Introduction to Histology - PowerPoint Presentation

Slide1

Introduction to HistologyLight and Electron MicroscopesTissue Preparation

E-mail: a.al-nuaimi@sheffield.ac.ukE. mail: abdulameerh@yahoo.com

Prof. Abdulameer Al-NuaimiSlide2

Histology :Is a branch of anatomy that deals with the minute structure of animal and plant tissues. The

fundamental aim of histology is to determine how tissues and organs are organized at all structural levels.  It is commonly performed by examining cells and tissues under light microscope

or studying the ultrastructure components of cells under Electron microscope.Steps taken to prepare tissue for histological study Specimen is usually taken from a selected organ, Fixed, E

mbedded, Cut into a thin cross section with a microtomeMounted on a microscope slide, Stained

E

xamined under the microscope Slide3

AnimalsAnimal

rights is the idea that some or all nonhuman animals are entitled to the possession of their own lives, and that their most basic interests (

such as an interest in not suffering) should be afforded the same consideration as the similar interests of human

beingsExperimental animals should be respectedSlide4

Dissecting

Board

Made of thick high-density polyethylene to retain shape without bending or swelling, and can be sterilized

All corners have rubber feet for stability Slide5

fixationSlide6

Tissue processing

1- Tissue FixationObjective of tissue fixation is to

preserve cells and tissue components and keep them as close to normal as possible and

allow for the preparation of thin, stained sections.

Fixation

is usually the first

step

in a process to prepare a sample of biological material for microscopy or other analysis. Slide7

Fixatives work in the following way.1-Fixative

usually acts to disable intrinsic biomolecules—particularly proteolytic enzymes—which otherwise digest or damage the sample.2-Fixative typically protects

a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample3-fixatives

often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This helps in preserving the morphology (shape and structure) of the sample as it is processed for further analysisSlide8

Choice of fixative and fixation protocol

It depend on the additional processing steps and final analyses that are planned. For

example, immunohistochemistry uses antibodies that bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically usedSlide9

Types of fixationHeat

fixation: preserves overall morphology but not internal structures.Freezing: Used to get rapid results but does not give fine details

Chemical fixationIn this process, structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative that can stabilise the

proteins, nucleic acids and mucosubstances of the tissue by making them insolubleSlide10

Target

Fixative of Choice

Fixative to Avoid

Proteins

Neutral Buffered Formalin, Paraformaldehyde

Osmium Tetroxide

Enzymes

Frozen SectionsChemical Fixatives

Lipids

Frozen Sections*,

Glutaraldehyde

/Osmium Tetroxide

Alcoholic fixatives, Neutral Buffered Formalin

Nucleic Acids

Alcoholic fixatives, HOPE

Aldehyde fixatives

Mucopolysaccharides

Frozen Sections

Chemical fixatives

Biogenic Amines

Bouin

Solution

,

Neutral

Buffered Formalin

Glycogen

Alcoholic based fixatives

Osmium TetroxideSlide11

Factors Affecting Fixation

1-pH of the fixativeShould be kept in the physiological range, between pH 4 to 9. The pH for the

ultrastructure preservation should be buffered between 7.2 to 7.42-Osmolarity of the fixative: Try to avoidHypertonic

solutions :give rise to cell shrinkage.Hypotonic solutions :

result

in cell swelling and poor fixation

.

10% neutral buffer formalin fixative (4% formaldehyde in phosphate buffered saline), is a very hypertonic solution, yet it has worked well as a general tissue fixation condition3-Size of the Specimen : Ideal thickness is 1-4mm 4-Volume of the Fixative :

At

least 15-20 times greater than tissue

volumeSlide12

5-Temperature: High temperature increases the speed of fixation. However, care is required to avoid cooking the specimen.

Fixation is routinely carried out at room temperature.6-Duration: As a general rule 1hr per 1mmFixation

is a chemical process, and time must be allowed for the process to complete. Although "over fixation" can be harmful, under-fixation

has been appreciated as a significant problem and may be responsible for inappropriate results for some assaysSlide13

2- Embedding

Embedding

means blocking tissue within hard medium (paraffin or resines)The aim of

Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Embedding materialFor

light microscopy

,

paraffin wax

is most frequently used. For electron microscopy, resins are the most commonly used.During embedding, the tissue samples are placed into molds along with liquid embedding material which is then hardened. Formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature,Slide14

Automatic Tissue ProcessorSlide15

Tissue Embedding MachineSlide16

3- Tissue Sectioning (Cutting)

For light microscopy, a steel knife mounted in a microtome is used to cut 3 -5 µm -thick

tissue sections which are mounted on a glass microscope slide. 1mm = 1000 µmFor transmission electron microscopy, a diamond knife

mounted in an ultramicrotome is used to cut 0.1-0.5 µm thick tissue sections which are mounted on a 3-mm-diameter copper grid. Then the mounted sections are treated with the appropriate stain.

Frozen

tissue

embedded in a freezing medium

and cut on a microtome in a cooled machine called a cryostat. It cuts 1-10 µm thick sections.Slide17

Paraffin sectioning MicrotomeSlide18

4- Mounting the sections on a microscope slideS

ections are mounted onto gelatin-coated histological slides. Slides are pre-coated with gelatin to enhance adhesion of the tissue.Sections are floated in a 56 °C

water bath and then mounted on the slidesMounted sections are dried on a hot plate

at 56 °C and thenKept overnight at room temperature.

Slides

with paraffin-embedded sections can be stored either at room temperature

or

at 2-8 °C for several years in slide storage boxes.Slide19
Slide20

5- Staining

Biological tissue has little contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest.

Hematoxylin and E

osin

(H&E stain)

is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus.Eosin, an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to add

contrast to tissue in the electron

microscope.

Histochemistry

: shows chemical

reactions between laboratory chemicals and components within tissue. Slide21

Histochemistry stainsPAS StainPAS stains carbohydrates and carbohydrate

rich macromolecules a deep red colour Masson's trichrome stain.This is often used to stain connective tissue.Collagen is stained green or blue

Van Gieson stainThis stains collagen red, nuclei blue, and erythrocytes and cytoplasm yellow. Slide22

immunohistochemistry.

Antibodies are used to specifically visualize proteins, carbohydrates, and lipids.

this photo shows some cells that have been immunofluorescently

stained for the protein actin.Slide23

History of stainingIn the 19th century, histology was an academic discipline in its own right. The 1906 Nobel Prize

in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y.

Cajal. They had given interpretations of the neural structure of the brain based in differing interpretations of the same images. Cajal won the prize for his correct theory and Golgi for the staining technique he invented to make it possible

Santiago Ramon y

CajalSlide24

Normal skinSlide25

Normal SkinSlide26

Normal LungSlide27

Light MicroscopeSlide28

Light MicroscopeSlide29

Electron MicroscopeSlide30

Thank you