Non-Standard Sample Environments Martin A. Schroer SAXS group - PowerPoint PPT Presentation

Non-Standard Sample Environments Martin A. Schroer SAXS group

Non-standard sample environments options at P12 Disclaimer • Might not work for all samples • Might not make sense for all samples • You will likely need more sample solution however • can give fundamental new insights • In situ • Time-resolved Contact the beamline scientists! • … See “S atellite: Designing Non- • demand a synchrotron source • Photon flux: weak signals, temporal resolution Standard Experiments ” • Small beam sizes: spatial resolution • Energy tunability: Penetration In yellow boxes: Advice to the users ! 2

Types of sample environments Robotic sample changer • Capillary holder Microfluidics (e.g. for THz) • Temperature cell • in vacuum capillary • continuous flow User setups : Need proper planning! • Cryo chamber (P12) • Infrastructure User setups: • X-ray parameters • High pressure cells • .... • Rheological cells  Contact in advance (when • Heating stages • … writing the proposal) SEC-SAXS/MALS Laser excitation Stopped Flow • Online purification and 28/02/2020 3 detection system

Non-standard sample environments at P12 • Heating • Stopped Flow • Laser light • Scanning SAXS • Collaborative project: THz-radiation 4

In air operation In vacuum: • Quarz capillary • Removing in-vacuum capillary / sample changer • Two sealing windows + BSA + buffer  Air gap + buffer • Place sample cell  Higher X-ray absorption by air  Higher background signal (air + windows + sample cell ) Try to reduce the air gap as much as possible! In air • Polystyrene cell Use proper window material! • Air • Kapton windows + BSA + buffer + buffer Do you really need in-air or better sample changer? Maybe two proposals? -> Contact & discuss in advance! 5

Heating stages (at P12) Sample changer temperature range • 5 – 60 ° C (80 ° C): with new sample unit (>2020) • Can Keep samples stored at different T (4 - ~40 ° C) • slow • for radiation sensitive samples • Proper buffer measurements • Lower background A good choice for: • Water-based samples • Moderate temperatures • Short notice temperature tests 6

Heating stages (at P12) Temperature controlled capillary holder • Peltier element • Quartz capillaries -> 1.5 mm diameter • different T range • fast T changes • highly viscous samples • tricky samples (toxic, corrosive, dirty,..) • Apolar solvent A good choice for: • Samples expierence same history • Non-water samples • Background is different • Large range temperature (phase diagram) • Exposure of similar spots (radiation • Strongly scattering samples damage) -> observation holes have been widened -> New options for heating gel samples 7

Temperature SAXS studies F. A. Facchini et al. J. Med. Chem. 61, 2895 (2018). Examples: • Biological relevant lipids: Têmp.-induced melting of lamellar structures • Ferro-nematics (liq crystal + nanoparticles) Phase transition 8 V. Gdovinova, M.A. Schroer et al. Soft Matter 13, 7890 (2017).

Time-resolved: Stopped Flow - Mixer Principle: • Mix to liquids rapidly • Observe the changing signal • Example: pH induced dissociation of apoferritin Dead time: ca. 7 ms load volume: (400 µl)...2-10 ml volume per shot: 100µl • Know that there is a reaction -> pre-testing • Have enough sample -> repeat & check • Explore the power of SAXS -> beyond simple kinetic 9

Why do I need so much sample? -> An example • Reaction of MsbA Nucleotide binding domain with ATP • 35 ms frames collected • Expected : Monomer – dimer transition H. Tidow 10 Josts et al, Structure 28, 348 (2020).

• Start reaction and directly probe  Continuous change of Rg 11

• Start reaction and directly probe  Continuous change of Rg • Start reaction, wait (delay time), then probe  Continuous change of Rg But NOT overlapping  Radiation Damage 12

• Start reaction and directly probe  Continuous change of Rg • Start reaction, wait (delay time), then probe  Continuous change of Rg But NOT overlapping  Radiation Damage • Actually only the first frames can be used  Pump – probe scheme 13

• Start reaction and directly probe  Continuous change of Rg • Start reaction, wait (delay time), then probe  Continuous change of Rg But NOT overlapping  Radiation Damage • Actually only the first frames can be used  Pump – probe scheme 14

Pump – probe approach • Mix/pump • Wait for time • Probe (use only first frame)  No unneeded exposure of sample Example: • Monomer -> Dimer -> Monomer formation • Fully corrupted by RD otherwise + Allows to determine the kinetics/reaction • Alternative option : - High sample consumption : Stroboscopic mode user P12 chopper! - Crucial for biological samples • Good planning needed! • However, already standard ‘non-standard’ experiment. 15

P12 – laser system To fibre • Tuneable Nd:YAG – laser ( Ekspla , Lithuania) Direct • Wave lengths: • 335 – 500 nm & 1065 – 2500 nm (fibre port to P12 hutch experiments) • Repetiton rate: 10 Hz • Pulse length: 6 ns Energy per pulse [mJ] 16

1 st laser triggered reactions H. Tidow 10 6 0 s 30 s • Reaction of nucleotide binding domain 60 s 120 s with caged ATP I(q) [arb. u.] • Excite @355 nm releases ATP 10 5 • Monomer-dimer-monomer reaction 10 4 • Reaction can be initalized after different number of pulses (here: 300, i.e. 30 s) 0 0.5 1 1.5 2 1 q [nm -1 ] 0.9  Triggering and synchronization works 0.8  Laser power via fiber too low for single 0.7 volume fraction 0.6 pulse excitation Monomer 0.5 Dimer  Checking options for optimization 0.4 0.3 0.2 • Under commissioning 0.1 • Looking for collaborative projects 17 19/01/2018 0 0 50 100 150 delay time [s]

Scanning SAXS • Scan samples with a small X-ray beam  Real space maps of SAXS patterns • Allows to reveal the spatial distribution on nanometer- sized structures (shape, size) and orientation • Applications: Heterogenuous samples • Hierarchical materials: e.g. Bones, wood, • tissue,... • Critical parameters (defining the resolution) • beam size, steps size, beam divergence 18

Scanning SAXS @ P12 F. Schmidt et al. Adv. Sci . 6, 1900287 (2019). Moderate µm focus -> low real space resolution Low divergence -> high reciprocal space resolution Example : Mineral particle  Eiger 4M at large detector distance: e.g. collagen distribution within a swordfish sword Old mirrors + cutting the beam Beam size : ~ 75 x 75 µm 2 (hor x vert) • New mirrors ( without cutting) • beam size: 200 x 25 µm 2 (hor x vert) From 2021: New SEU - piezo stage • Moderate field-of-view scanning SAXS • Screening of more specimen -> more (clinical) impact • Resolution of large structures 19

Collaborative experiment: Studying the effect of THz- radiation on proteins Different steps of an experiments • Concept • Design • Conduction THz radiation • Electromagnetic radiation • Sensitive to large molecular vibrations (collective) / low in energy -> THz spectroscopy • Strong absorption in water • Non-ionizing but thermal & athermal effects -> possible risks are discussed in literature 20 L. Wei et al. Frontiers in Laboratory Medicine (2019).

THz-excitation of proteins Fröhlich‘s prediction • THz-radiation can excite collective motions within biological macromolecules by coupling to their dipole moment ( Fröhlich condensation ) Such collective vibrations ( normal modes ) may lead to long-range conformational changes . Such changes can be probed by SAXS.  THz excitation & SAXS probe 21 A. Panjkovich, D.I. Svergun. PCCP 18, 5707 (2016).

THz-SAXS - Experiment Such a noval type of experiment needs • THz sources (cw + pulsed): complementary spectra -> Excite the sample • Dedicated microfluidic cell Setup I -> small channel width • Sample delievery system • Small, asymmetric X-ray beam: (80 x 120 µm 2 ) & smaller now! • Precise positioning (sub-micron) (hexapod) • Synchronization (data collection) Setup II M. Roessle (TH Lübeck) 22 G. Katona (U Gothenburg)

S. Schewa, M. Roessle et al. THz-SAXS - microfluidic cell (TH Lübeck) Combined THz-SAXS measurements demand dedicated sample environment Microfluidic chips: • • Flowing of sample → reduce radiation damage • Transparent for THz → enough sample excitation • Narrow channel (500 µm) as THz absorption in water is strong → enough sample excitation • Low X-ray background → record SAXS signal  3D printed Polystyrene cell with mylar X-ray windows W = 0.2 mm <-> optimized for THz T = 2 mm <-> optimized for X-rays 23 S. Schewa, M.A. Schroer, et al., Rev. Sci. Instrum. 91 , 084101 (2020).

THz-SAXS cell: Properties • THz-absorption in PS: Low as PS apolar • THz-spectra of PS: No absorption lines • SAXS from proteins: Access to proteins of different molecular weight S. Schewa, M.A. Schroer, et al., Rev. Sci. Instrum. 91 , 084101 (2020). 24 10/10/2018