Part 2

Question 1

Why does fluorescence occur?

Answer 1

When molecules are made, atomic orbitals mix to form molecular orbitals filled from the lowest energy upwards by the electrons from each of the atoms. Each orbital is described by a set of quantum numbers and these tell us about where the electrons will probably reside and what there spins will be. When a molecule absorbs a photon which corresponds to a transition between orbitals, electrons can be promoted from the highest occupied molecule orbital (HOMO) to the lowest (LOMO). This represents a transition between the electronic ground state and an electronic excited state of the molecule. Electrons relax after a given time (excited state lifetime). This can be radiative such as photons or a non-radiative decay process.

Question 2

What is Stokes shift?

Answer 2

The difference in energy wavenumber or frequency between positions of the band maxima and absorption and emission spectra of the same electronic transition.

Question 3

What is a Jablonski diagram?

Answer 3

A diagram of absorption and emission processes. For each process in the Jablonski diagram, a rate constant k can be given which is the probability per unit time that a process will occur.

Question 4

What is the quantum efficiency of a fluorescence?

Answer 4

The number of photons emitted by the molecule as fluorescence divided by the number of photons absorbed by the molecule. A high quantum efficiency is important for a dye to be useful. It is a measurement of the likelihood of a certain process occurring in a molecule.

Question 5

What is intrinsic fluorescence?

Answer 5

The presence of certain amino acids in a biomolecule can lead to absorption around 280m. Excitation at 280nm can then lead to an intrinsic fluorescence of that biomolecule

Question 6

What is the naturally occurring protein found in jellyfish?

Answer 6

GFP which has an absorption at 400nm and fluoresces at 510nm

Question 7

What is the aim of fluorescence microscopy?

Answer 7

Certain molecules form pores in lipid membranes. From this we can build a model system and use this to understand the mechanism by which this occurs.

Question 8

What do four melittin peptides associate together to form?

Answer 8

Tetramer pore which causes the cell to leak and ultimately die.

Question 9

how can we use a fluorescence assay to test which type of pore is formed in the presence of melittin?

Answer 9

• We create an artificial lipid membrane (a liposome) and fill it with dye
• Inside the liposome, the dye is at very high concentration which causes the individual dye molecules to quench via collision (be less fluorescent than it should be)
• We can create liposomes that contain a percentage of type I or type II lipids and see which releases the dye faster
• This tells us about the type of pore being formed
• Each trace shows the amount of dye leakage against time of different lipid mixtures

Question 10

What is Forster Resonance Energy Transfer (FRET)?

Answer 10

A type of molecular, fluorescent ruler that can tell us about nanoscale distances between biomolecules or structures. It works on the principle of the transfer of excitation energy from an electronically excited molecule (the donor, D) to an acceptor molecule (A) in the electronic ground state.

Question 11

When you have two overlaid pairs of fluorescent absorption and emission spectra for two different dyes in FRET, where can you get energy transfer?

Answer 11

Where the emission of dye 1 overlaps with the absorption of dye 2. The donor and acceptor molecules must be close together in space for energy transfer to occur.

Question 12

What is the Forster distance in FRET?

Answer 12

The distance at which the energy transfer from donor to acceptor is 50% efficient. It is typically in the range of 20 to 60 Angstroms.

Question 13

What is the efficiency of energy transfer in FRET?

Answer 13

The fraction of photons absorbed by the donor that are transferred to the acceptor.

Question 14

What is the process of imaging with fluorescence microscopy?

Answer 14

• The sample is labelled with a fluorescent dye and then illuminated through the lens with a high energy excitation source
• This light is absorbed by the dye and causes it to emit a longer and lower energy wavelength light
• This fluorescent light can be separated from the surrounding radiation with filters designed for that specific wavelength allowing imaging only of the fluorescence
• The microscope has a filter than only lets through radiation with the specific wavelength that matches the fluorescing material
• The fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter
• This works because the light emitted is of lower energy and has a longer wavelength

Question 15

Why use confocal microscopy over conventional fluorescence microscopy?

Answer 15

In conventional fluorescence microscopy, the whole sample is illuminated and the fluorescence is collected including the background fluorescence that may not be in focus. This limits the resolution of the image. By eliminating the out-of-focus light from the detection, only light from close to the focal plan is detected in confocal microscopy.

Question 16

How does confocal microscopy work?

Answer 16

A collimated laser is expanded to a diameter matching that of the back aperture of the microscope objective. Fluorophores in the focal region are excited by the laser beam. Fluorescence is then collected by the same objective and leaves the back aperture as a collimated beam. This light is again focused and passed through a pinhole meaning the only light collected is that originating from the sample within the focal excitation region. This leads to a greatly enhanced imaging and allows detection down to the single molecule level.

Question 17

What is the downside of confocal microscopy? What can be done to resolve this?

Answer 17

Resolution is increased but at the expense of intensity so often long exposure times are needed. To get 3D images, multiple images in the z direction must be collected and stacked on top of each other to reconstruct the image. Spinning disk confocal instruments greaty speed up capture by rapidly cycling the location of the pinhole.

Question 18

How does Total Internal Reflection Fluorescence Microscopy (TIRF) differ to confocal microscopy?

Answer 18

In confocal we limit our detection of the mitted fluorescence using a pinhole. In TIRF, only fluorescence from molecules in the direct vicinity of the microscope slide and the sample are imaged

Question 19

How does TIRF work?

Answer 19

• When light reaches a surface between two optically transparent but different materials (e.g. glass and water) under a narrow angle, total internal reflection can occur
• The angle at which this occurs us dependent on the refractive indices of the two materials n1 and n2 where n1 > n2

Question 20

How does the evanescent wave decay in TIRF?

Answer 20

Exponentially

Question 21

What is the main benefit of TIRF?

Answer 21

By limiting the volume from which the fluorescence an be detected, the effective illumination path experienced by TIRF is sub-wavelength in size. This means it is able to resolve single molecules.

Question 22

What is the main benefit of TIRF?

Answer 22

By limiting the volume from which the fluorescence can be detected, the effective illumination path experienced by TIRF is sub-wavelength in size. This means it is able to resolve single molecules.

Question 23

What is photobleaching?

Answer 23

Where fluorescent dyes undergo electronic rearrangement to reach an excited state. Excited molecules are more reactive so your dyes g dark after a few seconds/minutes of illumination.

Question 24

What is the time constant of a process?

Answer 24

The inverse of the rate constant

Question 25

What is the overall lifetime of an electronic state?

Answer 25

A time constant that can be calculated from the inverse sum of all of the rate constants of the processes which depopulate this state.

Question 26

What do you measure in Fluorescence Lifetime Imaging (FLIM)?

Answer 26

• Viscosity: More viscus solutions glow for longer
• Polarity: Introduce more opportunities for excited molecules to pass on energy therefore more polar environments only fluoresce briefly
• Temperature: Higher temperatures means more vibrations so ore collisions.
• Also anything that can contribute to non-fluorescence decay

Question 27

How does stimulated emission work?

Answer 27

If a photon illuminates a molecule in the excited state and this photon has an energy that exactly corresponds to to a fluorescence transition then the photon can induce the transition resulting in a photon which is identical to the photon which induced the transition. This de excites the fluorescence marker.

Question 28

On is an induced dipole?

Answer 28

• When EM waves fall on an atom or molecule, the electric field disturbs the electron cloud around the nuclei
• When the field is applied from the top, the positive charge density is attracted towards it and negative density repelled creating a dipole
• In the situation where the field oscillates between upper and lower positions, the induced dipole will oscillate with the frequency of the field

Question 29

How does the induced dipole cause stimulated emission?

Answer 29

• The dipole moment is created in the direction of the Electric vector of light
• If the frequency of light is resonant with the energy levels of the molecule, the energy can be absorbed, creating oscillating dipole moment µ
• In the classical explanation of stimulated emission, the photon which is incident on the excited state electron induces a transition state dipole moment (which oscillates with the E field of the radiation), which “encourages” the atom / molecule to produce a second photon identical to the incident photon

Question 30

Stimulated emission can be thought of as the opposite of what process?

Answer 30

Induced absorption

Question 31

Stimulated emission can be thought of as the opposite of what process?

Answer 31

Induced absorption

Question 32

How does Stimulated Emission Depletion Microscopy (STED) work?

Answer 32

In STED the shorter wavelength beam excites the sample and the simulated emission depletion donut beam is of a longer wavelength turned to the fluorescence maxima to force molecules in it excitation volume back to ground state
- Now two lasers are used, a pump beam for absorption and a STED beam to de-excite the markers
- Optics are arranged such that the STED beam has a Bessel profile
- As such, all markers that are not in the hole are depleted, leave a very small central spot, which only depends on the STED beam, and the properties of the dye, and can be resolved down to 20 nm
- The resolution is a function of the STED intensity and the properties of the dye

Question 33

What is Photo Activated Localisation Microscopy (PALM)?

Answer 33

A high resolution technique that uses specially designed fluorescent proteins which can be switched between two states each of which has different fluorescence properties

Question 34

How do you use PALM?

Answer 34

A sample labelled with a fluorescent protein is irradiated with a small amount of switching light. If this s done in a diffraction limited area the only one protein will switch in that space. Thus only the light coming from the few activated proteins can be seen. As the wavelength of the emitted light is different to that of the switch, using appropriate filters means this can be observed.. The spots seen will have a Gaussian intensity distribution but the protein will be at the centre of this Gaussian distribution. In the first step, a few proteins are switched on and their position is found to a few nanometres. Once this has been done, these can be switched off by photobleaching. The procedure is repeated until a total image is reconstructed with sub-diffraction spatial resolution from positions of all the localized proteins

Question 35

What are the limitations of STED and PALM?

Answer 35

• They both need labelling of the sample and any label can potentially perturb the sample so that it is not true to the actual sample under investigation
• Until recently PALM measurements were nt fast enough to look at cellular dynamics
• Recent advances in label free super resolution microscopy have used auto fluorescent structures in cells themselves and use two different laser wavelengths to resolve closely spaced objects

Question 36

What is Fluorescence Correlation Spectroscopy (FCS)?

Answer 36

It uses the focus formed in a confocal microscope to measure diffusion of fluorescently labelled particles. Its advantage over conventional laser light scattering is that diffusion coefficients can be measured for molecules in very dilution solutions (nM) and in small volumes (µl). A fluorescently labelled molecule in solution emits photons as long as it moves through a laser spot.

Question 37

What does the number of photons emitted per unit time in FCS depend on?

Answer 37

• The number of molecules
• The diffusion time of the molecule
• Spot size
• Quantum yield

Question 38

What is the benefit of using FCS?

Answer 38

• It is a high resolution technique which can monitor spontaneous intensity fluctuations of fluorescently labelled molecules caused by minute deviations of the small system from thermal equilibrium
• In general all physical parameters that give rise to fluctuations in the fluorescence signal are accessible by FCCS?

Question 39

What are the measurements in FCS?

Answer 39

The measured signal is a constant mean intensity and a fluctuating contribution. The measured intensity fluctuations are converted to an auto correlation function

Question 40

What is anomalous sub-diffusion?

Answer 40

Any behavior that cannot be described any homogeneous diffusion and for which no other explanation holds

Question 41

When may anomalous sub-diffusion be suspected?

Answer 41

When non-Brownian behaviour behaviour is observed in a diffusing system, i.e. that the mean square displacement of the molecules is not proportional to the measurement time.

Question 42

What is cross-correlation FCS?

Answer 42

When two different molecules are labelled with two different fluorescent dyes (with different excitation/emission spectra). Each is excited with a different laser line and autocorrelations for each of the individually dyed molecules can be observed independently. If these molecules associate, the double labelled associated molecule can be distinguished from the singly labelled ones by cross-correlation of data.

Question 43

What is two photon FCS?

Answer 43

Nonlinear 2 photon excitation is based on the simultaneous absorption of two photons. Since the energy of a photon is inversely proportional to its wavelength, the two absorbed photons must have a wavelength which is about twice that for one-photon excitation.

Question 44

What are the requirements for two photon FCS work?

Answer 44

• Two-photon excitation requires that absorption of two photons of theoretically double the wavelength usually required for the excitation, within the tiny time interval of about one femtosecond. In order to get a reasonable probability of such three-particle events, the photon flux must be extremely high (i.e high output power and pulsed excitation is needed).

Question 45

What are the advantages and disadvantages of two photon FCS?

Answer 45

Advantages:
- Good for intracellular –measurements, better resolution.
- Can use IR excitation, which is generally better for biological systems.
- Less autofluorescence and light scattering.
- Better signal-to-noise ratio.

Disadvantages:
- More photobleaching.
- Data analysis is difficult.

Question 46

What are the uses of X-rays in biophysics?

Answer 46

• Diffraction: atomic structure of proteins and protein-ligand complexes
• Scattering: ordering of soft systems, for example lipid phases

Question 47

What are the pros and cons of using X-ray diffraction in biophysics?

Answer 47

Pros: atomic resolution, allows high resolution understanding of structure-function relationships
Cons: requires a crystal (not always easy with a protein), is a “snapshot” which may not reflect biological reality, the phase problem (see later)

Question 48

What are the pros and cons of using X-ray scattering in biophysics?

Answer 48

Pros: resulting patterns give easy analysis of structural phase and spacing in an ordered soft system, can be done under a range of sample conditions
Cons: no atomic structure, prone to beam damage, not always a realistic model

Question 49

What are the conditions for constructive interference of waves?

Answer 49

• They must have the same wavelength
• They must be in phase

Question 50

How can Bragg’s law be used in diffraction?

Answer 50

• Two waves (parallel, monochromatic) hit atoms in our crystal at an angle Θ – they will be scattered by the electrons of the atoms
• If they leave in phase, we get constructive interference; out of phase, destructive interference
• To be in phase the path difference they travel must be equal to an integer number of wavelengths
• The spot that arises from constructive interference upon diffraction of the x-rays is called a reflection
• The diffraction pattern is related to the atomic positions of the atoms (via the electron density) by the Fourier transform – the inverse Fourier transform of the diffraction pattern can be used to build an electron density map

Question 51

What are the problems with X-ray diffraction?

Answer 51

• The amount of scattering from any atom experienced by x-rays is directly proportional to its atomic number
• Many of the types of atoms commonly biological molecules (e.g. C, H, O) will provide weak scattering due to their low atomic numbers.
• Specialised equipment is therefore needed to enable one to detect the weak signals produced by scattered x-rays.
• Bragg’s Law shows how the molecular aggregates which make up soft condensed matter systems (like a lipid bilayer) will only produce scattering at extremely low angles