A Method for Micrometer Resolution Patterning of Primary Culture Neurons for SPM Analysis

P. Degenaar (1) , Bruno Le Pioufle (2) , L. Griscom (3) , A. Tixier (3) , Y. Akagi (3) , Y. Morita (3) , Y. Murakami (1) , K. Yokoyama (1) , H. Fujita (3) , E. Tamiya (1)

1 JAIST - Japan Advanced Institute of Science and Technology
2 SATIE - Systèmes et Applications des Technologies de l'Information et de l'Energie
3 LIMMS - Laboratory for Integrated Micro Mechatronics Systems

In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis is useful to the study of long term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-l-lysine was selectively deposited using a silicone based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8 day old chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.

Domaines: Micro et nanotechnologies/Microélectronique

P. Degenaar, Bruno Le Pioufle, L. Griscom, A. Tixier, Y. Akagi, et al.. A Method for Micrometer Resolution Patterning of Primary Culture Neurons for SPM Analysis. Journal of Biochemistry, 2001, 130, pp.367-376. ⟨hal-00786168⟩