453052

ng Flow Cytometry: Review of the MethodBarteneva et al.

2012 The Author(s) 2010

JHCXXX10.1369/0022155412453052Imagi

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Review
Journal of Histochemistry & Cytochemistry 60(10) 723733
The Author(s) 2012
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DOI: 10.1369/0022155412453052
http://jhc.sagepub.com

Imaging Flow Cytometry: Coping with Heterogeneity in Biological

Systems
Natasha S. Barteneva, Elizaveta Fasler-Kan, and Ivan A.Vorobjev

Immune Disease Institute and Program in Cellular and Molecular Medicine, Childrens Hospital Boston and Department of Pediatrics, Harvard Medical
School, Boston, Massachusetts (NSB); University of Applied Sciences Northwestern Switzerland (FHNW), Institute of Chemistry and Bioanalytics,
Muttenz, Switzerland (EF-K); A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia (IAV); and Department of
Cell Biology and Histology, Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia (IAV).

Summary
Imaging flow cytometry (IFC) platforms combine features of flow cytometry and fluorescent microscopy with advances in
data-processing algorithms. IFC allows multiparametric fluorescent and morphological analysis of thousands of cellular events
and has the unique capability of identifying collected events by their real images. IFC allows the analysis of heterogeneous
cell populations, where one of the cellular components has low expression (<0.03%) and can be described by Poisson
distribution. With the help of IFC, one can address a critical question of statistical analysis of subcellular distribution of
proteins in a cell. Here the authors review advantages of IFC in comparison with more traditional technologies, such as
Western blotting and flow cytometry (FC), as well as new high-throughput fluorescent microscopy (HTFM), and discuss
further developments of this novel analytical technique. (J Histochem Cytochem 60:723733, 2012)
Keywords
imaging flow cytometry, flow cytometry, biological heterogeneity, fluorescence, cellular morphology, Poisson distribution,
single cell, high-throughput fluorescent microscopy

Several types of high-throughput instrumentation for analyzing and quantifying different aspects of cell biology are
now available. They include plate readers, sequencing platforms, DNA, RNA and protein microarrays, Western blotting, flow cytometers, and so on. However, many platforms
allow readouts only on the population level. Technologies
that allow readout at the single-cell level include flow
cytometry (FC), imaging cytometry, and different automated microscopy setups. These platforms are essential for
assaying diversity within cell populations and searching for
rare cells with specific features (stem cells, etc.).
Cytometry assays are sensitive, fluorescence-based
methods aimed at determining a molecular phenotype of
single cells. They can be multiparametric, multiplexed,
quantitative, and qualitative. They can also be extracted as
a result of kinetic or single end-point measurements. FC
allows for the simultaneous quantification of multiple fluorescent emissions and the scattered light of single cells
acquired in the laminar flow of cell suspension. FC is a

technology that measures the cellular properties (protein

expression, siRNA expression, etc.) in a snapshot of the
entire population (Shapiro 2005; Huang S 2009). Imaging
cytometry (IC) is represented by two different types of technology: 1) high-throughput microscopy and laser scanning
Received for publication May 14, 2012; accepted June 5, 2012.
Supplementary material for this article is available on the Journal of
Histochemistry & Cytochemistry Web site at http://jhc.sagepub.com/
supplemental.
Corresponding Authors:
Natasha Barteneva, Immune Disease Institute and Program in Cellular
and Molecular Medicine, Childrens Hospital Boston and Department
of Pediatrics, Harvard Medical School, D-239, 200 Longwood Avenue,
Boston, 02115, MA, USA.
E-mail: barteneva@idi.harvard.edu
Elizaveta Fasler-Kan, University of Applied Sciences Northwestern
Switzerland (FHNW), Institute of Chemistry and Bioanalytics,
Gruendenstrasse 40, CH-4132, Muttenz, Switzerland.
E-mail: elizaveta.fasler@fhnw.ch

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Barteneva et al.

cytometry, which interrogates cells or tissue specimens

in situ positioned on the microscope slide or in the microplate wells (Kamentsky and Kamentsky 1991; Darzynkiewicz
et al. 1999; Gerstner et al. 2004; Terjung et al. 2010; Henriksen
et al. 2011; Rimon and Schuldiner 2011), and 2) imaging
flow cytometry (IFC), which interrogates cells and cellular
aggregates in the laminar flow (McGrath et al. 2008).
The most important difference between FC and IFC
depends on whether the fluorescence data of the cell suspension are obtained with cell morphology or from fluorescence
pulse-analysis. IFC allows for the acquisition and identification of tens of thousands of cellular events based on their
fluorescent and morphological parameters. The first and
unique IFC instrumentImagestream 100 (IS100)was
introduced in 2005, and the next generation of Imagestream
imaging flow cytometers (IS-X) was recently launched by
Amnis Corp. (Seattle, WA) (Basiji et al. 2007).

IFC in the Evaluation of Cellular

Heterogeneity
IFC allows for the evaluation of morphological and fluorescent data at a single-cell as well as at a population level
(Figure 1). IFC combines the statistical advantage of FC
with the ability to identify each event based on a real image,
which allows it to analyze protein expression in single cells
in heterogeneous cell populations, where the level of
expression of one of the proteins is low and could be
described by Poisson distribution (rare cell subpopulations
with <0.01% of expression). The multiple applications of
IFC include analysis of nuclear-cytoplasmic translocation
(Arechiga et al. 2005; Fanning et al. 2006; Danis et al.
2008), quantification of apoptosis based on the changes in
nuclear morphology (George et al. 2004; Henery et al.
2008; Khuda et al. 2008), and quantitative analysis of internalized bacteria and protozoan parasites (Muskavitch et al.
2008; Bisha and Brehm-Stecher 2009; Ploppa et al. 2011).
In recent years, IFC was also employed for the evaluation
of asymmetric cell division (Filby et al. 2011), internalization of CypHer5E-conjugated antibodies and PKH-labeled
exosomes (Xu et al. 2010; Vallhov et al. 2011), intercellular
communication by exchange of cytoplasmic material
(Domhan et al. 2011), analysis of cell interactions and
immune synapse (Ahmed et al. 2009; Ouk et al. 2011), and
some other experimental applications (Ponomarev et al.
2011).
The cell populations are heterogeneous with respect to
cell cycle phase, size, volume, physiological state, and their
individual development history (Lloyd et al. 2000; Kaern et
al. 2005; Pilborough et al. 2009). In a clonal population,
large variations in phenotype may be the result of fluctuating gene expressions (Kim et al. 1998; Pilborough et al.
2009; Dietmair et al. 2012). Emerging fundamental research
on bacteria (Elowitz et al. 2002; Ozbudak et al. 2002; Yu

et al. 2006) and, more recently, on yeast and mammalian

cells (Newman et al. 2006; Raj et al. 2006; Sigal et al. 2006;
Chang et al. 2008) shows that protein expressions can have
significant variations inside clones of genetically identical
cells (intraclonal variation).
The important advantage of cytometric methods over
Western blotting and gel-shift assay is that they efficiently
overcome the heterogeneity drawback, allowing the data
collection of a number of cell populations without averaging the signal intensities. When measuring the average signal intensity, information regarding cell subpopulations of
heterogeneous populations can be missed (Huang S 2009).
For example, when a 25% decrease in signal intensity is
observed with Western blotting, it is impossible to tell, if
this results from a 25% reduction in 100% of the cell population or a 50% reduction in only 50% of the population. To
overcome this drawback, a combination of Western blotting
and cell sorting is used.
Also, the amount of cells needed for Western blotting
analysis is in the range of 5 105106 cells per sample,
making it practically useless for the analysis of small and
rare subpopulations (a rare population being <0.03%).
Microarrays and two-dimensional gels are also biased
toward more abundant genes (Lu and King 2009). Although
FC can identify and sort rare cell populations with high
speed, dealing with rare cell detection using this approach is
plagued by the contamination of false-positive events due
to autofluorescence, nonspecific immunostaining, and cell
aggregates (Radbruch and Recktenwald 1995). For rare cell
detection, the following parameters are considered important: 1) the ability of the instrument to process large numbers of cells, 2) the number of cells analyzed by instrument
per unit of time, 3) the sensitivity of the instrument, 4) the
specificity of the assay, and 5) the consistency of the instrument performance.
We had difficulties analyzing the large, 100K files using
the IDEAS software that came with the IS-100. However,
IFC is very helpful in identifying and characterizing small
populations of cells, such as the low percentage population
of cells that forms an immune synapse in our research of
CTL-macrophage interaction of an HIV infection (Ahmed
et al. 2009).
Recently, IFC, combined with cell sorting preenrichment, was successfully employed for the identification and
characterization of a novel pluripotent population of very
small embryonic-like stem cells (VSELs) in human and animal tissues (Kucia et al. 2006; Zuba-Surma, Klich, et al.
2009; Zuba-Surma, Kucia, Rui, et al. 2009). According to
Zuba-Surma, Kucia, Ratajczak, et al. (2009), the frequency
of VSELs in murine adult spleen (1 108 cells) was 3.9
103, which corresponds to approximately one cell per 2.5
104 (0.25% of cell population) and even lower in other adult
murine organs. IFC is also helpful for the analysis of rare
cell populations (<0.01%) after cell sorting.

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Imaging Flow Cytometry: Review of the Method

IFC in Signal Transduction Research

Strategies to monitor and manipulate individual signaling
pathways and the modification of target protein properties
by reversible phosphorylation can offer valuable tools to
investigate the precise role of signal transduction pathways
in the pathogenesis and progression of human diseases. The
phosphorylation of a protein is the principal cellular mechanism regulating protein function and is controlled by protein kinases and phosphatases. Therefore, monitoring
kinase and phosphatase activities is crucial to understanding signal transduction pathways. The Western blot is currently regarded as the most versatile and convenient assay
for quantifying signaling activities, partly due to the successful use of short synthetic peptides to produce epitopetargeted antibodies for this platform. However, using
Western blot, multiple signaling activities cannot be simultaneously quantified, and the signal intensities of various
subpopulations can not be differentiated. Other drawbacks
of the Western blotting approach are the necessity of cell
lysis and solubilization of the samples. The solubilization
step is required to release the protein of interest from its
membrane environment and may lead to artificial associations and oligomerization of proteins and other artifacts.
Also, Western blotting requires a significant amount of
cells (5 105106 per line), and in many cases, preliminary
immunomagnetic purification and/or fluorescent-activated
cell sorting (FACS) is needed. Many proteins involved in
signal transduction pathways are expressed at low levels
and cannot be evaluated by standard Western blotting techniques. Recently, new approaches, such as antibody colocalization microarrays and reverse protein assays, have
been developed (Kornblau et al. 2009; Brennan et al. 2010;
Pla-Roca et al. 2012). Reverse protein arrays can determine
the levels of multiple phosphoproteins in cell lysates in a
high-throughput fashion on chips, but it is impossible to
differentiate signal intensities of individual cells and discrete cell subsets using these techniques (Wu et al. 2010).
An increasingly common technique to characterize signal transduction pathways relies on FC. FC has been shown
to provide quantitative data for measuring signaling activities similar to the Western blot (Krutzik and Nolan 2003;
Krutzik et al. 2004, 2011) and is also capable of measuring
multiple signaling activities in single cells on a population
level. FC has been most commonly used for the analysis of
multiple kinases (such as the JAK-STAT pathway) and
phosphatases in response to different signaling (Perez et al.
2004; Heller et al. 2006; Teofili et al. 2007; Kotecha
et al. 2008; Oh et al. 2010). The major drawback in using
FC to characterize different signal transduction pathways is
the lack of a reliably wide panel of antibodies with high
specificity. Antibodies are frequently cross-reactive, but in
FC, there is no way to filter out cross-reactivity

(Oberprieler and Tasken 2011). Currently, numerous antibodies against phosphoepitopes are under development in
several laboratories (Kotecha et al. 2008; Lee et al. 2008;
Oberprieler et al. 2010).
The signal-induced nuclear-cytoplasmic translocation of
key signal transduction molecules (such as NF-B, FOXp3, Smad, and others) is a common phenomenon in many
pathways. FC is unable to provide information about the
subcellular localization of the fluorescent signal. To obviate
the requirement for determining nuclear localization for
molecules in the NF-B pathway, the FC approach relies on
the detection of phospho-specific intermediaries
(Phospho-p65) (Armstrong et al. 2006). Also, FC is not
capable of providing information about the interrelationship
of signaling activities at the intracellular level. The FC
study of signaling in adhesive cells is at a disadvantage in
that cells have to be detached into a single suspension
before fixation. Also, the Western blotting of adherent cells
in many cases requires preliminary cell sorting to enrich for
a certain cell population; this situation is quite common in
the field of signal transduction.
Traditionally, transport between the nucleus and the
cytoplasm has been studied using subcellular fractionation
followed by Western blotting and/or fluorescent microscopy (Shakulov et al. 2000; Zhou et al. 2006) and, recently,
by automated high-throughput microscopy systems (Rimon
and Schuldiner 2011). For quantitative measurements of
protein distribution, the fluorescent intensities of the
nucleus and cytoplasm in the fluorescent microscope are
usually limited to 50 to 100 cells per experiment due to the
small frame size. However, the major advantage of the light
microscopy approach is spatial precision, which is being
further developed with the implementation of super-resolution techniques (reviewed by Huang B et al. 2009). Also,
measurements can be made in live, functioning cells and
directly correlated with single molecules trajectories in real
time (Tu and Musser 2011).
IFC allows for the quantitation of bidirectional trafficking of transcriptional regulators from the nucleus to the
cytoplasm inside the cell, with a maximum 60 magnification (for IS-X; 40 for IS-100). The approach of image
analysis to nuclear-cytoplasmic translocation used in IFC
includes the initial identification and segmentation of single
cells, and then the signal intensities are summed in the
nuclear and cytoplasmic regions; the latter is defined as the
whole cell body minus the nuclear region. This is important
for the precise identification of individual cells in nuclear
translocation assays. On the other hand, it is different from
conventional high-throughput imaging, where signal intensities are summed in the nuclear regions plus the cytoplasmic regions, defined by an annulus set around each cell
(Ding et al. 1998; Ozaki et al. 2010). IDEAS software,
developed by Amnis Corp, is capable of calculating

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Barteneva et al.

nuclear-cytoplasmic translocation based on the degree of

co-localization of DNA markers and the protein of interest
(George et al. 2006). The similarity score is derived from
Pearsons correlation coefficient (pixel-by-pixel correlation
of the NF-B and nuclear dye-image pair within the nuclear
morphology mask dilated by one pixel), and the cross-correlation methods have been used to estimate the translocation of NF-B and other transcription factors (FOX-p3,
p65, interferon regulatory factor-7 [IRF-7]) (Arechiga et al.
2005; Fanning et al. 2006; Danis et al. 2008; Maguire et al.
2011). IFC is particularly valuable for signal transduction
analysis in 1) experiments employing cells with low levels
of expression of the protein of interest and 2) cellular populations with a particularly wide range of responses to a signal (George et al. 2006).

Elimination of False-Positive and

False-Negative Events with IFC
A limitation of FC as a method is that it can create falsepositive heterogeneity due to staining and sample preparation artifacts resulting from cross-reactivity of antibodies,
as well as the absence of morphological information
(Lambert and Desbarats 2007; Kuonen et al. 2010). Dead
cell exclusion and multiple-event discrimination using gating on light-scatter dual-parameter dot blots alleviate but do
not completely eliminate the problem. IFC should be a
method of choice for the validation of antibody specificity
and cross-reactivity. Ouk et al. (2011) used IFC to demonstrate that adhesion of platelets on the surface of leukocytes
is likely to be responsible for false-positive expressions of
platelet markers on leukocytes determined by FC. Standard
gating exclusions of multiaggregates in FC analyses
(FSC-W/FSC-H) do not work in this case because of the
relatively small size of the platelets attached (23 ).
Most false-positive events are known to be associated
with apoptosis, cytotoxicity, and cell differentiation, including kinases and protein phosphatases (Crisman et al. 2007).
During apoptosis, detection of false-positive events can
occur due to several factors: 1) Apoptotic bodies and microparticles can adhere to the surface of live, non-apoptotic
cells; 2) cells stained with an apoptotic marker could adhere
to a normal cell; 3) cells can phagocytize a fluorescently
labeled apoptotic body and/or cell fragments; and 4) apoptotic bodies and/or cell fragments can be counted as apoptotic cells (Bedner et al. 1998, 1999; Darzynkiewicz et al.
2001). Because IFC includes real imaging of each fluorophore distribution in the cell, it is possible to determine if
fluorophores are located outside the area of the cell or at the
expected location (membrane for Annexin V assay, nucleus
for terminal deoxynucleotidyl transferase dUTP nick end
labeling [TUNEL] assay, cytoplasm for caspase-3 substrate) (Henery et al. 2008). Another example of falsenegative events is that some cells in the population can lose

the apoptotic marker due to the loss of membrane integrity.

It is also important that most if not all FC techniques for the
detection of apoptosis (such as TUNEL method, conventional apoptosis assays using propidium iodide, subG1
DNA content determination, etc.) also detect necrosis and
other cellular processes (Ansari et al. 1993; Li et al. 1995;
Frankfurt et al. 1996; Rieger et al. 2010; Bucur et al. 2012).
IFC is capable of discriminating false events on the basis of
cell images and providing morphological information on
these cells (nuclear fragmentation, membrane blebbing,
etc.).

Advantages of IFC
The FC statistical advantage comes at the price of missing
information because FC measures total fluorescence of
individual cells selected by sophisticated gating from a cell
population. As a result, fluctuations in the protein and gene
expression may be due to a difference in cell volumes
(Newman et al. 2006; Volfson et al. 2006; Tsuru et al.
2009). A common method to reduce cell variability relies
on gating (i.e., creating sequential gates in the FSC-A/
SSC-A and FSC-H/FSC-W dot blots and discarding all the
cells that fall outside the chosen gates, hence reducing variability in the cellular optical properties). In practice, FC
does not allow for the discrimination between multiple
events and large cells, masking potentially relevant cells
and biasing results. Excluding events outside scattered light
gates can drastically reduce the analyzed sample size, particularly in the analysis of detached or digested cells, enzymatically treated with trypsin or collagenase, and ignore the
ungated cell population by masking potentially relevant
cellular events. Recently, Knijnenburg et al. (2011) introduced a correction for phenotypic variability based on the
regression model that uses all the cells in the sample to
normalize cell size and granularity effects on fluorescence
intensity. On the other hand, IFC offers an alternative to
gating on the basis of light scattering such that gating uses
an aspect ratio of cells (a ratio of cell diameters), diameter
and cell volume, and other morphological features.
The pool of antibodies that can be used for IFC includes
FC-quality antibodies, as well as polyclonal antibodies used
for immunoprecipitation and immunohistochemistry assays
(such as rabbit polyclonal NFB p65 antibody and antiIRF-7 [Santa Cruz Biotechnology, Santa Cruz, CA])
(Fanning et al. 2006; Tibrewal et al. 2008).
Image processing needs to be adapted to each new assay
developed and therefore is the bottleneck of high-throughput imaging technology (Starkuviene and Pepperkok 2007).
The IS-instruments analyze cells flowing in suspension and
collect dark field, bright field, and multiple fluorescent
parameters (Basiji et al. 2007). The greatest advantage of
IFC compared with the FC is its unique ability to identify
collected events by their real images: For each

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Imaging Flow Cytometry: Review of the Method

event displayed on a two-dimensional dot as a dot, IDEAS
software retrieves a real image of the event. The reverse
identification of cellular events on a dot blot is also possible. This capability is crucial for characterization and identification of cellular subpopulations and for identifying
false-positive and false-negative events. IFC allows for
simultaneous analysis of fluorescent and morphological
parameters, which is important for monitoring different
processes together such as apoptosis and autophagy (De la
Calle et al. 2011). The resolution of images is limited by
0.5- pixels in IS-100 and 0.3- pixels in IS-X (when using
60 objective), which allows for the quantification of many
cellular parameters, such as size, volume, shape, contrast,
spot count, and texture. The IDEAS software gives researchers the capability to analyze acquired cellular images while
using bivariate plots and histograms, as well as a sequential
gating strategy. It is also possible to create new, complex
features using Boolean logic and the combination of twodimensional masks (such as morphology masks, intensity
threshold masks, spot masks, and others). The detailed
description of individual masks and features can be found
on the company site (www.amnis.com). Moreover, the
IDEAS software has the additional option of defining populations using a gallery of individual images. Overall, the
IDEAS software used in IS instruments provides a unique
approach allowing the investigator to quickly change critical parameters during the analysis of cellular populations. It
is more user-friendly than other currently available image
analysis programs that are used in microscopy (Metamorph,
ImageJ, etc.), and analysis performed by IDEAS does not
require creating scripts or using special programming skills.
Because of the ability to collect real images, IFC also has
an advantage in the statistical analysis of not only single
cellular events but also cell aggregate content and the study
of cell-cell interactions. IFC allows for the analysis of tens
of thousands of cellular events versus high-throughput
automated fluorescent microscopy, which is usually limited
by parallel analysis of several hundred to a few thousand
events (Terjung et al. 2010). For example, quantitative analysis of microaggregates of leukocytes and platelets (Goetz
et al. 2005) achieved with light microscopy was limited by
analysis of 50 aggregates per experiment. FC analysis of
aggregates, the most widely used method of aggregate
detection, provides information only about the level of fluorescent intensity of the aggregate subpopulation defined by
the coexpression of platelet and leukocyte (or monocyte)
markers (Harding et al. 2007; Izzi et al. 2007; Alberti et al.
2009; Singh et al. 2012). FC is not able to provide information about morphology and quantitative cellular content of
mixed aggregates. Ouk et al. (2011) used IFC to quantitatively assess the number of adherent platelets to neutrophils
and monocytes, to visualize and further confirm mixed
aggregates forming during different experimental conditions. The IFC approach could be a method of choice in the

study of platelet-leukocyte interactions leading to the formation of mixed aggregates.

Analysis of cell interactions, such as forming immunological synapses with the help of IFC, also allows overcoming limitations of current imaging techniques, such as
difficulty in objectively finding rare events, limited statistics, and sample photobleaching during fluorescent microscopy sessions (Ahmed et al. 2009). Using IFC, we were
able to analyze thousands of T cellmacrophage conjugates
for each experimental point (Ahmed et al. 2009). The same
methodology could also be used to evaluate the co-localization
and movement of molecules in the interactions between
antigen-specific cells and target cells and/or between pathogen and immune cells.

Challenges Associated with IFC

Studies
The IFC research discussed in the previous sections provided a lot of new information about protein expression,
signaling pathways, and cellular interactions. Nevertheless,
there appears to be a number of technical and experimental
limitations, all of which should be considered when planning IFC experiments.
Traditional microscopic methods have many advantages
compared with IFC; they have better spatial resolution and
allow analysis of spatial-temporal organization of the samples, time-lapse experiments with single cells, and so on
(Rimon and Schuldiner 2011). However, a limited number
of cells are evaluated, and until recent introduction of automated systems, operator bias always had to be considered.
IFC has common traits with high-throughput microscopic
systems, such as moderate resolution (0.51.0 ) and the
capability of analyzing thousands of cellular events (Table 1).
On the other hand, high-throughput fluorescent microscopy
(HTFM) has advantages of time-lapse analysis of cells and
parallel analysis of hundreds of markers, and no perturbation is used for adherent cells. The major advantages of IFC
include a hardware that is oriented to analysis without perturbation of cells into suspension and well-developed software that does not require programming skills and is not
limited by a few prearranged analysis algorithms. The
choice between the currently available microscopic imaging software is influenced mainly by the programming
expertise and the hardware resources (Terjung et al. 2010).
Although technical limitations continue to be addressed
through constant developments, the multicolor experiments
developed for flow cytometers or microscopes are not
immediately transferable to IS instruments (IS-X or early
model IS-100). Some differences need to be taken into
account when you are planning the multicolor assays using
an imaging flow cytometer. Two major considerations are
the following: 1) wide bandpass of the optical filters and
related significant spectral compensation and 2) optimal

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Table 1. Summary of Imaging Flow Cytometry (IFC) and Other Common Techniques for Cell Population Analysis

IFC

Laser Scanning
Cytometry

Flow Cytometry

High-throughput
Fluorescent
Microscopy

Feature

Western Blotting

Capable of analyzing
heterogeneous cell
population
Light scatter

No

Yes

Yes

Yes

Yes

No

Yes (side scatter)

Yes

No

Brightfield
No
Capable of analyzing rare No
subpopulation (<0.01%)

Yes
Yes

Yes (forward and

side scatter)
No
Yes

Needs spectral
compensation
Cell morphology data
Statistics (representative
experiment)

NA

Yes

Yes

No
Averaged data from
5 105106 cells
per slot
5 105106 cells
per slot up to
1013 101
slots/hr
No

Yes
103104 events

No
Up to 106107
events

Yes
102105 cells

3001000
events/sec

300010,000
events/sec

No

No

Up to 100 cells/sec Parallel acquisition;

(5000 cells/min)
rate depends on
(Pozarowski et al.
the exposure time
2006)
Yes
Yes

No

Yes

Yes

Yes

Yes

Not significant

Not significant

Not significant

Not significant

Not significant

Speed of acquisition

Single-cell analysis (time

lapse)
Cell population analysis
(time lapse)
Operator bias

Yes
Yes
Capable of analyzing No
1:104 cells
(Megyeri et al.
2004)
No
No
Yes
102104 cells

NA, not applicable.

speed that relies on cell concentrations and is relatively low.

For example, maximal speed (up to 300 events/sec for
IS-100 and up to 1000 events/sec for IS-X) can only be
achieved with a relatively high sample concentration that is
about 107 cells per milliliter (500,000 cells per 50 l of volume for IS-100 and 3 less for IS-X). Another problem that
stems from the contamination of the sample is a large number of non-target fluorescent particles and microparticles,
which are acquired by IS systems as separate events.
The acquisition timescale in IFC ranges in minutes, all
the way from tenths of a minute to 100 min needed to push
through tens of thousands of events from samples with a
low cellular concentration. Several issues arise when trying
to automatically image multiple live cells with the help of
the IS series in IFC, such as the lack of availability of nutrients and oxygen over long time periods, because samples
presumably are run in phosphate buffer solution (PBS).
Focusing is a large contributor to the length of time required
for analysis, because a large percentage of cells will be
excluded from analysis due to focusing problems. Moreover,
if fluorescence labeling with cell tracking and vital dyes is
used, additional experiments are required to exclude

phototoxicity as a reason for observed differences between

the experimental and control cells.
Postacquisition image analysis of data from IS instruments requires pixel-by-pixel spectral compensation,
described in detail in the study published by Ortyn et al.
(2006). The IS instruments have wide, non-interchangeable
optical filters with the width in ranges from 25 to 85 nm
(channel 3, 500560 nm; channel 4, 560595 nm; channel 5,
595660 nm; channel 6, 660730 nm for IS-100) (see Suppl.
Table S1 for IS-X). Many dyes may require a significant
spectral compensation (e.g., dihydroethidium [DHE] may
require 73.9% compensation in channel 4 and 40.6% compensation with DRAQ 5; cited by Ploppa et al. 2011 for the
IS-100). This leads to the low detection signal of quantum
dots (QDs) and such standard violet dyes as Pacific Blue and
Pacific Orange. These dyes are practically undetectable in
the multiparameter panels partly due to the significant spectral compensation (Chattopadhyay et al. 2012; our unpublished data). The preferential violet fluorochromes for
antibody conjugates imaged with IS instruments are Brilliant
Violetbased tandem dyes (Chattopadhyay et al. 2012). The
QD combinations for IS instruments provide a good staining

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Figure 1. A typical workflow for image flow cytometry.

only in the absence in the panel overlapping bright fluorochromes with wide spectra; that is, only as a combination of
different QDs together (such as QD525 and QD605 in the
study by Domhan et al. 2011).
It is useful to perform IFC in combination with other methodologies (Western blotting, FC, fluorescent microscopy) and
verify, if the different platforms can give comparable results.
Consequently, it will help to overcome drawbacks and ambiguities associated with data interpretation.

Conclusions
Recent advances in fluorescent technologies have made the
collection of multiparametric imaging files routine, but efficient analysis of these data remains a challenge. IFC offers
a solution by allowing a collection of tens of thousands of
cellular events and the choice of gated statistical analysis
on the basis of fluorescent and morphological features

combined with the capability of identifying collected

objects on the basis of real images. The principal enhancements of IFC over the past 5 years have been the following:
1) the introduction of a higher resolution (60 objective); 2)
using variable objectives, allowing the throughput options
to be tailored to the requirements of a specific application
(the speed of acquisition can be increased up to 1000 events/
sec); 3) introducing additional functionality to the instrumentation (switching between FC and IFC modes); and 4)
increasing laser line versatility (to enhance reagent compatibility and multiplexing capability). The advantage of IFC
compared with high-throughput microscopy and laser scanning cytometry systems designed to work with adherent
cells is its well-developed experiment-approached software
and its ability to image cells in suspension. Thus, IFC takes
its own unique place in biomedical research. Also, as the
interest in performing IFC systems grows, the necessity of
combining this technique with cell sorting becomes evident.

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Barteneva et al.

Acknowledgements
We are grateful to Aleksandra Gorelova for help with the preparation of the manuscript. Space considerations limited us to a
selected list of available literature on IFC.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.

Funding
The authors disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article: The work
was supported in part by NIH S10 RR023459 grant and the Immune
Disease Institute to NSB and RFBR grant 11-04-01749a to IAV.

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