (DVS Science)

cytometry
standart
(FCS)
Flow
The CyTOFTM cell analyzer instrument is a high

3.0
3.0
NI
NI
throughput mass cytometer for individual cell analysis

manufacturers)
(according to
rate - event/s
based on a novel elemental mass-spectrometry detec-

tion technology. The instrument detects the stable
70.000
70.000
70.000
70.000
isotopic tags attached to antibodies using labeling kits.

Sort
Because there are many (up to 100) available stable

manufacturers)
isotopes, and the mass spectrometer provides highly

(according to
rate - event/s
Acquisition
precise resolution between detection channels, many

100.000
100.000
100.000
parameters can be measured—and typically without

NI
requiring compensation. Each individual cell is ana-

lyzed for any number of parameters that the user
Number
decades
requires, and the data is downloaded into conventional

NI
NI
of
flow cytometry software for examination.

Electronic
Number
of bits
Perspectives
NI
32
32
14
Reflection
Astrios
Other optical developments appear such as color-

Synergy
MoFlo
MoFlo
BTS/
XDP
BSC
space-time (COST) coding, which is a new way to

Table 2 continued
detect multiple fluorescent wavelengths using a single

NI no indication
photodetector (Cho et al. 2010b). This method of

discriminating multiple fluorescent colors with a
single photomultiplier holds great promise to signif-
icantly reduce the cost and size of the total system. By
123
Cytotechnology (2012) 64:109–130 129
using COST technology, user can differentiate eleven De Rosa SC, Herzenberg LA, Roederer M (2001) 11-color,
commonly used fluorochromes in flow cytometry by 13-parameter flow cytometry: identification of human
naive T cells by phenotype, function, and T-cell receptor
using a single PMT, a major step toward the realiza- diversity. Nat Med 7:245–248. doi:10.1038/84701
tion of compact, cost effective and multicolor flow Dittrich W, Gohde W (1969) Impulse fluorometry of single cells
cytometers for point-of-care applications. in suspension. Z Naturforsch B 24:360–361
For the future, users can expect that flow cytom- Fulwyler MJ (1965) Electronic separation of biological cells by
volume. Science 150:910–911
eters will continue to decrease in size and energy Fulwyler MJ (1977) Hydrodynamic orientation of cells. J His-
consumption and to increase in detection and precision tochem Cytochem 25:781–783
measurements; but, in regard to the healthcare of Goddard G, Kaduchak G (2005) Ultrasonic particle concentra-
users, biosafety is one of the major points which will tion in a line-driven cylindrical tube. J Acoust Soc Am
117:3440–3447
require improvement. Goddard G, Martin JC, Graves SW, Kaduchak G (2006)
Ultrasonic particle-concentration for sheathless focusing of
Conflict of interests The authors state that they have no particles for analysis in a flow cytometer. Cytometry A
conflict of interest. 69:66–74. doi:10.1002/cyto.a.20205
Goddard GR, Sanders CK, Martin JC, Kaduchak G, Graves SW
(2007) Analytical performance of an ultrasonic particle
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Cytotechnology (2012) 64:109–130

Flow cytometry: retrospective, fundamentals and recent

Keywords Flow cytometry Retrospective

J. Picot C. Le Van Kim As flow cytometry is a technology that combines

Fig. 1 Flow cytometry

Mechanical cell sorters Manufactured since 1994, FACSCaliburTM uses a

Benchtop A50 Micro/Universal No No 0.5, 1, 1.5, 2, 5 No Hydrodynamic Quartz

AccuriCSampler 2 488, 640 4 Diode F Direct 24 7 10.000 3.1

SynergyTM BTS/BSC (Sony) High-throughput acquisition is achieved by using a

Holder Fluidic Optic

Mechanical CyFlow Yes 96 5 No No Quartz No 2 355, 375, 407, 3 Diode F NI

Electronic Collect Biosafety

Mechanical CyFlow 16 4 10.000 300 3.0 No 1 No NI NI NI NI

a brightfield lamp, five excitation lasers (405, 488,

ImageStreamx system to replicate the RGB brightfield

imagery of a microscope. The MultiMag option

extended depth of field technology uses a combination

of specialized optics and unique image processing

one crisp plane of focus.

0.6, 1.5, 5, 15,

0.5, 1.0, 1.5,

technology, combines a brightfield lamp, four excita-

fluorescence colors per cell at a rate of 2,000 cells/s.

The FlowSight operates with a pixel size of one

For high throughput analysis, the ImageStreamX

and the FlowSight are equipped with a 96-well plate

autosampler. These systems use the IDEAS software

Mass cytometry (DVS Science)

The CyTOFTM cell analyzer instrument is a high

throughput mass cytometer for individual cell analysis

based on a novel elemental mass-spectrometry detec-

isotopic tags attached to antibodies using labeling kits.

Because there are many (up to 100) available stable

isotopes, and the mass spectrometer provides highly

precise resolution between detection channels, many

parameters can be measured—and typically without

requiring compensation. Each individual cell is ana-

requires, and the data is downloaded into conventional

flow cytometry software for examination.

Other optical developments appear such as color-

space-time (COST) coding, which is a new way to

detect multiple fluorescent wavelengths using a single

photodetector (Cho et al. 2010b). This method of

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