![Blooddraw](https://upload.wikimedia.org/wikipedia/commons/9/95/Blooddraw.jpg)
Cytometry is the
measurement
Measurement is the quantification of attributes of an object or event, which can be used to compare with other objects or events.
In other words, measurement is a process of determining how large or small a physical quantity is as compared ...
of number and characteristics of
cells
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
. Variables that can be measured by cytometric methods include
cell size
Cell growth refers to an increase in the total mass of a cell, including both cytoplasmic, nuclear and organelle volume. Cell growth occurs when the overall rate of cellular biosynthesis (production of biomolecules or anabolism) is greater than t ...
,
cell count
Cell counting is any of various methods for the counting or similar quantification of cells in the life sciences, including medical diagnosis and treatment. It is an important subset of cytometry, with applications in research and clinical prac ...
, cell morphology (shape and structure),
cell cycle
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...
phase,
DNA content, and the existence or absence of specific
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s on the cell surface or in the
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
. Cytometry is used to characterize and count
blood cell
A blood cell, also called a hematopoietic cell, hemocyte, or hematocyte, is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), ...
s in common
blood test
A blood test is a laboratory analysis performed on a blood sample that is usually extracted from a vein in the arm using a hypodermic needle, or via fingerprick. Multiple tests for specific blood components, such as a glucose test or a cholester ...
s such as the
complete blood count
A complete blood count (CBC), also known as a full blood count (FBC), is a set of medical laboratory tests that provide cytometry, information about the cells in a person's blood. The CBC indicates the counts of white blood cells, red blood cell ...
. In a similar fashion, cytometry is also used in cell biology research and in medical diagnostics to characterize cells in a wide range of applications associated with diseases such as
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
and
AIDS
Human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV/AIDS) is a spectrum of conditions caused by infection with the human immunodeficiency virus (HIV), a retrovirus. Following initial infection an individual m ...
.
Cytometric devices
Image cytometers
Image cytometry is the oldest form of cytometry. Image cytometers operate by statically imaging a large number of
cells using
optical microscopy
Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultraviole ...
. Prior to analysis, cells are commonly stained to enhance contrast or
to detect specific molecules by labeling these with
fluorochrome
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...
s. Traditionally,
cells are viewed within a
hemocytometer
The hemocytometer (or haemocytometer) is a counting-chamber device originally designed and usually used for counting blood cells.
The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a ...
to aid manual counting.
Since the introduction of the
digital camera
A digital camera is a camera that captures photographs in digital memory. Most cameras produced today are digital, largely replacing those that capture images on photographic film. Digital cameras are now widely incorporated into mobile device ...
, in the mid-1990s, the automation level of
image cytometers has steadily increased. This has led to the commercial availability of automated image cytometers, ranging from simple cell counters to sophisticated
high-content screening High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a ...
systems.
Flow cytometers
Due to the early difficulties of automating microscopy, the
flow cytometer
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of Cell (biology), cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and inject ...
has since
the mid-1950s been the dominating cytometric device.
Flow cytometers operate by aligning single cells using flow techniques. The cells are characterized
optically or by the use of an
electrical impedance
In electrical engineering, impedance is the opposition to alternating current presented by the combined effect of resistance and reactance in a circuit.
Quantitatively, the impedance of a two-terminal circuit element is the ratio of the comp ...
method called the
Coulter principle
A Coulter counter is an apparatus for counting and sizing particles suspended in electrolytes. The Coulter counter is the commercial term for the technique known as resistive pulse sensing or electrical zone sensing, the apparatus is based on ...
.
To detect specific molecules when optically characterized, cells are in most cases stained with the same
type of fluorochromes that are used by image cytometers. Flow cytometers generally provide
less data than image cytometers, but have a significantly higher throughput.
Cell sorters
Cell sorters are flow cytometers capable of sorting cells according to their characteristics.
The sorting is achieved by using technology similar to what is used in
inkjet printers
Inkjet printing is a type of computer printing that recreates a digital image by propelling droplets of ink onto paper and plastic substrates. Inkjet printers were the most commonly used type of printer in 2008, and range from small inexpensi ...
.
The fluid stream is broken up into droplets by a mechanical vibration.
The droplets are then electrically charged according to the characteristics of the cell contained
within the droplet. Depending on their charge, the droplets are finally deflected by an electric field into
different containers.
Time-lapse cytometers
A key characteristic of time-lapse cytometers is their use of non heat-generating light sources such as
light-emitting diode
A light-emitting diode (LED) is a semiconductor device that emits light when current flows through it. Electrons in the semiconductor recombine with electron holes, releasing energy in the form of photons. The color of the light (cor ...
s.
This allows a time-lapse cytometer to be placed inside a conventional
cell culture incubator
to facilitate continuous observation of cellular processes without heat building up inside the incubator.
History
Hemocytometer
The early history of cytometry is closely associated with the development of the blood cell counting.
Through the work of
Karl von Vierordt
Karl von Vierordt (July 1, 1818 – November 22, 1884) was a German physiologist.
Vierordt was born in Lahr, Baden. He studied at the universities of Berlin, Göttingen, Vienna, and Heidelberg, and began a practice in Karlsruhe in 1842. In ...
,
Louis-Charles Malassez
Louis-Charles Malassez (21 September 1842 – 22 December 1909) was a French anatomist and histologist born in Nevers, department of Nièvre.
He studied medicine in Paris, where he worked as an ''interne'' from 1867. He served with the 5th Ambul ...
,
Karl Bürker and others blood cell
concentration could by the late 19th century be accurately measured using a blood cell counting chamber,
the
hemocytometer
The hemocytometer (or haemocytometer) is a counting-chamber device originally designed and usually used for counting blood cells.
The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a ...
, and an
optical microscope
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microsco ...
.
Until the 1950s the hemocytometer was the standard method to count blood cells.
In blood cell counting applications the hemocytometer has now been replaced by
electronic cell counters.
However, the hemocytometer is still being used to count cells in cell culture laboratories.
Successively the manual task of counting, using a microscope, is taken over by small automated image cytometers.
Fluorescence microscope
In 1904,
Moritz von Rohr
Moritz von Rohr (4 April 1868 – 20 June 1940) was an optical scientist at Carl Zeiss in Jena, Germany.
A street in Jena is named after him: Moritz-von-Rohr-Straße, near Carl-Zeiss-Promenade and Otto-Schott-Straße.
Life
Moritz von Ro ...
and
August Köhler
August Karl Johann Valentin Köhler (4 March 1866 – 12 March 1948) was a German professor and early staff member of Carl Zeiss AG in Jena, Germany. He is best known for his development of the microscopy technique of Köhler illumination, an imp ...
at
Carl Zeiss
Carl Zeiss (; 11 September 1816 – 3 December 1888) was a German scientific instrument maker, optician and businessman. In 1846 he founded his workshop, which is still in business as Carl Zeiss AG. Zeiss gathered a group of gifted practica ...
in Jena constructed the first ultraviolet microscope.
The intent of the microscope was to obtain higher optical resolution by using illumination with a shorter wavelength than visual light.
However, they experienced difficulties with
autofluorescence
Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores) ...
when observing biological material. Fortunately,
Köhler saw the potential of fluorescence.
A filtering technique for fluorescence excitation light was developed by
Heinrich Lehmann at Zeiss in 1910, based on work by
Robert Wood Robert Wood may refer to:
Art
* Robert E. Wood (painter, born 1971), Canadian landscape artist
* Robert William Wood (1889–1979), American landscape artist
* Robert Wood (artist), accused and acquitted of the Camden Town murder
Military
* R ...
. However, the "Lumineszenzmikroskop" he developed was only second on the market, after the one independently developed by
Oskar Heimstädt who worked at C Reichert, Optische Werke AG in Vienna, which today is a part of
Leica Microsystems
Leica Microsystems GmbH is a German microscope manufacturing company. It is a manufacturer of optical microscopes, equipment for the preparation of microscopic specimens and related products. There are ten plants in eight countries with distribu ...
.
Cytophotometry
By the early 1930s various firms manufactured ultraviolet fluorescent microscopes. The stage was
set for cytometry to now go beyond the now established hemocytometer. At this time,
Torbjörn Caspersson
Torbjörn Oskar Caspersson (15 October 1910 – 7 December 1997) was a Swedish cytologist and geneticist. He was born in Motala and attended the University of Stockholm, where he studied medicine and biophysics.
Contributions
Caspersson made sev ...
,
working at the Karolinska Institute in Stockholm, developed a series of progressively more sophisticated
instruments called ''cytophotometers''. These instruments combined a fluorescent microscope with a
spectrophotometer
to quantify cellular nucleic acids and their relation to cell growth and function. Caspersson’s early
apparatus now seems hopelessly primitive. But, even this primitive apparatus got results, and attracted
the attention of other researchers. Many of the advances in analytical cytology from the 1940s and on-wards
were made by people who made the pilgrimage to Stockholm.
Pulse cytophotometry
![Model A COULTER COUNTER from Advertisement](https://upload.wikimedia.org/wikipedia/commons/7/70/Model_A_COULTER_COUNTER_from_Advertisement.jpg)
The first attempts to automate cell counting were made around World War II.
Gucker et al. builds a device to detect bacteria in aerosols. Lagercrantz builds an automated
cell counter based on microscopy and identifies the difficulties
in aligning cells to be individually counted using microscopy, as Moldavan had proposed in 1934.
Joseph and
Wallace Coulter circumnavigates these difficulties by inventing the principle
of using electrical impedance to count and size microscopic particles suspended in a fluid.
This principle is today
known as the
Coulter principle
A Coulter counter is an apparatus for counting and sizing particles suspended in electrolytes. The Coulter counter is the commercial term for the technique known as resistive pulse sensing or electrical zone sensing, the apparatus is based on ...
and was used in the automated blood cell counter released by
Coulter Electronics
in 1954. The “
Coulter counter
A Coulter counter is an apparatus for counting and sizing particles suspended in electrolytes. The Coulter counter is the commercial term for the technique known as resistive pulse sensing or electrical zone sensing, the apparatus is based on T ...
” was the first commercial flow cytometer.
During the 1960s Dittrich, Göhde and Kamentsky improves the design pioneered by Caspersson 30 years earlier.
Dittrich and Göhde’s ''pulse cytophotometer'' was built around a Zeiss fluorescent microscope and commercialized
as the ICP 11 b
Partec GmbHin 1968.
Kamentsky’s device was commercialized by Bio/Physics Systems Inc. as the Cytograph in 1970.
These devices were able to count cells, like the earlier Coulter counter. But more importantly, they were also capable of measuring cellular characteristics.
However, these early cytophotometers where microscopy-based.
Flow cytometry
In 1953 Crosland-Taylor published an unsuccessful attempt to count red blood cells using microscopy in which he solved the problem of aligning cells by using
sheath fluid to
hydrodynamically focus the cells.
In the late 1960s, Van Dilla at
Los Alamos National Laboratory
Los Alamos National Laboratory (often shortened as Los Alamos and LANL) is one of the sixteen research and development laboratories of the United States Department of Energy (DOE), located a short distance northwest of Santa Fe, New Mexico, ...
built the first non microscopy-based cytophotometer. He did this by combining Crosland-Taylor's breakthrough with the fluorescent dyes originally developed for microscopy and a laser-based fluorescent detection system — the flow cytometer as we know it today. Fulwyler, at Los Alamos as well, combines the Coulter principle with
continuous inkjet printer technology to create the first cell sorter in 1965.
In 1973 Steinkamp and the team at Los Alamos follow up with a fluorescence-based cell sorter.
In 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name ''pulse cytophotometry''
was changed to ''flow cytometry'', a term which quickly became popular.
At that point pulse cytophotometry had evolved
into the modern form of flow cytometry, pioneered by Van Dilla ten years earlier.
See also
*
Mass cytometry
Mass cytometry is a mass spectrometry technique based on inductively coupled plasma mass spectrometry and time of flight mass spectrometry used for the determination of the properties of cells (cytometry). In this approach, antibodies are conju ...
References
External links
Cytometry Volume 10by Purdue University Cytometry Laboratories
{{DEFAULTSORT:Cytometry
Blood tests
Medical tests
Cell biology
Clinical pathology
Flow cytometry
Laboratory techniques
Laboratory equipment
Biological techniques and tools