Histological techniques

Principles for light microscopy

Beam of visible light concentrated by condenser lens Light refracted by objective, forming a magnified image. Segment of image is magnified by eyepiece (ocular) The best resolving power is about 0.2 micrometer Parafin-embedded sections o Most often used in laboratory classes o Use a microtome (instrument that cut extremely thin slices) with optimum resolution of 0.6 micrometer. o In order to earn better resolutions, thinner (resin-embedded) sections (0.50.2) must be used. o Epoxy resin sections are cut with special microtome, and stained with toluidine blue. These epoxy resin slides are mostly for comparison for example of cellular details. Light source Visible light Condenser lense (consentrates light) Plane with tissue section Objective Forming magnified image Eyepiece Magnifies image again eye

Principles of the transmission electron microscopy (TEM)

Use beams of electrons instead of beams of light. Use electromagnetic lenses instead of glass lenses o Electromagnets create electromagnetic field that bens and thereby forcus the beam of electrons. Use very thin objects (specimens) e.g. ultrathin epoxy resin sections. o The beam of electrons is transmitted through the specimen thereby the name Use high vacuum produced by pumps o Inside theres a tungsten (wolfram) cathode which emits electrons. Electrons become accelerated in a electric field between cathode and anode (metallic plate with a small hole in its center) and focused on object by the condenser lenses Electrons are transmitted through the specimen, and scattered (=electron dispersion = spredt). Some gets absorbed though. Objective lense forms magnified image

o A segment of this image gets projected by to projective lens to a screen (fluorescent) or a photographic film) Tungsten cathode heated, eemitted acceleration anode (metallic plate) Condenser lense Focus beam on Specimen e- transmitted or scattered Objective lense magnified projective lense Structures on specimen that scatter electrons appear in the fluorescent screen as dark (electron dense structures) o Electron reflection capacity depends on the molecular weight of components of the specimen. o Heavy atoms cause higher deflection of electrons from the straight course than light atoms. o All biogenic atoms (H,C,O,N,P,S) are light and therefor no good contrast. Heavy metals (like uranium or lead) are therefore used to increase contrasts of ultrathin sections. The energy of the electrons are not very high so they get easily absorbed by the specimen o Solution for this is to use very thin objects (they are ultrathin about 5090 nm thick) Use of electrons instead of light allow structures as small as 1 nm to be seen. o To preserve the structure it is necessary to use fixatives. Current fixation procedures are: Gluteraldehyde to retain protein constituents (bestanddeler) of the cell Osmium tetroxide to retain the lipid components especially phospholipids. o Since it contains heavy metal it also plays a role in electron deflection and consequent image formation. Instead of paraffin, tissues are embedded in plastic e.g. epoxy resin. o This is harder than paraffin and makes sure that the tissue can be cut extremely thin. Special microtomes with glass or diamond cutter are used. Thereafter the sections are placed on a coppermesh grid and stained. o Stain contains heavy metals, e.g. lead citrate. In light microscopy the paraffin must be removed before staining but in the plastic preparations its not needed to remove the embedding medium. Best practical resolving power for biological objects is about 1.0 nm but the resolving power in theory is about 0.25 nm

Basic histological techniques histological stainings

Directions how to prepare preparations for microscopic analysis Individual cells o Use cytological technique Heamatological smears From the blood From the bone marrow The other body fluids (cerebrospinal fluid, ascites, pleural exsudate) Smears following centrifugation Chambers for spontaneous sedimentation Cell tissues o Use tissue sections Taking from either: In vivo (living body=BIOPSY) or in autopsy (NECROPSY) Either: o Rapid (during operation) o Normal way of processing Expiration (e.g. tumor, the whole organ or its portion = in operation) Probationary excision a piece of tissues is cut from the organ Probationary puncture The sample is taken with a thick needle form an organ (liver, kidney) or the bone marrow = sternal puncture Curettage Scrape (e.g. of the endometrium) Abrasion of some inner surfaces of the body o E.g. from vagina, cervix uteri Staining solution act the same time as a fixative

Preparation of histological sections

1. Fixation a. Denaturation of tissue proteins i. Prevents dead tissue from spontaneous decay / autolysis (where their own enzymes causes a rotteing process) 1. Achieved by chemical fixatives b. The most common fixative is 10% formalin (40% formaldehyde diluted ;fortynnet; in water) c. Two was of fixation: i. Immersion (dipping) ii. Perfusion (pumping the stuff into the body via blood vessels, e.g. via the left heart ventricle

2. Cutting the tissue blocks a. Block size is usually 1x1x0.5 cm due to optimal proper diffusion of fixative 3. Tissue sections a. Cut with microtomes by frozen section technique or after embedding a supportive medium such as paraffin b. Frozen sections i. Advantage 1. Detection of lipids is possible 2. Enzymes and antigens remain intact 3. Slides are suitable for enzyme histochemistry and immunofluorescence ii. Disadvantage 1. Impossible to prepare thin sections 2. Cant be stored permanently c. Paraffin-embedded sections i. Advantage 1. Elative thin sections can be prepared 2. Can be stained with many methods 3. Microscopic structure is well preserved 4. Sections can be archived without changes for decades ii. Disadvantages 1. Shrinkage of tissue 2. Inactivation of enzymes 3. Lipids cant be detected iii. Paraffin embeddin 1. Pass through a series of graded ethanols up to 100% and acetone due to its embedding paraffin 2. Ethanol must be replaced by a non-polar solvent of paraffin such as: Xylen or Benzene a. This also render the tissue translucent (gjennomskinnelig) 3. Tissue is then placed with melted 56 degrees celcius paraffin and in the infiltration chamber it is embedded by paraffin. 4. After cooling the paraffin hardens and gives rise to the paraffin block which is now ready for sectioning. 5. Sections are put on the glass plates, stretched and attached with gelatin or a mixture of white egg and glycerol (1:1) d. Processing mineralized tissues i. Ground sections 1. Thin plate cut from the bone or tooth 2. Held against a rotating base plate with abrasive (slipende) surfaces 3. After reach of required thickness sections are polished 4. Mounted in solid Canada balsam or are embedded in methacrylate

ii. Decalcification 1. Minerals are dissolved in diluted acids (fortynnede syrer) (e.g. formic acid, HCL or 5% nitric acid and tricholacetic acid) 2. Or by using chelating agent that capture Ca2+ and Mg2+ (EDTA) a. EDTA is more suitable than acids, but decalcification may last up to 8 weeks. b. Then decalcification liquid is removed by a proper washing in solution of 5% natrium sulphate or so. 4. Staining the sections a. Nucleic (basic) stains i. Bind to acidic tissue structures, basophilic structures 1. Chromatin of nucleus 2. Ergastoplasm 3. Ground substance of cartilage ii. Hematoxylins 1. Stain nuclei dark iii. Nuclear red 1. Stain nuclei red iv. Basic aniline dyes 1. Stain ergastoplasm in a different shade than nuclei. 2. E.g. Cresyl violet, toluidine blue 3. Nissl staining (in nervous system) a. Nuclei = greenish blue b. Nissl substance = Reddish b. Plasmatic aniline dyes (acidic) i. Bind to basic tissue structures, acidophilic structures 1. Cytoplasm 2. Collagen 3. Mucus 4. Fibrin 5. Elastic fibers ii. Eosin 1. Stain in pink. These are stained intensively (red) a. Granules of eosinophilic leukocytes b. Acidophilic cells in the pituitary gland c. Eryhtrocytes iii. Picric acid 1. In van Giesons stain only iv. Haematocylin and eosin (HE) staining are general and most used staining method

c. Elective stains of the connective tissue i. Collagen and hyaline 1. Acidic fuchsin = deep red 2. Van Giesons stain a. Mixture of picric acid and acidic fuchsin (picrofuchsin) b. Collagen = red c. Muslce = yellow d. Nuclei with haematoxylin = dark 3. Massons trichrome stainings a. Blue trichrome i. Collagen with aniline blue = blue ii. Muscle with a cytoplasmic dye = red iii. Nuclei with haematoxylin = dark b. Green trichrome i. Collagen with the light green = green ii. Muscle with a cytoplasmic dye = red or orange iii. Nuclei with haematoxylin = dark ii. Elastin 1. Stained with a. Weigert resorcin-fuchsin technique i. Gives a purble-black color to elastin ii. Nuclei is counterstained with nuclear red = red iii. Reticular (argyrophylic) fibers 1. Stain black (with ammoniac silver nitrate) 2. Nuclei with nucler red = red d. Neutral lipids i. Stained with Sudan III = red ii. Stained with Sudan IV = black iii. Nuclei with haematoxylin = dark e. Polysaccharides i. Glycogen 1. Stained with best carmine a. Purple red b. Nuclei with haematoxyline = dark ii. Neutral mucopolysaccharides, mucoproteins and glycogen 1. Stained with PAS-method (Periodic acid and Schiffs reagent) a. Purple red b. Nuclei with haematoxylin = dark iii. Acidic mucopolysaccharides 1. Hale-Mller colloid oron = blue 2. Nuclei with nuclear read = red iv. Mucus (mucin) 1. With mayers mucicarmine technique 2. Pink 3. Nuclei with haematoxylin = dark

f. Neurohistology i. Neurofibrillary impregnation of axons 1. With ammoniac silver nitrate (protargol) 2. Black 3. Nuclei with nuclear red = red ii. Myelin sheaths (phospholopides) 1. With Luxol blue 2. Blue-green nuclei 3. Nissl substance with cresyl violet = red iii. Impregnation of astrocytes 1. With auric chloride and sublimate 2. Dark purple iv. Block impregnation of NEURONS 1. Including axons and ramification of dendrites 2. Black g. Pigment melanin i. Argentaffin reaction = black ii. Haemosiderin 1. With pearls reaction = Blue 5. Sections covering a. Stained sections are dehydrated in increasing concentrations of ethanol and xylene (=clearing) and mounted in Canada balsam or DPC mounting medium.