Cytometry

Cytometry is the measurement of the characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology (shape and structure), cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm. Cytometry is used to characterize and count blood cells in common blood tests such as the complete blood count. 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 and AIDS. Cytometry is the measurement of the characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology (shape and structure), cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm. Cytometry is used to characterize and count blood cells in common blood tests such as the complete blood count. 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 and AIDS. Image cytometry is the oldest form of cytometry. Image cytometers operate by statically imaging a large number ofcells using optical microscopy. Prior to analysis, cells are commonly stained to enhance contrast or to detect specific molecules by labeling these with fluorochromes. Traditionally,cells are viewed within a hemocytometer to aid manual counting. Since the introduction of the digital camera, in the mid-1990s, the automation level ofimage 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 systems. Due to the early difficulties of automating microscopy, the flow cytometer has sincethe mid-1950s been the dominating cytometric device.Flow cytometers operate by aligning single cells using flow techniques. The cells are characterizedoptically or by the use of an electrical impedance method called the Coulter principle.To detect specific molecules when optically characterized, cells are in most cases stained with the sametype of fluorochromes that are used by image cytometers. Flow cytometers generally provideless data than image cytometers, but have a significantly higher throughput. 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.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 containedwithin the droplet. Depending on their charge, the droplets are finally deflected by an electric field intodifferent containers. A key characteristic of time-lapse cytometers is their use of non heat-generating light sources such as light-emitting diodes.This allows a time-lapse cytometer to be placed inside a conventional cell culture incubatorto facilitate continuous observation of cellular processes without heat building up inside the incubator. The early history of cytometry is closely associated with the development of the blood cell counting.Through the work of Karl von Vierordt, Louis-Charles Malassez, Karl Bürker and others blood cellconcentration could by the late 19th century be accurately measured using a blood cell counting chamber,the hemocytometer, and an optical microscope. 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. In 1904, Moritz von Rohr and August Köhler at Carl Zeiss 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 when observing biological material. Fortunately,Köhler saw the potential of fluorescence.A filtering technique for fluorescence excitation light was developed byHeinrich Lehmann at Zeiss in 1910, based on work byRobert Wood. 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.