Ld lci plan apochromat 25x 0.8 imm corr dic m27

In all experiments, 8-10 somite stage zebrafish embryos were mounted in 1% low-melting point agarose in a glass bottom petri dish (MatTek Corporation) for a dorsal view of the tailbud and imaged at 25 ${}^{\circ }$ C using an inverted Zeiss Laser Scanning Confocal (LSM 710, Carl Zeiss Inc.). Confocal stacks through the tailbud were acquired with a step size of 2 $\mathrm{\mu }\text{m}$ and time interval of 2 minutes for 2 hours, using a 25x water immersion objective (LD LCI Plan-Apochromat 25x/0.8 Imm Corr DIC M27, Carl Zeiss Inc.).

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.

Cleared tissue samples were incubated in RIMS solution for one day. The samples were then mounted in the respective solutions using 7.0 mm or 3.0 mm spacers (iSpacer, SunJin Lab) with cover glasses. The mounted samples were stored at room temperature and shielded from light prior to imaging. Fluorescent images and initial sample visualization were performed on a conventional confocal microscope (Zeiss LSM 880) with either a Plan-Apochromat 5X/0.45 M27 objective (working distance 6.0 mm), 10 × 0.45 N.A. Plan-Apochromat objective, LD LCI Plan-Apochromat 25X/0.8 Imm Corr DIC M27 multi-immersion objective (working distance 0.57 mm) and 63x/1.3 glycerol DIC M27 (working distance 0.30 mm). After imaging, samples were embedded in RIMS at room temperature and protected from light for storage.