Autophagy
Autophagy is the natural, regulated mechanism of the cell that disassembles unnecessary or dysfunctional components. It allows the orderly recycling of cellular components. Three forms of autophagy are described: macroautophagy and chaperone-mediated autophagy. In macroautophagy, targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome; the autophagosome fuses with lysosomes and the contents are degraded and recycled. In disease, autophagy has been seen as an adaptive response to stress, which promotes survival, whereas in other cases it appears to promote cell death and morbidity. In the extreme case of starvation, the breakdown of cellular components promotes cellular survival by maintaining cellular energy levels; the name "autophagy" was coined by Belgian biochemist Christian de Duve in 1963 based on his discovery of the functions of lysosome. The identification of autophagy-related genes in yeast in the 1990s allowed researchers to deduce the mechanisms of autophagy, which led to the award of the 2016 Nobel Prize in Physiology or Medicine to Japanese researcher Yoshinori Ohsumi.
Autophagy was first observed by Keith R. Porter and his student Thomas Ashford at the Rockefeller Institute. In January 1962 they reported an increased number of lysosomes in rat liver cells after the addition of glucagon, that some displaced lysosomes towards the centre of the cell contained other cell organelles such as mitochondria, they called this autolysis after Christian de Duve and Alex B. Novikoff; however Porter and Ashford wrongly interpreted their data as lysosome formation. Lysosomes could not be cell organelles, but part of cytoplasm such as mitochondria, that hydrolytic enzymes were produced by microbodies. In 1963 Hruban and colleagues published a detailed ultrastructural description of "focal cytoplasmic degradation," which referenced a 1955 German study of injury-induced sequestration. Hruban and colleagues recognized three continuous stages of maturation of the sequestered cytoplasm to lysosomes, that the process was not limited to injury states that functioned under physiological conditions for "reutilization of cellular materials," and the "disposal of organelles" during differentiation.
Inspired by this discovery, de Duve christened the phenomena "autophagy". Unlike Porter and Ashford, de Duve conceived the term as a part of lysosomal function while describing the role of glucagon as a major inducer of cell degradation in the liver. With his student Russell Deter, he established that lysosomes are responsible for glucagon-induced autophagy; this was the first time the fact that lysosomes are the sites of intracellular autophagy are established. In the 1990s several groups of scientists independently discovered autophagy-related genes using the budding yeast. Notably, Yoshinori Ohsumi and Michael Thumm examined starvation-induced non-selective autophagy, they soon found that they were in fact looking at the same pathway, just from different angles. The genes discovered by these and other yeast groups were given different names. A unified nomenclature was advocated in 2003 by the yeast researchers to use ATG to denote autophagy genes; the 2016 Nobel Prize in Physiology or Medicine was awarded to Yoshinori Ohsumi, although some have pointed out that the award could have been more inclusive.
The field of autophagy research experienced accelerated growth at the turn of the 21st century. Knowledge of ATG genes provided scientists more convenient tools to dissect functions of autophagy in human health and disease. In 1999, a landmark discovery connecting autophagy with cancer was published by Beth Levine's group. To this date, relationship between cancer and autophagy continues to be a main theme of autophagy research; the roles of autophagy in neurodegeneration and immune defense received considerable attention. In 2003, the first Gordon Research Conference on autophagy was held at Waterville. In 2005, Daniel J Klionsky launched a scientific journal dedicated to this field; the first Keystone Symposia Conference on autophagy was held in 2007 at Monterey. In 2008, Carol A Mercer created a BHMT fusion protein, which showed starvation-induced site-specific fragmentation in cell lines; the degradation of betaine homo-cysteine methyltransferase, a metabolic enzyme, could be used to assess autophagy flux in mammalian cells.
There are three main types of autophagy, namely macroautophagy and Chaperone mediated autophagy. They are mediated by their associated enzymes. Macroautophagy is divided into bulk and selective autophagy. In the selective autophagy is the autophagy of organelles. Macroautophagy is the main pathway, used to eradicate damaged cell organelles or unused proteins. First the phagophore engulfs the material that needs to be degraded, which forms a double membrane known as an autophagosome, around the organelle marked for destruction; the autophagosome travels through the cytoplasm of the cell to a lysosome, the two organelles fuse. Within the lysosome, the contents of the autophagosome are degraded via acidic lysosomal hydrolase. Microautophagy, on the other hand, involves the direct engulfment of cytoplasmic material into the lysosome; this occurs by invagination, meaning the inward fold
Protein
Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, transporting molecules from one location to another. Proteins differ from one another in their sequence of amino acids, dictated by the nucleotide sequence of their genes, which results in protein folding into a specific three-dimensional structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are considered to be proteins and are called peptides, or sometimes oligopeptides; the individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, encoded in the genetic code.
In general, the genetic code specifies 20 standard amino acids. Shortly after or during synthesis, the residues in a protein are chemically modified by post-translational modification, which alters the physical and chemical properties, stability and the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can work together to achieve a particular function, they associate to form stable protein complexes. Once formed, proteins only exist for a certain period and are degraded and recycled by the cell's machinery through the process of protein turnover. A protein's lifespan covers a wide range, they can exist for years with an average lifespan of 1 -- 2 days in mammalian cells. Abnormal or misfolded proteins are degraded more either due to being targeted for destruction or due to being unstable. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in every process within cells.
Many proteins are enzymes that are vital to metabolism. Proteins have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, the cell cycle. In animals, proteins are needed in the diet to provide the essential amino acids that cannot be synthesized. Digestion breaks the proteins down for use in the metabolism. Proteins may be purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation and chromatography. Methods used to study protein structure and function include immunohistochemistry, site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance and mass spectrometry. Most proteins consist of linear polymers built from series of up to 20 different L-α- amino acids. All proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group, a carboxyl group, a variable side chain are bonded.
Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation. The side chains of the standard amino acids, detailed in the list of standard amino acids, have a great variety of chemical structures and properties; the amino acids in a polypeptide chain are linked by peptide bonds. Once linked in the protein chain, an individual amino acid is called a residue, the linked series of carbon and oxygen atoms are known as the main chain or protein backbone; the peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone; the end with a free amino group is known as the N-terminus or amino terminus, whereas the end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus.
The words protein and peptide are a little ambiguous and can overlap in meaning. Protein is used to refer to the complete biological molecule in a stable conformation, whereas peptide is reserved for a short amino acid oligomers lacking a stable three-dimensional structure. However, the boundary between the two is not well defined and lies near 20–30 residues. Polypeptide can refer to any single linear chain of amino acids regardless of length, but implies an absence of a defined conformation. Proteins can interact with many types of molecules, including with other proteins, with lipids, with carboyhydrates, with DNA, it has been estimated. Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on the order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more pro
Micrograph
A micrograph or photomicrograph is a photograph or digital image taken through a microscope or similar device to show a magnified image of an object. This is opposed to a macrograph or photomacrograph, an image, taken on a microscope but is only magnified less than 10 times. Micrography is the art of using microscopes to make photographs. A micrograph contains extensive details of microstructure. A wealth of information can be obtained from a simple micrograph like behavior of the material under different conditions, the phases found in the system, failure analysis, grain size estimation, elemental analysis and so on. Micrographs are used in all fields of microscopy. A light micrograph or photomicrograph is a micrograph prepared using an optical microscope, a process referred to as photomicroscopy. At a basic level, photomicroscopy may be performed by connecting a camera to a microscope, thereby enabling the user to take photographs at reasonably high magnification. Scientific use began in England in 1850 by Prof Richard Hill Norris FRSE for his studies of blood cells.
Roman Vishniac was a pioneer in the field of photomicroscopy, specializing in the photography of living creatures in full motion. He made major developments in light-interruption photography and color photomicroscopy. Photomicrographs may be obtained using a USB microscope attached directly to a home computer or laptop. An electron micrograph is a micrograph prepared using an electron microscope. Micrographs have micron bars, or magnification ratios, or both. Magnification is a ratio between the size of an object on its real size. Magnification can be a misleading parameter as it depends on the final size of a printed picture and therefore varies with picture size. A scale bar, or micron bar, is a line of known length displayed on a picture; the bar can be used for measurements on a picture. When the picture is resized the bar is resized making it possible to recalculate the magnification. Ideally, all pictures destined for publication/presentation should be supplied with a scale bar. All but one of the micrographs presented on this page do not have a micron bar.
The microscope has been used for scientific discovery. It has been linked to the arts since its invention in the 17th century. Early adopters of the microscope, such as Robert Hooke and Antonie van Leeuwenhoek, were excellent illustrators. After the invention of photography in the 1820s the microscope was combined with the camera to take pictures instead of relying on an artistic rendering. Since the early 1970s individuals have been using the microscope as an artistic instrument. Websites and traveling art exhibits such as the Nikon Small World and Olympus Bioscapes have featured a range of images for the sole purpose of artistic enjoyment; some collaborative groups, such as the Paper Project have incorporated microscopic imagery into tactile art pieces as well as 3D immersive rooms and dance performances. Close-up Digital microscope Macro photography Microphotograph Microscopy USB microscope Make a Micrograph – This presentation by the research department of Children's Hospital Boston shows how researchers create a three-color micrograph.
Shots with a Microscope – a basic, comprehensive guide to photomicrography Scientific photomicrographs – free scientific quality photomicrographs by Doc. RNDr. Josef Reischig, CSc. Micrographs of 18 natural fibres by the International Year of Natural Fibres 2009 Seeing Beyond the Human Eye Video produced by Off Book - Solomon C. Fuller bio Charles Krebs Microscopic Images Dennis Kunkel Microscopy Andrew Paul Leonard, APL Microscopic Cell Centered Database - Montage Nikon Small World Olympus Bioscapes Other examples
Intercalation (biochemistry)
In biochemistry, intercalation is the insertion of molecules between the planar bases of deoxyribonucleic acid. This process is used as a method for analyzing DNA and it is the basis of certain kinds of poisoning. There are several ways molecules can interact with DNA. Ligands may interact with DNA by electrostatically binding, or intercalating. Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA; these ligands are polycyclic and planar, therefore make good nucleic acid stains. Intensively studied DNA intercalators include berberine, ethidium bromide, daunomycin and thalidomide. DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in growing cancer cells. Examples include doxorubicin and daunorubicin, dactinomycin. Metallointercalators are complexes of a metal cation with polycyclic aromatic ligands; the most used metal ion is ruthenium, because its complexes are slow to decompose in the biological environment.
Other metallic cations that have been used include iridium. Typical ligands attached to the metal ion are dipyridine and terpyridine whose planar structure is ideal for intercalation. In order for an intercalator to fit between base pairs, the DNA must dynamically open a space between its base pairs by unwinding; the degree of unwinding varies depending on the intercalator. This unwinding causes creating an opening of about 0.34 nm. This unwinding induces local structural changes to the DNA strand, such as lengthening of the DNA strand or twisting of the base pairs; these structural modifications can lead to functional changes to the inhibition of transcription and replication and DNA repair processes, which makes intercalators potent mutagens. For this reason, DNA intercalators are carcinogenic, such as the exo 8,9 epoxide of aflatoxin B1 and acridines such as proflavine or quinacrine. Intercalation as a mechanism of interaction between cationic, polycyclic aromatic systems of the correct size was first proposed by Leonard Lerman in 1961.
One proposed mechanism of intercalation is as follows: In aqueous isotonic solution, the cationic intercalator is attracted electrostatically to the surface of the polyanionic DNA. The ligand displaces a sodium and/or magnesium cation present in the "condensation cloud" of such cations that surrounds DNA, thus forming a weak electrostatic association with the outer surface of DNA. From this position, the ligand diffuses along the surface of the DNA and may slide into the hydrophobic environment found between two base pairs that may transiently "open" to form an intercalation site, allowing the ethidium to move away from the hydrophilic environment surrounding the DNA and into the intercalation site; the base pairs transiently form such openings due to energy absorbed during collisions with solvent molecules. Molecular tweezers Intercalation
Eukaryote
Eukaryotes are organisms whose cells have a nucleus enclosed within membranes, unlike prokaryotes, which have no membrane-bound organelles. Eukaryotes belong to Eukarya, their name comes from the Greek εὖ and κάρυον. Eukaryotic cells contain other membrane-bound organelles such as mitochondria and the Golgi apparatus, in addition, some cells of plants and algae contain chloroplasts. Unlike unicellular archaea and bacteria, eukaryotes may be multicellular and include organisms consisting of many cell types forming different kinds of tissue. Animals and plants are the most familiar eukaryotes. Eukaryotes can reproduce both asexually through mitosis and sexually through meiosis and gamete fusion. In mitosis, one cell divides to produce two genetically identical cells. In meiosis, DNA replication is followed by two rounds of cell division to produce four haploid daughter cells; these act as sex cells. Each gamete has just one set of chromosomes, each a unique mix of the corresponding pair of parental chromosomes resulting from genetic recombination during meiosis.
The domain Eukaryota appears to be monophyletic, makes up one of the domains of life in the three-domain system. The two other domains and Archaea, are prokaryotes and have none of the above features. Eukaryotes represent a tiny minority of all living things. However, due to their much larger size, their collective worldwide biomass is estimated to be about equal to that of prokaryotes. Eukaryotes evolved 1.6–2.1 billion years ago, during the Proterozoic eon. The concept of the eukaryote has been attributed to the French biologist Edouard Chatton; the terms prokaryote and eukaryote were more definitively reintroduced by the Canadian microbiologist Roger Stanier and the Dutch-American microbiologist C. B. van Niel in 1962. In his 1937 work Titres et Travaux Scientifiques, Chatton had proposed the two terms, calling the bacteria prokaryotes and organisms with nuclei in their cells eukaryotes; however he mentioned this in only one paragraph, the idea was ignored until Chatton's statement was rediscovered by Stanier and van Niel.
In 1905 and 1910, the Russian biologist Konstantin Mereschkowski argued that plastids were reduced cyanobacteria in a symbiosis with a non-photosynthetic host, itself formed by symbiosis between an amoeba-like host and a bacterium-like cell that formed the nucleus. Plants had thus inherited photosynthesis from cyanobacteria. In 1967, Lynn Margulis provided microbiological evidence for endosymbiosis as the origin of chloroplasts and mitochondria in eukaryotic cells in her paper, On the origin of mitosing cells. In the 1970s, Carl Woese explored microbial phylogenetics, studying variations in 16S ribosomal RNA; this helped to uncover the origin of the eukaryotes and the symbiogenesis of two important eukaryote organelles and chloroplasts. In 1977, Woese and George Fox introduced a "third form of life", which they called the Archaebacteria. In 1979, G. W. Gould and G. J. Dring suggested that the eukaryotic cell's nucleus came from the ability of Gram-positive bacteria to form endospores. In 1987 and papers, Thomas Cavalier-Smith proposed instead that the membranes of the nucleus and endoplasmic reticulum first formed by infolding a prokaryote's plasma membrane.
In the 1990s, several other biologists proposed endosymbiotic origins for the nucleus reviving Mereschkowski's theory. Eukaryotic cells are much larger than those of prokaryotes having a volume of around 10,000 times greater than the prokaryotic cell, they have a variety of internal membrane-bound structures, called organelles, a cytoskeleton composed of microtubules and intermediate filaments, which play an important role in defining the cell's organization and shape. Eukaryotic DNA is divided into several linear bundles called chromosomes, which are separated by a microtubular spindle during nuclear division. Eukaryote cells include a variety of membrane-bound structures, collectively referred to as the endomembrane system. Simple compartments, called vesicles and vacuoles, can form by budding off other membranes. Many cells ingest food and other materials through a process of endocytosis, where the outer membrane invaginates and pinches off to form a vesicle, it is probable that most other membrane-bound organelles are derived from such vesicles.
Alternatively some products produced by the cell can leave in a vesicle through exocytosis. The nucleus is surrounded with pores that allow material to move in and out. Various tube- and sheet-like extensions of the nuclear membrane form the endoplasmic reticulum, involved in protein transport and maturation, it includes the rough endoplasmic reticulum where ribosomes are attached to synthesize proteins, which enter the interior space or lumen. Subsequently, they enter vesicles, which bud off from the smooth endoplasmic reticulum. In most eukaryotes, these protein-carrying vesicles are released and further modified in stacks of flattened vesicles, the Golgi apparatus. Vesicles may be specialized for various purposes. For instance, lysosomes contain digestive enzymes that break down most biomolecules in the cytoplasm. Peroxisomes are used to break down peroxide, otherwise toxic. Many protozoans have contractile vacuoles, which collect and expel excess water, extrusomes, which expel material used to deflect predators or capture prey.
In higher plants, most of a cell's volume is taken up by a central vacuole, whi
Optical microscope
The optical microscope referred to as the light microscope, is a type of microscope that uses visible light and a system of lenses to magnify images of small objects. Optical microscopes are the oldest design of microscope and were invented in their present compound form in the 17th century. Basic optical microscopes can be simple, although many complex designs aim to improve resolution and sample contrast. Used in the classroom and at home unlike the electron microscope, used for closer viewing; the image from an optical microscope can be captured by normal, photosensitive cameras to generate a micrograph. Images were captured by photographic film, but modern developments in CMOS and charge-coupled device cameras allow the capture of digital images. Purely digital microscopes are now available which use a CCD camera to examine a sample, showing the resulting image directly on a computer screen without the need for eyepieces. Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy and scanning probe microscopy.
On 8 October 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner and Stefan Hell for "the development of super-resolved fluorescence microscopy," which brings "optical microscopy into the nanodimension". There are two basic types of optical microscopes: compound microscopes. A simple microscope is one. A compound microscope uses several lenses to enhance the magnification of an object; the vast majority of modern research microscopes are compound microscopes while some cheaper commercial digital microscopes are simple single lens microscopes. Compound microscopes can be further divided into a variety of other types of microscopes which differ in their optical configurations and intended purposes. A regular microscope uses a lens or set of lenses to enlarge an object through angular magnification alone, giving the viewer an erect enlarged virtual image; the use of a single convex lens or groups of lenses are found in simple magnification devices such as the magnifying glass and eyepieces for telescopes and microscopes.
A compound microscope uses a lens close to the object being viewed to collect light which focuses a real image of the object inside the microscope. That image is magnified by a second lens or group of lenses that gives the viewer an enlarged inverted virtual image of the object; the use of a compound objective/eyepiece combination allows for much higher magnification. Common compound microscopes feature exchangeable objective lenses, allowing the user to adjust the magnification. A compound microscope enables more advanced illumination setups, such as phase contrast. There are many variants of the compound optical microscope design for specialized purposes; some of these are physical design differences allowing specialization for certain purposes: Stereo microscope, a low-powered microscope which provides a stereoscopic view of the sample used for dissection. Comparison microscope, which has two separate light paths allowing direct comparison of two samples via one image in each eye. Inverted microscope, for studying samples from below.
Fiber optic connector inspection microscope, designed for connector end-face inspection Traveling microscope, for studying samples of high optical resolution. Other microscope variants are designed for different illumination techniques: Petrographic microscope, whose design includes a polarizing filter, rotating stage and gypsum plate to facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation. Polarizing microscope, similar to the petrographic microscope. Phase contrast microscope, which applies the phase contrast illumination method. Epifluorescence microscope, designed for analysis of samples which include fluorophores. Confocal microscope, a used variant of epifluorescent illumination which uses a scanning laser to illuminate a sample for fluorescence. Two-photon microscope, used to image fluorescence deeper in scattering media and reduce photobleaching in living samples. Student microscope – an low-power microscope with simplified controls and sometimes low quality optics designed for school use or as a starter instrument for children.
Ultramicroscope, an adapted light microscope that uses light scattering to allow viewing of tiny particles whose diameter is below or near the wavelength of visible light. Microscopes can be or wholly computer-controlled with various levels of automation. Digital microscopy allows greater analysis of a microscope image, for example measurements of distances and areas and quantitaton of a fluorescent or histological stain. Low-powered digital microscopes, USB microscopes, are commercially available; these are webcams with a high-powered macro lens and do not use transillumination. The camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor, they offer modest magnifications without the need to use eyepieces, at low cost. High power illumination is provided by an LED source or sources adjacent to the camera lens. Digital microscopy with low light levels to avoid damage to vulnerable biological samples is available using sensitive photon-counting digital
Fluorescence microscope
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image. On 8 October 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner, Stefan Hell for "the development of super-resolved fluorescence microscopy," which brings "optical microscopy into the nanodimension"; the specimen is illuminated with light of a specific wavelength, absorbed by the fluorophores, causing them to emit light of longer wavelengths. The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter.
Typical components of a fluorescence microscope are a light source, the excitation filter, the dichroic mirror, the emission filter. The filters and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. In this manner, the distribution of a single fluorophore is imaged at a time. Multi-color images of several types of fluorophores must be composed by combining several single-color images. Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path; these microscopes are used in biology and are the basis for more advanced microscope designs, such as the confocal microscope and the total internal reflection fluorescence microscope. The majority of fluorescence microscopes those used in the life sciences, are of the epifluorescence design shown in the diagram. Light of the excitation wavelength illuminates the specimen through the objective lens.
The fluorescence emitted by the specimen is focused to the detector by the same objective, used for the excitation which for greater resolution will need objective lens with higher numerical aperture. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence method therefore gives a high signal-to-noise ratio; the dichroic beamsplitter acts as a wavelength specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any remaining excitation light back towards the source. Fluorescence microscopy requires intense, near-monochromatic, illumination which some widespread light sources, like halogen lamps cannot provide. Four main types of light source are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, supercontinuum sources, high-power LEDs. Lasers are most used for more complex fluorescence microscopy techniques like confocal microscopy and total internal reflection fluorescence microscopy while xenon lamps, mercury lamps, LEDs with a dichroic excitation filter are used for widefield epifluorescence microscopes.
By placing two microlens arrays into the illumination path of a widefield epifluorescence microscope uniform illumination with a coefficient of variation of 1-2% can be achieved. In order for a sample to be suitable for fluorescence microscopy it must be fluorescent. There are several methods of creating a fluorescent sample. Alternatively the intrinsic fluorescence of a sample can be used. In the life sciences fluorescence microscopy is a powerful tool which allows the specific and sensitive staining of a specimen in order to detect the distribution of proteins or other molecules of interest; as a result, there is a diverse range of techniques for fluorescent staining of biological samples. Many fluorescent stains have been designed for a range of biological molecules; some of these are small molecules which are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains such as DAPI and Hoechst and DRAQ5 and DRAQ7 which all bind the minor groove of DNA, thus labeling the nuclei of cells.
Others are drugs or toxins which bind specific cellular structures and have been derivatised with a fluorescent reporter. A major example of this class of fluorescent stain is phalloidin, used to stain actin fibres in mammalian cells. There are many fluorescent molecules called fluorophores or fluorochromes such as fluorescein, Alexa Fluors, or DyLight 488, which can be chemically linked to a different molecule which binds the target of interest within the sample. Immunofluorescence is a technique which uses the specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the molecule of interest. A fluorophore can be directly conjugated to the primary antibody. Alternatively a secondary antibody, conjugated to a fluorophore, which binds to the first antibody can be used. For example, a primary antibody raised in a mouse which recognises tubulin combined with a secondary anti-
