Rapid prototyping of 3D micro- nanostructures to explore cell - PowerPoint PPT Presentation

Rapid prototyping of 3D micro- nanostructures to explore cell behavior Herbert Schuck (F. Bauerfeld, D. Sauer, R. Le Harzic, E. Lai, T. Velten, I. Riemann, K. König) October 3, 2007 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Outline • Introduction (IBMT) • State of the art • Two Photon polymerisation (2PP) • Experimental Setup • Material/Biological Results 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Fraunhofer Institute for Biomedical Engineering (FhG-IBMT) • Cell Differentiation Itzehoe • Cell Technology Rostock Lübeck Lübeck Bremen Hanover Berlin Potsdam Golm Brunswick Teltow Paderborn Magdeburg Oberhausen Potzdam-Golm • Cryobiophysics Sulzbach Cottbus Dortmund Duisburg Halle Aachen Schmallenberg • Cellular Dresden St. Augustin Jena Euskirchen Biotechnology Chemnitz Ilmenau • Microsystems • Biochips Frankfurt • Laser Medicine Darmstadt Würzburg • Molecular Kaiserslautern Erlangen • NMR St. Ingbert Bioanalytics Wertheim Saarbrücken Nuremberg Karlsruhe • Ultrasound St. Ingbert • Bioelectronics Pfinztal • Neuroprostetics Stuttgart Freising • Biohybrid System Munich Freiburg Oberpfaffenhofen Holzkirchen • Telemedicine Telematics Efringen-Kirchen Garmisch- Partenkirchen � � � Interface between technology and biology (medicine) � Shenzhen, China 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

State of the art Technology Biology ~ 100 x 100 nm ~ 20 µm ~ 100 nm Intel P4 Processor Electron micrograph SEM of a human fibroblast cell built on 90 nm technology of an influenza virus on structured Si surface � � � � adhesion } � � molecular surface interactions � � surface properties: � � attachment � � � � � � morphology } � � topography � � � � orientation � � 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Biological problems/ requirements • cell growth } � � similarity to in vivo conditions � � • cell differentiation � � culture container � � } � bioreactors � � � � � with reproducible in vivo surface conditions � � � � � matrices � . . Problem: � many different cell types requires � � investigate cells � � different environment conditions � produce tissue � � � � � develop therapeutic � � � surface production method agents � � considering the different biological conditions � � � � � � flexible change of geometry (real 3D) � � rapid � � 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Two Photon Polymerisation (2PP) h h h h ν 365nm = 2 = 2 h ν = 2 = 2 ν ν 365nm ν ν ν ν 730nm One photon excitation Two photon excitation (TPE) 365nm 365nm e.g. 365 nm e.g. 730 nm 15 s � t t t t abs ≤ 10 10 - 10 10 -15 - - 15 15 s s s abs ≤ ≤ ≤ � � � abs abs S1 S1 � photon flux density ≥ 10 24 ½E 1 [photons cm -2 s -1 ] E 1 ½E 1 � fs laser pulses (e.g. Ti:Sa) S0 S0 Photoresist: abs. at 365 nm Laserbeam trans. at 730 nm lens � polymerised ellipsoid volume in space focal Photoresist � real 3D structures volume with undercuts TPE � CAM volume φ xy ~ 100 nm 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Experimental setup Si-wafer Photo resist 2PP volume Cover slip oil high NA objectives Specimen Specimen (1,3 oil/ 40x) Scanning Scanning Scanning Scanning Optics Optics Mirrors Mirrors Objective Lens Objective Lens AOTF AOTF Coherent Coherent non amplified system Ti:Sa Dichroic Beamsplitter (DBS) Dichroic Beamsplitter (DBS) Chameleon Chameleon DBS DBS (720 - 930 nm/ 90 MHz/ 150 fs/ Zeiss LSM 510 DBS DBS PMT PMT Meta NLO 1,6 W avg / Chameleon/ Coherent ) Emission Emission META Detector META Detector Filters Filters (linear PMT Array) (linear PMT Array) Grating Grating PMT PMT � � � � Processing of photo resists with this “cheap” experimental setup 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Material results SU8 (negative resist) AZ 9260 Problem: SU8 with fluorescent marker (positive resist) 3D-Structuring by maskless non destructive analysis ? nano lithography 300 nm (50µm high) � Aspect ratio 150:1 6 µm 10 µm Exposure time depends on complexity of structure, resist, equipment, laser….. 3D cell container (SU8) (e.g. 250x250x50µm ~ 5min) 3D cell container (FIB cut & SEM by IZM Berlin) (with undercuts) 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Biological results Concentric circles test pattern (SU8) on silicon ��� ������������ ���������� (CHO [Chinese Hamster Ovary] 48h ) ��� ��� ��� �� ��� ��� �� �� Spirals ������������������������� r ~ 480 µm ��� ������������ ���������� ��� 100 µm ��� �� �� �� �� �� ��� ��� ���� ������������������������� Spiraled test pattern (SU8 on glass) 5 µm (DPSC) (CHO (96h) ) [DentalPulpaSteamCells]/96h 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Summary/ Outlook • Alignment depends on structure and cell species • Structures with positive and negative resist • High aspect ratio150:1 • Structures down to 100 nm • Structures with undercuts • Electroplating/ hot embossing experiments • Rapid production of 3D prototype structures 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour

Thank you 4M Conference Rapid prototyping of 3D micro- nanostructures to 3.10.2007 Borovets explore cell behaviour