Rapid prototyping of 3D micro- nanostructures to. explore cell behavior. Herbert Schuck. October 3, 2007

Transcription

1 explore cell behavior Herbert Schuck (F. Bauerfeld, D. Sauer, R. Le Harzic, E. Lai, T. Velten, I. Riemann, K. König) October 3, 2007

2 Outline Introduction (IBMT) State of the art Two Photon polymerisation (2PP) Experimental Setup Material/Biological Results

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

4 State of the art Technology Biology ~ 100 x 100 nm ~ 100 nm ~ 20 µm Intel P4 Processor built on 90 nm technology Electron micrograph of an influenza virus SEM of a human fibroblast cell on structured Si surface surface properties: adhesion attachment morphology orientation } } molecular surface interactions topography

5 Biological problems/ requirements cell growth cell differentiation culture container bioreactors matrices. Problem: } } similarity to in vivo conditions with reproducible in vivo surface conditions many different cell types requires different environment conditions surface production method considering the different biological conditions flexible change of geometry (real 3D) rapid investigate cells produce tissue develop therapeutic agents

6 Two Photon Polymerisation (2PP) S1 S0 Photoresist One photon excitation e.g. 365 nm E 1 focal volume Two photon excitation (TPE) e.g. 730 nm S1 S0 Laserbeam φ xy lens TPE volume ~ 100 nm ½E 1 ½E 1 h ν 365nm = 2 h ν 730nm 365nm t abs abs s photon flux density [photons cm -2 s -1 ] fs laser pulses (e.g. Ti:Sa) Photoresist: abs. at 365 nm trans. at 730 nm polymerised ellipsoid volume in space real 3D structures with undercuts CAM

7 Experimental setup oil Si-wafer Photo resist 2PP volume Cover slip high NA objectives Objective Lens Specimen Scanning Optics Scanning Mirrors (1,3 oil/ 40x) Dichroic Beamsplitter (DBS) Zeiss LSM 510 Meta NLO META Detector (linear PMT Array) DBS DBS AOTF Emission Filters PMT Coherent Chameleon non amplified system Ti:Sa ( nm/ 90 MHz/ 150 fs/ 1,6 W avg / Chameleon/ Coherent ) Grating PMT Processing of photo resists with this cheap experimental setup

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

9 Biological results Concentric circles 80% 60% 40% 20% 0% Spirals r ~ 480 µm (CHO [Chinese Hamster Ovary] 48h) 10 Frequency (normalized) test pattern (SU8) on silicon Difference in Orientation 40% 20% > % µm (normalized) Frequency 60% Difference in Orientation 5 µm (DPSC) (CHO (96h)) Spiraled test pattern (SU8 on glass) [DentalPulpaSteamCells]/96h

10 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

11 Thank you