Week 6: Experimental techniques Flashcards
Sketch an experimental set-up for infrared spectroscopy.
(89)
How does infrared spectroscopy work?
Different wavenumbers of light will have different levels of absorbance as the light passes through the sample.
Bond-stretching, bending and vibration will have specific energies or wavenumbers. Will be seen when measuring absorbance against wave number.
Different H-bonds will lead to different environments and red and blue shifts.
What are Amide I peaks?
Absorbance peaks around 1650 cm-1.
What is circular dichroism?
A spectroscopic technique that measures the difference in the absorption of left-handed and right-handed circularly polarised light by a molecule.
Why may circular dichroism be used?
As the differential absorption of left and right-handed light is affected by the adopted molecular conformation and local packing, e.g. alpha-helix or beta-sheet of a peptide.
What is Fluorescence resonance energy transfer (FRET)?
FRET is a distance-dependent process by which energy is transferred non-radiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-dipole coupling.
At what range can FRET measure molecular proximity?
10 - 100 Angstrom
When is FRET efficient?
When the donor and acceptor are positioned within a few nm.
What are the components of a fluorescence microscope?
Sample
Cover slip
Objective lens
Dichroic mirror
Excitation filter
Detector
Emission filter
Mercury light source
Sketch a fluorescence microscope setup.
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Sketch a confocal microscope setup.
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What is the Abbe theory?
It tells us what the best resolution that can be achieved by a lens.
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What formula gives the resolution in the depth (z) direction?
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What is scanning confocal microscopy?
The object is illuminated by image of point source and pinhole detection, enabling depth scanning.
What advantage does scanning confocal microscopy have over simple microscopy?
Higher resolution
Sketch a scanning confocal microscopy setup.
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What is the formula for the force from optical tweezers?
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How do laser tweezers work?
A laser beam is tightly focussed, with high NA lens.
Forces due to refraction are directed back along negative z-direction, act against radiation pressure, resulting in a z-trapping force.
Combing with a transverse trapping force results in a 3D trap (or optical tweezers).
How does an atomic force microscope work?
An AFM generates images by scanning a small cantilever over the surface of a sample.
The sharp tip on the end of the cantilever contacts the surface, bending the cantilever and changing the amount of laser light reflected into the photodiode.
The height of the cantilever is then adjusted to restore the response signal, resulting in the measured cantilever height tracing the surface.
Sketch a basic AFM setup.
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What is the formula for bond force for an AFM?
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What is the momentum transfer for light scattered by a sample?
(98)
What is the scattered intensity for light scattered by a sample?
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How can X-ray diffraction be used to determine crystalline structures?
Braggs law
Varying the scattering angle of X-ray beams produces diffraction peaks that match the d-spacing within a crystal.
What causes problems in x-ray diffraction?
Defects cause complications and so does the presence of different atoms.
Conversion of scattering intensities back to structure suffers from phase uncertainties and weak signals to some peaks, making the experiments difficult.
What is Braggs law?
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What result is gained from X-ray fibre diffraction?
X-ray diffraction from helical macromolecules gives a characteristic cross pattern.
What is the formula for the angle of the cross gained from X-fibre diffraction?
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Why are UV and soft X-rays not typically used for imaging living organisms?
UV and soft X-rays are strongly absorbed by water.
What challenges need to be overcome for routine X-ray microscopy in biology?
Hard X-rays are transmitted through most matter, but it is hard to make optical components.
Experiments must probe reciprocal space rather than direct space images.
Dense tissues, such as bones, absorb X-rays more strongly than soft tissues, such as fat or muscle.
How to use X-ray diffraction patterns of a DNA double helix to estimate the pitch?
1/distance between layer lines gives the pitch of the helices.
How to use X-ray diffraction patterns of a DNA double helix to estimate the diameter?
The angle of the cross can be used to measure the width of the helix.
Neutrons scatter differently from hydrogen and deuterium nuclei. Explain how this effect can be used for structural analysis in biology.
Can perform H/D contrast variation experiments to produce higher resolution structural information by undertaking parallel runs of the same chemical compositions but under different H/D contrasts.
Analysis to these parallel runs improves structural resolution. In particular, it is very useful to calculate the structure of hydrogen bonds associated in neutron diffraction.
What are the pros and cons of an X-ray scattering for the study of proteins?
High quality X-ray beam in coherence improves the information for solving the phase problem.
High brightness or intensity of the beam can help study smaller crystallites in structural determination.
Proteins are vaporised on picosecond time scales, so the challenge is to collect a diffraction pattern before the sample disintegrates.
