Nd72z2laq pifoc objective scanning system

A mode locked Ti:Sapphire laser (Chameleon, 120 fs pulse width, 90 MHz repetition rate, Coherent, CA) was coupled into a custom-made scanning system based on a pair of galvanometric mirrors (VM500+, Cambridge Technologies, MA). The laser was focused onto the specimen by a water immersion 20x objective lens (XLUM 20, NA 0.95, WD 2 mm, Olympus, Japan) for uncleared (PBS) sample imaging or a tunable 20x objective lens (Scale LD SC Plan-Apochromat, NA 1, WD 5.6mm, Zeiss, Germany) for cleared (47% TDE/PBS) sample imaging. The system was equipped with a motorized xy stage (MPC-200, Shutter Instrumente, CA) for axial displacement of the sample and with a closed-loop piezoelectric stage (ND72Z2LAQ PIFOC objective scanning system, 2 mm travel range, Physik Instrumente, Germany) for the displacement of the objective along the z axis. The fluorescence signals were collected by two photomultiplier modules (H7422, Hamamatsu Photonics, NJ). The instrument was controlled by custom software, written in LabView (National Instruments, TX).

Fluorescence images were obtained using a commercial confocal microscope (Nikon Eclipse TE300 C2 LSCM, Nikon, Japan) equipped with a Nikon 60 × or 100 × immersion oil objective (Apo Plan, NA 1.4), and a custom-made two-photon fluorescence microscope (TPFM). Briefly, a mode-locked Ti: Sapphire laser (Chameleon, 120 fs pulse width, 90 MHz repetition rate, Coherent, CA) operating at 800 nm was coupled into a custom-made scanning system based on a pair of galvanometric mirrors (LSKGG4/M, Thorlabs, USA). The laser was focused onto the specimen by a refractive index tunable 25 × objective lens (LD LCI Plan-Apochromat 25X/0.8 Imm Corr DIC M27, Zeiss, Germany). The field of view was 450 × 450 μm², the resolution employed was 0.44 × 0.44 μm² or 1.75 × 1.75 μm² for high- and low-resolution reconstruction, respectively. The system was equipped with a closed-loop XY stage (U-780 PILine XY Stage System, Physik Instrumente, Germany) for the radial displacement of the sample and with a closed-loop piezoelectric stage (ND72Z2LAQ PIFOC objective scanning system, 2 mm travel range, Physik Instrumente, Germany) for the axial displacement of the objective. The fluorescence signal was collected by an independent GaAsP photomultiplier module (H7422, Hamamatsu Photonics, NJ). Emission filters of 482/35 nm and 618/50 nm were used for fibers and cell body detection, respectively.