Light and Fluorescence Microscopy

Question 1

Resolution definition

Answer 1

The smallest distance between two structures at which they can be seen as two separate entities. Determines the quality of the image, its clarity and richness of detail.

Question 2

Resolution of a light microscope

Answer 2

approximately 0.2 micro-metres

Question 3

What can be viewed?

Answer 3

Nuclei, mitochondria. ribosome, storage granules and bacteria

Question 4

Three steps to prepare specimens for light microscopy

Answer 4

Fixation, sectioning and staining

Question 5

Fixation definition

Answer 5

pieces of organ are placed as soon as possible after removal from the body into solutions of stabilising and cross-linking fixatives.

Question 6

Why are tissues cut into fine fragments?

Answer 6

Fixative must diffuse fully into tissue

Question 7

What fixative is used in light microscopy?

Answer 7

37% formaldehyde

Question 8

Function of fixation

Answer 8

Preserve tissue structure and prevent degradation by enzymes released from cells or microorganisms

Question 9

Sectioning definition

Answer 9

Cutting the tissue into thin specimen

Question 10

What occurs before sectioning?

Answer 10

The fixed tissue is embedded in a material that imparts firm consistency, such as paraffin.

Question 11

Stages of sectioning

Answer 11

Embedded, fixed tissue is placed into a microtome.

Paraffin sections cut between 3-10 micrometers in thickness. Specimen then placed on a slide.

Question 12

Staining definition

Answer 12

Adding a dye to differentiate between different materials in the cells

Question 13

What dye is often used?

Answer 13

Hematoxylin and eosin H&E

Question 14

Basophilic vs acidophilic

Answer 14

Hematoxylin binds to basophilic organelles with a net negative charge

Eosin binds to acidophilic organelles, with a net positive charge

Question 15

What colour is hematoxylin and what does it bind?

Answer 15

Purple- DNA, RNA, ribosomes, GAGs and cartilage matrix

Question 16

What colour is eosin and what does it bind?

Answer 16

Pink- other cytoplasmic structures and collagen. Most proteins stain pink, especially cytoplasm of muscle, red blood cells and epithelial cells

Question 17

Immunohistochemistry definition

Answer 17

Based on specific reactions between an antibody and antigen labeled with visible markers, often fluorescent compounds. Then viewed using light microscopy.

Question 18

Two types of immunohistochemistry explained

Answer 18

Direct- labelled antibody binds to protein of interest

Indirect- Sequential application of two antibodies and washing steps. Antibody added, then washed, another antibody added with tag, then washed, enzyme added that binds with tag

Question 19

Hybridisation definition

Answer 19

Implies the specific binding between two single strands of nucleic acid, which occurs under appropriate conditions if the strands are complementary.

Question 20

In situ hybridisation definition

Answer 20

Nucleic acid sequences in solution applied directly to prepared tissues and cells, where they diffuse into the nuclei and bind to RNA/DNA.

Question 21

What form of nucleic acid is used?

Answer 21

Probes

Question 22

Fluorescence microscopy explained

Answer 22

Specimen illuminated with a light at a specific wavelength, which is absorbed by fluorophores, causing them to emit light at a longer wavelength (at a different colour than absorbed light)