Aplicacions biomèdiques de la llum de sincrotró (Seminaris Tecnològics 2017)

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APLICACIONS BIOMÈDIQUES DE LA

LLUM DE SINCROTRÓ

Núria Benseny Cases

Postodoc at Infrared Beamline (MIRAS)

ALBA Synchrotron

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1. Com funciona un sincrotró

2. MIRAS: Microscopia d’Infraroig

1. Microscopia dInfraroig

2. Estudis d’Infraroig en la malaltia d’Alzheimer 3. Altres Exemples

3. MISTRAL: Tomografia de rajos X

4. CLAES: Absorció de rajos X

5. NCD I Xaloc: Difracció de Rajos X

6. Demanar beamtime

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Interacció de la llum amb la matèria

XALOC: Línia de cristalografia NCD: Difracció no cristal·lina MISTRAL: Tomografia de rajos X CLAES: Espectroscopia d’absorció de rajos X MIRAS: Microscopiqa d’IR

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X - H

≡

=

-

Symmetric stretching Antisymmetric stretching Scissoring N-H O-H C-H C≡C C≡N C=O C≡N C=C 𝐸 = ℎ 2𝜋 𝑘 𝜇

Chemical bonds – springs & mass

Vibrational modes: k: spring constant 𝜇: reduces mass Fingerprint Martin Kreuzer

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Globar vs Synchrotron light source

Spectra with 10 x 10 µm2 _Aperture

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0 1.1e+07 5.4e+06 5e+06 4.6e+06 4.2e+06 3.8e+06 3.4e+06 3e+06 2.6e+06 2.2e+06 1.8e+06 1.4e+06 1e+06 6e+05 5.8e+06 6.2e+06 6.6e+06 7e+06 7.4e+06 7.8e+06 8.2e+06 8.6e+06 9e+06 9.4e+06 9.8e+06 1.02e+07 1.06e+07 0 1.1e+07 5.4e+06 5e+06 4.6e+06 4.2e+06 3.8e+06 3.4e+06 3e+06 2.6e+06 2.2e+06 1.8e+06 1.4e+06 1e+06 6e+05 5.8e+06 6.2e+06 6.6e+06 7e+06 7.4e+06 7.8e+06 8.2e+06 8.6e+06 9e+06 9.4e+06 9.8e+06 1.02e+07 1.06e+07

Globar vs Synchrotron light source

10 x 10 µm2_Aperture: GLOBAR Synchrotron y-axis [µm] x-axis [µm] y-axis [µm] Tr ans mit ted ph ot ons [ a.u .] x-axis [µm] Martin Kreuzer

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Alzheimer Disease

Cortex shrivels up:

Damaging areas involved in thinking, planning and remembering.

Severe Hippocampus shrinkage

Damaging areas play a key role in formation of new memories.

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Amyloid peptides

Presence of amyloid plaques

in the affected regions.

Plaques present in Alzheimer Disease brain

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APP and Aβ(1-40)

…EVKM AEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVVIVITLVMLKK…

Sticht, H. et al. (1995) Structure of amyloid Aβ-(1-40)-peptide of Alzheimer's disease, Eur J Biochem. 233, 293-8.

A(1-40) A(1-

a) b)

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Time

Fibril

fraction

Benseny-Cases N, Cócera M, Cladera J.

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Amyloid plaques in situ

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 1700 1650 1600 Wavenumber (cm-1) 0.0005 1630/1650: β structure/α helices

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1630

1650 1740

Protein Aggregation Ratio: 1630/1650 Lipid Oxidation Ratio: 1740/total lipid

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Plaques Tiss ue ou tside the pl aq ues

β-sheet content C-OOH ( Lipid Oxidation)

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plaque no plaque -0.1 0.0 0.1 0.2 0.3 0.4 Ratio 163 0/1650 plaque no plaque 0.4 0.6 0.8 1.0 Ratio 174 0/2960

Brain with Plaques but without

Alzheimer Disease symptoms

No co-localization of oxidation and plaques

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Main Conclusions

• Lipids in the plaques and surroundings are

oxidized.

• Control brains, with plaques but without

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Sitges in the Barcelona area

Photo by Rich2012

Painting by Ferrer Bassa (1346) Saint Michael's Chapel in Barcelona

Martin Kreuzer

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Painting by Ferrer Bassa (1346) Saint Michael's Chapel in Barcelona

How to refurbish/restore the paintings? What are the origins of the dark spots?

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1 fungi layer 1 2 blue azurite 2 3 calcium carbonate 3

4 substrate 4

 The salts are directly related to the reaction compounds secreted by fungis.  Black marks result from fungi attacks.

 The dark colour observed is due to the presence of melanin secreted by the fungi.  Melanin is water-soluble, and therefore, can be easily removed.

 This treatment has been successfully applied in the restoration wall paintings. Anal. Methods, 2016,8, 1637-1645

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Example: Drug Delivery in Dermatology

Stratum corneum Stratum granulosum Stratum spinosum Stratum basale Epidermis

J Drug Discov Develop and Deliv. 2014;1(2): 8.

Cross section of the outer layers of the skin.

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Example: Drug Delivery in Dermatology Stratum corneum Stratum granulosum Stratum spinosum Stratum basale Epidermis

J Drug Discov Develop and Deliv. 2014;1(2): 8.

Cross section of the outer layers of the skin. Drug

in lipid vesicle

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Colloids and Surfaces B: Biointerfaces 131 (2015) 102–107

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Colloids and Surfaces B: Biointerfaces 131 (2015) 102–107

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Cryo SXT

Tomography of vitrified samples Working at water window 520eV: natural contrast

Ø cryo system (sample < 130K -148C) – Frozen Hydrated state

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ICCB 2016 Visible/ Fluorescence Light Microscope Transmission X-Ray Microscope Select the region of interest Tomogram -70, +70 Reconstruction Select the cell

Select the cell

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Hepatocyte control cells A whole cell Natural contrast Without any addition of chemical compounds for fixation or staining Without artifacts Mitochondria Mitochondrial Cristae Nuclear Envelope Nucleus Cytoskeleton Endoplasmic Reticulum

Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection. Pérez-Berná AJ et al, ACS Nano. 2016 Jun 28

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0 Projection Accumulation of vesicles 1 mm HCV Infected cells 1 mm Accumulation of vesicles Membranous web Membranous vesicles

Membranous vesicles are tubes in different orientation

3D map

Neck-like structures connecting endoplasmic reticulum extrusions to the

tubular network Close Compartments

for RNA replication

Functional RNA replicase complexes

Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection. Pérez-Berná AJ et al, ACS Nano. 2016 Jun 28

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X-ray absorption spectroscopy

• When an x-ray is absorbed by an atom, its

energy is transferred to a core-level

electron (K, L, or M shell) resulting in its

ejection from the atom.

• The atom is left in an excited state with an

empty electronic level (a core-hole).

• Any excess energy from the x-ray is given

to the ejected photoelectron.

Cu K-edge ~9000 eV

Cu L-edges ~930 eV Ag K-edge ~25500 eV Ag L-edges ~3500 eV S K-edge ~2400 eV

S L-edges ~160-230 eV

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XANES

_EXAFS

Cu K edge (8979 eV)

The ejected

photoelectron is

backscattered by the

neighboring atoms.

 local structure

 frequencies ~ bond distances

9000 9300 9600 0,0 0,7 1,4 x m (E) E (eV)

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Am. Min. 95, 200 (2010)

An example studied in Claess:

the spinach ferrodoxine

In collaboration with prof P. Postorino, University of Roma Sapienza Ferredoxins are iron-sulfur proteins that mediate electron

transfer in a range of metabolic reactions. Ferredoxin is

involved in both cyclic and non-cyclic photophosphorylation

reactions of photosynthesis. In non-cyclic

photophosphorylation, ferredoxin is the last electron acceptor thus reducing the enzyme NADP+_{reductase. These biological}

"capacitors" can accept or discharge electrons, with the effect of a change in the oxidation state of the iron atoms between +2 and +3.

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XANES

• Oxidation state

• Unoccupied electronic states

• Spin state

• Local structure

• direct information about bond angles.

EXAFS

• Bond distances

• Coordination number

• Static and dynamic disorder

Am. Min. 95, 200 (2010) 9030 9100 9170 1.4 1.6 1.8 x m (E) E (eV)

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NCD: Non cristalline difraction

Xaloc: Cristalline Difraction

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Liposomes

Multilamelar

Unilamelars

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SAXS

Material no cristal·lí

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2 CONVOCATÒRIES - Febrer