SAXS and SANS facilities and experimental practice Clement Blanchet - - PowerPoint PPT Presentation

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SAXS and SANS facilities and experimental practice Clement Blanchet - - PowerPoint PPT Presentation

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SAXS and SANS facilities and experimental practice Clement Blanchet SAS experiment Detector Sample X-ray or neutron Beam 2 s Buffer SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet X-rays Roengten, 1895

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SLIDE 1

SAXS and SANS facilities and experimental practice

Clement Blanchet

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SAS experiment

2

s

X-ray or neutron Beam

Sample Buffer Detector

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 3

X-rays

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

Roengten, 1895

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Electromagnetic wave

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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The electromagnetic spectrum

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 6

Why these wavelength?

  • λ<<dimension of the object

– Transmission

  • λ ≈ dimension of the object

– Diffraction

  • λ >object, object invisible

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 7

Neutrons

  • Matter wave (De Broglie, 1924)

=h/mv

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 8

SAXS and SANS

  • Common analysis methods but:

– The scattered particles are different – Different interactions

  • X-rays interact with electrons via electromagnetic

forces

  • Neutrons interact with nucleus via nuclear forces

 Different instruments

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 9

Outlines

  • X-rays and neutrons sources
  • SAXS and SANS instruments

– Optics – Sample area – Detectors

  • Sample requirements and collection strategy

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 10

X-rays and neutron sources

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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X-rays: how are they produced?

  • Principle

– Maxwell equation: accelerated charge -> radiation

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 12

X-ray sources

  • Synchrotrons

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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X-ray sources

  • Synchrotron radiation

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 14

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Insertion devices

Dipole bending magnet (APS) Undulator (PetraIII)

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Synchrotrons around the world

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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X-ray sources

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Lab sources

  • Principle : electron beam send on a target

– Brehmstrahlung – Fluorescence

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 19

X-ray sources

  • Lab source (rotating anode, liquid jet)

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 20

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Neutron production

  • Nuclear reactor

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Neutron sources

  • Spallation source

– Accelerated protons hit a target.

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 23

Neutron Sources

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SAXS and SANS Instruments

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Optics

  • Prepare the beam coming from the source
  • Monochromatic beam
  • Focus/collimated beam

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 26

Monochromatic X-ray

  • Bragg diffraction on a crystal

n = 2dsin

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Monochromator

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

  • Before
  • Polychromatic
  • After
  • One wavelength + harmonics
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SLIDE 28

Focusing/low divergence

  • Small beam at the detector position
  • Small beam at the sample position

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

2

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Focusing X-ray

  • Focussing mirror
  • Reflectivity

Energy [eV]

10000

Transmission

0,0 0,2 0,4 0,6 0,8 1,0

0.15 Degree 0.25 Degree 1 Degree

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 30

Focussing mirror – harmonics filter

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Monochromatic neutrons

  • De Broglie equation

– λ=h/mv – The wavelength of a neutron is related to its velocity.

  • Velocity selector

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Collimation neutrons

  • A collimator is used to obtain a parallel beam

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Sample environment

  • As many sample environment as there is

sample

  • For biological macromolecules in solution:

– Liquid containing cell – Preferably in vacuum – thermostated

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Sample cell

  • Cell material, low absorption and low

scattering

– Mica, polycarbonate

  • Cell thickness: compromise between

absorption and scattering

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Sample environment

  • On dedicated beamline, Sample handling is

now automated:

– Faster measurement – Better cleaning – Unattended operation

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Flight tube

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Beamstop

  • Prevent the direct beam from hitting the

detector

– Big enough to stop the direct beam – Small enough to collect the small angle

  • Measure transmitted beam

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Detectors

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Single photon counting detector principle

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Single photon counting detector Pilatus

– High dynamic range – No background noise – Fast framing

 Ideal for SAXS

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Neutron detection

  • He3 detector:

n + 3He → 3H + 1H + 0.764 MeV

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Experimental practice

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Experiment

  • SAS applicable to many type of samples.
  • Biological macromolecules in solution

– Isotropic scattering – Weakly scattering

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SAS Experiment

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Buffer subtraction

  • Biological sample scatters very weakly
  • Care should be taken for the buffer

subtraction

– Exactly matching buffer (dialysis, elution buffer) – Sample and buffer measured in the same cell

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Monodispersity

  • SAS is very sensible to aggregation, the sample

should be monodisperse

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Monodispersity

  • Check the monodispersity of your sample

before coming to the beamline.

(native gel, dynamic light scattering, ultracentrifugation,…)

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Monodispersity

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

Improving monodispersity:

  • nline size exclusion column
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SEC + SAXS

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet Defined buffer region

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Inter-particle interactions

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 51

Inter-particle interactions

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

  • Change solution (pH, salt concentration) to

limit interactions

  • Measure different concentrations and

extrapolate

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SLIDE 52

Measure also water and/or standard protein…

  • … to estimate the molecular mass of your

sample using the forward scattering

– For data on an absolute scale (water measurement)

  • M=I(0)*NA/(C*

– Using a protein standard

  • M=MBSA*I(0)/IBSA(0)

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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X-rays - Radiation damage!!!

  • With intense third generation synchrotons:

creation of free radicals in solution, that degrades protein and causes aggregation.

  • Monitor radiation damage: collect several

frames and compare them.

  • Limit the radiation damage

– Use of scavengers DTT, Glycerol. – Flow measurements.

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Contrast in neutron

  • Neutrons interact with the nucleus of atoms
  • Each atoms has its own scattering length:

H D C N O P S

  • .3742 0.6671 0.6651 0.940 0.5804 0.517 0.2847

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SLIDE 55

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Labeling

Deuteration of sample

– Expression of the protein in deuterated medium

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Collection strategy

  • Solvent matching

– Find the solvent composition that match the contrast of the component you want to hide – Measure the sample in this solvent

  • Contrast variation

– Measure the sample in solvent with different contrast – Using Stuhrmann analysis you can access the curves of the different components.

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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Conclusion - Instruments

  • SAXS and SANS facilities: great high tech

instruments.

  • Apply for beamtime (dedicated bioSAXS

beamline in PetraIII/EMBL, ESRF, Soleil, SSRL, Australian synchrotron, spring8, diamond)

  • Lab source also available for SAXS

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SAS sample

  • Protein concentration: 1-10 mg/ml
  • Volume: 5-50 microliter (SAXS), 200-300

microliter (SANS)

  • Time:
  • lab source: 5-60 min
  • Synchrotron: seconds
  • Neutrons: 30 minutes - hours

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet

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SAS sample

  • Pure and monodisperse sample
  • Exactly matching buffer
  • Measure concentration series
  • For SAXS:

– Be aware of radiation damage

  • For SANS:

– Carefully design your experiment, think of your collection strategy.

11/30/2012 SAXS and SANS facilities and experimental practice - C. Blanchet