HISTOLOGY & HISTOCHEMISTRY - PowerPoint Presentation

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HISTOLOGY & HISTOCHEMISTRY By Professor Abdel-Majeed Safer

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Professor Abdel-Majeed Safer Histology & Histochemistry Slide2

Professor Abdel-Majeed Safer Histology & Histochemistry 2Chapter 1Slide3

What is HISTOLOGY ?

3Professor Abdel-

Majeed Safer Histology &Histochemistry

Histology

is the study of the tissue of the body and how these tissues are arranged to constitute organs

. Histology involves

all aspects of tissue biology

with the focus on how cells’ structure and arrangement optimize functions specific to each organs.

Histochemistry

& Cytochemistry is the study of the chemical composition of the tissue and cell of the body. Or precisely, the microscopic study of the chemical characteristics of tissues and cells, through the use of substances (dyes etc.) producing identifying chemical reactions.

The branch of science that deals with the chemical composition of the cells and tissues of the body.Slide4

HISTOLOGY AND HOW IT IS STUDIED4Professor Abdel-Majeed Safer Histology & Histochemistry Slide5

5Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Preparation of Tissues for Study

Fixation

Embedding & Sectioning

Light Microscopy

Bright field microscopy

Fluorescence microscopy

Phase-contrast microscopy

Interference microscopy Confocal microscopy Polarizing microscopyElectron Microscopy

Transmission Electron Microscopy TEM

Scanning Electron Microscopy SEMSlide6

6Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Autoradiography

Cell & Tissue culture

Histochemistry

&

Cytochemistry

Detection Methods using specific Interactions between molecules

Immunohistochemistry

Hybridization Techniques

Problems in the study of Tissue SectionsSlide7

7Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Histology

is the study of the tissue of the body and how these tissues are arranged to constitute organs

. Histology involves

all aspects of tissue biology

with the focus on how cells’ structure and arrangement optimize functions specific to each organs.

Tissues are made of two interacting components:

cells and extracellular matrix

. The ECM consists of many kinds of molecules, such as

collagen fibrils

. there is, thus, an intense interaction between cells and matrix, with many components of the matrix . Most organs are formed by an orderly combination of several tissues, except the CNS, which is formed almost solely by nervous tissue. The precise combination of these tissues allows the functioning of each organ and of the organism as a whole.

The small size of cells and matrix components makes histology

depedent

on the use of microscope.Slide8

8Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Preparation of Tissues for Study

Fixation

see Fig 1

Is to avoid tissue digestion by enzymes within the cells (autolysis) or by

bateria

and to preserve structure and molecular composition.

Chemical methods - the tissues are immersed in solution of stabilizing or cross-linking agents called fixatives. or less frequently

Physical methods (deep freeze).

In routine LM

, formalin ; a buffered isotonic solution of 37% formaldehyde.Formaldehyde and glutaraldehyde are commonly used and known to react with amine groups NH2- of tissue proteins. Glutaraldehyde, is reinforced by virtue of being a dialdehyde which croos

-links proteins.

In EM, where high resolution is require, a buffered

glutaraldehyde

followed by a second fixation in buffered OsO4 for fine structural studies. OsO4 is used to preserve and stain lipids and proteins.Slide9

Tissue Fixation and Processing for Light MicroscopySlide10

10Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Dehydration

To extract the water from the tissue, through graded series of alcohol (70% - 100% ethanol).

Clearing

Ethanol is then replaced by a clearing agent that is miscible with both alcohol and the embedding medium. E.g.

xylene

Paraffin

Melted paraffin 52- 60oC in an oven.

Embedding and Sectioning

Tissues are embedded in a solid medium to facilitate sectioning. To obtain thin sections with the

microtomes, tissues must be infiltrated after fixation with embedding solutions that impart a rigid consistency to the tissue. Such as paraffin and plastic resins.Slide11

11Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Tissues to be embedded in plastic resin are also dehydrated in ethanol, cleared and embedded in plastic solvent, which are then hardened by means of cross-linking polymerizes.

Sectioning and

Microtomy

see Fig.2

The hard blocks containing the tissues are placed in microtome. Cut 1-10 um (

ium

= 1/1000 mm.

1nm = 0.001um = 10-6 mm. 1A = 0.1nm 10-4um.

Sections are are floated on water and then transferred to glass slides to be stained .Frozen sections are also performed by rapid freezing (physical , not chemical fixation), using a freezing mictotome or cryostat. This is mainly for rapid

invistigation

in hospitals and for

histochemical

studies, as enzymes and small molecules and lipids are very sensitive.Slide12

UltramicrotomeSlide13

Columnar epithelium lining the small intestineMicrograph stained with hematoxylin and eosin (H&E).Tissue stained with

hematoxylin and eosin (H&E).

Tissue stained with the periodic acid-Schiff (PAS) reaction for glycoproteins

. Slide14

14Professor Abdel-Majeed Safer Histology & Histochemistry

HISTOLOGY and Its Method of Study

Histological Stains

For routine histological work it is customary to use two dyes, one stains certain components, and the other stains different ones (counterstain).

Toluidine blue

Basophilic and Acidophilic Staining

Basophilic substance is the one that takes up basic stain, acidophilic is the one that takes up an acidic stain.

Hematoxylin is a basic stain or cationic stain, while Eosin is an acidic stain or anionic stain.

What do the color seen in Hematoxylin sections indicate?

Ordinary stains such H&E provide only general information about the chemical composition of the components that they color.Slide15

Professor Abdel-Majeed Safer Histology & Histochemistry 15Light Microscopy

Bright field microscopyStained slides are examined by means of ordinary light that passes through the specimen.

See Fig.1-3.

for details.

Flourescence

Microscopy

When certain substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength. This phenomenon is called

Flourescence

.

In

Flourescence Microscopy, tissue sections are irradiated with ultraviolet UV light and the emission is in the visible portion of the spectrum. The fluorecent substances appear brilliant on a dark background. Thus, the microscope has a strong UV light source and special filters that select rays of different wavelengths by the substances. Fluorescent compounds with affinity for specific cell macromolecules may be used as Fluorescent stains. E.g. Acridine

Orange

(bind to DNA and RNA).

See Fig.1-4a

e.g..

Hoechst stain and DAP1

specifically bind to DNA giving a blue

flourecence

under UV

.Slide16

Bright Field MicroscopeSlide17

Fluorescent MicroscopyKidney cells

acridine orange

Culture of kidney cells

DAPI

(4’,6-diamino-2-phenylindole) Slide18

Professor Abdel-Majeed Safer Histology & Histochemistry 18

Phase-contrast microscopy and Interference microscopy

Unstaned

biological specimens are usually transparent and difficult to view in detail,

beacause

all parts of the specimen have the same optical density.

Phase-contrast microscopy

uses a

lense

system that produces visible images from transparent objects

(Fig. 1-5).

Principle:

Light changes its speed when passing through cellular and extracellular structures with different refractive indices.

Thses

changes are used by Phase-contrast system to cause the

strctures

to appear lighter or darker in relation

ro

each other. So, living cells and tissue cultures can be observed ( no need for fixation or staining). Phase-contrast microscope is essential tool in tissue culture labs.

also,

differential Interference microscopy,

which produces images with a more apparent 3-D aspect than routine Phase-contrast microscopy

(Fig. 1-5).Slide19

Bright field microscopyInterference microscopy

Phase contrast microscopySlide20

Professor Abdel-Majeed Safer Histology & Histochemistry 20Confocal microscopyWith a regular bright-field microscopy the beam of light is relatively large and fills the specimen.

Stray light reduces contrast within the image;

confocal microscopy avoids stray light and achieves greater resolution by using:

A small point of high intensity light providing by a laser .

A plate with a pinhole aperture

infront

of the image detector.

the point light source, the focal point of the lens, and the detector’s pinpoint aperture are all optically conjugated or

alighned

to each other in the focal plane (CONFOCAL) and unfocused light does not pass through the pinhole. This greatly improves resolution of the object in focus and allows the localization of specimen components with much greater precision than with bright-filed microscope.

Fig. 1-6.Slide21

Principle of confocal microscopy Slide22

Figure 1–6.Principle of confocal microscopy. Although a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the blind. Thus, only one very thin plane of the specimen is focused at a time. The diagram shows the practical arrangement of a confocal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pinhole and a detector. Light from components of the specimen that are above or below the focused plane is blocked by the blind. The laser scans the specimen so that a larger area of the specimen can be observed.Slide23

Professor Abdel-Majeed Safer Histology & Histochemistry 23Polarizing microscopyAllows the recognitio

, in of structures made of highly organized molecules.When normal light passes through a polarizing filter, it exists vibrating in only one direction.

If a second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through. If, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repetitive structure rotates the axis of the light emerging from the polarizer and they appear as bright structures against a dark background

Fig.1-7.

So, crystalline substances or substances containing highly oriented molecules such as cellulose, collagen,

micromolecules

and microfilaments have the property of

birefringes

. Slide24

bright-field and polarizing microscopy

Bright-field microscopy

Plarizing microscopySlide25

Figure 1–7.Tissue appearance with bright-field and polarizing microscopy. Polarizing light microscopy produces an image only of material having repetitive, periodic macromolecular structure; features without such structure are not seen. Shown here is a piece of thin mesentery that was stained with red picrosirius, orcein, and hematoxylin

, and was then placed directly on a slide and observed by bright-field and polarizing microscopy. (a): Under routine bright-field microscopy collagen fibers appear red, along with thin dark elastic fibers and cell nuclei. (b): Under polarizing light microscopy, only collagen fibers are visible and these exhibit intense birefringence and appear bright red or yellow; elastic fibers and nuclei lack oriented macromolecular structure and are not visible.Slide26

Professor Abdel-Majeed Safer Histology & Histochemistry 26Electron microscopyTEM and SEM are based on the interaction of electrons and tissue components.The wavelength in electron beam is much shorter than of light. Allowing a thousand-fold increase in resolution.

Transmission Electron Microscopy TEM

with a resolution of 0.3nm

(Fig.1-8a).

Which allows magnification of up to 800,000 times to be viewed with details. (

1nm = 0.001um = 10-6 mm. 1A = 0.1nm 10-4um).

Principle:

A beam of electrons can be deflected by

electromagnitic

fields. E beam is produced by a cathode and passes down the column in a vacuum. The beam can be focused by means of electric coils (electromagnetic lenses).

First lens is Condenser lens, which focus beam on the specimen section.

Second lens is Objective lens, when electrons pass the specimen without interacting with it, it reaches the Objective lens. This form a focused, magnified image, and reach a viewing screen. The image is white and black and gray. (bright areas indicate electron passing through, darker areas – electron dense- indicate electrons were absorbed or deflected).Slide27

Schematic view of a transmission electron microscope (TEM) Slide28

Professor Abdel-Majeed Safer Histology & Histochemistry 28TEM requires very thin sections (40-90 nm), so plastic embedding blocks are cut with glass or diamond knives. Sections are collected on small copper grids and transferred into the microscope to be analyzed.Freeze-fractyre

, cryofracture

, freeze etched combined with TEM have been useful for examining membrane structure.

Scanning Electron Microscopy SEM

It permits 3-D views of the surfaces of cells, tissues, and organs.

Beam does not pass through the specimens

(Fig.1-8a).

The sample is dried and coated with a very thin layer of metal atoms through which electrons do not pass readily. When the beam is scanned from point to point across the specimen it interact with the metal atoms and produces reflected electrons or secondary electrons emitted from the metal. These are captured by a

detedtor

and the resulting signal is processed to produce a black-and-white image on a monitor

(Fig.1-8b).

Slide29

Schematic view of a scanning electron microscope (TEM) Slide30

Professor Abdel-Majeed Safer Histology & Histochemistry 30AutoradiographyIs a method of localizing newly synthesized macromolecules (DNA, RNA, protein, glycoproteins

, and polysaccharides) in cells or in tissue sections. Radioactively labled

metabolites (nucleotides, amino acids) incorporated into the macromolecules emit weak radiation that is restricted to the cellular regions where the molecules are located. Radiolabel cells or mounted tissue sections are coated in a darkroom with photographic emulsion containing silver bromide crystals, which acts as

microdetectors

of this radiation in the same way that they respond to light in common photographic film. After some times, the slides are developed photographically. The silver bromide crystals reduced by the radiation are reduced to small black grains of metallic silver, indicating location of

radiolabled

macromolecules in the tissue.

This tech can be used for both LM and EM

(Fig.1-9).

Practically, it is possible to know which cells is a tissue produce more protein and which cell produce less, because the number of silver grains formed over the cells is proportional to the intensity of protein synthesis. Slide31

Autoradiographs are tissue preparations in which particles called silver grains indicate the regions of cells in which specific macromolecules were synthesized just prior to fixationBlack “silver grains” are visible over regions with secretory granules and the duct indicating glycoprotein locations. X1500. Slide32

The same tissue prepared for TEM autoradiography shows silver grains with a coiled or amorphous appearance again localized mainly over the granules (G) and in the gland lumen (L). X7500. Autoradiographs are tissue preparations in which particles called silver grains indicate the regions of cells in which specific macromolecules were synthesized just prior to fixationSlide33

Professor Abdel-Majeed Safer Histology & Histochemistry 33Cell & Tissue CultureLive cells and tissues can be maintained and studied outside the body. cell culture has been very useful in isolating the effects of single molecules on specific types of cells. It also allows the direct observation of the

behavior of living cells under the phase contrast microscope. Many experiments that cannot be performed in the living animal can be accomplished

in vitro.

The cells and tissues are grown in complex solutions of known composition (salts, amino acids, vitamins) to which serum components or specific growth factors are added.

Cells must be dispersed mechanically or

enzymatically

.

The cells are then cultivated in a clear dish to which they adhere, as single-layer

cellss

(Fig.1-5).

This is called primary cell culture, which makes cell line. Normal or pathologic tissue have been maintained in vitro ever since they have immortalized and now constitute a permanent cell line. Cell culture has been widely used for study of the metabolism of normal and cancerous cells and for the development of new drugs. Slide34

Professor Abdel-Majeed Safer Histology & Histochemistry 34Histochemistry &

Cytochemistry

They are methods for localizing cellular structures in tissues using enzyme activity present in these structures. Usually it is applied to unfixed or mildly fixed tissue, sectioned on a cryostat or freezing microtome to avoid adverse effects of heat, paraffin and chemicals on enzymatic activity.

1. Tissue sections are immersed in a solution containing the substrate of the enzyme to be localized.

2. The enzyme is allowed to work on the substrate.

3. The section is put in contact with the marker compound.

4. This compound reacts with a molecule produced by enzymatic action on the substrate.

5. The final reaction product (insoluble and visible in LM or EM) only if it is colored of electron- dense, precipitate over the site that contains the enzyme.

e.g.

Phosphatases

,

split the bond between a PO4 group and an alcohol residue of phosphorylated molecules. The visible insoluble reaction product of phosphateses is lead phosphate or lead sulphide. At alkaline pH and cid phosphatese. (Fig.1-10).Slide35

Professor Abdel-Majeed Safer Histology & Histochemistry 35DehydrogenasesRemove H from one substrate and transfer it to another. Mitochondria can be specifically identified by this method, dehydrogenases are key enzymes in the citric acid cycle.

Peroxidases

Present in several types of cells, promotes the oxidation of certain substates with the transfer of H ions to H2O2, forming H2O.

Fixed sections are incubated in a solution of H2O2 and 3,3”-diamino-azobenzinedine DAB. DAB is oxidized in the presence of peroxidaze, resulting in an insoluble , brown, electron-dense ppt w permit the localization of peroxidase activity by LM and EM. Slide36

Professor Abdel-Majeed Safer Histology & Histochemistry 36Artifacts

Kind of flaw that is due to faulty technique:Postmortem degeneration

ShrinkagePrecipitants

Wrinkles and Folds

Nicks in the microtome knife

Rough Handling

Problems in the study of Tissue Sections

(Fig.1-15)Slide37

Professor Abdel-Majeed Safer Histology & Histochemistry 37END of Chapter 1