Plan apo 60x

The laser system (Fig. 1A) is similar to that previously used for local heat shock^{24 (link),25 (link)}, and is built around an inverted microscope (Nikon TE-2000). A thermoelectrically cooled, fiber-coupled diode laser (Fitel FOL1425RUZ-317, 1480 nm wavelength) generates a beam that emerges from the fiber end and is collimated by a lens. The beam reflects from a dichroic beam splitter, enters an objective lens (Nikon PLAN APO 60X, NA 1.4, oil immersion), and is focused onto a sample slide (Fig. 1B). A computer, pulse generator, and laser current source (Opto Power OPC-PS03-A) drive the laser to generate pulses of the specified frequency and duration.
We made several modifications to the previous heat shock system^{24 (link),25 (link)} to adapt it for laser ablation. First, we replaced the original collimating lens (focal length f = 75 mm) with one of smaller focal length (f = 11 mm) in order to create a smaller diameter beam, allowing a larger fraction of the beam power to transmit through the objective and reach the sample. Second, the laser power output was increased from 260 to 400 mW. Third, we adjusted the pulse duration and count as described above.

Experiments were performed in a 6-channel microfluidic flow-cell (LUMICKS B.V.) that was mounted on an automated XY-stage housed within a custom-made inverted microscope that combines dual-trap optical tweezers with wide-field fluorescence microscopy^{28 (link)}. In brief, two orthogonally polarized optical traps were generated using a 1064 nm fibre laser (YLR-10-LP, 10 W, IPG Photonics) via a water-immersion microscope objective (Plan Apo 60X, NA 1.2, Nikon). Biotinylated dsDNA (λ-DNA or pKYB1) was tethered between two streptavidin-coated optically trapped microspheres (Spherotech Inc) in situ within the flow-cell. Single TRR gate opening events (Fig. 2d) were recorded using pKYB1-ssDNA and 1.75 µm beads. For all other experiments, λ-ssDNA and 4.6 µm beads were used. All experiments were performed at room temperature. The optical tweezers were controlled using a custom-written programme written in LabVIEW 2011^{28 (link)}. Fluorescence from TRR-mCherry, BLM-SNAP649, and SYBR Gold intercalator dye was generated by excitation with a 561 nm (Cobolt Jive 25 mW CW), a 639 nm (Coherent Cube 50 mW CW) and a 491 nm (Cobolt Calypso 50 mW CW) laser, respectively. Fluorescence images were recorded using an EMCCD camera (iXon + 897E, Andor Technology). Fluorescence excitation lasers were controlled with an AOTF (Acousto-optical tunable filter, AA Opto Electronic).

Electroporated zygotes were culture overnight and observed using a confocal microscope (Nikon A1RSi, Minato-ku, Tokyo, Japan; oil-immersion objective Plan Apo 60X, NA = 1.4). A 543 nm laser was used to excite the rhodamine 123 fluorophore. Localization of labeled DNA was analyzed for multiple Z-stacks in each zygote. The images were recorded with NIS Element software (Version 4.2, Nikon, Tokyo, Japan).

Fluorescence imaging was conducted on the Nikon A1R® confocal imaging system equipped with a Nikon® oil immersion Plan‐APO 60x numerical aperture 1.40 lens. The Nikon® NIS Elements software was used for imaging control and projections. With acquisition settings kept constant, Z‐stacks of images at 0.2‐mm intervals were acquired, and maximum intensity projections were created.¹⁶, ¹⁷, ¹⁸, ¹⁹

Apoptosis was analysed using a Muse Cell Analyzer and Muse 1.3 Analysis software (Merck Millipore). Cell cycle distributions were analysed using a BD FACSCanto™ II analyser and FACSDiva 6.0 software (Becton Dickinson Biosciences). Fluorescence microscopy was performed using a Nikon TE300 inverted microscope using a Plan Apo 60x or 100x DIC oil immersion objective (NA 1.4). Images were obtained using an ORCA-R² camera (Hamamatsu) using Velocity software, v6.0.1 (PerkinElmer), and images processed using Adobe Photoshop 7. Alternatively, microscopy was performed on a Leica TCS SP5 laser scanning confocal microscope equipped with a Leica DMI 6000B inverted microscope. Images were captured and processed using Leica LAS AF software.

The images of cells and chromosomes were acquired using the Nikon Plan Apo 60x or 100×/1.4 oil objective lens on a TE2000-U microscope (Nikon) with a Retiga SRV CCD camera (QImaging) operated with Meta Morph imaging software (MetaMorph Inc.) at room temperature. In addition, the confocal images of cells were documented with Leica TCS SPE Laser Scanning Confocal DM6-Q microscope using the ACS Apo 63x/Oil objective lens operated by Leica LAS X Imaging software.