Multispectral FLIM

In practice, the only feasible solution is often to transfer the light to the poly-chromator slit plane by an optical fibre. The slit is removed, and the numerical aperture at the input of the fibre is reduced to match the numerical aperture of the polyehromator. Because only moderate wavelength resolution is required, a relatively thick fibre (up to 1 mm) can be used. Therefore a reasonably high coupling efficiency with a single fibre can be obtained, even for nondescanned detection systems. The fibre should be not longer than 50 cm to avoid broadening of the IRF by pulse dispersion. 

Another possibility is to use a fibre bundle. The input side of the bundle is made circular, the out side is flattened to match the polyehromator slit. The large area of the fibre bundle makes it relatively simple to collect the light from the nondescanned detection path of a scanning microscope. However, the aperture of the microscope objective lens must be correctly imaged onto the input of the bundle. Otherwise the illuminated spot scans over the bundle and causes the fibre structure to appear in the image. 

Multispectral TCSPC FLIM by using a polyehromator at the fibre output of a Zeiss LSM510 NLO was demonstrated in [35]. One-photon excitation with a frequency-doubled Ti Sapphire laser was used. The spectmm was detected by a 

16-channel detector head containing an R5900L16 PMT and the routing electronics. An SPC-730 TCSPC module recorded the photons into 16 wavelength and 64 time channels. The image size was 64 x 64 pixels. Multispectral FLIM images of an HEK (human embryonic kidney) cell transfected with CPF and YFP are shown in Fig. 5.81. 

The 64 time channels were binned into four consecutive 1.25-ns intervals, the 16 wavelength channels into eight consecutive 20-nm intervals. The fluorescence decay results in considerable intensity differences over the later time intervals. To display the images in the time windows, the intensities were normalised. The same normalisation factor was used for each row of images. The normalisation factor was calculated so as to display equal total intensities of the images in the 500-to-520-nm channel. 

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Fig. 5.81 Multispectral FLIM images of an HEK cell transfected with CFP and YFP. Images in successive time and wavelength windows. Normalised for constant total intensity in the time windows of the 500-to-520-nm channel...

A two-photon microscope with multispectral FLIM and nondescanned detection is described in [60]. An image of the back aperture of the microscope lens is projected into the input plane of a fibre. The fibre feeds the light into a polychro-mator. The spectrum is detected by a PML-16 multianode detector head, and the time-resolved images of the 16 spectral channels are recorded in an SPC-830 TCSPC module. Spectrally resolved lifetime images obtained by this instrument are shown in Fig. 5.82. 

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