Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument; the sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are labeled with fluorescent markers so that light is first absorbed and emitted in a band of wavelengths. Tens of thousands of cells can be examined and the data gathered are processed by a computer. Flow cytometry is used in basic research, clinical practice, clinical trials. Uses for flow cytometry include: Cell counting Cell sorting Determining cell characteristics and function Detecting microorganisms Biomarker detection Protein engineering detection Diagnosis of health disorders such as blood cancersA flow cytometry analyzer is an instrument that provides quantifiable data from a sample.
Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties. The first impedance-based flow cytometry device, using the Coulter principle, was disclosed in U. S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. Mack Fulwyler was the inventor of the forerunner to today's flow cytometers - the cell sorter. Fulwyler developed this in 1965 with his publication in Science; the first fluorescence-based flow cytometry device was developed in 1968 by Wolfgang Göhde from the University of Münster, filed for patent on 18 December 1968 and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time, absorption methods were still favored by other scientists over fluorescence methods. Soon after, flow cytometry instruments were developed, including the Cytofluorograph from Bio/Physics Systems Inc. the PAS 8000 from Partec, the first FACS instrument from Becton Dickinson, the ICP 22 from Partec/Phywe and the Epics from Coulter.
The first label-free high-frequency impedance flow cytometer based on a patented microfluidic "lab-on-chip", Ampha Z30, was introduced by Amphasys. The original name of the fluorescence-based flow cytometry technology was "pulse cytophotometry", based on the first patent application on fluorescence-based flow cytometry. At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola in 1976 - eight years after the introduction of the first fluorescence-based flow cytometer - it was agreed to use the name "flow cytometry", a term that became popular. Modern flow cytometers are able to analyze many thousand particles per second, in "real time," and, if configured as cell sorters, can separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers high-throughput, automated quantification of specified optical parameters on a cell-by-cell basis.
To analyze solid tissues, a single-cell suspension must first be prepared. A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, a computer for analysis of the signals; the flow cell has a liquid stream, which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system use measurement of impedance and optical systems - lamps; the detector and analog-to-digital conversion system converts analog measurements of forward-scattered light and side-scattered light as well as dye-specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be logarithmic; the process of collecting data from samples using the flow cytometer is termed'acquisition'. Acquisition is mediated by a computer physically connected to the flow cytometer, the software which handles the digital interface with the cytometer; the software is capable of adjusting parameters for the sample being tested, assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly.
Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in acquisition such as fluorescently labeled antibodies has developed. Modern instruments have multiple lasers and fluorescence detectors; the current record for a commercial instrument is 30 fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, can more identify a target population by their phenotypic markers. Certain instruments can take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells. Cells must pass uniformly through the center of focused laser beams to measure optical properties of cells in any flow cytometer; the purpose of the fluidic system is to move the cells one by one through the lasers beam an
Red blood cell
Red blood cells known as RBCs, red cells, red blood corpuscles, erythroid cells or erythrocytes, are the most common type of blood cell and the vertebrate's principal means of delivering oxygen to the body tissues—via blood flow through the circulatory system. RBCs take up oxygen in the lungs, or gills of fish, release it into tissues while squeezing through the body's capillaries; the cytoplasm of erythrocytes is rich in hemoglobin, an iron-containing biomolecule that can bind oxygen and is responsible for the red color of the cells and the blood. The cell membrane is composed of proteins and lipids, this structure provides properties essential for physiological cell function such as deformability and stability while traversing the circulatory system and the capillary network. In humans, mature red blood cells are oval biconcave disks, they lack most organelles, in order to accommodate maximum space for hemoglobin. 2.4 million new erythrocytes are produced per second in human adults. The cells develop in the bone marrow and circulate for about 100–120 days in the body before their components are recycled by macrophages.
Each circulation takes about 60 seconds. A quarter of the cells in the human body are red blood cells. Nearly half of the blood's volume is red blood cells. Packed red blood cells are red blood cells that have been donated and stored in a blood bank for blood transfusion. All vertebrates, including all mammals and humans, have red blood cells. Red blood cells are cells present in blood; the only known vertebrates without red blood cells are the crocodile icefish. While they no longer use hemoglobin, remnants of hemoglobin genes can be found in their genome. Vertebrate red blood cells consist of hemoglobin, a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules in the lungs or gills and release them throughout the body. Oxygen can diffuse through the red blood cell's cell membrane. Hemoglobin in the red blood cells carries some of the waste product carbon dioxide back from the tissues. Myoglobin, a compound related to hemoglobin, acts to store oxygen in muscle cells.
The color of red blood cells is due to the heme group of hemoglobin. The blood plasma alone is straw-colored, but the red blood cells change color depending on the state of the hemoglobin: when combined with oxygen the resulting oxyhemoglobin is scarlet, when oxygen has been released the resulting deoxyhemoglobin is of a dark red burgundy color. However, blood can appear bluish when seen through skin. Pulse oximetry takes advantage of the hemoglobin color change to directly measure the arterial blood oxygen saturation using colorimetric techniques. Hemoglobin has a high affinity for carbon monoxide, forming carboxyhemoglobin, a bright red in color. Flushed, confused patients with a saturation reading of 100% on pulse oximetry are sometimes found to be suffering from carbon monoxide poisoning. Having oxygen-carrying proteins inside specialized cells was an important step in the evolution of vertebrates as it allows for less viscous blood, higher concentrations of oxygen, better diffusion of oxygen from the blood to the tissues.
The size of red blood cells varies among vertebrate species. The red blood cells of mammals are shaped as biconcave disks: flattened and depressed in the center, with a dumbbell-shaped cross section, a torus-shaped rim on the edge of the disk; this shape allows for a high surface-area-to-volume ratio to facilitate diffusion of gases. However, there are some exceptions concerning shape in the artiodactyl order, which displays a wide variety of bizarre red blood cell morphologies: small and ovaloid cells in llamas and camels, tiny spherical cells in mouse deer, cells which assume fusiform, lanceolate and irregularly polygonal and other angular forms in red deer and wapiti. Members of this order have evolved a mode of red blood cell development different from the mammalian norm. Overall, mammalian red blood cells are remarkably flexible and deformable so as to squeeze through tiny capillaries, as well as to maximize their apposing surface by assuming a cigar shape, where they efficiently release their oxygen load.
Red blood cells in mammals are unique amongst vertebrates. Red blood cells of mammals cells have nuclei during early phases of erythropoiesis, but extrude them during development as they mature; the red blood cells without nuclei, called reticulocytes, subsequently lose all other cellular organelles such as their mitochondria, Golgi apparatus and endoplasmic reticulum. The spleen acts as a reservoir of red blood cells. In some other mammals such as dogs and horses, the spl
Supravital staining is a method of staining used in microscopy to examine living cells that have been removed from an organism. It differs from intravital staining, done by injecting or otherwise introducing the stain into the body, thus a supravital stain may have a greater toxicity, as only a few cells need to survive it a short while. The term "vital stain" is used by some authors to refer to an intravital stain, by others interchangeably with a supravital stain, the core concept being that the cell being examined is still alive; as the cells are alive and unfixed, outside the body, supravital stains are temporary in nature. The most common supravital stain is performed on reticulocytes using new methylene blue or brilliant cresyl blue, which makes it possible to see the reticulofilamentous pattern of ribosomes characteristically precipitated in these live immature red blood cells by the supravital stains. By counting the number of such cells the rate of red blood cell formation can be determined, providing an insight into bone marrow activity and anemia.
This is in contrast to vital staining, when the dye employed is one, excluded from the living cells so that only dead cells are stained positively. Supravital staining can be combined with cell surface antibody staining for applications such as FACS analysis. Immunofluorescence can be done within the interior of live cells by reversible cell permeabilization using the detergent Triton X-100. Adjusted to the appropriate concentration for the number of cells, the pretreatment can permit access of molecules between 1 and 150 kilodaltons to the interior of the cell. Although antibodies may be used in a similar way in this context, the term "supravital stain" is reserved for smaller chemicals which possess suitable properties intrinsically. Supravital dyes for RBCs: New methylene blue Brilliant cresyl blue Crystal violet Methyl violet Nile blue Hoechst stain
Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development and reproduction of all known organisms and many viruses. DNA and ribonucleic acid are nucleic acids; the two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases, a sugar called deoxyribose, a phosphate group; the nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two separate polynucleotide strands are bound together, according to base pairing rules, with hydrogen bonds to make double-stranded DNA; the complementary nitrogenous bases are divided into two groups and purines. In DNA, the pyrimidines are cytosine. Both strands of double-stranded DNA store the same biological information.
This information is replicated as and when the two strands separate. A large part of DNA is non-coding, meaning that these sections do not serve as patterns for protein sequences; the two strands of DNA are thus antiparallel. Attached to each sugar is one of four types of nucleobases, it is the sequence of these four nucleobases along the backbone. RNA strands are created using DNA strands as a template in a process called transcription. Under the genetic code, these RNA strands specify the sequence of amino acids within proteins in a process called translation. Within eukaryotic cells, DNA is organized into long structures called chromosomes. Before typical cell division, these chromosomes are duplicated in the process of DNA replication, providing a complete set of chromosomes for each daughter cell. Eukaryotic organisms store most of their DNA inside the cell nucleus as nuclear DNA, some in the mitochondria as mitochondrial DNA, or in chloroplasts as chloroplast DNA. In contrast, prokaryotes store their DNA only in circular chromosomes.
Within eukaryotic chromosomes, chromatin proteins, such as histones and organize DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed. DNA was first isolated by Friedrich Miescher in 1869, its molecular structure was first identified by Francis Crick and James Watson at the Cavendish Laboratory within the University of Cambridge in 1953, whose model-building efforts were guided by X-ray diffraction data acquired by Raymond Gosling, a post-graduate student of Rosalind Franklin. DNA is used by researchers as a molecular tool to explore physical laws and theories, such as the ergodic theorem and the theory of elasticity; the unique material properties of DNA have made it an attractive molecule for material scientists and engineers interested in micro- and nano-fabrication. Among notable advances in this field are DNA origami and DNA-based hybrid materials. DNA is a long polymer made from repeating units called nucleotides.
The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes. In all species it is composed of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the same axis, have the same pitch of 34 angstroms; the pair of chains has a radius of 10 angstroms. According to another study, when measured in a different solution, the DNA chain measured 22 to 26 angstroms wide, one nucleotide unit measured 3.3 Å long. Although each individual nucleotide is small, a DNA polymer can be large and contain hundreds of millions, such as in chromosome 1. Chromosome 1 is the largest human chromosome with 220 million base pairs, would be 85 mm long if straightened. DNA does not exist as a single strand, but instead as a pair of strands that are held together; these two long strands coil in the shape of a double helix. The nucleotide contains both a segment of the backbone of a nucleobase. A nucleobase linked to a sugar is called a nucleoside, a base linked to a sugar and to one or more phosphate groups is called a nucleotide.
A biopolymer comprising multiple linked nucleotides is called a polynucleotide. The backbone of the DNA strand is made from alternating sugar residues; the sugar in DNA is 2-deoxyribose, a pentose sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings; these are known as the 3′-end, 5′-end carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond. When imagining DNA, each phosphoryl is considered to "belong" to the nucleotide whose 5′ carbon forms a bond therewith. Any DNA strand therefore has one end at which there is a phosphoryl attached to the 5′ carbon of a ribose and another end a
Vitamin B12 known as cobalamin, is a water-soluble vitamin, involved in the metabolism of every cell of the human body: it is a cofactor in DNA synthesis, in both fatty acid and amino acid metabolism. It is important in the normal functioning of the nervous system via its role in the synthesis of myelin, in the maturation of developing red blood cells in the bone marrow. Vitamin B12 is one of eight B vitamins, it consists of a class of chemically related compounds. It contains the biochemically rare element cobalt positioned in the center of a corrin ring; the only organisms to produce vitamin B12 are certain bacteria, archaea. Some of these bacteria are found in the soil around the grasses; because there are no common vegetable sources of the vitamin, vegans must use a supplement or fortified foods for B12 intake or risk serious health consequences. Otherwise, most omnivorous people in developed countries obtain enough vitamin B12 from consuming animal products including meat, milk and fish. Staple foods those that form part of a vegan diet, are fortified by having the vitamin added to them.
Vitamin B12 supplements are available in single multivitamin tablets. The most common cause of vitamin B12 deficiency in developed countries is impaired absorption due to a loss of gastric intrinsic factor, which must be bound to food-source B12 in order for absorption to occur. Another group affected are those on long term antacid therapy, using proton pump inhibitors, H2 blockers or other antacids; this condition may be characterised by limb neuropathy or a blood disorder called pernicious anemia, a type of megaloblastic anemia. Folate levels in the individual may affect the course of pathological changes and symptomatology. Deficiency is more after age 60, increases in incidence with advancing age. Dietary deficiency is rare in developed countries due to access to dietary meat and fortified foods, but children in some regions of developing countries are at particular risk due to increased requirements during growth coupled with lack of access to dietary B12. Other causes of vitamin B12 deficiency are much less frequent.
B12 is the most chemically complex of all the vitamins. The structure of B12 is based on a corrin ring, similar to the porphyrin ring found in heme; the central metal ion is cobalt. Four of the six coordination sites are provided by the corrin ring, a fifth by a dimethylbenzimidazole group; the sixth coordination site, the reactive center, is variable, being a cyano group, a hydroxyl group, a methyl group or a 5′-deoxyadenosyl group (here the C5′ atom of the deoxyribose forms the covalent bond with cobalt to yield the four vitamers of B12. The covalent C-Co bond is one of the first examples of carbon-metal bonds to be discovered in biology; the hydrogenases and, by necessity, enzymes associated with cobalt utilization, involve metal-carbon bonds. Vitamin B12 is a generic descriptor name referring to a collection of cobalt and corrin ring molecules which are defined by their particular vitamin function in the body. All of the substrate cobalt-corrin molecules from which B12 is made must be synthesized by bacteria.
After this synthesis is complete, the human body has the ability to convert any form of B12 to an active form, by means of enzymatically removing certain prosthetic chemical groups from the cobalt atom and replacing them with others. The four vitamers of B12 are all red-colored crystals and water solutions, due to the color of the cobalt-corrin complex. Cyanocobalamin is one form of B12 because it can be metabolized in the body to an active coenzyme form; the cyanocobalamin form of B12 does not occur in nature but is a byproduct of the fact that other forms of B12 are avid binders of cyanide which they pick up in the process of activated charcoal purification of the vitamin after it is made by bacteria in the commercial process. Since the cyanocobalamin form of B12 is easy to crystallize and is not sensitive to air-oxidation, it is used as a form of B12 for food additives and in many common multivitamins. Pure cyanocobalamin possesses the deep pink color associated with most octahedral cobalt complexes and the crystals are well formed and grown up to millimeter size.
Hydroxocobalamin is another vitamer of B12 encountered in pharmacology, but is not present in the human body. Hydroxocobalamin is sometimes denoted B12a; this is the form of B12 produced by bacteria, and, converted to cyanocobalmin in the commercial charcoal filtration step of production. Hydroxocobalamin has an avid affinity for cyanide ions and has been used as an antidote to cyanide poisoning, it is supplied in water solution for injection. Hydroxocobalamin is thought to be converted to the active enzymic forms of B12 more than cyanocobalamin, since it is little more expensive than cyanocobalamin, has longer retention times in the body, has been used for vitamin replacement in situations where added reassurance of activity is desired. Intramuscular administration of hydroxocobalamin is the preferred treatment for pediatric patients with intrinsic cobalamin metabolic diseases, for vitamin B12 deficient patients with tobacco amblyopia.
In cell biology, the nucleus is a membrane-bound organelle found in eukaryotic cells. Eukaryotes have a single nucleus, but a few cell types, such as mammalian red blood cells, have no nuclei, a few others including osteoclasts have many; the cell nucleus contains all of the cell's genome, except for a small fraction of mitochondrial DNA, organized as multiple long linear DNA molecules in a complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these chromosomes are structured in such a way to promote cell function; the nucleus maintains the integrity of genes and controls the activities of the cell by regulating gene expression—the nucleus is, the control center of the cell. The main structures making up the nucleus are the nuclear envelope, a double membrane that encloses the entire organelle and isolates its contents from the cellular cytoplasm, the nuclear matrix, a network within the nucleus that adds mechanical support, much like the cytoskeleton, which supports the cell as a whole.
Because the nuclear envelope is impermeable to large molecules, nuclear pores are required to regulate nuclear transport of molecules across the envelope. The pores cross both nuclear membranes, providing a channel through which larger molecules must be transported by carrier proteins while allowing free movement of small molecules and ions. Movement of large molecules such as proteins and RNA through the pores is required for both gene expression and the maintenance of chromosomes. Although the interior of the nucleus does not contain any membrane-bound subcompartments, its contents are not uniform, a number of nuclear bodies exist, made up of unique proteins, RNA molecules, particular parts of the chromosomes; the best-known of these is the nucleolus, involved in the assembly of ribosomes. After being produced in the nucleolus, ribosomes are exported to the cytoplasm where they translate mRNA; the nucleus was the first organelle to be discovered. What is most the oldest preserved drawing dates back to the early microscopist Antonie van Leeuwenhoek.
He observed the nucleus, in the red blood cells of salmon. Unlike mammalian red blood cells, those of other vertebrates still contain nuclei; the nucleus was described by Franz Bauer in 1804 and in more detail in 1831 by Scottish botanist Robert Brown in a talk at the Linnean Society of London. Brown was studying orchids under the microscope when he observed an opaque area, which he called the "areola" or "nucleus", in the cells of the flower's outer layer, he did not suggest a potential function. In 1838, Matthias Schleiden proposed that the nucleus plays a role in generating cells, thus he introduced the name "cytoblast", he believed that he had observed new cells assembling around "cytoblasts". Franz Meyen was a strong opponent of this view, having described cells multiplying by division and believing that many cells would have no nuclei; the idea that cells can be generated de novo, by the "cytoblast" or otherwise, contradicted work by Robert Remak and Rudolf Virchow who decisively propagated the new paradigm that cells are generated by cells.
The function of the nucleus remained unclear. Between 1877 and 1878, Oscar Hertwig published several studies on the fertilization of sea urchin eggs, showing that the nucleus of the sperm enters the oocyte and fuses with its nucleus; this was the first time. This was in contradiction to Ernst Haeckel's theory that the complete phylogeny of a species would be repeated during embryonic development, including generation of the first nucleated cell from a "monerula", a structureless mass of primordial mucus. Therefore, the necessity of the sperm nucleus for fertilization was discussed for quite some time. However, Hertwig confirmed his observation in other animal groups, including amphibians and molluscs. Eduard Strasburger produced the same results for plants in 1884; this paved the way to assign the nucleus an important role in heredity. In 1873, August Weismann postulated the equivalence of the maternal and paternal germ cells for heredity; the function of the nucleus as carrier of genetic information became clear only after mitosis was discovered and the Mendelian rules were rediscovered at the beginning of the 20th century.
The nucleus is the largest organelle in animal cells. In mammalian cells, the average diameter of the nucleus is 6 micrometres, which occupies about 10% of the total cell volume; the contents of the nucleus are held in the nucleoplasm similar to the cytoplasm in the rest of the cell. The fluid component of this is termed the nucleosol, similar to the cytosol in the cytoplasm. In most types of granulocyte, a white blood cell, the nucleus is lobated and can be bi-lobed, tri-lobed or multi-lobed; the nuclear envelope, otherwise known as nuclear membrane, consists of two cellular membranes, an inner and an outer membrane, arranged parallel to one another and separated by 10 to 50 nanometres. The nuclear envelope encloses the nucleus and separates the cell's genetic material from the surrounding cytoplasm, serving as a barrier to prevent macromolecules from diffusing between the nucleoplasm and the cytoplasm; the outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum, is studded with ribosomes.
The space between the membranes is called the perinuclear space and is continuous with the RER lumen. Nuclear pores, which provide aqueous cha
A microscope is an instrument used to see objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small structures using such an instrument. Microscopic means invisible to the eye. There are many types of microscopes, they may be grouped in different ways. One way is to describe the way the instruments interact with a sample to create images, either by sending a beam of light or electrons to a sample in its optical path, or by scanning across, a short distance from the surface of a sample using a probe; the most common microscope is the optical microscope, which uses light to pass through a sample to produce an image. Other major types of microscopes are the fluorescence microscope, the electron microscope and the various types of scanning probe microscopes. Although objects resembling lenses date back 4000 years and there are Greek accounts of the optical properties of water-filled spheres followed by many centuries of writings on optics, the earliest known use of simple microscopes dates back to the widespread use of lenses in eyeglasses in the 13th century.
The earliest known examples of compound microscopes, which combine an objective lens near the specimen with an eyepiece to view a real image, appeared in Europe around 1620. The inventor is unknown. Several revolve around the spectacle-making centers in the Netherlands including claims it was invented in 1590 by Zacharias Janssen and/or Zacharias' father, Hans Martens, claims it was invented by their neighbor and rival spectacle maker, Hans Lippershey, claims it was invented by expatriate Cornelis Drebbel, noted to have a version in London in 1619. Galileo Galilei seems to have found after 1610 that he could close focus his telescope to view small objects and, after seeing a compound microscope built by Drebbel exhibited in Rome in 1624, built his own improved version. Giovanni Faber coined the name microscope for the compound microscope Galileo submitted to the Accademia dei Lincei in 1625; the first detailed account of the microscopic anatomy of organic tissue based on the use of a microscope did not appear until 1644, in Giambattista Odierna's L'occhio della mosca, or The Fly's Eye.
The microscope was still a novelty until the 1660s and 1670s when naturalists in Italy, the Netherlands and England began using them to study biology. Italian scientist Marcello Malpighi, called the father of histology by some historians of biology, began his analysis of biological structures with the lungs. Robert Hooke's Micrographia had a huge impact because of its impressive illustrations. A significant contribution came from Antonie van Leeuwenhoek who achieved up to 300 times magnification using a simple single lens microscope, he sandwiched a small glass ball lens between the holes in two metal plates riveted together, with an adjustable-by-screws needle attached to mount the specimen. Van Leeuwenhoek re-discovered red blood cells and spermatozoa, helped popularise the use of microscopes to view biological ultrastructure. On 9 October 1676, van Leeuwenhoek reported the discovery of micro-organisms; the performance of a light microscope depends on the quality and correct use of the condensor lens system to focus light on the specimen and the objective lens to capture the light from the specimen and form an image.
Early instruments were limited until this principle was appreciated and developed from the late 19th to early 20th century, until electric lamps were available as light sources. In 1893 August Köhler developed a key principle of sample illumination, Köhler illumination, central to achieving the theoretical limits of resolution for the light microscope; this method of sample illumination produces lighting and overcomes the limited contrast and resolution imposed by early techniques of sample illumination. Further developments in sample illumination came from the discovery of phase contrast by Frits Zernike in 1953, differential interference contrast illumination by Georges Nomarski in 1955. In the early 20th century a significant alternative to the light microscope was developed, an instrument that uses a beam of electrons rather than light to generate an image; the German physicist, Ernst Ruska, working with electrical engineer Max Knoll, developed the first prototype electron microscope in 1931, a transmission electron microscope.
The transmission electron microscope works on similar principles to an optical microscope but uses electrons in the place of light and electromagnets in the place of glass lenses. Use of electrons, instead of light, allows for much higher resolution. Development of the transmission electron microscope was followed in 1935 by the development of the scanning electron microscope by Max Knoll. Although TEMs were being used for research before WWII, became popular afterwards, the SEM was not commercially available until 1965. Transmission electron microscopes became popular following the Second World War. Ernst Ruska, working at Siemens, developed the first commercial transmission electron microscope and, in the 1950s, major scientific conferences on electron microscopy started being held. In 1965, the first commercial scanning electron microscope was developed by Profess