HistologyHistology

HistologyHistology

The study of tissue is large avisual science that The study of tissue is large avisual science that relies on microscopy to reveal relies on microscopy to reveal cells,tissue&organcells,tissue&organ

--Establishing basis for learning histopathology Establishing basis for learning histopathology which involves the relationship between which involves the relationship between abnormal tissue structures&functional defectabnormal tissue structures&functional defect

33 different methods,for different methods,for preparing histological slidespreparing histological slides

11--Paraffin technique(common used)Paraffin technique(common used)22--Celloidin techniqie(less common)Celloidin techniqie(less common)

33--Freezing technique by using cryostateFreezing technique by using cryostate

Steps of paraffin Preparing TissueSteps of paraffin Preparing Tissue

SpecimenSpecimen11--Chemical fixation:Chemical fixation: Formalin,Carnoy,bouins Formalin,Carnoy,bouins

solution,steeph ,normal salinesolution,steeph ,normal salineTime of fixationTime of fixationSmall specimenSmall specimenLarge specimenLarge specimen

22--DehydrationDehydration::

Eases pentration of tissue by clearing agent Eases pentration of tissue by clearing agent replaces water in tissue with organic solvent replaces water in tissue with organic solvent common (Ethyl) ascending grades of alcoohol common (Ethyl) ascending grades of alcoohol 70%,95%,100%(absolute)70%,95%,100%(absolute)

33--ClearingClearing::

prepares fixed tissue for prepares fixed tissue for infiltration,dehydrating agent is replaced with infiltration,dehydrating agent is replaced with clearing agentclearing agent(Xylene)paraffin(Xylene)paraffin solvent is solvent is common used for ligt microscopycommon used for ligt microscopy

Propylene oxide(plastic solvent)Propylene oxide(plastic solvent)in commonly in commonly used for electron microscop (EM)used for electron microscop (EM)

44--Impregnation Impregnation soft paraffin in 60-65 Csoft paraffin in 60-65 C

55--EmbeddingEmbedding:: prepares infiltrated tissue for prepares infiltrated tissue for sectioning makes tissue firm&prevents sectioning makes tissue firm&prevents

crushing or other tissue during section permits crushing or other tissue during section permits thin,uniform sectioningthin,uniform sectioning

66--SectioningSectioning:histological sections by:histological sections by Standard rotary micrtomeStandard rotary micrtome

77--put the paraffin sections in hot plate to put the paraffin sections in hot plate to drydry

77--Staining:With (H&E)Staining:With (H&E)

a-Xelenea-Xeleneb-decending grades of alcool 100% 90%,70%b-decending grades of alcool 100% 90%,70%

(hydration)(hydration)c-stain hematoxelene 4-6mc-stain hematoxelene 4-6m

d-running water 5md-running water 5mE-eosin 2mE-eosin 2mF-wash 2mF-wash 2m

Dehydrate the sections70%-100%Dehydrate the sections70%-100%

88--Mounting:Mounting: Eases handling and decrease Eases handling and decrease damage to specimen during examinationdamage to specimen during examination

Canda balsam&put the coverCanda balsam&put the cover

Acid stain(Eosin)bind basic(acidophilic,)Acid stain(Eosin)bind basic(acidophilic,)Basic stain(hematoxylin)bind acidic(basophilic Basic stain(hematoxylin)bind acidic(basophilic

tissuecomponenttissuecomponent((H&EH&E))most common stain mixture for light most common stain mixture for light

microscopy distinguishes nucleus from microscopy distinguishes nucleus from cytoplasmcytoplasm

Light Microscopy – Hematoxylin & Eosin )H&E( Stain

Basophilic )blue( Nuclei

Eosinophilic )red(

cytoplasm

44--Units of measuresUnits of measures

Measurement of cells &tissue component Measurement of cells &tissue component provide useful the metric system is used provide useful the metric system is used exclusively for the common units of measure exclusively for the common units of measure in histology are thein histology are the

millimetermillimeter , ,micrometersmicrometers&&

nanometernanometer

Advantage of paraffin sectionAdvantage of paraffin section11--gives serial sectiongives serial section

22--easy to prepare sectioneasy to prepare section33--it takes short timeit takes short time

44--slides can be stained easyslides can be stained easy

DisadvantageDisadvantage11--Not ideal for histochmistryNot ideal for histochmistry

22--can not used for demostration of fatcan not used for demostration of fat33 - -in process we use heatin process we use heat

MicroscopyMicroscopy

Many types of microscopes used in study Many types of microscopes used in study biological materialbiological material

I-Light microscope(LM) max. magnification is I-Light microscope(LM) max. magnification is 100 times100 times

II-Electron Microscope(Transmition) II-Electron Microscope(Transmition) Magnification power is up 100,000 timesMagnification power is up 100,000 times

III-Scanning Electron MicroIII-Scanning Electron Micro

IV- Ultraviolet MicroscopicIV- Ultraviolet MicroscopicThe image is received on fluorescent The image is received on fluorescent

screen,magn. Power is 400 timesscreen,magn. Power is 400 timesV-phase contrast microscopeV-phase contrast microscope

Colorized low mag TEM of a cell

Red = Mitos

Blue = Ribos

Green = RER

Colorized TEM of nuclear pores )blue( en face

High mag TEM of a nucleolus

Pars fibrosa/granulosa )dense black( and

fibrillar center )arrow(

Low mag TEM of a nuclei

Cell structure & function Cell structure & function *Introduction :*Introduction :

The cell is the functioning unit of all living tissues :The cell is the functioning unit of all living tissues :

--DifferentiationDifferentiation

-The nucleus + the cytoplasm-The nucleus + the cytoplasm

-Organelles-Organelles

-Light microscope + EM + histochemistry + -Light microscope + EM + histochemistry + immunohistochemistry immunohistochemistry

cells are the basic structural and functional units of the cells are the basic structural and functional units of the human body & there are many different types of cells (e.g., human body & there are many different types of cells (e.g.,

muscle, nerve, blood, and so on)muscle, nerve, blood, and so on)

Chemical Constituents of Cells and Tissues

CellCell

The cells divides into two major The cells divides into two major compartments composed of compartments composed of nucleus&cytoplasmnucleus&cytoplasm

ShapeShape(round,oval,cubical or columnar(round,oval,cubical or columnar sizesize .small (cerebellum) .small (cerebellum)

large (skeletal muscle)large (skeletal muscle)

CytoplasmaCytoplasma

Consist of cytoplasmic matrix formed of Consist of cytoplasmic matrix formed of proteinprotein,,

CarbohydrateCarbohydrateMineralsMinerals

EnzymeEnzymeCytoplasmic organellesCytoplasmic organelles

Cytoplasmic inclusionCytoplasmic inclusion

Cytoplasmic OrganellesCytoplasmic Organelles

****Membranous cell organelles have membraneMembranous cell organelles have membrane

****Nonmembranous without membranesNonmembranous without membranes

Membranous cell OrganellesMembranous cell Organelles

11--Cell MembraneCell Membrane22--MitochondriaMitochondria33--Golgi ApartusGolgi Apartus

44--PeroxisomePeroxisome55--Endoplasmic reticulumEndoplasmic reticulum

66--LysosomesLysosomes77--coated vesiclescoated vesicles

88--EndosomesEndosomes

Plasma MembranePlasma Membrane

**The outer cell membrane is called the plasm The outer cell membrane is called the plasm membrane or plasmlemmamembrane or plasmlemma

**Cell Membrane can not be seen by LMCell Membrane can not be seen by LM**Very thin can be stained by (PAS)Very thin can be stained by (PAS)

Composed of Phospholipid &cholestrolComposed of Phospholipid &cholestrol

tissue is a group of cells that perform a specific function and tissue is a group of cells that perform a specific function and the basic types of tissues in the human body include the basic types of tissues in the human body include

epithelial, muscle, nervous, and connective tissuesepithelial, muscle, nervous, and connective tissues

22--proteins:proteins: may make up more than 50% of may make up more than 50% of membrane weightmembrane weight

Most membrane proteins are globular and Most membrane proteins are globular and belong to one of the following two groupsbelong to one of the following two groups

a-a-integral membrane proteinsintegral membrane proteinsbb-peripheral membrane protieins-peripheral membrane protieins

33--carbohydrate:carbohydrate:ocure on plasm membranes ocure on plasm membranes mainly as oligosaccharidesmainly as oligosaccharides

Membrane FunctionMembrane Function11--selective permeability: is essential to selective permeability: is essential to

maintaining the functional homestasismaintaining the functional homestasisa-passive diffusiona-passive diffusion

b-facilitated diffusionb-facilitated diffusionc-active transportc-active transport

A-Biochemical Component

1-Lipid Molecules(30%):present in cell membrane as phospholipids,&cholesterol

1-Phospholipid molecule is formed of two partsa-Hydrophilic (its likes water)b-hydrophobic non charged (its dislikes water)Function: facilitate enternce of fat-soluble materials into the cell

2-proteins (60%):

may make up more than 50%of membrane weightMost membrane proteins are globular and belong to one of the following two groupsa-Integral membrane proteins(Extrinsic)b-peripheral membrane protieins(Intrinsic)

Active transportActive transport

Molecules(10%):3-Carbohydrate

3-carbohydrate ocure on plasm membranes mainly as oligosaccharides present in external surface of the cell membrane

Membrane Function:

1-Active transport of material(sugar,amino acid&fatty acids) 2-passive diffusion of small molecules

Potassium ions&oxygen to pass into the cellsCarbohydrate to leave the cells

3-selective permeability: is essential to maintaining the functional homestasis

4-transport of solid material by phagocytosis5-transport of fluid material by pinocytosis

6-explusion of residual by exocytosis7-sodium-potasium pump function

(Na pumping out side the cells accumulate of Na in the tissue fluid than cytoplasm

Potassium accumulate in the cytoplasm than tissue fluid )

transport across plasma membrane :

1)PASSIVE TRANSPORT

2)FACILATED DIFFUSION needs “Carriers”

3)Active transport needs energy in form of ATP

4)bulk transport Pinocytosis

Phagocytosis

Needs membrane –bound organelles called lysosomes Endocytosis

Exocytosis

transcytosis

Active transport : movement of a substance across a cell membrane from Active transport : movement of a substance across a cell membrane from an area of low concentration to an area of high concentration using a an area of low concentration to an area of high concentration using a

carrier moleculecarrier molecule