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

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

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

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

Why these wavelength? • λ<<dimension of the object – Transmission • λ ≈ dimension of the object – Diffraction • λ >object, object invisible SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Neutrons • Matter wave (De Broglie, 1924) =h/mv SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

Outlines • X-rays and neutrons sources • SAXS and SANS instruments – Optics – Sample area – Detectors • Sample requirements and collection strategy SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

X-rays and neutron sources SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

X-rays: how are they produced? • Principle – Maxwell equation: accelerated charge -> radiation SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

X-ray sources • Synchrotrons SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

X-ray sources • Synchrotron radiation SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

Insertion devices Undulator (PetraIII) Dipole bending magnet (APS) SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Synchrotrons around the world SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

Lab sources • Principle : electron beam send on a target – Brehmstrahlung – Fluorescence SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

X-ray sources • Lab source (rotating anode, liquid jet) SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

Neutron production • Nuclear reactor SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Neutron sources • Spallation source – Accelerated protons hit a target. SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

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

Optics • Prepare the beam coming from the source • Monochromatic beam • Focus/collimated beam SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Monochromatic X-ray • Bragg diffraction on a crystal n = 2 d sin SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Monochromator • Before • Polychromatic • After • One wavelength + harmonics SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Focusing/low divergence 2 • Small beam at the detector position • Small beam at the sample position SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Focusing X-ray • Focussing mirror • Reflectivity 1,0 0,8 Transmission 0,6 0.15 Degree 0.25 Degree 0,4 1 Degree 0,2 0,0 10000 Energy [eV] SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Focussing mirror – harmonics filter SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Monochromatic neutrons • De Broglie equation – λ=h/mv – The wavelength of a neutron is related to its velocity. • Velocity selector SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Collimation neutrons • A collimator is used to obtain a parallel beam SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Sample environment • As many sample environment as there is sample • For biological macromolecules in solution: – Liquid containing cell – Preferably in vacuum – thermostated SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Sample cell • Cell material, low absorption and low scattering – Mica, polycarbonate • Cell thickness: compromise between absorption and scattering SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Sample environment • On dedicated beamline, Sample handling is now automated: – Faster measurement – Better cleaning – Unattended operation SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

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

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

Single photon counting detector principle SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Single photon counting detector Pilatus – High dynamic range – No background noise – Fast framing  Ideal for SAXS SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Neutron detection • He3 detector: n + 3 He → 3 H + 1 H + 0.764 MeV SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

Experiment • SAS applicable to many type of samples. • Biological macromolecules in solution – Isotropic scattering – Weakly scattering SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

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

Monodispersity • SAS is very sensible to aggregation, the sample should be monodisperse SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Monodispersity • Check the monodispersity of your sample before coming to the beamline. (native gel, dynamic light scattering, ultracentrifugation,…) SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

Monodispersity Improving monodispersity: online size exclusion column SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

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

Inter-particle interactions • Change solution (pH, salt concentration) to limit interactions • Measure different concentrations and extrapolate SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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)*N A /(C* – Using a protein standard • M=M BSA *I(0)/I BSA (0) SAXS and SANS facilities and experimental 11/30/2012 practice - C. Blanchet

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

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

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