Lesson 4 - Fluorescence and confocal microscopy Flashcards
(22 cards)
1
Q
What is fluorescence microscopy?
A
- uses a property of certain molecules to fluoresce at a specific wavelength
- can visualize more than one protein or cell structure
2
Q
What are fluorochromes?
A
- they absorb energy kicking electrons into a higher, unstable orbital
3
Q
What happens when blue light is used in fluorescence?
A
- blue light excites electrons and drops down to ground state, it emits different wavelength of light which is green
4
Q
How are fluorescence images obtained?
A
- light source is intense and has a broad range of lights/wavelengths that it emits
5
Q
Excitation vs. emission light
A
- excitation (green) and emission (red) light goes through objective
- red light goes through projection lens and goes to emission filter which further refine the optimally excited light
6
Q
Short vs long wavelength in terms of strength
A
- short = stronger
- long = weaker
7
Q
What is immunofluorescent staining?
A
- dyes can be conjugated with antibodies to localize any molecules of interest in cells
8
Q
How are mitochondria stained?
A
- labelled with a mitotracker and will only fluoresce red
9
Q
What are HAT medium?
A
- toxic for myeloma cells (which have mutation of specific gene)
10
Q
What are hybrid cells?
A
- survive in HAT medium because they obtain a missing gene product from spleen cells
- like myeloma cells and produce antibodies
11
Q
What happens if a rat is injected with protein?
A
- generates an immune response that will result in cells that produce antibodies that recognize antigen or ptoein
- antibodies proliferate and reside within the spleen and cause clusters
12
Q
What is the issue with spleen cells?
A
- they are primary cells
- you can only use them for a short period of time (not good for harvesting)
- but fusing spleen cells with mouse myeloma cells, can use for a longer period of time because hayflick is overcome with myeloma cells
13
Q
What will kill myeloma cells?
A
- HAT
- fused cells will survive in HAT medium
14
Q
What is immunofluorescence microscopy?
A
- antibodies are added to cells which bind their specific protein
- secondary antibodies are added and bind primary antibody
- each fluorochrome has a unique excitation and emission wavelength that can be detected in microscope
- chemically treated with formaldehyde (freezes) and detergent is added for antibody to cross over pm and bind to protein of interest and fixed cell
- kills cells in order to look at structure
15
Q
What is Duel Label Microscopy?
A
- appropriate microscope filter set for each fluorochrome and images are digitally overlayed
- Rhodamine binds to actin and is conjugated with rhodamine which fluoresces red when excited
- filter cubes moved to different settings to get different wavelengths of emission
16
Q
What is Green Fluorescent Protein (GFP)?
A
- protein contains a short sequence of aa (chromophore) that are capable of fluorescing when excited with blue light
- GFP is fused with gene of interest to create hybrid DNA and hybrid DNA is introduced inside of culture mammalian cell
- Expression plasmid is recognized by transcription and apparatus of cell codes GFP-fusion protein
- fusion protein expressed in cell
- should emit green light when blue light is absorbed
17
Q
What is the Principle of Confocal Microscopy?
A
- uses a laser which passes through a pinhole that hits a dichroic mirror and is reflected down to objective and excites the point of focus
- light emitted will be above and below focal point
- any light above the focal plane will not pass through the pinhole
18
Q
What is Deconvolution microscopy?
A
- captures multiple images in Z plane
- considers point spread function which determines the degree of blurriness by comparison to a reference set of fluorescent beads
- can take images quickly
- image analysis can be done only after multiple hours
19
Q
What is Two-Photon Excitation Microscopy?
A
- used for deep tissues
- longer wavelength = you need to pulse it, followed by a second illumination
- 2 photons with half the energy will generate the same emission wavelength
- longer wavelengths = penetrate deeper into tissues
20
Q
What is FRET - Fluorescence resonance energy transfer?
A
- measures protein interactions in live cells
21
Q
What provides better resolution? Fluorescence or Electron Microscopy?
A
EM
22
Q
What are the different kinds of Electron Microscopy’s?
A
- transmission electron microscope : images formed from electrons that pass through a specimen
- Scanning/ scatter electron microscopy : images are formed from electrons that are scattered from a metal - coated specimen
