A method for micrometer resolution patterning of primary culture neurons for SPM analysis

In this work we present a method for ultra-fine patterning of primary cultu re neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near-field optics to b reak the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neu rons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method co nsists of chemical guidance with an element of physical confinement and all ows for ultra-fine patterning of neural growth on transparent glass substra tes. Substrates consist of microfabricated perfluoropolymer barrier structu res on glass. Poly-L-lysine was selectively deposited using a silicone-base d microfluidic stencil aligned to the perfluoropolymer/glass substrate. Pri mary culture neurons were extracted from 8-day-old chicks and grown for 3 d ays to form good networks. This patterning system shows very specific growt h with patterning separations down to the level of individual neurites. Flu orescent imaging was carried out on both cell viability during growth and i mmuno-tagged microtubule-associated proteins on the neurites. Neurons insid e the patterned structures were imaged and analyzed with a tapping mode SNO AM.