Histology & Its Methods of Study (A-D) - PDF

 UNIT I: Histology & Its Methods of Study A. Preparation of Tissues for Study B. Light Microscopy C. Electron Microscopy D. Interpretation of Structures in Tissue Sections Mescher (2021) WHAT IS HISTOLOGY? Histo = “tissue” or “...

 UNIT I: Histology & Its Methods of Study A. Preparation of Tissues for Study B. Light Microscopy C. Electron Microscopy D. Interpretation of Structures in Tissue Sections Mescher (2021) WHAT IS HISTOLOGY? Histo = “tissue” or “web” 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 Mescher (2021) BRIEF BACKGROUND Tissues have two interacting components: 1. CELLS =produce the (ECM) 2. EXTRA CELLULAR MATRIX (ECM)  BRIEF BACKGROUND 1. CELLS Animal cells are eukaryotic (Gr. eu, good, + karyon, nucleus), cytoplasm basic structural and functional containing various membrane-limited units; organelles the smallest living parts of the body Bacteria are prokaryotic cell wall around Cells produce the ECM locally the plasmalemma and lack other and are in turn strongly membranous structures, including an influenced by matrix molecules envelope around their DNA Mescher (2021) BRIEF BACKGROUND 2. EXTRA CELLULAR MATRIX The connective tissues non cellular portion is known as the extracellular matrix (ECM) giving the physical scaffolding for the cells hold water provide appropriate hydration of the tissue form part of a selective barrier to the external environment. supports the cells and contains the fluid transporting nutrients to the cells, and carrying away their wastes and secretory products. Mescher (2021)  BRIEF BACKGROUND 2. EXTRA CELLULAR MATRIX In human, the main components of the extracellular matrix are: Fibrous elements (e.g. collagen, elastin, reticulin), Link proteins (e.g. fibronectin, laminin), and Space filling molecules (e.g. proteoglycans, glycosaminoglycans) Schematic illustrating the ECM composition Mescher (2021) BRIEF BACKGROUND Marie François Xavier Bichat Father of modern histology and descriptive anatomy first did the study of tissues the one who used the term tissue in an anatomical sense discovered different weaves and textures in the body and named them layers of tissues  Preparation of A Tissues for Study understand fixation, embedding & sectioning, staining A. Preparation of Tissues for Study Mescher (2021) A. Preparation of Tissues for Study Most tissues studied histologically are prepared as shown, with this sequence of steps: from Junqueira’s Basic Histology Text and Atlas Mescher (2021) A. Preparation of Tissues for Study Bruce-Gregorios (2017) 1. Fixation 7. Trimming 2. Decalcification (optional) 8. Section-Cutting (Microtomy) 3. Dehydration 9. Staining 4. Clearing 10. Mounting 5. Impregnation (Infiltration) 11. Labeling 6. Embedding Bruce-Gregorios (2017)  A. P 1. FIXATION r e p a Fixation of tissues is the most crucial step r a t Goal: Preserve the sample's structure and prevent decomposition. i o n Specifically, o f 1. to stop metabolism; T i ü (prevent degradation by enzymes released from the cells or s s microorganisms causing an autolytic activity) u e s 2. to fix structures of organelles and molecules in their current f position; and o r 3. to make material accessible and stable during further processing. S t u d y https://www.ou.edu/research/electron/bmz5364/prepare.html#:~:text=Fixation%20of%20tissues%20is%20the,of%20the%20tissue%20(preservation).  A. P 1. FIXATION r e p a Examples of fixatives (liquid fixing r a t agent): i o n o f Formalin (buffered isotonic solution T i of 37% formaldehyde) – light s s microscopy u e s f o Glutaraldehyde – electron r microscopy S t u d y https://www.ou.edu/research/electron/bmz5364/prepare.html#:~:text=Fixation%20of%20tissues%20is%20the,of%20the%20tissue%20(preservation).  A. P 1. FIXATION r e p a Fixation of tissues is the most crucial step r a t Goal: Preserve the sample's structure and prevent decomposition. i o n o f T i s s u e s f o r S t u d y https://www.ou.edu/research/electron/bmz5364/prepare.html#:~:text=Fixation%20of%20tissues%20is%20the,of%20the%20tissue%20(preservation).  A. P 1. FIXATION r e p a r Ideally, specimens should remain in fixative long enough for it to a t i penetrate the tissue and then for an additional period in order to o n allow the chemical reactions of fixation to reach equilibrium o f (fixation time). T i s s u e s f o r S t u d y https://www.ou.edu/research/electron/bmz5364/prepare.html#:~:text=Fixation%20of%20tissues%20is%20the,of%20the%20tissue%20(preservation).  A. P 2. Dehydration r e p a Goal: Remove water from the sample to prepare it for embedding. r a t The tissue is transferred through a series of increasingly concentrated i o alcohol solutions, ending in 100%, which removes all water. n o f T i s s u e s f o r S t u d y  A. P 2. Dehydration r e p a extract water gradually through increasing concentrations of ethanol r a solution, ending in 100% ethanol t i o immersing specimens in a series of ethanol (alcohol) solutions with n increasing concentration to avoid excessive distortion of tissue until a o f water-free tissue in alcohol is reached T i s s u e s f o r S t u d y  A. P 2. Dehydration r e p a r a A typical dehydration sequence for specimens not more than t i 4mm thick would be: o n o f 1. 70% ethanol 15 min T i 2. 90% ethanol 15 min s s 3. 100% ethanol 15 min u e 4. 100% ethanol 15 min s 100% ethanol 30 min f 5. o r 6. 100% ethanol 45 min S t u d y  A. P 2. Dehydration r e p a r a A typical dehydration sequence for specimens not more than t i 4mm thick would be: o n o f 1. 70% ethanol 15 min T i 2. 90% ethanol 15 min s s 3. 100% ethanol 15 min u e 4. 100% ethanol 15 min s 100% ethanol 30 min f 5. o r 6. 100% ethanol 45 min S t u d y  A. P 3. Clearing r e p a r Goal: Replace alcohol with a substance that is transparent to a t i allow light to pass through. o n o f to remove a substantial amount of fat from the tissue which T i otherwise presents a barrier to wax infiltration s s u e s f o r S t u d y  A. P 3. Clearing r e p a r a the ethanol is gradually replaced with xylene t i o A typical clearing sequence for specimens not more than 4mm thick n would be: o f 1. Xylene 20 min T i 2. Xylene 20 min s s 3. Xylene 45 min u e s f o r S t u d y  A. P 3. Clearing r e p a r a t i o n o f T i s s u e s f o r S t u d y  A. P 4. Infiltration r e p a r a To completely displace the clearing agent, t i o a typical paraffin infiltration sequence of n paraffin for specimens not more than o f 4mm thick would be: T i 1. Paraffin wax 30 min s s u 2. Paraffin wax 30 min e s 3. Paraffin wax 45 min f o r S t u d y  A. P 5. Embedding r e p a Goal: Provide support and r a orientation for sectioning. t i o n hardening of infiltrated tissue with o f paraffin T i s Correct orientation of tissue in a mold is s u the most important step in embedding e s Incorrect placement of tissues may f o result in diagnostically important tissue r elements being missed or damaged S during microtomy t u d y  A. P 6. Section-cutting r e p a r a Goal: Produce thin sections for t i microscopy o n the hardened block with tissue o and surrounding embedding f medium is trimmed and placed for T i sectioning in a microtome s s u e s Light Microscopy 3-10 um Microtome f o Electron Microscopy