Iq software

Imaging was done using an Olympus IX-81 inverted microscope as has been described previously [24] (link). Fluorescence images were acquired at 10 ms integration time using a 100 W Hg arc lamp, a 470/40 nm bandpass excitation filter at 150X magnification with a 1.45 NA objective (Olympus) focusing on apical membranes of the lamella of the cells. Detection of all colors was done simultaneously through a QuadView emission splitter (dichroic mirrors at 585, 630, and 690 nm, and emission bandpass filters 535/30, 605/20, 655/20, and empty position) and an Andor EMCCD camera at 25 Hz. The camera has a pixel size of 16 µm, such that the projected pixel size in our case was 107 nm. The spectral overlap of the QD605, QD655, and QD705s among the image channels is such that less than 5% of the QDs are detected in the wrong image channel [24] (link). Image acquisition was controlled by Andor IQ software and movies of 1200 frames (∼48 s) were recorded at RT. The signal-to-noise in the image channel of the YFP-KRas2 fusion protein under the chosen imaging conditions (10 ms camera integration, 25 Hz imaging rate) is very low on an image frame by frame basis. Hence we have so far used these images only to provide a detailed image of the footprint of the plasma membrane of each cell for the duration of the time lapse sequence by generation of a Sum Intensity Projection image in ImageJ.

Loading of cells with Oregon Green BAPTA 1 (Invitrogen) and time-lapse fluorescence imaging was done as described previously (Nash et al., 2010 ). All experiments were performed at 25 ± 0.5°C in a continuous flow of medium (sEBSS). Images were captured at 0.1 Hz using a ×40 oil immersion objective and a Q Imaging Rolera-XR cooled CCD camera or Andor Ixon 897 EMCCD camera controlled by iQ software (Andor Technology, Belfast, UK; Nash et al., 2010 ).
KIKKK and scrambled KIKKK were applied by addition to the perfusion header at 5 µM, a concentration that provides optimal loading of mammalian spermatozoa with CPP within minutes without compromising membrane integrity/cell viability and distinguishes clearly between peptides with high and low translocation efficiency (Jones et al., 2013 (link)).

Images following immunocytochemical staining as well as during FM uptake experiments were acquired on a spinning disc confocal microscope (Zeiss Axio Observer.Z1 with Andor spinning disc unit and cobolt, omricron, i-beam lasers (405, 490, 562 and 642 nm wavelength)) using a 63 × 1.4 NA Plan-Apochromat oil objective and an iXon ultra (Andor, Belfast, UK) camera controlled by iQ software (Andor, Belfast, UK). For live cell imaging, neurons (DIV14–16) growing on 22 × 22 mm coverslips were mounted in a custom-built chamber designed for perfusion, heated to 37°C by forced-air blower and perfused with Tyrode’s saline solution (25 mM HEPES, 119 mM NaCl, 2.5 mM KCl, 30 mM glucose, 2 mM CaCl₂, 2 mM MgCl₂, pH 7.4).
Images from FM unloading experiments were acquired on a Olympus IX83 microscope (Olympus, Hamburg, Germany) equipped with a 60 × 1.2 NA UPlanSApo water objective, a CoolLED system (405 nm, 470 nm, 555 nm, 640 nm wavelength) (Acal^bfi, Göbenzell, Germany) and a Andor Zyla camera (Andor, Belfast, UK).

Recordings were made as described previously (Nash et al., 2010 ) but using Fluo-4. All experiments were performed at 25 ± 0.5°C in a continuous flow of medium. Images were captured at 0.2 Hz using a 40× oil objective and Andor Ixon 897EMCCD camera controlled by iQ software (Andor Technology, Belfast, UK). Fluorescence from the sperm posterior head/neck was background-corrected and normalized to give % change in intensity (Nash et al., 2010 ).
To assess [Ca²⁺]_i oscillations, paired experiments were conducted using cells from the same sample exposed to hFF or P4. Traces were examined by eye for the occurrence of cyclical [Ca²⁺]_i oscillations following the initial [Ca²⁺]_i transient.

Amniotic cells, glass-bottom 35 mm u-dishes (Ibidi GmbH, Germany) coated with fibronectin, were co-transfected with H2B-GFP and pmRFP-tubulin expression plasmids (Addgene, MA, USA) using Lipofectamine 3000 transfection reagent and according to the manufacturer's instructions. Live cell imaging was performed 48 hr following transfection under a spinning-disk confocal system Andor Revolution XD (Andor Technology, UK) coupled to an Olympus IX81 inverted microscope (Olympus, UK) equipped with an electron-multiplying CCD iXonEM Camera and a Yokogawa CSU-22 unit based on an Olympus IX81 inverted microscope. Two laser lines at 488 and 561 nm were used for the excitation of GFP and pmRFP and the system was driven by IQ software (Andor Technology, UK). Z-stacks (0.8–1.0 μm) covering the entire volume of the mitotic cells were collected every 1.5 min with a PLANAPO 60×/1.4 NA objective. ImageJ was used to process all the videos.

Live analysis of mitosis was done in S2 cell lines expressing the indicated constructs. 4D datasets were collected at 25°C with a spinning disc confocal system (Revolution; Andor) equipped with an electron multiplying charge-coupled device camera (iXonEM+; Andor) and a CSU-22 unit (Yokogawa) based on an inverted microscope (IX81; Olympus). Two laser lines (488 and 561 nm) were used for near-simultaneous excitation of EGFP and mCherry. The system was driven by iQ software (Andor). Time-lapse imaging of z stacks with 0.8 μm steps covering the entire volume of the cell were collected and image sequence analysis and video assembly done with ImageJ and iQ software. For quantification of EGFP-Mad1 fluorescence at kinetochores, the mean intensity was calculated within the area corresponding to kinetochores and corrected for cytosolic signal. The changes in fluorescence intensity with time were plotted as normalized signal relative to the signal measured at NEB.