Cell tracking Intracellular Polysilicon Barcodes for Cell Tracking Elisabet Fernandez-Rosas, Rodrigo Go ´mez, Elena Iban ˜ez, Leonardo Barrios, Marta Duch, Jaume Esteve, Carme Nogue ´s, * and Jose ´ Antonio Plaza* During the past decade, diverse types of barcode have been designed in order to track living cells in vivo or in vitro, but none of them offer the possibility to follow an individual cell up to ten or more days. Using silicon microtechnologies a barcode sufficiently small to be introduced into a cell, yet visible and readily identifiable under an optical microscope, is designed. Cultured human macrophages are able to engulf the barcodes due to their phagocytic ability and their viability is not affected. The utility of the barcodes for cell tracking is demonstrated by following individual cells for up to ten days in culture and recording their locomotion. Interestingly, silicon microtechnology allows the mass production of reproducible codes at low cost with small features (bits) in the micrometer range that are additionally biocompatible. 1. Introduction An increasing demand for tracking smaller items has driven the exploration for novel methods of barcoding at much smaller scales. [1] Individual cell tracking is of great interest in cell biology [2] to evaluate individual cell behavior (cell survival, cell movement, relationship with other cells, etc.) under different conditions (exposure to toxic gases or compounds, to a chemical or light stimulus (chemotaxy, phototaxy) or to X-ray micro- beams, for example). Therefore, in the last decade there has been a growing interest in developing different types of barcodes to track living cells either in vivo or in vitro. Encoded particles from different materials and patterns have been proposed to follow cells in culture. Quantum-dot-tagged microbeads, [3] metallic barcodes, [4,5] porous-silicon photonic crystals, [6] acrylic-encoded carriers, [7] iron oxide magnetic nanoparti- cles, [8,9] poly(dimethylsiloxane) (PDMS) particles, [10] nano- disk codes, [11] and diamond nanoparticles [12] have been used among other devices. While some of these particles act as a barcode because of their own nature, [5] others need to be coupled to different fluorochromes, [3,13] and this extra step makes barcode manufacturing more expensive and difficult. Additionally, the use of fluorochromes requires a fluorescence microscope or a confocal scanning laser microscope to visualize the barcode but UV light and laser beams have been reported to be harmful for living cells. [14] Codes can be introduced into cells, [15] attached to the plasma membrane, immobilized on a solid substrate via complementary single-stranded DNA or immunocytochemical reaction systems, [16] or can even be used as a chamber containing the cell. Until now, codes have been envisaged to follow different cell populations mixed in the same culture or to follow a subpopulation of cells in vivo. To track individual cells during several days in culture and using an optical microscope, the codes have to fulfill special biological, optical, and fabrication requirements. First, from the biological point of view, codes must be designed to the micrometer size, sufficiently small to be introduced into a cell. In addition, the number of uniquely identifiable barcodes must be large enough to track a sufficient number of cells to perform biological studies, and the devices have to be made of a biocompatible material in order to ensure cell viability. Second, from the optical point of view, the codes have to be simple and visible under light microscopes. Thus, the structures that represent every single bit have to be larger than the resolution limit of these microscopes, which is in practice close to 1 mm. Finally, the fabrication technology of the codes Intracellular Polysilicon Barcodes for Cell Tracking [  ] Dr. C. Nogue ´s, Dr. E. Iban ˜ez, Dr. L. Barrios Departament Biologia Cellular, Fisiologia i Immunologia Facultat Biocie `ncies. Universitat Auto `noma de Barcelona 08193-Bellaterra, Barcelona (Spain) E-mail: carme.nogues@uab.cat Dr. J. A. Plaza, E. Ferna ´ndez-Rosas, R. Go ´mez, M. Duch, Prof. J. Esteve Instituto de Microelectro ´nica de Barcelona, IMB-CNM (CSIC) Campus UAB, 08193-Bellaterra, Barcelona (Spain) E-mail: joseantonio.plaza@ultra.cnm.es DOI: 10.1002/smll.200900733 Keywords:  barcodes  cell tracking  micromachining  optical microscopy  polysilicon small 2009, x, No. x, 1–7 ß 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Final page numbers not assigned