Bx50wi upright microscope

Optical recording with biosensor was performed as previously described (Polito et al., 2013 (link)). Wide-field images were obtained with Olympus BX50WI upright microscope with a 20× 0.5 NA or a 40× 0.8 NA water-immersion objective and an ORCA-AG camera (Hamamatsu). Images were acquired with iVision (Biovision). The excitation and dichroic filters were D436/20 and 455dcxt. Signals were acquired by alternating the emission filters, HQ480/40 for CFP and D535/40 for YFP, with a filter wheel (Sutter Instruments). Image acquisition was triggered manually, except for kinetics measurement where images were acquired automatically within 3–5 s intervals. Pseudocolor images display the ratio value coded in hue and the fluorescence intensity coded in intensity. A calibration square indicates the intensity values from left to right and the ratio values from bottom to top. The size of the square indicates the scale of the image in microns.

Kinetic of annexin fusion-tagged protein translocation to plasmalemma was monitor in live cells or fixed trophoblasts using a laser scanning confocal microscope (SP5; Leica) equipped with a 63x N.A. 1.2 water-immersion objective in the line-scan mode. Trophoblasts were incubated with ionomycin (10 μM). For live cells, images were acquired before incubation and automatically within 20 s intervals for 10 min after incubation.
Optical recording of intracellular Ca²⁺ transient was performed by loaded trophoblasts with the membrane-permeant Fluo-4 AM as recommended by the manufacturer protocol. Wide-field images were obtained with Olympus BX50WI upright microscope with a 40 × 0.8 NA water-immersion objective and an ORCA-AG camera (Hamamatsu)50 (link). Images were acquired automatically within 60 s intervals. Pseudo-color images display the intracellular Ca²⁺ value coded in hue and the fluorescence intensity coded in intensity.