Lec 14 -15 (Pathology, Assays, and Microscopy) Flashcards
Histopathology definition
Pathology using thin tissue slices viewed under the microscope
Histopathology protocol
1- Harden tissue via wax or freezing
2- Cut tissue into sections
3- Stain tissue sections
Name of compound used to stain DNA a in blue colour
Hematoxylin
Name of compound used to stain cytoplasm and proteins an orange-pink colour
Eosin
Protein assay types
- ELISA (main)
- Gel electrophoresis
- Microarrays
Nucleic acid assay types
- PCR (amplification)
- FISH (location)
- Gel electrophoresis (size ranking)
- Microarrays
Cellular assay types
- Flow cytometry (counting)
- Microscopy (imaging)
- Microarrays
Tissue diagnostic types
- Microscopy
- Nonlinear imaging
ELISA objective
Measuring protein concentrations in a sample using enzyme-linked antibodies
- Enzymes fluoresce –> Light intensity proportional to protein concentration
ELISA types
- Sandwich
- Direct
- Indirect
Sandwich ELISA steps
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
Direct ELISA steps
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
Indirect ELISA steps
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
ELISA limitation
Limited multiplexing ability: Low number of antigens that can be measured at once
High-Throughput screening goal
Measure different antigens at once using fluorescent tags of different colours
- Colour ratio of microbead gives the concentration of each antigen
High-Throughput screening steps
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
Gel electrophoresis objective
Separation of negatively charged nucleic acids using an electric field
Gel electrophoresis steps
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
PCR objective
Exponentially double size of nucleic acid sample using thermal cycling
High-Throughput screening limitation
If fluorescent tags of too many colours are used, the accuracy of the test decreases.
PCR steps
1- Denaturation
2- Annealing
3- Elongation
PCR ingredients
- DNA primers
- Original DNA
- Nucleotides
- DNA polymerase
Nucleic acid assays (NAA) objective
Determine if gene is present in sample
Nucleic acid assays (NAA) steps
1- Extracton extracts nucleic acid from sample
2- Amplification (ex: PCR)
3- Detection
Nucleic acid assays (NAA) limitation
Gives no indication on location of gene in sample
Microarray objective
Simultaneously analyse expression of multiple genes
FISH objective
Determine if gene is present in sample and its location
FISH steps
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
Flow Cytometry objective
Cell counting using lasers
Flow Cytometry steps
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 `
Coulter Counter objective
Cell counting using electrical methods
Coulter Counter steps
1- Sample flows through an aperture
2- Analyte molecules block ion flow –> increase in resistivity
3- Resistivity change measured by electronics
Contrast formula
(I - I_b)/I_b
I: Sample intensity
I_b: Background intensity
Phase contrast steps
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
Phase contrast distinctive feature
Distinctive feature: Uses refraction and destructive interference to produce high image resolution
Bright fields distinctive feature
Uses absorption to produce images –> Objects dimmer than background
Dark field distinctive feature
Uses scattering to produce images –> Smallest objects shine brightest
Confocal microscopy advantages
Pinhole added –>
Light from under sample unable to reach detector (out of focus light can’t reach) –>
Increased resolution
