Plan apochromat 63 1.40 oil dic m27 objective

For time-lapse imaging of ovulation and observations of live or fixed animals, partially synchronized populations were obtained by egg prep and animals were grown at 23°C for ∼54 h, around the time of the first ovulation. Live animals were immobilized with 0.01% tetramisole and 0.1% tricaine in M9 buffer (Kirby et al., 1990 ; McCarter et al., 1997 ) and mounted on 2% agarose pads or with 0.05 μm Polybead microspheres (Polysciences) diluted 1:2 in water and mounted on 5% agarose pads (Wang and Audhya, 2014 (link)). Confocal microscopy was performed on an LSM 710 confocal microscope (Zeiss) equipped with Zen software (Zeiss) using a Plan-Apochromat 63×/1.40 oil DIC M27 objective. A 488-nm laser was used for GFP and DyLight 488, and a 561 nm laser was used for mKate2 and TexasRed. For movies, 40 or 20 z-slices were acquired at 14- or 10-s intervals for imaging actin labeled with GFP and MLCK-1 labeled with mKate2, respectively. Illumination of the spermathecae from animals expressing actin labeled with GFP (GFP::ACT-1) with the 488-nm laser for ∼5 min prior to oocyte entry frequently caused the valve to remain partially closed during ovulation, increasing oocyte dwell time. Live animals were imaged for ∼ 30 min total. For still images of live and fixed animals and tissue, z-slices were acquired at 0.38-μm intervals, with each slice representing the average of two or four scans.

Cultured cells and brain tissue slices were imaged with a fluorescence confocal microscope LSM 780 (Zeiss, Jena, Germany) using a plan apochromatic oil‐immersion objective 40×/numerical aperture 1.3 or 63×/numerical aperture 1.4 (Zeiss). Confocal images were obtained with a 488 nm argon laser (BODIPY^493/503), 561 nm diode‐pumped solid‐state laser (Alexa Fluor⁵⁴⁶, Nile Red), and 405 nm (DAPI) diode laser excitation. The fluorescence emissions were filtered using 500–550 nm, 565–630 nm, and 413–463 nm band‐pass filters.
Images of Drosophila brains were recorded with an inverted Zeiss LSM800 confocal microscope with a Plan‐Apochromat 63×/1.40 Oil DIC M27 objective (Zeiss) using 488 nm and 561 nm diode laser excitation. Emission spectra were acquired sequentially with 410–514 nm (EGFP or BODIPY^493/503) and 564–700 nm (Nile Red) bandpass emission filters. For LD analysis, Z‐stacks with 10 μm interval and 50 μm pinhole were recorded for each brain hemisphere.
Live‐cell imaging was performed on an LSM 510 META laser scanning microscope (Zeiss) equipped with a Plan‐Neofluar 63×/1.4 oil DIC immersion objective (Zeiss). Confocal images were obtained with a 488 nm Ar‐ion laser (LysoTracker, MitoTracker Green) and a 543 nm He‐Ne laser (Nile Red), and the fluorescence emissions were collected using 505–530 nm band‐pass and 560 nm long‐pass emission filters, respectively.