Imaging Practical Flashcards
Give an overview of fluorescence microscopy.
- Fluorescence microscopy is an imaging technique used in light microscopes that allows the excitation of fluorophores and subsequent detection of the fluorescence signal.
- Fluorophores are compounds that re-emit light upon excitation.
- Fluorescence is produced when light excites an electron to a higher energy state, immediately generating light of a longer wavelength than that which excited the fluorophore.
- To visualize labeled molecules in the sample, fluorescence microscopes use a powerful light source and a dichroic mirror to reflect and split up light at the desired wavelength.
- The filtered excitation light then passes through the objective to be focused onto the sample and the emitted light from the sample is filtered back onto the detector.
List 2 types of microscopes used by fluorescence microscopy.
Fluorescence microscopy uses light microscopes such as:
1 - Confocal microscopes.
2 - Multiphoton microscopy.
Superresolution techniques:
3 - Total internal reflection microscopy.
4 -Stimulated emission depletion.
What is confocal microscopy?
What is the advantage of confocal microscopy?
What is the disadvantage of confocal microscopy?
- Confocal microscopy is an optical imaging technique that uses a small pinhole to capture light.
- The advantage of confocal microscopy is that it can perform optical sectioning (creating a 3D image from 2D slices, known as Z stacking).
- It is able to do this because it has a high Z resolution, meaning it has a narrow plane of focus because of the small pinhole through which light enters the microscope.
- The disadvantage of this is that only one small point can be visualised at a time. To produce an image as described above, the microscope must scan the focal point on the Z axis across the X/Y plane to produce a 2D image. ‘Line scanning’ can also be carried out to produce an image of a line in 2D space.
What is multiphoton microscopy?
What are the advantages of multiphoton microscopy?
What is the disadvantage of multiphoton microscopy?
- Multiphoton microscopy is an optical imaging technique used in conjunction with an intense pulsed laser.
- In other forms of fluorescent microscopy, a relatively less powerful light source is constantly applied to fluorophores to produce fluorescent light. In multiphoton microscopy, the objective is to have two photons excite the fluorophore at the same time, hence the high intensity and pulsatile nature of the laser.
- If this occurs, photons of double the wavelength (and hence half the energy) are required to excite the fluorophore.
- The advantages of multiphoton microscopy are:
1 - Longer wavelengths are attenuated less by tissue through which light must pass to reach the fluorophore. This improves tissue penetration.
2 - Longer wavelengths scatter less than high frequency wavelengths.
- The disadvantage of using multiphoton microscopy is that the effective intensity is halved, since 2 photons are required to excite a fluorophore compared to the usual 1 in other forms of light microscopy.
- This means that the optical plane of multiphoton microscopy is much narrower than conventional microscopy, because there is a specific point on the z axis at which the intensity of light is sufficient to excite a fluorophore.
What is total internal reflection microscopy?
Whats is the advantage of total internal reflection microscopy?
Whats is the disadvantage of total internal reflection microscopy?
- Total internal reflection microscopy is a superresolution light microscopy technique used to detect cell-surface processes.
- A sample is placed on a glass surface and labelled with a fluorophore.
- An excitation wave is projected onto the glass at an angle which causes total internal reflection.
- This generates an evanescent wave at the point of reflection (the glass), which excites all fluorophores within a small distance of the point of total internal reflection.
- All other fluorophores in the sample that are not near the point of reflection are not excited, and so only the surface of the sample is visualised.
- Advantages of total internal reflection microscopy are:
1 - It allows for visualisation of cell surface structures..
2 - It allows for the resolution of objects that are at within 100 nm of the reflection boundary. Normally the maximum resolution of light microscopy is less than half of the wavelength of light emitted by the fluorophore (i.e. ~200 nm).
- It is therefore good for neurotransmitter release.
- The disadvantage is that it cannot be used for non-cell surface processes.
What is stimulated emission depletion?
What is it useful for?
What is the advantage of stimulated emission depletion?
What is the disadvantage of stimulated emission depletion?
- Stimulated emission depletion is a superresolution light microscopy technique.
- It involves firstly using a strong light source to inactivate the fluorophores.
- The fluorophores begin to recover from this inactivated state in a random pattern.
- As the fluorophores recover, they emit light and many frames are recorded, capturing the fluorophores as they reactivate.
- Since the fluorophores recover randomly, two adjacent fluorophores that are of a very small distance apart emit light at separate times, meaning they can be distinguished.
- In neuroscience, this is useful for measuring small structures such as vesicles.
- The advantage is its high resolution.
- The disadvantage is that it can only measure static processes (cannot move a distance more than the resolution).
What is the concentration of intracellular Ca2+?
The concentration of intracellular Ca2+ is 100 nM.
What is the meaning of the -AM suffix on Ca2+ indicators?
What is the problem with using these Ca2+ indicators on nervous tissue?
How can this be overcome?
- The -AM suffix on Ca2+ indicators means that the drug is an esterified form of the active component of the drug.
- The esterification means it is able to freely diffuse into the cell and be cleaved by an endogenous esterase enzyme to release the drug from the ester bond in the cell.
- This is one way of getting a hydrophilic drug into a cell without having to perforate the membrane.
- The problem with using these Ca2+ indicators on nervous tissue is that some cells, particularly glia and fibroblasts, more readily uptake the indicator than neurones. This is because of their relatively high expression of esterase enzymes.
- If the indicator needs to be administered into a synapse, this cannot be done precisely because of the size of the synapse. This can be overcome by administering the indicator into the neurone cell body, and the indicator will be transported to the synapse through the axon.
Describe the mechanism for short-term potentiation (AKA frequency facilitation).
Short-term potentiation mechanism (AKA frequency facilitation):
- Following an action potential, intracellular Ca2+ concentration remains high for a few seconds (Ca2+ influx is rapid but Ca2+ efflux is slow). For context, the axon is able to repolarise within 1 microsecond.
- The high Ca2+ concentration following an action potential facilitates subsequent depolarisation because the positive charge of the Ca2+ increases the resting membrane potential, making it closer to the threshold potential.
Why is Ca2+ influx used to optically detect neurotransmitter release instead of Na+?
Ca2+ is used to optically detect neurotransmitter release instead of Na+ because even though the absolute Na+ flux is larger than Ca2+ flux, the relative change in intracellular Ca2+ is much greater than that of Na+. For context:
Resting Na+:
- Extracellular: 140mM.
- Intracellular: 15mM.
Resting Ca2+:
Ca2+:
- Extracellular: 2.4mM
- Intracellular: 0.1uM
- Intracellularly, the Ca2+ change is about 0.1 micromole, and the Na+ change is 1 micromole. The relative change in intracellular Ca2+ is very large, whereas the relative change in intracellular Na+ is very small.
Why is neurotransmitter release from varicosities thought to be ‘intermittent’?
Neurotransmitter release from varicosities is thought to be intermittent because a vesicle is only released in response to 1-2% of all action potentials (50-100 APs required for a single vesicle release).
