Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low- and high-molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one- or two-colour lasers avoids the need for ultraviolet-transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E-C1 or E-C2) for versatile use with Ti-sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.Lay Description
Cells contain proteins and compounds that may be visualized using ultra-violet (UV) excitation. Generation of fluorescence from these chromophores requires excitation in the UV. However most commercial microscopes do not have UV transmitting optics for high resolution imaging. 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. 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 visible confocal technique. The advantages include the use of near infra-red light leading to reduced phototoxicity and improved tissue transparency. 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.Lay Description
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 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.