We demonstrate multiphoton NDD FLIM of tissue samples stained with near-infrared dyes. For the experiments we used a Zeiss LSM 7MP multiphoton microscope with a Coherent Chameleon OPO (optical parametric oscillator) as an excitation source. The excitation wavelengths range from 1000 nm to 1300 nm. The fluorescence was detected by an HPM‑100-50 NIR hybrid detector attached to the NDD (non-descanned detection) port of the microscope; the FLIM data were recorded by a standard bh TCSPC FLIM system. We demonstrate the performance of the system for tissue samples stained with Methylene Blue, Indocyanin Green (ICG), and 3,3’-Diethylthiatricarbocyanine (DTTCC). All three dyes could be efficiently excited at wavelengths from 1200 nm to 1300 nm. The dyes showed remarkable variability in their fluorescence lifetimes. The lifetimes clearly depended on the tissue structures the dyes were located in. For more details please see application note ‘Multiphoton NDD FLIM at NIR Detection Wavelengths with the Zeiss LSM 7MP and OPO Excitation‘.
Pig skin stained with 3,3’-Diethylthiatricarbocyanine. bh Simple-Tau 152 TCSPC FLIM system. Left: Lifetime image, amplitude-weighted lifetime of double-exponential decay model, 512 x 512 pixels, 256 time channels. Right: Decay curves in selected spots of the image.