We demonstrate two-photon FLIM (2p-FLIM) with a femtosecond fibre laser. Our system consists of a bh DCS-120 MP scanner attached to a Nikon TE microscope, a 785 nm Toptica Femto Fibre Pro laser, HPM-100 hybrid detectors in a non-descanned beam configuration, and a TCSPC FLIM system with two SPC-150NX TCSPC modules, a GVD-120 scan controller, and a DCC-100 detector controller.
We show that the system is perfectly suited to record NADH FLIM data from cells and tissue. It provides high-quality decay data suitable for double-exponential decay analysis. The system is thus capable of recording the metabolic state of cells and tissues via analysis of the amplitudes of the bound and unbound NADH components. It also records high-resolution autofluorescence images from small organisms. FLIM images are also obtained from stained specimens, such as the Invitrogen BPAE cell and mouse kidney samples. The available excitation power is absolutely sufficient for all these applications. All FLIM data were recorded with no more than 6 mW in the sample plane. Higher power was not only unnecessary, but even caused destructive effects in the samples.
For more information, please see application notes, ‘Two-Photon FLIM with a Femtosecond Fibre Laser‘.