Comparative study of two-photon fluorescent bio-markers at nanosecond and femtosecond pulsed excitation

In this study we investigate visible fluorescence of cytotoxic bio-markers (molecular probes) based on the derivatives of piperidone at femtosecond infrared pulsed laser excitation. The subject of this investigation is the origin of the fluorescence. Does it originate from the excited state absorption, which occurs only at slow, nanosecond excitation, or is it due to intrinsic multi-photon absorption? In the past, it has been shown indirectly, through the laser photolysis study, that the contribution of the excited state absorption is minimal for several compounds of such type. The results of direct experiments with an infrared femtosecond fiber laser as an excitation source described here support this hypothesis. The observed dependence of the fluorescence on the pump power indicated the contribution of not only two-photon, but multi-photon routes of excitation. Additionally, it was shown that the spectral features of the fluorescence correlate with the presence of glycine, an amino acid that is one of the building blocks of proteins in a cell. The implication of this result is, in addition to their anticancer action, the compounds can possibly be used for fluorescent diagnostics of cancer and multi-photon fluorescent microscopy of malignant cell cultures using portable infrared fiber lasers as excitation sources.