Falck-Hillarp method of fluorescence

Last updated June 27, 2019

The Falck-Hillarp method of fluorescence (the F-H method) is a technique that makes it possible to demonstrate and study, with unique precision and susceptibility, certain monoamines, among those the three catecholamines dopamine, noradrenaline, and adrenaline, as well as serotonin and related substances.^[1]^[2]

A catecholamine is a monoamine neurotransmitter, an organic compound that has a catechol and a side-chain amine.

Dopamine is an organic chemical of the catecholamine and phenethylamine families. It functions both as a hormone and a neurotransmitter, and plays several important roles in the brain and body. It is an amine synthesized by removing a carboxyl group from a molecule of its precursor chemical L-DOPA, which is synthesized in the brain and kidneys. Dopamine is also synthesized in plants and most animals. In the brain, dopamine functions as a neurotransmitter—a chemical released by neurons to send signals to other nerve cells. The brain includes several distinct dopamine pathways, one of which plays a major role in the motivational component of reward-motivated behavior. The anticipation of most types of rewards increases the level of dopamine in the brain, and many addictive drugs increase dopamine release or block its reuptake into neurons following release. Other brain dopamine pathways are involved in motor control and in controlling the release of various hormones. These pathways and cell groups form a dopamine system which is neuromodulatory.

Adrenaline hormone, neurotransmitter and medication. Epinephrine is normally produced by both the adrenal glands and certain neurons

Adrenaline, also known as epinephrine, is a hormone, neurotransmitter, and medication. Adrenaline is normally produced by both the adrenal glands and certain neurons. It plays an important role in the fight-or-flight response by increasing blood flow to muscles, output of the heart, pupil dilation response, and blood sugar level. It does this by binding to alpha and beta receptors. It is found in many animals and some single cell organisms. Napoleon Cybulski first isolated adrenaline in 1895.

The method is based on the important and decisive discovery that these compounds are able to react with formaldehyde – in near complete absence of water – to form fluorophores, i.e. molecules that, when irradiated with light invisible to the eye, will emit visible light. This happens in a “dry” state, without extracting the monoamines from the cells during the entire procedure, a process that starts with separation of a tissue sample and ends with a thin tissue slice that can be examined in a fluorescence microscope.

Formaldehyde Widely used toxic organic compound

Formaldehyde (systematic name methanal) is a naturally occurring organic compound with the formula CH₂O (H−CHO). It is the simplest of the aldehydes (R−CHO). The common name of this substance comes from its similarity and relation to formic acid.

A fluorophore 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 several π bonds.

The F-H method allowed, for the first time, the examiner to watch these monoamines light up in the microscope and to precisely determine in which cells they were present, and thereby understanding their functions.^[3] The method was developed by Bengt Falck and Nils-Åke Hillarp in the 1960s at the Department of Histology, University of Lund. For intense neurobiological research it became possible to demonstrate the presence of monoamines in nerve cells belonging to the central and the peripheral nervous system and for the first time comprehend that these substances act as signal substances, i.e. transmitters.

Bengt Olof Torsten Falck is a Swedish scientist, who is professor emeritus at the Faculty of Medicine at Lund University, Sweden. Falck has published numerous works in the fields of histology, endocrinology and neurobiology.

Nils-Åke Hillarp was a Swedish scientist and a prominent force in research on the brain's monoamines.

The central nervous system (CNS) is the part of the nervous system consisting of the brain and spinal cord. The CNS is so named because it integrates the received information and coordinates and influences the activity of all parts of the bodies of bilaterally symmetric animals—that is, all multicellular animals except sponges and radially symmetric animals such as jellyfish—and it contains the majority of the nervous system. Many consider the retina and the optic nerve, as well as the olfactory nerves and olfactory epithelium as parts of the CNS, synapsing directly on brain tissue without intermediate ganglia. As such, the olfactory epithelium is the only central nervous tissue in direct contact with the environment, which opens up for therapeutic treatments. The CNS is contained within the dorsal body cavity, with the brain housed in the cranial cavity and the spinal cord in the spinal canal. In vertebrates, the brain is protected by the skull, while the spinal cord is protected by the vertebrae. The brain and spinal cord are both enclosed in the meninges. Within the CNS, the interneuronal space is filled with a large amount of supporting non-nervous cells called neuroglial cells.

The initial publication, written already in 1961,^[4] described a wide-ranging examination of nerves supplying a large number of organs in the body. This work validated the concept of Ulf von Euler, the Nobel prize winner, that noradrenaline is the signal substance in peripheral autonomic nerves. In the same year, this first publication was followed by an explanation of the chemical background of the F–H method.

Ulf Svante von Euler was a Swedish physiologist and pharmacologist. He shared the Nobel Prize in Physiology or Medicine in 1970 for his work on neurotransmitters.

Very thin membranes, such as the rat iris or mesentery, do not have to be sectioned for microscopic studies but may simply be spread on glass, dried, and then exposed to gaseous formaldehyde for subsequent study with a fluorescence microscope.

The publication on the chemical background was later named among "The 200 Most-Cited Papers of All Time".^{[ citation needed ]}

In 2012, the Faculty of Medicine at the University of Lund arranged a symposium “From Nerve to Pills” celebrating the 50th anniversary of the initial publication of the F-H method.

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Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy which looks at larger structures visible without a microscope. Although microscopic anatomy may be divided into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.

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 objects and structures using such an instrument. Microscopic means invisible to the eye unless aided by a microscope.

A nerve is an enclosed, cable-like bundle of nerve fibres called axons, in the peripheral nervous system. A nerve provides a common pathway for the electrochemical nerve impulses called action potentials that are transmitted along each of the axons to peripheral organs or, in the case of sensory nerves, from the periphery back to the central nervous system. Each axon within the nerve is an extension of an individual neuron, along with other supportive cells such as Schwann cells that coat the axons in myelin.

In biology, tissue is a cellular organisational level between cells and a complete organ. A tissue is an ensemble of similar cells and their extracellular matrix from the same origin that together carry out a specific function. Organs are then formed by the functional grouping together of multiple tissues.

The optical microscope, often referred to as the light microscope, is a type of microscope that commonly uses visible light and a system of lenses to magnify images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. Often used in the classroom and at home unlike the electron microscope which is used for closer viewing.

Staining is a technique used in the field of histology and microscopy to enhance contrast in samples examined under the microscopic. Stains and dyes are frequently used in and histopathology, the medical field that studies and diagnoses disease at a microscopic level. Stains may be used to define biological tissues, cell populations (classifying different blood cells, or organelles within individual cells.

A neuroeffector junction is a site where a motor neuron releases a neurotransmitter to affect a target—non-neuronal—cell. This junction functions like a synapse. However, unlike most neurons, somatic efferent motor neurons innervate skeletal muscle, and are always excitatory. Visceral efferent neurons innervate smooth muscle, cardiac muscle, and glands, and have the ability to be either excitatory or inhibitory in function. Neuroeffector junctions are known as neuromuscular junctions when the target cell is a muscle fiber.

Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue micro-fragments.

A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, scattering, reflection, 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.

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in depth. It differs from traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, as the wavelengths of the two exciting photons are longer than the wavelength of the resulting emitted light. Two-photon excitation microscopy typically uses near-infrared excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.

A malignant peripheral nerve sheath tumor (MPNST) is a form of cancer of the connective tissue surrounding nerves. Given its origin and behavior it is classified as a sarcoma. About half the cases are diagnosed in people with neurofibromatosis; the lifetime risk for an MPNST in patients with neurofibromatosis type 1 is 8–13%. MPNST with rhabdomyoblastomatous component are called malignant triton tumors.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

Vertico spatially modulated illumination (Vertico-SMI) is the fastest light microscope for the 3D analysis of complete cells in the nanometer range. It is based on two technologies developed in 1996, SMI and SPDM. The effective optical resolution of this optical nanoscope has reached the vicinity of 5 nm in 2D and 40 nm in 3D and surpasses the 200 nm resolution limit predicted by Abbe‘s law. Abbe postulated in 1873 the theoretical limit of resolution of optical microscopy.

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

The catecholamines comprise the endogenous substances dopamine, noradrenaline (norepinephrine) and adrenaline (epinephrine) as well as numerous artificially synthesized compounds such as isoprenaline. Their investigation constitutes a prominent chapter in the history of physiology, biochemistry and pharmacology. Adrenaline was the first hormone extracted from its endocrine gland and obtained in pure form, before the word hormone was coined. It was also the first hormone the structure and biosynthesis of which were clarified. Apart from acetylcholine, adrenaline and noradrenaline were the first neurotransmitters to be discovered and the first intercellular biochemical signals to be found in intracellular vesicles. The β-adrenoceptor was the first G protein-coupled receptor the gene of which was cloned. Goal-directed catecholamine research began with the preparation by George Oliver and Edward Albert Sharpey-Schafer of a pharmacologically active extract from the adrenal glands.

Calcium imaging is a scientific technique usually carried out in research which is designed to show the calcium (Ca²⁺) status of an isolated cell, tissue or medium. Calcium imaging techniques take advantage of so-called calcium indicators, fluorescent molecules that can respond to the binding of Ca²⁺ ions by changing their fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). Calcium imaging can be used to optically probe intracellular calcium in living animals. This technique has allowed studies of calcium signalling in a wide variety of cell types and neuronal activity in hundreds of neurons and glial cells within neuronal circuits.

References

↑ Falck, B. (1962). "Observations on the possibilities of the cellular localization of monoamines by a fluorescence method". Acta Physiol. Scand. 56 (suppl. 197): 1–25.
↑ Falck, B.; Hillarp, N. A.; Thieme, G.; Torp, A. (1982). "Fluorescence of catechol amines and related compounds condensed with formaldehyde". Brain Research Bulletin. 9 (1–6): xi–xv. doi:10.1016/0361-9230(82)90113-7. PMID 7172023.
↑ "The Falck-Hillarp Method - a revolution in the study of monoamine systems in the CNS". Wallenberg Neuroscience Center, University of Lund. 2019-01-09.
↑ FALCK B; TORP A (1962). "New evidence for the localization of noradrenalin in the adrenergic nerve terminals". Medicina Experimentalis. 6 (3): 169–72. doi:10.1159/000135153. PMID 13891409.

External links

The Falck-Hillarp Fluorescence Method,

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[1] Falck, B. (1962). "Observations on the possibilities of the cellular localization of monoamines by a fluorescence method". Acta Physiol. Scand. 56 (suppl. 197): 1–25.

[2] Falck, B.; Hillarp, N. A.; Thieme, G.; Torp, A. (1982). "Fluorescence of catechol amines and related compounds condensed with formaldehyde". Brain Research Bulletin. 9 (1–6): xi–xv. doi:10.1016/0361-9230(82)90113-7. PMID 7172023.

[3] "The Falck-Hillarp Method - a revolution in the study of monoamine systems in the CNS". Wallenberg Neuroscience Center, University of Lund. 2019-01-09.

[4] FALCK B; TORP A (1962). "New evidence for the localization of noradrenalin in the adrenergic nerve terminals". Medicina Experimentalis. 6 (3): 169–72. doi:10.1159/000135153. PMID 13891409.