Multiphoton Microscopy (MPM) has enabled an unprecedented level of dynamic exploration within living cells and organisms. The physical principle behind this imaging technique involves the use of near-infrared femtosecond lasers to excite optical processes in fluorescent molecules using two or more photons¹. The use of long wavelengths (700 nm – 1000 nm) enables deep tissue penetration (up to 1000 μm) without inducing harmful biological effects. Previous studies include the examination of membrane potentials on the single-molecule scale², the non-invasive observation of embryo development³, and the simultaneous multiplane imaging of calcium transportation in transgenic mice⁴. An excellent review of this field can be found in the literature⁵.

            Herein we demonstrate a novel multiphoton-based technique for imaging non-fluorescent carbon-based nanomaterials such as C₆₀ fullerenes, single-walled carbon nanotubes, and graphene. More specifically, we demonstrate optical and photoluminescent activity in water-soluble C₆₀-serinol, highly enriched (95 %) metallic and semiconducting single-walled carbon nanotubes, and functionalized graphene nanoribbons. This multiphoton imaging technique was also applied to in vitro and in vivo animal model studies. Various concentrations of the above mentioned nanomaterials were given to human pancreatic ductal adenocarcinoma (Panc-1) and hepatocellular carcinoma (Heb3B) cancer cell lines, as well as normal, healthy pancreatic ductal epithelial cells (HPDE). In all cases, imaging of these nanomaterials was possible using the MP imaging technique even though the nanomaterials exhibit no inherent fluorescent properties. We also examined the ability to image these nanomaterials, real-time, in vivo, using mice with orthotopic 4T1 breast tumors.  Finally, we also include a range of spectroscopy data to examine the specific optical characteristics of the carbon nanomaterials.

References

1.         Denk, W.; Strickler, J.; Webb, W. Science 1990, 248, (4951), 73-76.

2.         Peleg, G.; Lewis, A.; Linial, M.; Loew, L. M. Proceedings of the National Academy of Sciences 1999, 96, (12), 6700-6704.

3.         Chu, S.-W.; Chen, S.-Y.; Tsai, T.-H.; Liu, T.-M.; Lin, C.-Y.; Tsai, H.-J.; Sun, C.-K. Opt. Express 2003, 11, (23), 3093-3099.

4.         Cheng, A.; Goncalves, J. T.; Golshani, P.; Arisaka, K.; Portera-Cailliau, C. Nat Meth 2011, 8, (2), 139-142.

5.         Hoover, E. E.; Squier, J. A. Nat Photon 2013, 7, (2), 93-101.