A series of flexible design adaptations to the Nikon E-C1 and E-C2 confocal microscope systems for UV, multiphoton and FLIM imaging

Multiphoton microscopy in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) is now a key technique in biomedical and life-science research for live cell and whole animal imaging providing several advantages over the one photon confocal technique. The advantages include the use of near infra-red light leading to reduced phototoxicity and improved tissue transparency. Furthermore, whilst fluorescence microscopy remains the most commonly used tool for biological imaging, it does pose a major disadvantage that for the majority of applications there is a need to add bulky labels to visualise the biomolecule of interest. This may lead to artefacts due to changes in molecular conformation and cytotoxicity from photodynamic effects although the technique may be improved and some of these drawbacks reduced or eliminated when using endogenous chromophores such as tryptophan, tyrosine and phenylalanine. However, generation of fluorescence from these chromophores requires excitation at ultraviolet wavelengths. Multiphoton excitation is a method providing the equivalent of UV excitation beneath UV absorbing surface materials permitting direct excitation of simple molecular systems that absorb below 350 nm. FLIM is a powerful technique that is fast becoming an indispensable imaging technique for biomedical and life-science research. FLIM may be performed using both one-photon as well as multiphoton excitation. The added benefit and advantage of FLIM over standard steady state fluorescence microscopy is that the measured lifetime of a chromophore is independent of concentration below ca. 1 mM and may be used as a reliable probe for distance-depended processes involving resonance energy transfer and also reports directly on the chemical environment of the chromophore. Although FLIM can be carried out on UV as well as visible absorbing and emitting chromophores, UV excitation and emission monitoring is rarely performed due to the lack of UV transmitting optics in almost all commercial confocal scanning systems and microscopes. Here we report a straightforward adaptation to a commercial Nikon confocal scanning system and microscope to allow simultaneous one-photon, steady state multiphoton imaging, FLIM and deep UV microscopy below 300 nm. We show that aromatic amino acid compounds and UV absorbing anticancer drugs can be detected and imaged in mammalian cells using these adaptations. The modifications also apply to FLIM studies and we show clear differentiation between UV absorbing and emitting chromophores added and those naturally present in live cells following multiphoton excitation with pulsed visible light. The wider spectral working region has significant potential for drug studies in cells where the main excitation and emission is in the UV region of the electro-magnetic spectrum