FULL ARTICLE Femtosecond laser nanosurgery of sub-cellular structures in HeLa cells by employing Third Harmonic Generation imaging modality as diagnostic tool George J. Tserevelakis 1; 2 , Stylianos Psycharakis 1 , Bojan Resan 3 , Felix Brunner 3 , Evagelia Gavgiotaki 1; 2 , Kurt Weingarten 3 , and George Filippidis * ; 1 1 Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, P.O. Box 1385, 71110, Heraklion, Crete, Greece 2 Department of Physics, University of Crete 3 Time-Bandwidth Products AG, Technoparkstrasse 1, 8005 Zurich, Switzerland Received 16 June 2011, revised 28 July 2011, accepted 28 July 2011 Published online 26 August 2011 Key words: nanosurgery, THG, imaging, HeLa cells, viability, fs laser 1. Introduction Femtosecond (fs) laser nanosurgery addresses the need for precise, minimally invasive dissection of cells and sub-cellular components (with sub-mm reso- lution), minimal alteration to cellular environment to living biological samples without using genetic methods or chemical agents. An fs laser acts like a # 2012 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Journal of BIOPHOTONICS Femtosecond laser assisted nanosurgery of microscopic biological specimens is a relatively new technique which allows the selective disruption of sub-cellular structures without causing any undesirable damage to the sur- rounding regions. The targeted structures have to be stained in order to be clearly visualized for the nanosur- gery procedure. However, the validation of the final na- nosurgery result is difficult, since the targeted structure could be simply photobleached rather than selectively destroyed. This fact comprises a main drawback of this technique. In our study we employed a multimodal sys- tem which integrates non-linear imaging modalities with nanosurgery capabilities, for the selective disruption of sub-cellular structures in HeLa cancer cells. Third Har- monic Generation (THG) imaging modality was used as a tool for the identification of structures that were sub- jected to nanosurgery experiments. No staining of the biological samples was required, since THG is an intrin- sic property of matter. Furthermore, cells’ viability after nanosurgery processing was verified via Two Photon Ex- citation Fluorescence (TPEF) measurements. THG image of HeLa cancer cells * Corresponding author: e-mail: filip@iesl.forth.gr, Phone: ++302810391320, Fax: ++302810391305 J. Biophotonics 5, No. 2, 200–207 (2012) / DOI 10.1002/jbio.201100055