The Becker & Hickl FLIM systems use a combined FLIM / PLIM technique to simultaneously record images of the oxygen concentration and of the NAD(P)H unbound/bound ratio in cells and tissues. The oxygen concentration is derived from the luminescence lifetime of a phosphorescent dye, the unbound/bound ratio from the amplitudes of the double-exponential fluorescence decay of NAD(P)H. The FLIM/PLIM technique is based on scanning with a high-frequency pulsed laser that is on/off modulated at a period in the microsecond range synchronously with the pixels of the scan. The signals are recorded by bh’s multi-dimensional TCSPC process. FLIM is obtained by building up a photon distribution over the times of the photons in the laser pulse period and the scan coordinates, PLIM by building up the distribution over the times of the photons in the laser modulation period and the scan coordinates. The technique records FLIM and PLIM simultaneously, avoids a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using high pulse energy for phosphorescence excitation. Compared with techniques which use only one laser pulse to for every phosphorescence excitation cycle it reaches a far higher PLIM sensitivity, avoids pile-up problems for the FLIM recording, and is perfectly compatible with two-photon excitation.
FLIM and PLIM image of SCC-4 cells stained with (2,2’-bipyridyl) dichlororuthenium (II) hexahydrate. FLIM shown left, PLIM shown right. Zeiss LSM 780 NLO with, bh Simple-Tau 152 FLIM/PLIM system, 2-photon excitation at 750 nm. Data analysis by bh SPCImage.
For more information please see application note ‘Simultaneous phosphorescence and fluorescence lifetime imaging by multi-dimensional TCSPC and multi-pulse excitation‘.
S. Kalinina, V. Shcheslavskiy, W. Becker, J. Breymayer, P. Schäfer, A. Rück, Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping. J. Biophotonics (2016)