Eclipse ti e inverted microscope

Monocyte-derived macrophages were first stained with 2.5 µg/ml of Vybrant DiO Cell-Labeling Solution (Thermo Scientific) diluted in PBS. The cells were then washed three times with PBS and incubated with eFluor 670 spermatozoa in the absence or presence of SEM fibrils using conditions described above. After 3 hr, macrophages were washed 3x with PBS, trypsinized, and stored in 1% PFA at 4°C in the dark until imaging. Confocal imaging was carried out using a Nikon Eclipse Ti-E inverted microscope equipped with a Yokogawa CSU22 spinning disk confocal scanner, a Sutter emission Lambda filter wheel adapter with ET460/50m, ET525/50m, ET645/65m and ET700/75m filters, a Prior motorized stage with Piezo Z-drive, and a Photometrics Evolve 512 Delta EMCCD Camera. Images were acquired with Micro-Manager software version 1.4.21. Image scanning was executed employing a Plan Apo VC 100x/1.4 Oil (DIC N2/100X I) objective using 488 nm and 640 nm, 100 mW Coherent OBIS lasers. The Piezo Z-drive was set for fast acquisition and z-steps at 0.40 µm. Image analysis was performed using ImageJ software version 1.48 with the Deconvolution lab (EPFL) and 3D viewer (B. Schmid) plugins installed. All acquisition and analysis parameters were maintained constant for all samples.

Prior to imaging, 1 mL cell culture was concentrated to 50 µL by centrifugation at 1500 x g, 30 s. 2 µL cell suspension was loaded under a 22 × 22 mm glass coverslip (VWR, thickness: 1.5).
Spinning-disk confocal images were captured with Eclipse Ti-E inverted microscope fitted with Yokogawa CSU-X1 spinning disk confocal scanning unit, equipped with ILE 405 nm 100 mW, ILE 488 nm 50 mW and ILE OBIS 561 nm 50 mW laser lines, Nikon CFI Plan Apo Lambda 100× (N.A. = 1.45) oil objective and Andor iXon Ultra U3-888-BV monochrome EMCCD camera. Imaging was performed at 30 °C with acquisition of 13 z-slices, 0.58 µm per slice, controlled by either Andor iQ 3.6.5 (Oxford Instruments) or Andor Fusion software 2.3.0.36 (Oxford Instruments). Temperature control was achieved by the Okolab cage incubator set at 30 °C.
Epifluorescence brightfield images were acquired using Zeiss Axio Observer Z1 fluorescence microscope fitted with α Plan-FLUAR 100×/1.45 NA oil objective lens (Carl Zeiss) and the Orca-Flash4.0 C11440 camera (Hamamatsu). All images were taken at the medial focal plane, controlled by Zen 2012 software (blue edition, Carl Zeiss Microscopy GmbH).

To visualize the proliferation of alpha, cells were collected and resuspended in 300 μl LPB (containing 4 to 22% sucrose) and placed in the wells of a chambered 8-well μ-slide (ibidi). Cells were imaged on a Nikon Eclipse Ti-E inverted microscope equipped with a confocal spinning disk unit (CSU-X1) operated at 10,000 rpm (Yokogawa), using a 40× Plan Fluor lens (Nikon), and illuminated in bright field. Images were captured every 2 min for 10 to 15 h by an Andor iXon Ultra 897 high-speed electron microscope charge-coupled device (EM-CCD) camera (Andor Technology). Z-stacks were acquired at 0.2- to 0.5-μm intervals using an NI-DAQ-controlled Piezo element. During imaging, wall-less cells were kept at 30°C using an INUG2E-TIZ stage top incubator (Tokai Hit).