Histology Intro

Why are stains used

• most tissue must be stained to visualise tissue components in the light microscope

Types of dyes

• acidic

• Basic

Basic dyes, name and what they stain

• Basophilic dyes

• RNA and DNA

Acidic dyes, name and what they stain

• acidophilic

• mitochondria

• collagen

What are the most common dyes used

1. Haematoxylin→ basic→ preferential affinity for the acidici components of cells→ stained dark blue

   • nucleus and the cytoplasmic regions→ rich in ribosomes

2. Eosin→ acidc→ stained basic components pink/red

   • reacts with a variety of cytoplasmic proteins and extra-cellular strucutres

   • avid for RBC and eosinophils

(H&E)

Other types of stains

Making only smear of cheek cells

1. firmly wipe around inside of mouth with cotton bud

2. spread product onto slide

3. allow to dry

4. Stain with one or two drops of 1% methylene blue→ 2 mins

5. rinse with water

6. allow to dry

7. coverslip with DPX (weird kinda gluey thing)

8. Examine

4 Main primary tissue types→ based on types of cells and arrangement

1. Connective cells form connective tissue

2. Nerve cells form nervous tissue

3. Muscle cells form muscle tissue

4. Epithelial cells form epithelial tissue

1. Connective tissue

• comprise a variety of cells that differ in morphology and function

  • fribroblasts, adipocytes, chrondrocytes

What they do:

• Secrete abundance extracellular matrix

• provide support for other cells/tissues

Examples

• collage

• bone

• elastin

• adipose

  1. White

     • unilocular, single large fat droplet per cell

  2. Brown

     • multilocular, many small droplets per cell

• blood

  1. RBC

  2. WBC

     • lymphocytes, neutrophils, monocytes, Eosinophils, Basophils

  3. Plateletes

• cartilage

1. Connective tissue→ pics

2. Nervous tissue

Function of nerve cells

• receiving, generating and transmitting nerve impulses

Different types but will see

• ganglia and nerve trunks

Support cells

• different types

• Schwann cells give rise to myelin

2. Nervous tissue→ longitudinal section of nerve trunk

3. Muscle tissue

• elongated

• with contractile properties

Smooth:

• tapered cells + central nuclei

Striated

1. cardiac

   • central nucleus

   • intercalated discs

   • Purkinje fibres

   • appear to branch

2. Skeletal

   • peripheral nuclei

   • motor end plates

4. Epithelial tissue

1. cover body surfaces

2. line body cavities

3. form solid glands→ e.g salivary glands

Layers:

1. Simple

2. stratified

3. pseudostratified (simple but seems 2+ layers)

4. Transitional (found in urinary system→ appearance changes if stretched/unstretched)

Shape

1. Squamous

2. Cuboidal

3. Columnar

Modifications

1. cilia

2. microvilli

3. secretory bodies (mucus droplets, secretory granules)

Light microscopy overview

• most widley used form of microscopy

• optical and mechnaical pats

1. Visual light passes through specimen

2. collected by image-forming optices

3. reveal strucutre of living cells and tissues (and non-living)

Optical components

1. Condenser lens

   • collects and focuses light from light source

   • onto specimen

2. Objective len

   • collect light from specimen

   • enlarge and project illuminated image to eyepeice

3. Eyepiece (ocular) lens

   • further magnified image

   • projects it onto viewer’s eye

Mechanical parts

1. stage

   • platform slides mounted

2. Illumination system

   • tungsten lamp for transmitted light

   • with varibale control

3. diaphragm

   • alters amount of light reaching condenser and passes to specimen

4. Control knobs

   1. focus image→ moving stage up and down

   2. stage control→ movement of speciment along x and y

   3. regulation of light intensity

5. Nosepiece

   • holds objective lenses

The types of microscope we used

• upright→ illumination system below stage and lens system above the stage

• binocular→ 2 eye pieces

• 3 objective lenses (x4,x10,x40

• two x10 eyepieces

• mechanical stage with x an y vernier scales

Total magnification oof light microscope

magnifying power of objective x magnifying power of the eyepieces

Using light microscope

1. examine slide with naked eye on white background

2. place on stage

3. make sure right way up→ coverslip on top

4. focus x4 with focus knob

5. increase magnification

6. x40 objective→ constantly adjust the fine focus control

   • high power→ depth of field is less than the thickness of section→ so it is possible to scan and assemble a picture of all available details

   • depth of field→ thickness of the layer which is brought to sharp focus

7. note: easier to search for structure in low power→ look for it in lower power first then increase power

How condenser is set up

• set up with top lens almost touching the under surface of the slide

  • should not be needed to vary its position unless frosted surface of the light source is exactly in focus and interfers with observations

• Use only lower-power lens of the condenser for→ lower-power x4 objective lens

Aperture diaphragm of condenser

• should be reset whenever objective is changed:

  • open then gradually close until contrast of object is adequate

  • not too dark as to produce distortions

Resolution

• ability of the objective lens to distinguish detail

When is the resolution optimum

• when the aperture of the diaphragm is set just to fill the back focal plane

What happens when the diaphragm aperture closes

• contrast increases

  • contrast→ relative difference in light intensity coming from different parts of the specimen

  • usually low in most sections

Therefore, the aperture of the diaphragm comprimisers between

1. structure being clearly and comfortably visible

2. ability to see as much fine detail as possible

Other features of mechanical stage

• has vernier scale

• used to record exact position of an object on the slide

  • read like map references with east-west given first

Objectives are par-focal

→ have the same focal length

Meaning:

• if one is corrently focused→ the others will also be approximately in focus when switched

• But→ working distances are very different (i.e the x40 is much closer than the x10)

  • working distance of the x40 is less than the thickness of a slide

    • so spaciment cannot be focused on x40 if slide upside down

Cleaning

• slides→ ordinary tissues

• lenses→ special lens tissue→ rub gently using moisture from breath

Estimating size of a structure

• imagine how many of the structures can fit into the diamter of a field

or

• compare object of known size

  • e.g RBC→ 7 um

Electron microscopy

• must higher resolution and magnification than light

  • enables to exam cells and sub-cellular strucutures

Resolution

• minimum distance between two points on a specimen

• than can still be distinguished as two separate entities

Electron vs light resolution and magnitudes

resolution

E→ 2.5-7.5nm

L→250nm

Magnification

E→x 100,000

L→ x1000

Cell structure: cell membranes

1. plasma membrane- >highly selective barrier between cytoplasm and environment

   • EM→ thin line 10nm thick enclosing cell

   • EM provides evidence for the fluid mosaic model

2. Eukaryotic membrane around organelles

Organelles

• most organelles can be resolved under EM

Mitochondria

• inner membrane→ cristae

• contains enzymes for ox phos

Endoplasmic reticulum

• tubular membrane strucutres

• faltterned sheets and sacs

Rough ER

• flattened sheets→ cisternae

• rough→ ribosomes

• protein synthesis and export

Smooth ER

• no ribosomes

• smooth

• tubular

• lipid synthesis and transport

• intracellular C2+ storage

Golgi apparatus

• stacked, faltterend sacs of membrane

• surrounded by numerous vesicles→ pinched off from golgi

• concentration, chemical mod and packaging of proteins from ER

• sorts and directs protesin to correct cellular compartment

Lysosomes

• membrane bound

• acid hydrolases

• intracellular digestion of macromolecules

• vary in size and shape

Glycogen granules

• form aggregates

• appear as black dots in cytosol

Secretory granules

• intracellular vesicles

• seen in protein secreting cells

Immunocytochemistry

• technique used to detect presence of specific moelcules in cells and tissues→ especilaly protein

How does it work

1. antibodies (immunoglobulins, Igs) bind to targets with high specificty and affinity

2. can be generated by immune reactions and isolated and purifed

3. tagged with→

   1. radioactive isotopes

   2. gold colloid

   3. fluoresent compounds

   4. enzymes that catalyse formation of coloured reaction

   5. electron sense protducts

4. Antibody binding sites viewed on LW or EM→ to find protein location

5. ALSO→ can be used as rough estimate for amount of protein

   1. → when calibrated→ e.g brightness of fluorescent light

In situ hybridisation

1. short nucleic acid segments of particular sequence synthesized chemically→ ‘probes’

2. tagged in similar way to antibodes

3. bind to

   1. complementary uncoiled DNA in nucleus or

   2. mRNA in cytosol

4. Shows gene expression→ cox uncoiled DNA (only in transciption and replication) and mRNA only in transciption

Therefore the use of in situ hybridisation

1. map location of genes in chromosome

2. localize the site of production of peptide hormones

3. detect presence of viruses

How is the probe localised

Use

• immunocytochemistry

or

• autoradiography

Preparing a tissue microscopic examination: aim

• preserve the normal tissue structure

how

• cut with microtome→ very thin sections to allow light pass through

• mount to glass slide

• stain coz most tissue is colourless

Observed without sectioning?

• living cells

• thin, transparent membrane→ e.g mesentery

→ observed directly

Preparation of a tissue for miscroscopic examination consists of

1. Fixation

   1. Embedding

   2. Sectioning

   3. Distortion

2. Mounting

3. Staining

1. Fixation

• in vivo cellular strucuture preserved

• Chemical fixation with fixatives

  • solutions of stabilising or cross-linking agents

  • e.g Formaldehyde and glutaraldehyde most common

    • protein cross-linker

    • produce less distortion than fixative that coagulate protein (alcohlic fixers)

  • Other fixative effects→ induce chemical changes to tissue

    • e.g alcohol or organic solvents→ extract fat→ fat droplets look empty

1. Fixation→ must be fixed in a way that it can survive

1. embedding→ in semi-rigid medium (wax/plastic)

2. Slicing→ into 5-10um sections)

3. staining→ with selective dyes

1.1 Embedding

• infiltrate tissue with small molecules

• which can then be cross-linked to form matrix hard enough to withstand thin sectioning with microtome

• Most common→ paraffin wax and acrylic resins

Because embedding media is water insoluble…

Must be dehydrated

How:

1. pass tissue through series of graded alcohol to 100% alcohol

2. then immersed in monomer plastic at room temp

or

2. melted paraffin wax in oven at 60 degrees

3. Cooled to room temp→ suitably hard for sectioning

1.2 Sectioning

• slice with microtome

• thin enough to transmit light

  • wax sections→ 7um thin

  • resin section→ 1-2 um thick

1.3 Distortion

Can be introduced to tissue during embedding and sectioning. How?

1. alcohol for dehydrating→

   • extracts fats

   • coagulates poorly fixed proteins

2. Paraffin wax and other embedding agents

   • shrink and sistort tissues

   • produces artefacts o nsections

2. Mounting

• stuck onto glass slide

3. Staining

Variety of histologic stains commonly used to show up particular biochemical components of the tissue

1. Alcain blue →mucopolysaccharides

2. Eosin (neg charged, stains acidic)→ mitochondria, collagen, some secretory granules

3. Haematoxylin (pos charge, stain basophilic)→ nuclei, ribosomes, DNA

4. Ponceau S→ elastin

5. Osmium tetroxide→ lipid

Trichome stain

• comprise three different dyes

• to stain different components in different colours