Two‐Photon Imaging of a Cellular Line Using Organic Fluorescent Nanoparticles Synthesized by Laser Ablation

Abstract

A comparative study of the optical properties of organic fl uorescent nanoparticles fabricated by laser ablation (NPs-LA), reprecipitation (NPs-RP), and microemulsion (NPs-ME) methods is presented. These nanoparticles contain a fl uorene-based p-conjugated molecule (BT2). Distinctive electronic transitions are observed in samples due to the specifi c way in which the molecule BT2 is assembled in each type of nanoparticles; for instance, transitions involved in absorption and emission spectra of NPs-LA result in blueshifting with respect to the molecular solution of BT2, whereas redshifting is observed in NPs-RP and NPs-ME. Further, the results show that under infrared excitation, the aqueous suspensions of NPs-LA exhibit the highest fl uorescence induced by two-photon absorption (≈790 GM at 740 nm), as well as the best photostability, compared with aqueous suspensions of NPs-RP and NPs-ME. The nanoparticles synthetized by the three aforementioned methods are employed as exogenous agents for the visualization of human cervical cancer cell line (HeLa) using confocal and two-photon microscopy. Under similar experimental conditions, it is found that microscopy images of the best quality are obtained with NPs-LA. These results show that laser ablation is a suitable technique for the fabrication of organic fl uorescent nanoparticles used as contrast agents for in vitro fl uorescence microscopy.O-NPs have been successfully exploited as contrast agents (fl uorescent markers) for in vitro and in vivo studies in cellular lines and organisms, [ 5 ] medical imaging, [ 6 ] diagnostic purposes, [ 7 ] and positive confi rmation of the site-specifi c delivery of a drug with the goal of obtaining effective treatment of diseases. [ 8,9 ] Some advantages obtained by using organic nanostructures are: i) stability of fl uorescent organic materials in aqueous suspensions, ii) reduced toxicity, iii) decreased degradation, and iv) selectivity toward specifi c targets.Simple and versatile strategies of fabrication have been used to obtain O-NPs with different morphologies and optical properties in aqueous media. Reprecipitation (RP), [ 10 ] self-assembly, [ 11 ] ion-association, [ 12 ] condensation, [ 13 ] microemulsion (ME), [ 14 ] and laser ablation (LA) [ 15 ] are some methods used to fabricate O-NPs. For the preparation of O-NPs comprising luminescent properties, the most commonly employed methods are RP and ME. [ 16,17 ] LA has also been used to obtain fl uorescent O-NPs, [18][19][20] and some basic characterization of their optical properties has been performed, but to the best of our knowledge, demonstration of their use in the context of biophotonic applications, i.e., imaging of biological samples, has