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A Review and Applications of Flow Cytometry

Daniel Heller

Department of Chemistry, University of Illinois at Urbana-Champaign

December 17, 2004

Abstract

A description and applications using flow cytometry are presented herein. Cytometry

uses fluorescence and scattering to analyze a population of cells, organelles, or other

similarly sized particles quantitatively. Flow cytometry can examine a multitude of

biological parameters, such as particle size, cell type, DNA content, and enzymatic

function at up to 7000 cells per second. New applications, such as the characterization of

cell apoptosis, as well as the function of drug-delivery devices, are also examined.

Introduction

Cytometry is a set of methods to quantitatively examine a population of cells and the

analytes inside of them. It exploits fluorescent-labeling techniques such as

immunohistochemistry in order to obtain information about a large number of cells.

Fluorescence microscopy, the standard practice for analyzing cellular components via the

use of a large number of chromophore probes, is preferred when observing a small

number of cells is adequate, or when solely qualitative information is required.

Cytometry is the fluorescence microscope’s quantitative counterpart.

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Cytometry can aid in the analysis of cell types, organelles, nucleic acids, enzymes, and

other moieties in a cell population. As in microscopy, fluorescent probes are used to bind

to the species under investigation. For instance, attaching probes to particular cell surface

antigens on the membrane allows cytometry to quantify cell types in a sample.

Intracellular antigens are used for applications such as measuring enzymes, organelles

and inclusion bodies, monitoring mitosis, as well as analyzing nucleic acid content in

cells. The most popular cytometric technique is flow cytometry of living cells. This

involves the movement of suspended cells flowing through a thin orifice, sequentially

probed by a light source to excite the attached fluorescent label. Flow cytometry is

typically used in configurations for either quantitative analysis or physical cell sorting.

The most common type of quantitative analysis using cytometry data involves creating a

histogram of fluorescence events to count the number of cells with the attached probe.

This effectively creates a set of data which gives a ratio of the cells in a population with a

particular surface protein, enzyme, or other analyte. Multiple analytes can be quantified

simultaneously. Cell sorting involves the physical separation of two cell populations by

creating tiny droplets of detected cell media which are separated according to the

droplet’s fluorescence. The end-result of such separations can be over 99% accurate.

Cytometry developed from the need to count blood cells as counts of both red and white

cells were of considerable interest to medical researchers by the 1930’s.

Hemocytometers, which allow microscopists to count cells via a microscope slide

containing a ruled grid, giving a rough approximation of the number of cells per volume

of blood, were known to be imperfect. In the 1950’s, the Coulter counter was developed

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to supplant the hemoctyometer in high-volume applications. This device pushed cells

single-file through a device which measured their resistance. Since cells possess a higher

amount of resistance than a saline solution in which the cells were suspended, an increase

in electrical impedance in the device would signal the presence of a cell. By counting

such events, the accuracy and speed of cell counts were improved. Fluorescence was

combined with flow cytometry in the 1960’s because one could measure more than the

number of cells. Cellular DNA content was quantified with fluorescence-based cytometry

by researchers in Germany and Los Alamos. Physical cell sorting was developed by

Kamentsky at IBM in the 1960’s using ink jet printer technology. He then started his own

company, Bio/Physics Systems, which sold the first fluorescence-based cytometer with

an argon ion laser, instead of an arc lamp, as its excitation source. In the early 1980’s,

flow cytometers became especially popular when it was found that the new mysterious

disease, AIDS, could be characterized by a decline in T-helper lymphocytes. The fast

quantification of flow cytometry was invaluable to early AIDS researchers.

Method and Analysis Techniques

Cytometry must be conducted on a free suspension of single cells sans large aggregates

or debris. Protocols for preparation of blood cells usually involves suspending in buffer

and centrifugating. Dense tissues are often disassociated by enzymatic digestion.

Fluorescent labels are then applied to tag a desired cell population or other cell-related

moiety such as nucleic acid or protein. Antibody-labeled dyes are popular for tagging cell

surface antigens, enzymes, the cytoskeleton, and organelles. Intercalating dyes are often

used to measure DNA content in cells, which can be used to detect cancer. The dyes

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fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE) are especially popular for

differentiating cells. Dye sets are chosen so they can be excited with the same laser and

have fluorescence maxima which are significantly different in order to minimize

crosstalk.

PMT
w/ 575 PMT w/ 620 PMT w/
nm BP nm BP Filter 680 nm
Filter BP Filter

PMT w/ 525
nm BP Filter

PMT

Lens
r s
ri ro
M
Laser roic
Excitation ch
Di

Flow Cell
Backscatter
Detector

Figure 1. Optical layout of a typical flow cytometer.

The typical flow cytometer uses sheath flow to force cells into a capillary by the process

of hydrodynamic focusing. A narrowing channel of the fluid forces cells into the center

of the chamber and into a capillary through which cells flow single-file. The capillary

usually ends in a flow cuvette where the sample is then illuminated by an arc lamp or

laser. Collection optics (Figure 1) split the emission into separate channels for detection

of multiple fluorophores by separate photomultiplier tubes. Optical filtration is used to

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separate the emission into separate detectors, often via dichroic mirrors and bandpass

filters. Certain analysis techniques require scattered light instead of fluorescence. Flow

cytometers carry an extra detector for forward-scattered light. Orthogonal (90°) scattered

(side-scattered) light is also measured, often simultaneously with forward-scattered light

during an experiment.

When a sample of cells stained for the presence of one or more species is run through a

cytometer, data is usually displayed as either a histogram or a cytogram (dot plot). A

histogram records the number of events versus their intensities. Cells which stain positive

a b

Figure 2. (a) Histogram of FITC-labeled CD8 cells showing unlabeled and
labeled cell populations. (b) Cytogram of bi-labeled cells showing four discrete
populations divided into regions and quantified as a percentage of the total.
for a species will emit at a higher intensity than negative cells (Figure 2a). Usually

displayed logarithmically, a typical data set will contain two major populations of events

denoting stained and unstained samples. The second common type of display, the

cytogram, plots the intensity of events hitting one detector versus the intensity at a second

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detector, where each event is represented by a dot. This produces a set of clusters which

correspond to the populations within the sample (Figure 2b). The cytogram is analyzed

via computer: boxes are drawn around regions of the plot and the percentage of total

fluorescence events is calculated in each of the regions.

Scattered light is often

measured along with

fluorescence. The forward-

scatter detector can be used

to determine the ratio of

dead cells, which are more

permeable to certain

fluorophores. These cells
Figure 3. Cytogram of light scattering off a sample
of canine bone marrow cells. Investigators divided
these into populations of mature erythroid cells (R2), have lower forward scatter
immature erythroid cells (R3), metamyelocytes (R4),
immature myeloid cells (R5), megakaryocytes (R7), than live cells. The amount of
and neutrophils (R8). Lymphocytes cannot be
separated in this plot because they overlap with forward (small angle) scatter
mature erythroid cells.
is also dependent upon

particle size, refractive index of the cells with respect to the medium, and the absorbance

of the cells. Researchers can use forward scatter to measure these and other parameters.

The total amount 90° (side) scatter from a sample is also used for characterization. It is

theorized that the amount of side scatter is related to its cytoplasmic granularity of a cell.

Although this is not confirmed, the difference in side scattering between granulocytes,

lymphocytes, monocytes, and other cell types is clear and can be used for measuring their

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ratios without the need for a stain. Figure 3 shows a plot of side scatter versus forward

scatter, allowing the quantification of many cell types simultaneously.

Flow sorting, the physical separation of

cells or organelles, or other particles, is

one of the primary uses of flow

cytometers (Figure 4). Sorting is

accomplished by equipment placed after

the flow cuvette used for detection. Using

technology derived from ink-jet printers,

the fluid exiting the cuvette is divided

into equally-spaced droplets by a

piezoelectric transducer which applies a

regular vibration to the flow chamber. As
Figure 4. Flow sorting scheme. Droplets
are charged as they break off from the
each droplet is forming, it is given a
stream and deflected by high voltage plates
into receptacles for collection
positive, negative, or neutral charge by a

voltage pulse. The charged droplets then fall between two high voltage plates which can

deflect them to the left or right of the original stream. The charge given to each droplet is

controlled by circuitry which gives each falling drop the correct charge based on

parameters set by the user. The sorting may be accomplished by telling the computer to

look for droplets with a particular fluorescence intensity, scattering property, or other

directive decided by the investigator.

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a
a

b b

Figure 5. Healthy and apoptotic cells Figure 6. Healthy and apoptotic
characterized by (a) forward scatter cells characterized by Hoeschst dye
measurements to show changes in and forward light scattering. (a)
size and (b) side scatter Healthy cells appear in a defined
measurements to show changes in grouping. (b) Apoptotic cells form a
granularity second population as they become
more permeable to the dye and their
decreased particle size attenuates
Application: Apoptosis
forward scattering intensity.
Researchers at the University of Illinois

Flow Cytometry Facility are studying apoptosis, or programmed cell death. This is a

particular kind of degradation in which cells deteriorate into several remnants with intact

membranes. Flow cytometry can be used to study this process during an experiment via

several methods. For instance, forward scatter, which can detect particle sizes in a flow

cytometer, is employed to measure the decrease in sizes of apoptotic cells (Figure 5a).

Additionally, granularity, which can be detected by side scattering, increases in apoptotic

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cells. This is shown in Figure 5b, which illustrates the increase in orthogonal scatter

caused by apoptotic cells.

The plasma membranes of apoptotic cells become more permeable to certain fluorescent

dyes. This is similar to the measurements conducted dead (necrotic) cells. The Hoechst

dye is used to illustrate the transformation of healthy cells to those which are undergoing

apoptosis. Figure 6a shows a dot plot of Hoechst dye fluorescence versus forward light

scatter. This gives a relatively localized region of dots which illustrates the distribution of

fluorescence intensities of the dye versus the relative range of sizes of the cells.

a b

Figure 7. (a) Fluorescence c
histogram showing
populations of unlabeled (M1)
and FITC-BSA tagged
water/oil/water droplet
emulsions. (b) Dot plot of
forward and side scattering of
droplets showing the size
(forward scattering) and
complexity (side scattering)
distributions. (c) Micrograph
of the droplets.

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It is apparent that fluorescence intensity increases monotonically with particle size, which

is to be expected because larger cells naturally contain more dye molecules. Figure 6b

illustrates the change in size and fluorescence intensity of apoptotic cells, which become

smaller but increase in permeability to the Hoechst dye.

Application: Oil Emulsions

Water/oil/water immersions are droplets of oil, immersed in water, which contain smaller

droplets of water in them. They are under investigation for biomimetic mineral formation

and as drug delivery devices. As delivery devices, a useful compound could be stored in

the water phase which is entrapped by oil. It is important to understand the homogeneity

of the droplets as well as the release rate of the compound contained in this phase.

Water/oil/water immersions containing a fluorescent biomarker (BSA-FITC) trapped in

the internal water phase were made to study these phenomena via flow cytometry. To

study the yield of preparation of fluorescent droplets, a blank and tagged emulsion were

fed through a flow cytometer. After two minutes, 100,000 droplets (events) were

measured and a histogram was generated which shows logarithmic fluorescence intensity

versus count number (Figure 7a). The M1 region is defined as the background, and

contains all events which produced a fluorescence intensity of under 100 arbitrary units.

The M2 region is defined as having high enough intensity to make the determination that

the droplet contained the FITC-BSA label. Using the histogram, one can determine that,

as 27,353 of 100,000 measured events are contained in the M2 region, that the yield of

marked droplets is 27.35%.

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Forward and side scatter are used to show the droplet complexity and size

inhomogeneity. The forward scatter axis (x-axis) in Figure 7b shows the relative range of

sizes of the droplets. The side scatter axis (y-axis) is related to droplet complexity

(granularity if it were a cell).

The plot illustrates that the

larger oil droplets are more

complex—they contain a

larger number of internal

water droplets. A micrograph

of the emulsion droplets is

shown in Figure 7c.

Release of the FITC-BSA
Figure 8. Release of the fluorescent marker FITC-
BSA from emulsion droplets over time. marker from the droplets is

studied over a period of days by repeatedly running the droplets through a flow

cytometer. The release fraction of the fluorescent marker from the droplet is defined as R

= (Y0 – Yt) x 100/Y0. where Y0 is the yield of the marker at time 0 and Yt is the yield of

the marker at time t in days. Figure 8 shows the increase of the release fraction over 7

days after preparation for two different initial marker concentrations.

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