Lecture 6: Paramagnetic Resonance and Microscopy Flashcards
What is electron paramagnetic resonance?
EPR is also known as electron spin resonance (ESR).It is very similar to NMR, expect it is for electrons. It detects signals from unpaired electrons (free radicals and transition metal ions).
• It uses applied magnetic fields which give resonances in the microwave range (0.2cm-10cm wavelength).
• It is used for mobility probes, transition metals in proteins and distance measured.
• We get a spectrum which shows magnetic field strength on the x-axis.
• We get an absorbance signal. But we convert it to its first derivative. As shown above.
What is site directed spin labelling?
Site directed spin labelling is a method to look at structure and conformational switching in soluble and membrane proteins.
• Nitroxide is used as a spin label. It is a stable radical. It has a reactive group X which allows it to be attached to a cysteine.
• The cysteine is engineered into a specific position and a spin label is attached.
• We can then look at the spectrum.
What is light microscopy?
Light is transmitted through the specimen and observed. Lenses cause light to focus at a point (distance f) from the centre of the lens.
- What we can see depends contrast. This is often based on how different regions absorb light differently. We can increase contrast with staining. The simplest method of inducing contrast is a bright field microscope, where we Illuminate the sample from below.
- Optical lenses have fewer stages. There are two lenses, objective and projector. The objective lens flips the image and magnifies it. Then the projector lens flips it again and allows the image to be viewed.
What is light microscopy?
Light is transmitted through the specimen and observed. Lenses cause light to focus at a point (distance f) from the centre of the lens.
- What we can see depends contrast. This is often based on how different regions absorb light differently. We can increase contrast with staining.
- The simplest method of inducing contrast is a bright field microscope, where we Illuminate the sample from below.
- Optical lenses have fewer stages. There are two lenses, objective and projector. The objective lens flips the image and magnifies it. Then the projector lens flips it again and allows the image to be viewed.
What issues do we have with aberrations?
There are two types of aberration which we find in microscopes.
• Spherical aberrations: Spherical aberrations occur when there are different focal lengths when light hits different parts of the lens. This can be corrected by shaping the lens carefully. It gives a blurry image.
• Chromatic aberrations: Different wavelengths are refracted differently. It gives a blurry edge and a rainbow edge. It can be stopped by using a lens made of layers of glass with different refractive indices.
We have essentially perfected our lenses in terms of problems with aberration.
How does the resolution limit work?
Another problem is that images are limited by light diffraction. This is a fundamental property of light that we can do nothing about.
Resolution depends on wavelength and aperture (α). Resolution = wavelength/aperture. The resolution limit is called the Abbé limit.
Abbe=0.61 λ/(n sin(θ))
nsin(θ) is called the numerical aperture. For light microscopes, sin(θ) is about 0.9. Using oil with n~ 1.5 gives NA of more than 1.
What are phase contrast microscopes?
We can increase contrast in a multitude of ways. A very clever way is with a phase contrast microscope. Phase contrast microscopes use light waves with different phases to cancel each other out which results in very high contrast.
• There are two types of rays in this system. The yellow light is scattered by the specimen. The green light is transmitted rays which don’t interact with the sample.
• The direct beam goes through the phase plate annulus and placed out of phase by 90 degrees.
• It then goes through the phase plate and is further knocked out of phase, so it is 180 degrees out of phase overall.
• Any scattered waves which don’t change destructively interfere and disappear. Any which are changed appear brighter in comparison. It is very sensitive to changes in the refractive index of the specimen.
• A similar technique is called differential interference contrast microscopy. It splits polarised light through a prism below the condenser lens. The path lengths of the two beams are altered as they pass through the sample. The optics then recombine the two beams. One side of an object appears bright while the other side appears darker, which gives a pseudo 3D effect.
How do we prepare samples and stain?
We want to interfere as little as possible when we’re doing sample prep. There are many steps and processes we can do.
- Chemical fixation: Prevents degradation and maintains structure especially in studies of tissues in clinical work. We usually use formaldehyde in phosphate-buffered saline.
- Cutting: The sample is dehydrated in alcohol and the water is replaced with paraffin wax that solidifies. We can therefore cut thinner sections. We can then cut with a microtome into 5 micrometre thick sections.
- Staining: we can highlight features of interest. Hematoxylin is a basic dye with a positive charge which stains nuclei blue as it binds to DNA (methylene blue also stains nucleic acids). Eosin is an acidic dye which stains the cytoplasm pink. Rhodamine can stain proteins. Nile red stains lipids.
How do fluorescence microscopes work?
Fluorescence microscopes are very powerful and sensitive because the sample emits light and gives very high contrast.
• We often use immunofluorescence as a technique. The antibodies are labelled fluorescently. Primary and secondary antibodies are used.
• The cells and tissues are permeabilised with detergent before labelling. We can use these antibodies to monitor used cells to create heterokaryons.
• GFP can also be fused to proteins. We can also use some stains such as DAPI for DNA (turns DNA blue).
What is photobleaching?
- FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) are two techniques which exploit this. Photobleaching is when a fluorophore is destroyed by applied light.
- FRAP monitors how long it takes for fluorescence to recover in a defined area. Sometimes the fluorescence doesn’t fully recover, which implies that the proteins are immobilised.
- FLIP is a complementary technique which measures the drop in fluorescence in the adjacent area. Both FRAP and FLIP can measure molecular mobility.
What is confocal microscopy?
CM is a method to reduce out of focus light by putting a pinhole in front of the image plane.
• A single 3D spot is focused on with an illuminating light.
• The spot is scanned and any unwanted light is eliminated, giving a clearer image and the opportunity to create a 3D construction.
What is total internal reflection fluorescence?
TIRF is a method which used to only detect fluorophores in a local environment. We use TIR to create an evanescent field by the coverslip. It produces a very thin slice (10 times thinner). It is very useful for looking at events near the surface of the cell. We can use it when looking at single molecules as well.
How can we get beyond the Abbé resolution limit?
The size of the spot (point spread function) produced by a lens is diffraction limited to ~200nm in the xy direction and ~500-700nm in the z direction. If the spot size could be reduced we would get better resolution in a scanned confocal image.
- We can produce spots with better PSF profiles. We can improve resolution with ~30nm resolution, much better than what we thought.
- This is done using a technique called stimulated emission depletion microscopy.
- In STED the sample has the normal radiation spot, however there is also a ring of light at a wavelength that stimulates the depopulation of some of the excited fluorophores.
- The light ring quenches fluorescence from that area so the combined irradiation from the ring and the objective lens gives a smaller spot.
- Images with ~20 nm resolution are possible. Higher resolution images take longer to collect.
