Advanced microscopy requires excitation light with coherence, monochromaticity and control of pulse duration. The ideal laser source should be tuneable with continuity over a wide spectral range, capable of emitting short pulses and amenable to fibre coupling. All these requirements are met by supercontinuum lasers that can generate highly coherent picosecond pulses at wavelengths spanning from blue light to NIR and beyond.
A supercontinuum laser (SuperK EXTREME - NKT photonics, Denmark) was used to perform fluorescence lifetime imaging of human osteosarcoma cells labelled with a new probe (DCHQ5).
DCHQ5, in addition to quantify the total intracellular Mg concentration in cell populations, allows one to visualize the intracellular Mg distribution in single cells. Using an acousto-optic filter, a 10 nm spectral band, centred at 480 nm was sliced from the white light exiting the supercontinuum source. The excitation light, made by a 20 MHz train of few-picosecond-long pulses, was coupled to a 400 μm fibre using a wide aperture microscope objective in order to excite all modes. The flat top light source consisting in the distal end of the fibre was imaged onto the object plane of the microscope (Leica DM-RM) to provide uniform illumination over the whole field of view. This approach, which required a home-made optical system, implemented the epifluorescence scheme with a 63X immersion objective lens, used to deliver the excitation light and collect the fluorescence signal through a dichroic mirror @500 nm and a long pass filter @505 nm.
The fluorescence images were acquired with a fast picosecond camera (Picostar, LaVision Germany) made by a light intensifier coupled to a low noise CCD.
A sequence of images was collected at different delays (in the interval 0-60 ns) with respect to the laser pulses, from each microscope field.
Data analysis was performed using a custom written software based on the Matlab engine. The dataset, made by all the gated images, was processed using a non-linear algorithm for bi-exponential fit, operated in parallel mode. Amplitude and lifetime maps were recovered for both the fluorescent components that characterise the emission of the DCHQ5 probe. Additionally, to better estimate a possible variation of the decay dynamics of the probe in different cell comportments with alike magnesium concentration, two AOIs were isolated on the basis of the fluorescence intensity. All the pixels within the AOIs were binned to a single decay curve, which was fitted with a non-linear algorithm based on the NAG mathematical package.
The fluorescence lifetime maps show a uniform pattern over the whole cell, for both the short and long living components of the fluorescence, while the amplitude maps reveal a minimum in correspondence of the nucleus and a high value in the cytoplasm. This result is confirmed by the analysis of the AOIs. Therefore, the intracellular distribution of the fluorescence intensity depends on amplitude variations of both DCHQ5 fluorescent components, and not on lifetime variations.
Thus, fluorescence intensity maps reflect the concentration of the complex DCHQ5-Mg, and can be effectively used to quantify intracellular Mg2+.