Lec 14 -15 (Pathology, Assays, and Microscopy)

Question 1

Histopathology definition

Answer 1

Pathology using thin tissue slices viewed under the microscope

Question 2

Histopathology protocol

Answer 2

1- Harden tissue via wax or freezing
2- Cut tissue into sections
3- Stain tissue sections

Question 3

Name of compound used to stain DNA a in blue colour

Answer 3

Hematoxylin

Question 4

Name of compound used to stain cytoplasm and proteins an orange-pink colour

Answer 4

Eosin

Question 5

Protein assay types

Answer 5

• ELISA (main)
• Gel electrophoresis
• Microarrays

Question 6

Nucleic acid assay types

Answer 6

• PCR (amplification)
• FISH (location)
• Gel electrophoresis (size ranking)
• Microarrays

Question 7

Cellular assay types

Answer 7

• Flow cytometry (counting)
• Microscopy (imaging)
• Microarrays

Question 8

Tissue diagnostic types

Answer 8

• Microscopy
• Nonlinear imaging

Question 9

ELISA objective

Answer 9

Measuring protein concentrations in a sample using enzyme-linked antibodies
- Enzymes fluoresce –> Light intensity proportional to protein concentration

Question 10

ELISA types

Answer 10

• Sandwich
• Direct
• Indirect

Question 11

Sandwich ELISA steps

Answer 11

Distinctive feature: Capture antibodies
- Primary antibody sandwiched between capture and secondary antibodies

1- Capture antibody placed at bottom and antigen placed on top
2 - Primary antibody binds to antigen
3- Secondary antibody binds to primary and is enzyme-linked
4- Substrate reacts with enzyme –> Fluorescence

Question 12

Direct ELISA steps

Answer 12

Distinctive feature: No capture or secondary antibodies
- Primary antibody binds to antigen and enzyme

1- Antigen placed at bottom
2- Primary antibody binds to antigen and is enzyme-linked
3- Substrate reacts with enzyme –> Fluorescence

Question 13

Indirect ELISA steps

Answer 13

Distinctive feature: Has secondary antibody
- Primary antibody binds to antigen; secondary antibody bound to enzyme

1- Antigen placed at bottom
2- Primary antibody binds to antigen
3- Secondary antibody binds to primary and is enzyme-linked
4- Substrate reacts with enzyme –> Fluorescence

Question 14

ELISA limitation

Answer 14

Limited multiplexing ability: Low number of antigens that can be measured at once

Question 15

High-Throughput screening goal

Answer 15

Measure different antigens at once using fluorescent tags of different colours
- Colour ratio of microbead gives the concentration of each antigen

Question 16

High-Throughput screening steps

Answer 16

Distinctive feature: Microbeads

1- Microbeads with probes put in sample
2- Antigen in sample binds to probes on microbead
3- Fluorescent tags bind to antigen
4- Tags fluoresce when lase shone on them

Question 17

Gel electrophoresis objective

Answer 17

Separation of negatively charged nucleic acids using an electric field

Question 18

Gel electrophoresis steps

Answer 18

1- Sample placed in a gel
2- Anode and cathode turned on
3- Nucleic acid fragments migrate towards anode
4- Larger fragments move slower –> Travel shorter distance

Question 19

PCR objective

Answer 19

Exponentially double size of nucleic acid sample using thermal cycling

Question 20

High-Throughput screening limitation

Answer 20

If fluorescent tags of too many colours are used, the accuracy of the test decreases.

Question 21

PCR steps

Answer 21

1- Denaturation
2- Annealing
3- Elongation

Question 22

PCR ingredients

Answer 22

• DNA primers
• Original DNA
• Nucleotides
• DNA polymerase

Question 23

Nucleic acid assays (NAA) objective

Answer 23

Determine if gene is present in sample

Question 24

Nucleic acid assays (NAA) steps

Answer 24

1- Extracton extracts nucleic acid from sample
2- Amplification (ex: PCR)
3- Detection

Question 25

Nucleic acid assays (NAA) limitation

Answer 25

Gives no indication on location of gene in sample

Question 26

Microarray objective

Answer 26

Simultaneously analyse expression of multiple genes

Question 27

FISH objective

Answer 27

Determine if gene is present in sample and its location

Question 28

FISH steps

Answer 28

1- Probes attached to target gene
2- Denaturation: DNA of sample denatured
3- Hybridization: Sample DNA mixed with probed gene. If target gene is in DNA sample, probed gene will bind to DNA sample
4- Detection: Probes fluoresce, giving away location of target gene in sample DNA

Question 29

Flow Cytometry objective

Answer 29

Cell counting using lasers

Question 30

Flow Cytometry steps

Answer 30

1- Fluidics: Sample flows through sheath fluid
2- Optics: Laser shone on sample is scattered due to analyte molecules; different light sent to different detectors using filters
3- Electronics: Detectors convert light into electrical signal to be analysed `

Question 31

Coulter Counter objective

Answer 31

Cell counting using electrical methods

Question 32

Coulter Counter steps

Answer 32

1- Sample flows through an aperture
2- Analyte molecules block ion flow –> increase in resistivity
3- Resistivity change measured by electronics

Question 33

Contrast formula

Answer 33

(I - I_b)/I_b

I: Sample intensity
I_b: Background intensity

Question 34

Phase contrast steps

Answer 34

1- Source shines light on annular ring;
2- Condenser focuses light onto sample ; Sample refract light –> Phase difference created;
3- Lens focuses light onto phase plate; Phase plate increases phase difference –> increased destructive interference –> increased resolution
4- Lens focuses light onto eye for observation

Question 35

Phase contrast distinctive feature

Answer 35

Distinctive feature: Uses refraction and destructive interference to produce high image resolution

Question 36

Bright fields distinctive feature

Answer 36

Uses absorption to produce images –> Objects dimmer than background

Question 37

Dark field distinctive feature

Answer 37

Uses scattering to produce images –> Smallest objects shine brightest

Question 38

Confocal microscopy advantages

Answer 38

Pinhole added –>
Light from under sample unable to reach detector (out of focus light can’t reach) –>
Increased resolution