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Key Concepts addressed:

Introduction to the use of the microscope and the virtual microscopy system How samples are prepared for microscopic examination Appearances of tissues using standard histological stains Comparison between normal and diseased tissues at a microscopic level.

You will be introduced to the microscopic examination of tissues. After some preliminary work using conventional microscopy, you will use the virtual microscopy system to examine both normal and diseased tissues, with examples from the gastrointestinal tract. At the end of the session, you should be capable of using the virtual microscopy system to view histological and histopathological specimens.


Tissue preparation for examination under the microscope To preserve the structural relationship between cells in tissues, it is necessary to cut very thin slices (sections) that are suitable for LM (light microscopy) or EM (electron microscopy). Sections must be thin enough to transmit sufficient light and to avoid visual superimposition of their various components.

  1. Tissue samples are obtained by biopsy or postmortem. If the sample is taken from a cadaver, it is essential to remove it promptly after death to avoid degeneration. As soon as possible after it has been removed, it should be immersed in a fixative.
  2. Fixation. Fixatives prevent postmortem degeneration and structural changes in cells and tissues, and they harden soft tissues. Formaldehyde, 4% in aqueous solution buffered to neutral pH (formalin), is routinely employed for LM fixation. Chemical fixatives either 1) cross-link protein molecules (present in cells and tissues) or 2) denature and precipitate them by replacing the water associated with them. Fixatives also prevent the degradation of tissue components by hydrolytic enzymes released from cells when they die. Most fixatives are strong antiseptics, killing pathogenic microorganisms present in infected tissues. Fixation can also enhance subsequent staining of the tissue.
  3. Dehydration and Clearing. Because a large fraction of the tissue is made of water, a graded series of alcohol baths (50% alcohol and progressing in graded steps to 100% alcohol), are used to remove water (dehydration). The tissue is then treated with xylene, a chemical that is miscible with melted paraffin. This is known as clearing, since the tissue becomes transparent in xylene (a benzene derivative).
  4. Embedding. The xylene-permeated tissue block is passed through several changes of warm paraffin under vacuum. The melted wax fills in all the spaces formerly occupied by water, and when it hardens on cooling,it renders the tissue block ready for sectioning. Wax also acts as a physicl support to the sample.
  5. Sectioning. Once the excess wax has been trimmed away, sections can be cut from the tissue block by means of a microtome and flattened in a water bath (to get rid of wrinkles). (7-8 um slices are produced: ribbons of tissue that have to be flattened. Sometimes wrinkles/folds remain, producing artefacts in the slide). Frozen sections can be cut when there is surgical urgency or when fresh tissue is required (e.g. lipid, antigenic or enzymatic investigations) (alcohols stop enzymatic activity). Moreover, virtually everything present in the living tissue is still present in the same place in frozen sections, but it is necessary to examine these sections promptly to avoid fixation. Supplemental fixation is essential for a permanent preparation. To cut frozen sections, the block of fresh tissue is frozen very rapidly using liquid nitrogen (-150 to -170 C). It is then sectioned (5-10 um thick) inside the refrigerated cabinet of a cryostat, designed to maintain the microtome knife and tissue at a subzero temperature throughout the entire cutting operation. The sections are placed on pre-cooled glass slides, permitted to come to room temperature, and stained with specific dyes or treated for histochemical or immunohistochemical studies. Section thickness. For routine purposes, sections are generall cut to a thickness of 5 to 8 um. If thinner (semi thin) sections are required, the tissue is embedded in plastic or epoxy resin instead of paraffin wax and usually cut to a thickness of 1 to 2 um.
  6. Staining and mounting (to highlight different things). Most staining is done in aqueous solution, so the wax that permeates the section needs to be replaced by water. This is achieved by attaching the section to a slide and passing it through xylene to remove the paraffin, through absolute alcohol to remove the xylene, through alcohol rinses of decreasing strength, and then through water. Once stained, it is passed through a series of increasing strengths of alcohol to absolute alcohol and then xylene. Finally, it is mounted in a mounted in a mounting medium dissolved in xylene.

Mounting eliminates unwanted diffraction of the light passing through the section. A protective glass coverslip is positioned over the section so that when the xylene in the mounting medium evaporates, the coverslip becomes tightly bonded to the slide.

Histological Staining

Until tissue components have been suitably stained, they are all of fairly uniform optical density; hence most of what is present in a section appears indistinct and fails to stand out in sufficient contrast to the remainder. Staining is essential if all the components of a section are to be distinguished under LM. Used in appropriate combinations, histological stains can act in 2 different ways. First, they enable the observer to recognise certain tissue components by differentially colouring them. Secondly, they can impart their colours to different extents, which results in graded depth of staining.

Stains may be grouped into 3 classes:

  1. Stains that differentiate between acidic and basic components of the cell
  2. Specialised stains that differentiate fibrous components of the extracellular matrix
  3. Metallic salts that precipitate on tissues, forming metal deposits on them.

The most common stains are Haematoxylin and Eosin (H&E).

  • Haematoxylin (from the log-wood tree) is a basic (positive charge at pH 7) stain.
    • Colours the acidic components of the cell a purple-bluish tint.
    • The most acidic components are DNA and RNA, so the nucleus and regions of the cytoplasm rich in ribosomes stain dark blue; these components are referred to as basophilic.
    • Also stains:
      • Nuclei/DNA/RNA
      • Acid mucopolysaccharides (cartilage e.g. hyaline cartilage in joints)
      • Calcium salts
        • Eosin is an acid (negative charge at pH 7)
    • Dyes the basic components of the cell a red-pinkish colour.
    • Many cytoplasmic constituents are basic, so regions of the cytoplasm stain pink; these components are said to be acidophilic
  • Histopathology = histology of diseased tissues.
  • Physiology = normal function

Slide 1: Normal liver (H&E)

  • DNA = purple/blue under the stain. Hence nuclei stain purple/blue.
    • Some cells appear to have no nucleus as they are on a different plane
  • Hepatocyte = the dominant cell here.
  • We see a string of spaces and circles with cells around them
    • Very thin 2D slice of a 3D structure
    • Holes could either be 1) spheres (or cavities) 2) tubes
    • Very few tubes in the body go in straight lines
    • Turning tube = oval (unless on the corner, when you get a circle) – See Lazer’s notes.
    • Strings of cells radiating out from central holes (in 2D)
      • In 3D: sheets or columns of cells radiating out from a central tube
      • These are a series of radiating hepatic plates
      • In between plates are blood vessels, providing a high SA for osmosis/active transport. Therefore good for this transport.
        • All of the blood from the gut goes to the liver for processing.
    • Hence the liver is fed by a VEIN – the hepatic portal vein (not an artery)
    • The liver has both veins in and veins out.
  • When we see a blood vessel with fingers coming off of it, this means the vessel is branching
  • Lumen = hole in the middle of a vessel (the bore)
  • Veins – thin walls, large lumina, low pressure
  • Arteries - thick walls, small lumina, high pressure.
  • Artefacts in slides:
    • Folds (dark patch where section has folded when wax ribbon was flattened out)
    • Score marks (imperfections in the microtome have produced non-uniform cuts)
  • Vertical/horizontal ratio of a vessel:
    • In a vessel wall, there is a basement membrane with columnar/cuboidal/squamous epithelium cells.
    • Unroll the vessel (in your mind) and flatten out the basement membrane, with the cells sticking up.
    • This ratio = height of epithelium cells / length of the base
  • Portal triad drains bile
    • A portal triad (also known as portal area) is a distinctive arrangement in the liver. It is a component of the hepatic lobule. It consists of the following five structures:
      • hepatic artery
      • hepatic portal vein
      • bile duct
      • lymphatic vessels
      • branch of the vagus nerve
    • The misnomer "portal triad" traditionally has included only the first three structures, and was named before lymphatic vessels were discovered in the structure.
    • Bile or gall is a bitter-tasting, dark green to yellowish brown fluid, produced by the liver of most vertebrates, that aids the process of digestion of lipids in thesmall intestine. In many species, bile is stored in the gallbladder and upon eating is discharged into the duodenum. Bile is a composition of the following materials: water (85%), bile salts (10%), mucus and pigments (3%), fats (1%), inorganic salts (0.7%) and cholesterin (0.3%)
  • Collagen between cells = pink stuff (eosin stains it)
    • Fibular protein glue and support

Slide 2: Normal liver, reticulin stain

  • Uses silver salts to highlight the collagen framework that you couldn’t see well originally
  • Collagen appears as black lines.
  • Thin collagen framework surrounding the liver cells
    • This is the structural framework
    • If there is necrosis, we need this framework to survive if the liver is to regenerate (and recover its normal function)
    • Some diseases can destroy collagen framework. Then the liver cells don’t grow back in the correct 3D structure. Thus we can’t restore a functional tissue in these cases.

Slide 3: Normal pancreas

Under a microscope, stained sections of the pancreas reveal two different types of parenchymal tissue. Lightly staining clusters of cells are called islets of Langerhans, which produce hormones that underlie the endocrine functions of the pancreas. Darker staining cells form acini connected to ducts. Acinar cells belong to the exocrine pancreasand secrete digestive enzymes into the gut via a system of ducts.

Structure Appearance Function
Islets of Langerhans Lightly staining, large, spherical clusters Hormone production and secretion (endocrine pancreas)
Pancreatic acini Darker staining, small, berry-like clusters Digestive enzyme production and secretion (exocrine pancreas)
  • Pancreas
    • Produces hormones (e.g. insulin that lowers blood sugar levels and glucagon that raises blood sugar levels)
    • Produces digestive enzymes
  • Blood vessels
    • Lumen (can see blood cells inside: 1) red blood cells 2) white blood cells [dark dots])
    • Thick walls = arteries (high pressure) – surrounded by smooth muscle
    • Thin walls = veins (low pressure)
  • Adipose
    • Chicken wire
    • Adipocyte = 1 cell
    • Holes = liquid droplets
    • White fat is more common than brown fat
    • Serve as an energy reserve, and as insulation
    • Dissolved in preparation of the slide
    • Nucleus squashed to the side of the cell by the lipid.
  • Nerves
    • Wavy
    • No lumen
    • Surrounded by collagen
    • 2 types of cells in visible:
      • Schwann cells – produce myelin (insulates axons, makes messages travel faster)
      • Fibroblasts – make collagen
  • The group of pale cells in the pancreas are the islets of Langerhans
    • Alpha cells = glucagon producers
    • Beta cells = insulin producers
    • (can see neither of these with H and E)
    • Have lots of blood vessels so that hormones can be exported straight out through the blood
  • The dark purple cells are acini (acinar cells)
    • Produce enzymes
    • The enzymes are pushed through the ducts
  • Duct
    • Lined by necklace of simple cuboidal cells
    • Inside the ducts, there are no nuclei, just pink stuff (enzymes)
    • The ducts join to form bigger ducts, and then drain to the duodenum

Slide 4: Normal thick skin (palm)

The skin
  • Keratin on the surface is produced by dead keratinocytes of the stratum corneum. These cells and keratin fall off constantly and are replaced by mitotic cells in the dermis.
  • Ridging between the dermis (dermal papillae) and epidermis (epidermal pegs) to form fingerprints. Holds down the epidermis
  • Sweat ducts appear as adjacent ovals in the epidermis (spiralling duct that is cut straight)
  • At the base of the sweat ducts are the sweat glands.
  • Keratinising stratified squamous epithelium
  • The keratinocytes are produced in the stratum basale (very mitotic layer—above: germinativum)
  • As they move up, they die at the stratum granulosum.
  • They then fall off and produce keratin.
  • Meissner’s corpuscle: sensory receptor (think of it like a spring). Sensitive to light pressure. Quite superficial (below the stratum basale)
  • Pacinian corpuscle: sensory receptor (onion that squashes). Sensitive to firm/heavy pressure. Very deep.
  • Pink material = collagen (structural protein). There’s a lot of it.
  • Black lines in the dermis = elastic fibres (we have an elastic stain)
    • Skin has to stretch (e.g. obesity, pregnancy)
    • Can stretch beyond elastic potential, when they’ll snap (fail to obey Hooke’s law)
    • Then you get stretch marks (which are irreversible)
  • In between the fibres are spaces:
    • Some shrinkage
    • Also ground substance between fibres
      • Some injuries – oil substance comes out
      • Changes viscosity (slows down bacteria)
  • Haluronic acid, glycosaminoglycans: big molecules that bind to water and make it viscous like honey/jelly
  • Buys time so that your leucocytes can come defend
  • Fat is in the hypodermis (deep to the dermis)
    • Dermis’ collagen is anchored down here (holding on the palm of your hand)

Slide 5 Early cirrhosis of the liver (due to hepatitis)

[Chronic active hepatitis, cirrhosis]

  • Leucocytes are migrating in (dark purple blobs – these are their nuclei)
    • They are lymphocytes (all nucleus and not much cytoplasm)
    • Trying to fight the hepatitis C infection
    • (Most cells don’t have much RNA [unless they are mass production cells] hence dark purple usually indicates DNA [nucleus])
  • Hepatitis C
    • Gets into liver and stays for a long time
    • Ongoing recruitment of lots of host defense
    • Results in inflammation: 1) vessels dilate and become congested 2) oedema 3) leucocytes recruited
    • –itis = inflammation
    • hepar- = liver
  • Tissue was damaged
  • We see round clear spaces [adipose]
    • Possibilities: 1) air 2) fat 3) just fluid
    • Alcohol and xylene from slide preparation dissolve the fat
    • Fat blobs are spheres (to minimise SA:V)
    • Liver normally processes lipids
    • Disease state means that the liver doesn’t work properly and liver accumulates fats. Hence we have fatty blobs
  • Cells with 2 nuclei: mitosis
    • Trying to regenerate after damage
  • However, there was tissue damage with liver cells showing fat accumulation
    • Lots of inflammation
    • Cannot regenerate properly
    • Instead, there is repair, with scarring (collagen)
    • Therefore the structured plating of the liver is damaged (and so these tissues are not properly functional)
    • This liver is made of blobs of cells surrounded by scarring (collagen)
    • This is called cirrhosis.
  • Liver (a blood cleanser) not functioning properly
    • Hence there will be changes in the blood composition
    • We can detect these changes in blood tests
      • Things that the liver removes aren’t removed (so these will be high)
      • Things that the liver makes won’t be made (so these will be low)

Slide 6: Adenocarcinoma (carcinoma of the pancreas)

  • Lots of bands of scar tissue (collagen w/ fibroblasts)
  • Acini are damaged
  • This is indicative of inflammation followed by scarring
  • Chronic pancreatitis
  • Arrangement of tissue absolutely lost and we’re left with a mass of cells:
    • Loose connective tissue with lots of collagen in between
  • We also see cells with a dark block where the nucleus should be:
    • Mitosis is occurring
    • The cells duplicate their DNA
    • The nuclear membrane disintegrates
    • The mitotic spindle forms
    • The chromosomes move apart, to put one set in each new daughter cell
  • Dividing cells that have “lost the plot” – they are dividing beyond reason.
  • Few islets of Langerhans and other functional structures are observable
  • Hence dividing cells are destroying pre-existing cells
    • CANCER
    • A carcinoma of the pancreas
  • The vacuoles we observe are mucin vacuoles
    • The cancer is a mucous-secreting carcinoma
    • Sometimes the mucous is intracellular, other times it is extracellular
    • The pancreas normally secretes mucous
    • Now the structure of the islets of Langerhans has been destroyed, so the mucous-secreting cells are “going crazy” secreting mucous. Hence we see mucous vacuoules everywhere.

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