HISTOLOGY

INTRODUCTION Definition: A science: study normal micro-structure & its related function of human body. 4 structural levels: Cell: the smallest structural & functional unit. Tissue: groups of cells (similar in morphology or related in function)＋intercellular materials 4 types of fundamental tissue 　　 epithelium 　　 connective tissue 　　 muscular tissue nervous tissue　

Organs: organizations of various kinds of tissues in particular ways & perform a specific function. System: formed by several function-related organs which together perform a continuous physiological function. For example: digestive system

Why to study histology ? To complete the knowledge of human body’s structures----from gross to microscopic Be able to understand how the different tissues function----the basis of physiology Can find the diseases only after the normal is known----the basis of pathology It is related to some modern science fields: cell apoptosis, cell recognition, implantation of embryo stem cells, eugenics and etc. It is also a foundation of clinic sciences—for a good doctor needed in futrue

Unit used in microscope 1μm=1/1000 mm 1 n m=1/1000μm Maximum resolution Light microscope: 0.2μm Transmission electron microscope: 0.2nm Scanning electron microscope: 5nm

Investigative methods of histology I. Light microscopy

1. Tissue preparation A. paraffin section preparation Specimen: as fresh as possible  Fixation: fixative: formalin solution; purpose: to preserve the structural organisation Dehydration: replace the water in the tissue by alcohol Clearing: replace the alcohol by xylene Embedding: replace the xylene w/ melted paraffin Sectioning & mounting l                      

B. Frozen section: Better for preserving chemical components (e.g. enzymes) Freezing→cryotomy→staining C. the others: Smear preparations: for blood etc; Grind preparations: for bone

Basophilic: components bonded by basic 2. Staining Purpose: To make tissue section pigment for observation. H-E Staining: Hematoxylin: basic dye, purple-blue Eosin: acid dye, pink color Basophilic: components bonded by basic dye (H); pruple-blue（nuclear chromatin & basophilic substance in cytoplasm） Acidophilic: components bonded by acidic dye (E); pink (cytoplasm & collagenous fiber)

Neutrophilic: do not stain w/ both basic and acid dyes Metachromasia: a dye stains tissue a different color from that of dye solution, e.g. toluidine blue stains mast cells in purple color Special staining: Argyrophilia Fluorescent staining

Silver staining of the neuron and the bile canaliculi HO (Hoechst 33258)-PI staining shows the apoptosis in human HL-60 cells

II. Electron Microscopy 1. Transmission Electron Microscope (TEM) Using a beam of electrons (short wave-lengths) instead of visible light. Section preparation: similar to those for L.M mainly, plastic instead of paraffin, 50-70nm thick, heavy metal salts instead of HE.  Resolution: 0.1-0.5nm (0.2nm) Ultrastructrue: The structure in EM Electron-dense / electron-lucent

Transmission Electron Microscope

Diagrams of TEM

2. Scanning Electron Microscope (SEM) Sowing the 3 dimensional surface Architecture of cells and tissues    Resolution: 5nm

III. Histochemistry & Cytochemistry Reveal the chemical composition in situ (e.g. proteins, a.a., nucleic acid, lipids, enzymes etc.) w/ chemical, biochemical methods. The product of chemical reaction should be insoluble / colored / electron- scattering, & be seen in LM or EM

For instance: PAS(Periodic Acid Schiff) reaction: for manifesting polysaccharide and proteoglycan (e.g. glycogen). polysaccharide + HIO4 (hydroxyl group) (oxidise) Aldehyde group + Shiff’s reagent (colorless) Purplish red depositor

PAS reaction in the hepatocytes

Immunocytochemistry Based on antigen binds to specific antibody. Tissue section w/ Antigen + labelled antibody labelled Ag-Ab complex Fluoresceinlabelling enzyme labelling colloidal gold labelling

NOS positive neuron in hypothalamus of rat NOS & GnRH positive Neurons in hypothalamus of rat NOS positive neuron in hypothalamus of rat

m-ATPase activity in skeletal muscles

VI. in situ hybridization (ISH) IV. tissue culture V. isotopic tracing VI. in situ hybridization (ISH) —nucleic acid molecular hybridization Use nucleotide probe to check target fragment of intracellular DNA or mRNA in situ, in order to study the gene expression.

Expression of PSA and PSAmRNA in human prostate (histochemistry & ISH)

The method in learning histology Combination of the 2 dimentional structure with 3 dimentional

Combination of the theory with practice Combination of the structure with function Concern the dynamic change