Session 1: Light Microscopy

Most human cells are ___ in diameter

10-20 µm

Order these cells from sizes (smallest to largest)

Keratinocyte, Oocyte, Neutrophil, RBCs

1) Smallest = RBCs

2) Neutrophils

3) Keratinocyte

4) Largest = Oocyte

Limit of resolution

The minimum distance at which two objects can be distinguished (resolved) as two separate entities

Resolution is ___ proportional to wavelength

inversely

What is the practical limit of resolution for light microscopy

200nm

What is the practical limit of resolution for electron microscopy

~0.1nm

Intestinal epithelium

Value of histology in diagnosis

- Malignancies are associated with characteristic histological changes

- Benign/malignant samples differences

- Cancer 'staging' allows for effective trreatment and prognosis

What is the arrow pointing to here. Are these cells dividing? What types of cells are they?

Dividing melanocytes

The top layer is the = granular layer (last living layer of the epidermis)

Breslow thickness

Depth of invasion of melanoma from the granular layer of the epidermis, the best predictor of 5-year survival rates

Breslow thickness <1mm

5-year survival rate

95-100%

Breslow thickness 1-2mm

5-year survival rate

80-96%

Breslow thickness 2.5mm

5-year survival rate

60-75%

Breslow thickness 4mm

5-year survival rate

60-75%

Breslow thickness 5mm

5-year survival rate

37-50%

Describe the process of histology from biopsy to microscopy...

1) Biopsy = collection of tissue sample

2) Fixation = chemical preservation of tissue

3) Embedding/processing = dehydration and solidification of tissue

4) Sectioning = cutting of solifided tissue with microtome

5) Staining = colouring of tissue section

6) Viewing/analysis = microscopic viewing of stained tissue

Biopsy

Removal of a small piece of tissue for microscopic examination

Smear biopsy locations

Cervix

Buccal cavity

Curettage biopsy location

Endometrial lining of uterus

Direct incision biopsy location

Skin

Mouth

Larynx

Needle biopsy location

Brain

Breast

Liver

Kidney

Muscle

Endoscopic biopsy location

Lung

Intestine

Bladder

Transvascular biopsy location

Heart

Liver

Fixation

Macromolecules are cross-linked, cellular structure is preserved.

Prevents autolysis or putrefaction.

Give some examples of fixatives

Glutaraldehyde

Formaldehyde

Alcohol

Embedding is carried out at what temperature?

56 degrees celsius

Dehydration is carried out in what solution?

Ethanol (70-100%)

Biopsy is impregnated with and embedded in what solution?

Paraffin wax

Give an example of two ways artefacts can be formed due to errors in tissue processing process?

1) Scoring and tearing of section = due to nick in microtome knife edge

2) Air bubble formation = during tissue mounting procedure

Haematoxylin

- Basic dye

- Stains acidic components of cells purple/blue

- Stains nucleolus/r-RNA purple/blue

Eosin

- Acidic dye

- Stains basic components of cells pink

- Stains cytoplasmic proteins and extracellular proteins (collagen) pink

Gastric pit of the stomach (stained with H&E)

Periodic acid-Schiff (PAS)

- Stains carbohydrates and glycoproteins magenta

Intestinal villi stained with Periodic acid-Schiff (PAS)

Note = the goblet cells are stained magenta (due to mucus)

Viewing and analysis in clinical settings - tools used and two examples

High-throughout digital slide scanners and image analysis softwares commonly used in clinical settings

- Aperio AT2

- Leica

H&E stained pancreas

Heterochromatin

- Highly condensed/tightly packed chromatin/DNA

- Transcriptionally SILENT/inactive

- Intense haematoxylin staining

Euchromatin

- Less condensed form of chromatin/looser

- Transcriptionally ACTIVE

- Mostly found at periphery of nucleus/nucleolus

Where are Nissl bodies found?

Abundant in large nerve cells such as motor neurones

Nissl bodies

RER and free ribosomes in neurons = sites of protein synthesis

What factors must be considered when taking histological sections?

- Planes of section = region of interest may lie in a different plane

- Shape of cell being sectioned = e.g., RBC is biconcave

Periodic acid-Schiff (PAS) stains are useful for revealing structures such as...

Basement membrane

Periodic acid-Schiff (PAS) stains are useful for revealing what pathologies?

Diabetes

Phase-contrast microscopy

- Allows examination of living cells

- Quick and easy method

- No need for cell staining (cells unstained)

- Very good contrast compared to unstained brightfield microscopy

Brightfield microscopy

- Objects are dark and the field is light; can be used to observe unstained microorganisms

- Poor contrast

Confocal microscopy

- High resolution images with no out-of-focus light

- Stain specific proteins with fluorescent dyes/fluorescently-tagged antibodies = many applications

- 3D image construction

Darkfield microscopy

- Light objects are visible against a dark background

- Similar set-up to Brightfield

- Useful for diagnosing Treponema palliudum (syphilis)

- Useful for detecting Vibrio cholerae (cholera) in stool sample

What two conditions is darkfield microscopy useful for identifying?

1) Treponema pallidum = syphilis

2) Vibrio cholerae = cholera

Why is it necessary to preserve fresh tissue? Give the most appropriate answer.

a) To preserve nucleic acids

b) All answers are correct

c) Prevent putrefaction

d) Preserve proteins

e) Maintain structure

b) All answers are correct

Why is it difficult to cut thin sections of fresh tissue? Give the most appropriate answer.

Select one:

a. Fresh tissue will blunt the microtome blade too quickly

b. Fresh tissue is too bloody

c. Fresh tissue is too fibrous to cut consistently

d. Fresh tissue is soft and will lose its integrity as the microtome blade passes through it

d) Fresh tissue is soft and will lose its integrity as the microtome blade passes through it

Which two of the following methods allow for the effective sectioning of tissues?

a. Heating

b. Embedding in a solid medium such as wax or plastic

c. Formalin fixation

d. Freezing (cryopreservation)

e. Clearing in xylene

f. Drying

b) Embedding in a solid medium such as wax or plastic

d) Freezing (cryopreservation)

Why is it difficult to infuse fresh tissue with wax?

Cells are mainly water, which doesn't mix with wax

Why might you wish to stain histological sections with haematoxylin and eosin?

a) To allow long-term storage

b) To preserve structure

c) To identify specific proteins

d) Thin tissues are translucent and must be stained to reveal structure

d) Thin tissues are translucent and must be stained to reveal structure

On which component of cells do fixatives primarily act?

Select one:

a) The cytoskeleton

b) Nucleic acids

c) Lipids

d) The plasma membrane

e) Proteins in general

e) Proteins in general

What is the most commonly used dehydrating agent?

Select one:

a) Formaldehyde

b) Wax

c) Xylene

d) Alcohol

e) Eosin

d) Alcohol

Order these cells from smallest to largest...

Keratinocytes

Neutrophils

RBCs

Oocytes

Smallest: RBC

Neutrophils

Keratinocytes

Largest: Oocytes