Week 6: Experimental techniques

Question 1

Sketch an experimental set-up for infrared spectroscopy.

Answer 1

(89)

Question 2

How does infrared spectroscopy work?

Answer 2

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.

Question 3

What are Amide I peaks?

Answer 3

Absorbance peaks around 1650 cm-1.

Question 4

What is circular dichroism?

Answer 4

A spectroscopic technique that measures the difference in the absorption of left-handed and right-handed circularly polarised light by a molecule.

Question 5

Why may circular dichroism be used?

Answer 5

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.

Question 6

What is Fluorescence resonance energy transfer (FRET)?

Answer 6

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.

Question 7

At what range can FRET measure molecular proximity?

Answer 7

10 - 100 Angstrom

Question 8

When is FRET efficient?

Answer 8

When the donor and acceptor are positioned within a few nm.

Question 9

What are the components of a fluorescence microscope?

Answer 9

Sample
Cover slip
Objective lens
Dichroic mirror
Excitation filter
Detector
Emission filter
Mercury light source

Question 10

Sketch a fluorescence microscope setup.

Answer 10

(90)

Question 11

Sketch a confocal microscope setup.

Answer 11

(91)

Question 12

What is the Abbe theory?

Answer 12

It tells us what the best resolution that can be achieved by a lens.

(92)

Question 13

What formula gives the resolution in the depth (z) direction?

Answer 13

(93)

Question 14

What is scanning confocal microscopy?

Answer 14

The object is illuminated by image of point source and pinhole detection, enabling depth scanning.

Question 15

What advantage does scanning confocal microscopy have over simple microscopy?

Answer 15

Higher resolution

Question 16

Sketch a scanning confocal microscopy setup.

Answer 16

(94)

Question 17

What is the formula for the force from optical tweezers?

Answer 17

(95)

Question 18

How do laser tweezers work?

Answer 18

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).

Question 19

How does an atomic force microscope work?

Answer 19

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.

Question 20

Sketch a basic AFM setup.

Answer 20

(96)

Question 21

What is the formula for bond force for an AFM?

Answer 21

(97)

Question 22

What is the momentum transfer for light scattered by a sample?

Answer 22

(98)

Question 23

What is the scattered intensity for light scattered by a sample?

Answer 23

(99)

Question 24

How can X-ray diffraction be used to determine crystalline structures?

Answer 24

Braggs law

Varying the scattering angle of X-ray beams produces diffraction peaks that match the d-spacing within a crystal.

Question 25

What causes problems in x-ray diffraction?

Answer 25

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.

Question 26

What is Braggs law?

Answer 26

(100)

Question 27

What result is gained from X-ray fibre diffraction?

Answer 27

X-ray diffraction from helical macromolecules gives a characteristic cross pattern.

Question 28

What is the formula for the angle of the cross gained from X-fibre diffraction?

Answer 28

(101)

Question 29

Why are UV and soft X-rays not typically used for imaging living organisms?

Answer 29

UV and soft X-rays are strongly absorbed by water.

Question 30

What challenges need to be overcome for routine X-ray microscopy in biology?

Answer 30

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.

Question 31

How to use X-ray diffraction patterns of a DNA double helix to estimate the pitch?

Answer 31

1/distance between layer lines gives the pitch of the helices.

Question 32

How to use X-ray diffraction patterns of a DNA double helix to estimate the diameter?

Answer 32

The angle of the cross can be used to measure the width of the helix.

Question 33

Neutrons scatter differently from hydrogen and deuterium nuclei. Explain how this effect can be used for structural analysis in biology.

Answer 33

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.

Question 34

What are the pros and cons of an X-ray scattering for the study of proteins?

Answer 34

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.