Histology and Methods PDF

HISTOLOGY AND ITS METHODS PREPARED BY: MARY ABIGAIL PAULAN, RMT HISTOLOGY study of the tissues of the body and how these tissues are arranged to constitute organs. involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to e...

HISTOLOGY AND ITS METHODS PREPARED BY: MARY ABIGAIL PAULAN, RMT HISTOLOGY study of the tissues of the body and how these tissues are arranged to constitute organs. involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ. two interacting components: cells and extracellular matrix (ECM). PREPARATION OF TISSUES FOR STUDY PREPARATION OF TISSUES FOR STUDY The Microtome Trimming  Cutting PREPARATION OF TISSUES FOR STUDY STAINING Acidic structures  basic dye = basophilic (e.g. nucleus) Basic structures  acidic dye = acidophilic (e.g. cytoplasm) Hematoxylin and eosin MICROSCOPY LIGHT MICROSCOPY Bright-Field Microscopy stained preparations are examined by means of ordinary light that passes through the specimen. Resolving power  defined as the smallest distance between two particles at which they can be seen as separate objects.  0.2 μm LIGHT MICROSCOPY Fluorescence Microscopy Tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant on a dark background. Fluorescence  Phenomenon wherein certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength. LIGHT MICROSCOPY Phase-Contrast Microscopy based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices. uses a lens system that produces visible images from transparent objects enables examination of unstained cells and tissues and is especially useful for living cells. LIGHT MICROSCOPY Phase-Contrast Microscopy Interference microscope allows quantification of tissue mass Differential interference microscope using Nomarski optics useful for assessing surface properties of cells and other biologic objects. Unstained cells’ appearance in three types of light microscopy. LIGHT MICROSCOPY Dark-field Microscopy no direct light from the light source is gathered by the objective lens. only light that has been scattered or diffracted by structures in the specimen reaches the objective. equipped with a special condenser that illuminates the specimen with strong, oblique light. field of view appears as a dark background on which small particles in the specimen that reflect some light into the objective appear bright. LIGHT MICROSCOPY Confocal Microscopy combines components of a light optical microscope with a scanning system to dissect a specimen optically. Uses (1) a small point of high-intensity light, often from a laser, and (2) a plate with a pinhole aperture in front of the image detector. point light source, the focal point of the lens, and the detector’s pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (confocal), and unfocused light does not A. Light from a laser source hits the specimen and is reflected. B. A beam splitter directs the reflected light to a pinhole and a detector. C. Light from components of the specimen that are above or below the focused plane is blocked by the blind. D. The laser scans the specimen so that a larger area of the specimen can be observed. LIGHT MICROSCOPY Polarizing Microscopy uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light. Birefringence ability to rotate the direction of vibration of polarized light ELECTRON MICROSCOPY based on the interaction of tissue components with beams of electrons. The wavelength in the electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution. 2 KINDS  TEM  SEM ELECTRON MICROSCOPY Transmission Electron Microscopy an imaging system that permits resolution around 3 nm. magnifications of up to 400,000 times to be viewed in detail uses the interaction of a beam of electrons with a specimen to produce an image. Cryofracture and freeze etching a special method of sample preparation for transmission electron microscopy it is especially important in the study of membranes. ELECTRON MICROSCOPY Scanning Electron Microscopy the electron beam does not pass through the specimen but is scanned across its surface. the surface of the specimen is first dried and spray- coated with a very thin layer of heavy metal (often gold) through which electrons do not pass readily. When the beam is scanned from point to point across the specimen, it interacts with the metal atoms and produces reflected electrons or secondary electrons emitted from the metal OTHER METHODS ENZYME HISTOCHEMISTRY a method for localizing cellular structures using a specific enzymatic activity present in those structures. IMMUNOHISTOCHEMISTRY A highly specific interaction between molecules is that between an antigen and its antibody HYBRIDIZATION TECHNIQUES a method of localizing messenger RNA (mRNA) or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe. In situ hybridization binding of the nucleotide probe to the DNA or RNA sequence of interest is performed within cells or tissues, such as cultured cells or whole embryos. A. B. N OF STRUCTURES IN TISSUE SECTIONS References: Junqueira’s Basic Histology Text and Atlas, 13th edition Histology, A text and Atlas 6th edition by Ross and Pawlina Urinalysis and Body fluids 5th edition by Strasinger and Di Lorenzo