Givan A.L. Flow Cytometry. First Principles

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Photodetector: A photodetector is a device that senses light and

converts the energy from that light into an electrical signal.

Within the operating range of the detector, the intensity of the

electrical signal is proportional to the intensity of the light.

Photomultiplier tubes and photodiodes are two types of photo-

detectors.

Photodiode: A photodiode is a type of photodetector used to detect

relatively intense light signals. It does not have a high voltage

applied to increase the current ¯ow at its anode (output) end.

Photomultiplier tube: A photomultiplier tube is a type of photo-

detector used to detect relatively weak light signals. Its output

current is increased by means of high voltage applied to it.

Phycoerythrin: Phycoerythrin is a ¯uorochrome derived from red sea

algae. It is particularly useful in ¯ow cytometric applications

requiring dual-color analysis because, like ¯uorescein, it absorbs

488 nm light from an argon laser. However, it has a longer

Stokes shift than ¯uorescein, and therefore the ¯uorescences of

the two ¯uorochromes can be distinguished.

Plankton: Plankton are the small organisms that ¯oat in lakes and

oceans and are moved passively, drifting with the tides and

currents. Small plankton are excellent objects for ¯ow cyto-

metric analysis.

Precursor frequency: The precursor frequency of cells that have

divided in response to a stimulus is the proportion of cells pres-

ent at the time of the addition of the stimulus that were destined

to begin division. It is a measure of the potential of a mixed

population of cells for response to any particular activator and

can be determined, by ¯ow cytometry, with the use of ¯uores-

cent tracking dyes that stain cells with stability but dilute by half

each time a cell divides.

Probe: Probe is a general term used, in ¯ow cytometry, to refer to

any chemical that ¯uoresces when it reacts or complexes with a

speci®c class of molecules and therefore can be used to assay

that molecule quantitatively. Propidium iodide and acridine

orange are probes for nucleic acid because they complex specif-

ically with nucleic acids and ¯uoresce brightly when they have

Flow Cytometry252

reacted in this way. Fluo-3 is a calcium probe because it chelates

calcium ions and ¯uoresces brightly when it is complexed with

this ion.

Propidium iodide: Propidium iodide is a probe that can be used to

measure quantitatively the amount of double-stranded nucleic

acid that is present in a cell. After treatment of cells with

RNase, it will measure the amount of DNA present. Because it

does not cross an intact cell membrane, cells need to be treated

with detergent or ethanol before it can be used to determine

their DNA content. It can also be used to assess the viability of

cells.

Quadrant: For dual-parameter analysis, a two-dimensional plot of

particles according to the correlated ¯uorescence intensities of

two colors is, traditionally, divided into four quadrants. The di-

vision is based on the background ¯uorescence of the unstained

control sample. The quadrants, from this division, will contain

(1) cells that have stained with the ®rst ¯uorochrome only; (2)

cells that have stained with both ¯uorochromes; (3) cells that

remain unstained; and (4) cells that have stained with the second

¯uorochrome only. In other words, the quadrants are de®ned so

that they delineate the two types of single positive cells (1 and

4), double-negative cells (3), and double-positive cells (2).

Rare event: De®ning a rare event in ¯ow cytometry is a bit like an-

swering the question ``How long is a piece of string?'' Particles

that are less than 0.1% of the total are considered somewhat

rare and require some care for enumeration in a ¯ow cytometer.

Really rare particles are less than 0.001% of the total and can

be detected, counted, and sorted with careful gating based on

multiparameters.

Region: A region is a description, using ¯ow cytometric character-

istics, that will de®ne a cluster of cells. A region, in the old days,

was always rectangularÐwith an upper and lower channel limit

for each parameter involved in the description. Now, a region

can take any shape, based on the channel numbers de®ning all

the vertices of an irregular polygon or ellipse. Multiple regions

can be combined by Boolean logic (AND, OR, or NOT) to de®ne

a gate that is used for cell analysis. See Gate.

Glossary 253

Rube Goldberg device: Reuben Lucius Goldberg (1883±1970) was

an American humorous artist. A Rube Goldberg device is

the American synonym for a Heath Robinson (see above) de-

vice. Mr. Goldberg and Mr. Robinson may or may not have

known each other, but surely would have enjoyed each other's

company.

S-FIT: An S-FIT approximation is a mathematical algorithm for

guessing at which cells in a DNA-content histogram are actually

in the S phase of the cell cycle. The S-FIT algorithm bases this

guess on the shape of the DNA histogram in the middle region

between the G0/G1 and the G2/M peaks.

S phase: S phase is the period of the cell cycle during which cells

are in the process of synthesizing DNA in preparation for cell

division. During S phase, cells have between the 2C amount of

DNA normal to their species and the 4C amount of DNA,

which is exactly double the 2C amount. It is the overlap of ¯u-

orescence intensity between cells in S phase and some of the cells

with the 2C and 4C amounts of DNA that leads to uncertainty

in the ¯ow cytometric estimation of the S-phase fraction.

Sheath: Sheath is the ¯uid within which the central sample core is

contained during coaxial ¯ow within and from the ¯ow cell of a

¯ow cytometer.

Side scatter: Side scatter is light of the same color as the illuminating

beam that bounces o¨ particles in that beam and is de¯ected to

the side. The ``side'' is usually de®ned by a lens at right angle

(orthogonal orientation) to the line of the laser beam. Side

scatter light (SSC) may alternatively be called right-angle light

scatter (RALS) or 90



LS. The intensity of this light scattered to

the side is related in a general way to the roughness or irregu-

larity or granularity of the surface or internal constituents of a

particle.

Signature: Signature is a term used by marine ¯ow cytometrists to

refer to the ¯ow cytometric characteristics of particular plank-

tonic species in a sample of water. The signature of plankton is

related, in particular, to the auto¯uorescent pigments that are

present. Signature has also been used to describe the distin-

Flow Cytometry254

guishing histogram pro®le that results from the DNA-fragment

analysis of di¨erent bacterial species.

SOBR: SOBR is a mathematical algorithm for guessing at which

cells in a DNA-content histogram are actually in the S phase of

the cell cycle. The SOBR (sum of broadened rectangles) algo-

rithm bases this guess on the use of Gaussian-broadened rectan-

gular distributions to attempt to ®t the shape of the DNA ¯uo-

rescence intensity histogram of the cells in question.

Stokes shift: The Stokes shift is the di¨erence in color (expressed as

either energy or wavelength) between the light absorbed by a

¯uorochrome and the light emitted when that ¯uorochrome

¯uoresces.

Tandem dye: Tandem dyes are conjugates of two di¨erent ¯uo-

rochromes. They use resonance energy transfer to pass the light

energy absorbed by the primary ¯uorochrome to the secondary

¯uorochrome, which then emits that energy as ¯uorescence.

Tandem dyes occur naturally (e.g., the peridinin:chlorophyll

complex or the aequorin:GFP complex) and can also be manu-

factured by organic chemists (e.g., PE-Cy5). In ¯ow cytometry,

tandem dyes are useful because they e¨ectively increase the

Stokes shift of a ¯uorochrome and thereby allow more ¯uo-

rescence parameters to be observed with a single excitation

wavelength.

Tetraploid: Tetraploid is a term used to describe cells with double the

amount of DNA normal for a particular species. Malignant cells

are frequently of the tetraploid type. Distinguishing this type of

ploidy from normal G2 or M cells or, indeed, from clumps of

two cells can be di½cult with ¯ow cytometry.

Threshold: The threshold is an electronic device by which an ADC

can be made to ignore signals below a certain intensity. A for-

ward scatter threshold is most commonly used in ¯ow cytometry

to exclude very small particles, debris, and electronic or optical

noise from acquisition into a data ®le.

Time: Time is a parameter that is being used more frequently in

conjunction with ¯ow cytometric analysis. It is being used in

relation to the functional analysis of cells for the rate of reaction

Glossary 255

(e.g., calcium ion ¯ux or changes in membrane potential) in re-

sponse to various stimuli. It is also being used, on a ®ner scale,

in phase-sensitive measurements of ¯uorescence decay.

TUNEL: The TUNEL assay represents a historical low point in

attempts to coin acronyms. The Terminal deoxynucleotidyl

transferase±mediated dU TP Nick End-Labeling assay labels the

ends of DNA with ¯uorescent UTP. Apoptotic cells often have

fragmented DNA; these fragments will provide more substrate

for the enzyme terminal deoxynucleotidyl transferase. Apoptotic

cells can therefore be enumerated by ¯ow cytometry according

to their increased intensity in the TUNEL assay.

Volume: Volume is a useful and de®nite characteristic of any particle

Ðbut one that is not easily amenable to ¯ow cytometric analy-

sis. ``Coulter volume'' does measure volume to a close approxi-

mation, but ``forward scatter'' does not.

Wavelength (1): Wavelength is a characteristic of light that is related

exactly to its energy content and also (with light to which our

eyes are sensitive) to its color. Light of short wavelength lies

toward the bluer region of the spectrum and has more energy

than light of longer wavelength. Wavelength is used to describe

the characteristics of ®lters, of dichroic mirrors, of laser beams,

and of the absorption and emission of ¯uorochromes.

Wavelength (2): In sorting ¯ow cytometry, wavelength is a term used

to describe the distance between drops as they form from a

vibrating ¯uid jet. This drop wavelength is determined by the

diameter of the stream as well as by its velocity.

Flow Cytometry256

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Flow Cytometry260

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