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Multi-photon properties of fluorescent beads excited at the 1 700 nm window
Summary
This study examines the multi-photon fluorescence behavior of polystyrene beads when excited at the third harmonic wavelength. The research characterizes how these commonly used laboratory microspheres respond to multi-photon excitation, which has implications for using fluorescent beads as calibration standards in advanced microscopy techniques.
Fluorescent beads have wide applications in fields of biomedicine and biotechnology. Characterisation and evaluation of the multi-photon properties of fluorescent beads is essential for ensuring their effectiveness and reliability in multi-photon imaging applications. However, at the 1 700 nm excitation window which is well-suited for deep biological tissue imaging, such characterization techniques remain limited, hindering practical selection and application of fluorescent beads. In this study, a custom-built multi-photon imaging system operating at the 1 700 nm is utilized to characterize the multi-photon properties of two types of commercially available fluorescent beads: Fluosphere and polystyrene quantum dot fluorescent beads. Using multi-photon imaging, one can determine the multi-photon excitation order and relative multi-photon action cross-section of fluorescent beads, compare the multi-photon signal level, and investigate the photobleaching properties of the fluorescent beads to evaluate their photostability. Meanwhile, one can further determine the spatial resolution of the imaging system through multi-photon imaging. The results show that the relative action cross section and stability of Fluosphere are superior to those of quantum dot fluorescent beads at the 1 700 nm window. Moreover, our imaging system has a sub-micron spatial resolution. These findings provide a reference for selecting fluorescent beads in multi-photon imaging and characterizing the spatial resolution of imaging systems.