Hcx pl ap 1.10 na water immersion objective lens

Live-cell movies were acquired on a Leica SP5 confocal microscope using a 40x HCX PL AP 1.10 NA water immersion objective lens (Leica Microsystems), or a Zeiss LSM880 AiryScan microscope using a C-Apochromat 40 × 1.20 NA water immersion objective lens. For live-cell imaging experiments, at least 3–5 oocytes were recorded per session.
Fixed oocytes were imaged on a Leica SP8 microscope equipped with the HC PL APO 1.40 NA 100x oil immersion objective according to Nyquist criteria. For STED imaging, suitable Abberior STAR 580 and Abberior STAR RED or Abberior STAR 635P secondary antibodies or nanobodies were used (Abberior, NanoTag). Samples were imaged on a Leica SP8 STED microscope, with the HC PL APO CS2 1.40 NA 100x oil immersion objective and using the 775 nm depletion laser. Alternatively, we used an Abberior Instruments STEDYCON scan head mounted onto a Nikon Ti2 microscope equipped with a 100x CFI Plan Apochromat Lambda NA 1.45 oil immersion objective lens, or with an Abberior Instruments Expert Line STED microscope using an Olympus 100x UPLSAPO 100XS NA 1.4 oil immersion objective. At least five oocytes were recorded per sample.
Live and fixed oocyte images were processed and deconvolved using the Huygens software (Scientific Volume Imaging) with either confocal, AiryScan or STED settings as appropriate.

Microscopy was done on a Leica SP5 or SP8 confocal microscope equipped with a fast Z-focusing device (SuperZ galvo stage) and using a 40x HCX PL AP 1.10 NA water immersion objective lens (Leica Microsystems). To record chromosome transport, starfish oocytes were imaged in 3D (a Z-step of 2 µm over 70 µm) over time (time step of 5 s) using a square frame of 256 × 256 pixels at a pixel size of 447 nm. To monitor F-actin network contraction, starfish oocytes were imaged in 3D centered on the median plane (plane of biggest surface) with a Z-step of 2 µm covering 10–20 µm over time (time step 10 s) using a square frame of 512 × 512 pixels at a pixel size of 223 nm. All imaging was performed at room temperature (20–22°C).
A pulsed two-photon laser (Chameleon, Coherent) was interfaced to a LSM780 confocal microscope (Zeiss) through a customized optical path aligned with the optical axis of the microscope. We sequentially imaged (a Z-step of 2 µm over 20 µm every 10 s) and performed 3D ablation (typical duration ~ 1 s) by imaging a stack of 1 × 50×60 µm, with a Z-step of 2 µm at 800 nm (30% laser power). Imaging was started ~ 1 min before the 3D ablation.