3 How To Examine Cells And Tissues

Question 1

The limit of resolution

Answer 1

The smallest distance by which two objects can be separated and still be distinguished as two separate entities.
Resolution (d)= wavelength/2

Question 2

Light Microscopy vs Electron Microscopy

Answer 2

LM:
Can see in natural colour, large field of view, view living organisms, X600 magnification, 0.25microm, cheap & easy, can see down to bacterial level.
EM:
Monochrome, limited field of view, dead or inert objects, X500,000 magnification, 0.25nm resolution, expensive & timely.

Question 3

Transmission electron microscopy

Answer 3

Fixed in glutaraldehyde
Embed in epoxy resin
Stain
Use microtome with diamond knife

Question 4

Scanning EM

Answer 4

Fixed with glutaraldehyde
Embed in epoxy resin
Stain (OT)

Question 5

Freeze Fracture EM

Answer 5

Freeze tissue and fracture it by hitting with edge of knife. The fracture line passes through the plasma membrane to expose the interior

Question 6

Requirements for LM

Answer 6

Use formalin to prevent rotting
Embed tissue in paraffin wax so can thinly slice
Stain using Haematoxylin and Eoisin

Question 7

Staining methods?

Answer 7

H&E: most cells
—> H stains acids in cell so nucleus as DNA and RNA are acidic BLUE
—> E stains mainly proteins so cytoplasm and extracellular matrix PINK
Massons trichrome: keratin, muscle fibres
Periodic acid- schiff stain: anything with sugar attached

Question 8

Frozen section vs paraffin embedded section

Answer 8

Frozen Section:

• Fresh tissues
• 10-20 mins
• Saving time: months
• Morphology:opacity
• Application: intraoperative consultation

Paraffin embedded section:

• Fixed tissue
• 24-48 hours
• Saving time: permanent
• Morphology: clarity
• Application: pathological diagnosis

Question 9

Immunohistochemistry

Answer 9

Tagging antigens with antibodies. Can use sencondary Ab tagged with horseradish peroxidase which changes colour when attached.
Could also be immunofluorescently tagged.

Question 10

Confocal microscopy

Answer 10

Laser excites fluorescent molecules and electrons in dye. Raised to higher energy level. As electrons relax back to ground state, light from higher wavelength is emitted which reflects in mirrors reaching the CMOS detector.

—> used for sharp in-depth image. Allows full section scanning.

Question 11

Cell culture

Advantages and Disadvantages

Answer 11

Must maintain homeostatic parameters
Advantages: absolute control over physical environment, homogeneity, less need for animal models.
Disadvantages: hard to maintain, instability of sample, dedifferentiation, 3D architecture lost, costly process.