Lesson 4 - Fluorescence and confocal microscopy

Question 1

What is fluorescence microscopy?

Answer 1

• uses a property of certain molecules to fluoresce at a specific wavelength
• can visualize more than one protein or cell structure

Question 2

What are fluorochromes?

Answer 2

• they absorb energy kicking electrons into a higher, unstable orbital

Question 3

What happens when blue light is used in fluorescence?

Answer 3

• blue light excites electrons and drops down to ground state, it emits different wavelength of light which is green

Question 4

How are fluorescence images obtained?

Answer 4

• light source is intense and has a broad range of lights/wavelengths that it emits

Question 5

Excitation vs. emission light

Answer 5

• 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

Question 6

Short vs long wavelength in terms of strength

Answer 6

• short = stronger

- long = weaker

Question 7

What is immunofluorescent staining?

Answer 7

• dyes can be conjugated with antibodies to localize any molecules of interest in cells

Question 8

How are mitochondria stained?

Answer 8

• labelled with a mitotracker and will only fluoresce red

Question 9

What are HAT medium?

Answer 9

• toxic for myeloma cells (which have mutation of specific gene)

Question 10

What are hybrid cells?

Answer 10

• survive in HAT medium because they obtain a missing gene product from spleen cells
• like myeloma cells and produce antibodies

Question 11

What happens if a rat is injected with protein?

Answer 11

• 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

Question 12

What is the issue with spleen cells?

Answer 12

• 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

Question 13

What will kill myeloma cells?

Answer 13

• HAT

- fused cells will survive in HAT medium

Question 14

What is immunofluorescence microscopy?

Answer 14

• 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

Question 15

What is Duel Label Microscopy?

Answer 15

• 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

Question 16

What is Green Fluorescent Protein (GFP)?

Answer 16

• 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

Question 17

What is the Principle of Confocal Microscopy?

Answer 17

• 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

Question 18

What is Deconvolution microscopy?

Answer 18

• 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

Question 19

What is Two-Photon Excitation Microscopy?

Answer 19

• 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

Question 20

What is FRET - Fluorescence resonance energy transfer?

Answer 20

• measures protein interactions in live cells

Question 21

What provides better resolution? Fluorescence or Electron Microscopy?

Answer 21

EM

Question 22

What are the different kinds of Electron Microscopy’s?

Answer 22

• 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