COMBINED SURFACE-SENSITIVE MICROSCOPIES FOR ANALYSIS OF BIOLOGICAL TISSUES AFTER NEURAL DEVICE IMPLANTATION

Presentation about usage of surface analysis techniques to probe biological samples

1. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Combined Surface-sensitive microscopies for analysis of biological tissues after neural device implantation

2. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Neurons: Main cells in the nervous system Have electrical and chemical activity Communicate between neurons Synapse: Point of communication between two cells Release of neurotransmitters Bind of neurotransmitters to its receptors change the voltage across the cell membrane Creation of action potentials Neurons and the action potential Synapse Neuron

3. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Brain computer interface (BCI) Recording of EEG and ECoG signals Signals translated using algorithms Paralytic patients can control external devices Neural Recording electrode technologies Electroencephalography Electrocorticography Deep brain stimulation (DBS) Electrical impulses sent inside the brain Inactive neurons produce neurotransmitters again Treatment of neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease

4. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Host response to foreign materials Castner , D. G. & Ratner, B.D. Surface Science 500 (2002) 28–60 G. Bao & S. Suresh. Nature Materials 2 (2003) 715-725. rigid materials! Electrical impedance Device signal loss

5. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Example of studies in biological surface science during the 2000s: Surface chemical state imaging Molecular recognition surfaces Adsorbed protein films Hydrated surfaces What about truly biological specimens? Tendency since the 2010s Biological surface science context Biological surface science is a broad, interdisciplinary subfield of surface science, where properties and processes at biological and synthetic surfaces and interfaces are investigated, and where biofunctional surfaces are fabricated. 1960s 1970-80s Kasemo , Bengt. Surface science 500 (2002) 656-677. Höök , Fredrik, Kasemo , Bengt, Grunze , Michael et al. ACS Nano 2 (2008) 2428-2436. Biological models

6. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 STATE OF the ART IN MICROSCOPY OF BIOLOGICAL SAMPLES TOF-SIMS Molecular analysis of carcinogenic human breast tissue A) TOF-SIMS image B) H&E image Gamble et al 2015 XPEEM & XPS Surface chemical mapping of blood cells (a) PEEM image filtered with energy (b) spatially resolved chemical map and (c) corresponding level spectrum C1s. Andreas Skalberg et al 2017 Optical and AFM images of HeLa cells. Zhibin Yin 2019 XPS images of a spot of an E. coli microarray printed on an amino-functionalized. Nora Graf 2009 µ-XPS Surface chemical analysis of DNA microarrays AFM Unicellular topographic imaging 100 µm 100 µm Cell membrane i.e. phospholipids Cytoplasm and cell nucleus Stromal regions Epithelial regions 100 µm 100 µm

7. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 New surface tools available for analysis of biological samples Instruments: C luster ion beam technology S ample preparation protocols: Embedded tissue for cutting Cryo -preserving/analysis Process of sample dehydration E xperimental protocols of analysis: Multi-technique approaches New methods for data analysis: Multivariate statistical analysis (MVA) “There is never just one technique that can provide a complete surface characterization” Baio , Joe E ., Graham , Daniel J ., Castner , David G . Chemical Society Reviews 49 (2020) 3278-3296. EAG’s Bubble Chart, 2016.

8. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Neural implants for BCI applications Last versions are already applied to human brains Work context Corinne S. Mestais et al., IEEE, vol. 23, No. 1, 2015. First versions of neural device initially implanted in the brain of non-human primates ECoG implant was placed here!

9. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Why did the device trigger such an acute inflammatory reaction? Work context O ne year after implantation… Fibrous tissue formed around the neural device Non- human primate brain Cu-frame embedded in Kapton matrix Ti - TiN -Si Ni- Ti - TiN -Si MWCNTs Silver Epoxy Kapton Kapton Epoxy Silver Epoxy Cu-Ni-Au Cu-Ni-Au 1.5 cm

10. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Time of flight secondary ion mass spectrometry ( ToF -SIMS) Combined microscopies for analysis of biological tissues Operational modes Ability : Chemical information based on fragments of molecules Chemical detection : all elements are detected Mass resolution : 10,000 ∆M/M Spatial resolution : 1-5 µm Depth resolution : 2 nm

11. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 X-ray photoelectron spectromicroscopies (XPS & XPEEM) Combined microscopies for analysis of biological tissues Ability : local chemical states Chemical detection : all elements except H and He Energy resolution : 0.42 eV Lateral resolution : 15-20 µm Depth resolution : 2-20 nm (d=3 λ cos θ ) / 3 nm for biological samples N. Barret et O. Renault, Matériaux & Techniques 97 , 101–122 (2009)

12. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Sample preparation CNTs Tissue section deposited on ITO/glass substrate 1 µm A B C D E

13. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 TRACES of metals found IN THE FIBROUS TISSUE by tof-sims Ni + , 60 Ni + C 2 H 6 N + Si + Ti + , 50 TiN + , TiH 2 + Ni + Si + Si + CNTs Macro images of slices Macro images Overlay of C5H15NO4P+, sum of: Ni+, 60Ni+, Si+

14. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Main molecules constituting the fibrous tissue Membrane lipids Proteins Polysaccharides Nuclei acids RGB overlay of principal components PC 1 ( red ) and PC 2 (green) Main peaks from tissue Main peaks from substrate Principal components analysis (PCA) of ToF -SIMS data BS09 Sosnik , Alejandro et al., Biomaterials , 2006. [2] Lhoest , J. B. et al., Journal of Biomedical Materials Research , 1998. Tidwell , Caren D. et al., Surface and Interface Analysis , 2001. Barnes , Christopher A. et al., Biomaterials , 2011 . [5] Shard , A. G., Langmuir, 1997. [6] Charbonneau , Cindy et al., Biomaterials , 2011 .

15. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Main molecules constituting the fibrous tissue Phosphatidylcholine (PC) Membrane lipids Amino acids Collagen , Vitronectin & Fibronectin Nucleobases Polysaccharides DNA & RNA Heparin ( HSGAGs ) Hyaluronic Acid (HA) Gly Arg Ala C G T PC Chondroitin Sulfate ( CSGAGs )

16. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Chemically distinct regions on fibrous tissue 2mm Overlay of PC7 and PC8 CN- CNO- PO4H2- C18H35O2- C18H33O2- C16H30O2- C16H31O2- C4H8NO7P- H&E-stained histological image C6H10NO- C7OH7- Amino acids + GAGs & proteins fragments Phospholipid fragments + fatty acids C2H3SO- C2H4O4P- BS18 BS18 High celullarized regions Collagen type I

17. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Quantification of Chemically distinct regions in the biological tissue Extracellular matrix (ECM)-rich region Phospholipid membrane (PM)-rich region

18. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Quantification of Chemically distinct regions on fibrous tissue Phospholipid membrane (PM)-rich region Extracellular matrix (ECM)-rich region

19. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Chemical environment differences observed in micrometer scale on tissue chemical environment imaging by Xpeem analysis O 1s C 1s hydrocarbons C-C/C-H/C=C Ether/Amine C-O/C-NH Amide C=O/CO-N/C=N Amide O=C/O=C-N + eV + eV O 1s C 1s C 1s C 1s SE image

20. GOMES DE CARVALHO Amanda | Grenoble, November 11th, 2020 Combination of ToF -SIMS , XPS & XPEEM for analysis of truly biological samples Traces of metals such as nickel and titanium found in the biological tissue by ToF -SIMS imaging Main molecules present in the biological tissue highlighted by ToF -SIMS H ighlighting of zones of chemical contrast in the tissue by the use of PCA analysis of ToF -SIMS data Identification and quantification of main elements present in tissue as well as the chemical environment of these elements Chemical constrasts based on chemical binding environment imaged by XPEEM In summary

21. Thank you for your attention!