Lsm 780 scanning unit

Fluorescence lifetime images were acquired using an inverted Zeiss (Axio Observer.Z1) LSM 780 microscope equipped with a 32 channel QUASAR GaAsP spectral array detector. In this setup the emitted photons were routed through the direct coupling (DC) confocal port of the Zeiss LSM 780 scanning unit and detected by a Becker & Hickl HPM‐100‐40 hybrid photomultiplier tube (PMT). The data were recorded by a Becker & Hickl Simple‐Tau 152 system (SPC‐150 TCSPC FLIM module) with the instrument recording software SPCM version 9.42 in the FIFO image mode using 256 time channels (Becker & Hickl GmbH, Berlin, Germany). For all acquisitions a Plan‐Apochromat 40×/1.3 Oil DIC objective lens was used, and the pinhole was set to 20.2 μm. For excitation at 405 nm a Laser diode 405 nm CW/PS with a repetition rate of 50 were utilized, whereas a pulsed tunable In Tune laser with a repetition rate of 40 MHz were used for excitation at 565 nm. Data was analyzed in SPCImage version 3.9.4 (Becker & Hickl GmbH, Berlin, Germany). Typically, decays fitted to a bi‐exponential decay and the associated life‐times and weights were used to calculate an intensity averaged life‐time for plots and comparison.^[27]

Spectral images of stained tissue sections were acquired on an inverted Zeiss (Axio Observer.Z1) LSM 780 microscope equipped with a 32 channel QUASAR GaAsP spectral array detector. For all imaging a Plan-Apochromat 40 × /1.3 Oil DIC objective lens was used. Excitation was done by simultaneous excitation of three laser lines; an argon laser at 458 nm and 488 nm, and a DPSS (561-10) laser at 561 nm. Emission spectra were collected between 416 and 687 nm. Fluorescence lifetime images were acquired using the same microscope system as for the spectral imaging of stained tissue sections. In this setup the emitted photons were routed through the Direct coupling (DC) confocal port of the Zeiss LSM 780 scanning unit and detected by a Becker & Hickl HPM-100-40 hybrid photomultiplier tube (PMT). The data were recorded by a Becker & Hickl Simple-Tau 152 system (SPC-150 TCSPC FLIM module) with the instrument recording software SPCM version 9.42 in the FIFO image mode using 256 time channels (Becker & Hickl GmbH, Berlin, Germany). For all acquisitions a Plan-Apochromat 40 × /1.3 Oil DIC objective lens was used, and the pinhole was set to 20.2 μm. For excitation at 490 nm and 565 nm the pulsed tunable In Tune laser with a repetition rate of 40 MHz were used. Data was analyzed in SPCImage version 3.9.4 (Becker & Hickl GmbH, Berlin, Germany).