Microscopy Flashcards
What fundamental questions would we ask about a protein?
Where is it? When is it expressed? What are its partners and how do they influence each other? Is it regulated – how and when?
What would we ask about a mutated protei?
Does the mutation alter the proteins targeting? Does the mutation affect expression/degredation? Does the mutation affect interactions with partners? Is it no longer regulated?
What questions would we ask about finding a cure?
Can we retarget the protein? Can we increase the proteins level or folding? Can we force interactions? Can we mimic its regulation?
Describe light
Light is a type of electromagnetic radiation
* Radiation waves have: energy, frequency and wavelength
* Wavelength: 380-760nm detected by the eye and perceived as visible light
* Different colours have different wavelengths.
Describe brightfield microscopy
Light “normal” microscopy. Contrast from absorbance. Gives low contrast, flat image
Describe phase contrast and differential interference contrast (DIC)
Interference of different light paths mean dense regions appear darker than background.
DIC uses prisms to focus light and highlights the 3D nature of the specimen
Describe darkfield microscopy
Illuminates the sample from the side. Only scattered rays hit the lens. White on dark background
Describe the pros and cons of brightfield and its applications
- Pros: easy to use. Dark image, bright background
- Cons: Low contrast, some cells and components almost invisible, stains can be toxic
Applications: High throughput, commonly used in medical blood analysis, define cell border/nucleus in fluorescence microscopy
Describe the pros and cons of phase contrast and its applications
- Pros: increase contrast (unstained) and observe structures not visible with brightfield
- Cons: slightly more expensive, requires some alignment, can have ‘halos’
Applications: research into more transparent cells
Describe fluorescence
Must be excited to glow, very specific, possibility to co-stain
Describe the principles of fluorescence microscopy
- Fluorescent molecules absorb photons at one wavelength and emit at a lower energy one
- Detected when illuminated at the excitation wavelength and viewed through a filter corresponding to the emission spectra
- Fluorescent molecules can be chemical compounds (fluorophores), proteins or others
- Using different combinations of fluorophores with different properties allows multiplex imaging
Describe GFP
Use standard molecular biology to tag protein with GFP and express in cells of interest
Mutagenesis of GFP has led to colour variants
Describe biomolecular fluorescence complementation Split-YFP
BiFC.
Used to determine if two proteins interact. Each protein tagged with half the fluorophore. Each half emits no light, only when they combine. If the 2 proteins interact, light is emitted
Describe forster resonance energy transfer
Used to measure protein interactions in cells. Energy transferred between two light sensitive molceules. How efficiently the energy is transferred tells you how close the molecules are together
Describe Fluorescence recovery after photobleaching
Used to measure protein dynamics, photobleaching = fading = fluorescence permananetly lost
A strong, focused laser beam irreversibly bleaches.
Recovery of fluorescence has to come from elsewhere. Provides a measure of diffusion coefficients or dissociation of a protein from its location.
Describe immonofluorescence
Use of antibodies. Sometimes proteins cannot be tagged. Specificity is achieved with antibodies. Combine multiple primary and secondary antibodies to co-localise molecules of interest, typically used in fixed tissue.
Primary antibody: direct to an antigen.
Secondary: marker-coupled antibodies directed against the primary antibody, with markers on
Describe the resolution of light microscopy
Resolution distance (R) depends on the wavelength of light and the ability/quality of the microscope objective to gather light (numerical aperture) from the lens.
Describe resolution in general
The limit of resolution is the limiting separation at which 2 objects can still be seen as distinct.
A smaller resolution distance means you can see smaller distances between objects
Resolving 2 objects less than 200nm apart is not possible with conventional light microscopy
Describe super resolution microscopy
Covers anything with <100nm resolution.
Objects like organelles/proteins can be as close as 100nm,
irresolvable by normal light microscopy, but may not interact
Describe light microscopes vs electron microscopes
Light:
- Light (photons)
- Limit of resolution 200 nm
- Fixed or live sample
Electron
- Electrons
- Limit of resolution 0.05 nm
- Fixed
Describe transmission electron microscopy
Electron wavelengths >1nm
Uses focused beam of electrons instead of light
Can use antibodies as well to highlight specific proteins
Instead of fluorophore, gold particles are used with secondary antibodies (as gold is electron dense)
Describe EM tomography
- The specimen is tilted to varying
angles along an axis perpendicular
to the electron beam - Each image is projected in 2D
- All 2D images are stacked into a
3D reconstitution
