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Basics of Microscopes


Excitation/absorption Excitation means to energize atoms and molecules, or shift from a low-energy state to a high-energy state. An "excited" state is produced, for example, by applying ultraviolet light to a fluorescent material. The fluorescent material emits fluorescence or phosphorescence when returning to the low-energy ground state after "absorbing" excited light.
Photobleaching A phenomenon where the fluorescent signal intensity irreversibly decays when a fluorescent protein is continuously illuminated. There are two techniques to observe the inside of a cell using photobleaching of fluorescence: fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP).
Numerical aperture (NA) An index to measure the resolution of a microscope's objective lens. Defined as a numeric value, it represents the range over which the objective lens can gather light. The greater the numeric value, the higher the resolution and the brighter and clearer the sample can be imaged. This value is usually marked on the tube of objective lenses.
Aberration The blurring or distortion of an image in the optical system of a microscope. It is classified into monochromatic and color aberration. Monochromatic has five types - spherical, comatic, astigmatism, field curvature, and distortion aberration. Color has two types- axial and magnification color aberration.
Immunostaining A technique to visualize antigen-antibody reaction, which is normally invisible. This clarifies the localization of an antigenic substance in a tissue or cell. For antibodies, fluorescent reagents, ferritins, and enzymes are commonly used. The major techniques include autoradiography technique, enzyme antibody technique, and fluorescent antibody technique.
iPS cell An induced pluripotent stem cell. This type of stem cell is produced through cultivation after induced pluripotent factors are introduced into differentiated and matured body cells. As a master cell, it can theoretically be differentiation-induced to a cell of any tissue. Its commercialization has advanced in fields such as drug discovery, gene therapy, and tissue regeneration treatment.
ES cell An embryonic stem cell. A fertilized egg reaches the stage called blastocyst about a week after fertilization. An ES cell can be obtained by cultivating an inner cell mass of an embryo during the blastocyst stage. As with a fertilized egg, this cell has the potential to differentiate into any cell (pluripotentiality), in addition to a high proliferation ability. While having attracted attention to its availability for regeneration treatment, this cell also has raised an ethical issue because of the use of an embryo, which has the potential to become a human life.
Hela cell The oldest human-derived cell line. This cancer cell has been cultivated after being derived from a cervical cancer patient, Henrietta Lacks, in the United States in 1951. This cell has been widely used in laboratories around the world for separation and proliferation of various viruses.
Localization Existence in a limited or partial space. For example, localization of biofunctional molecules such as proteins in cells can be visualized using a confocal laser microscope.
FISH method An acronym for fluorescence in situ hybridization. This is a kind of chromosomal analysis and is used to locate genes in chromosomes through hybridization with the genes using nucleic-acid probes. Compared with autoradiographic immunostaining, this method can analyze chromosomes more safely, easily, and in a shorter time.
FRET An acronym for fluorescence resonance energy transfer. This is a phenomenon where excitation energy moves from a fluorescent molecule (donor) to another fluorescent molecule (accepter) when they are located close to each other. In this process, the latter emits fluorescence. FRET is used, for example, to detect interactions between proteins.
Apoptosis Programmed cell death. This occurs when a tissue is formed or an abnormal/unnecessary cell is removed. Unlike external injury, this cell death does not cause an inflammatory response because intracellular fluid is contained by a phagocytic or adjacent cell. This process exhibits distinctive changes such as nuclear fragmentation and changed formation of cell membrane lipid, and thus can be observed at a relatively high sensitivity by detecting these changes. Detection methods include the TUNEL (TdT-mediated dUTP nick end labeling) method and FACS analysis with annexin-V staining.
Autophagy Also called autophagocytosis. A function of a cell that digests proteins and other components in it. Originally, it referred to a phenomenon where cytoplasmic components such as proteins and mitochondria are transported and decomposed by lysosomes during nutrient starvation. Recently, it has been revealed that autophagy is related to physiological functions such as the generation and differentiation of cells and suppression of diseases like cancers and infections.
in vivo Latin for "within the living". This refers to experiments using living animals such as mice and rabbits. It usually means nonclinical tests in which test substances are administered to animals. This is used in contrast to "in vitro", which refers to experiments in test tubes. In the fields of organic chemistry and biochemistry, however, some experiments in test tubes are also called in vivo experiments if they use living cells.
in vitro Latin for "within the glass". This refers to experiments in test tubes or dishes. It usually means experiments where the body's internal environment is artificially created in a test tube using cultivated cells. Roughly speaking, experiments using tissues or cells derived from living organisms are called in vitro, but the terms "in vitro" and "in vivo" are used differently depending on the academic field. In molecular biology, for example, experiments using cultivated cells may be called in vitro. In organic chemistry and biochemistry, in vitro may also refer to experiments in test tubes without using cells.
Stem cell A cell that has self-replication and differentiation abilities, which will then form each tissue of a living body. These cells can roughly be divided into somatic stem cells (tissue stem cells) and pluripotent stem cells. The former can only create specific tissues such as blood and nerves while the latter can create any cell in the body. Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are classified into the latter category.
Nucleus A spherical structure enveloped in double nuclear membranes in a eukaryotic cell. It mainly consists of DNA and proteins, and functions to store and transmit genetic information. Nuclear membranes have many pores to transport mRNA and other substances between the nucleus and the cytoplasm.
Mitochondria An organelle with a diameter of 0.5 to 1 µm that generates ATP through oxidative phosphorylation in the electron transport chain (respiratory chain). This soft organelle can change its shape for fusion, and many mitochondria exist around energy-consuming cells. It consists of an outer membrane that contains a large number of transport proteins and an inner membrane (crista) that is rich in cardiolipin. Cytochrome c, which is emitted as the membrane permeability increases or when membranes are depolarized, causes internal apoptosis. This is where membrane potential and redox reactions occur. Various staining methods are used for this organelle, including the rhodamine family and Mito Tracker Green.
Live imaging A method to observe the movement of a body tissue or cell in its living state. By introducing GFP (green fluorescent protein) into a cell, it is possible to visualize its movement with a fluorescence microscope. Live imaging using living cells is specifically called live cell imaging.
Working distance (WD) The distance from the tip of the objective lens to the sample when in focus. The greater the numerical aperture of the lens, the shorter the working distance.
Parfocalizing distance of the objective (PFD) The distance from the revolver-attached end of the objective lens to the sample surface when in focus (e.g. 45 mm, 60 mm, and 75 mm).
Depth-of-focus The range in which the sample (or the objective lens) can be moved from the focused point and still remain focused on a microscope. The greater the numerical aperture, the shallower the depth of focus.

Latest developments in Microscope technology 4-part guide / Click here to download

Latest developments in Microscope technology 4-part guide / Click here to download

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