Cfi plan apo vc 60x wi

For live fluorescent imaging of the spindle, we used a spinning disk confocal microscope (Nikon TE2000, Yokugawa CSU-X1), equipped with 488 nm and 561 nm diode lasers, an EMCCD camera (Hamamatsu), and a 60X water-immersion objective (CFI Plan Apo VC 60X WI, NA 1.2, Nikon). We used a home-developed LabVIEW program (LabVIEW, National Instruments) to control the parameters of the imaging.

Images were captured by inverted epifluorescence microscope (NIKON TI-E) equipped with Photometrics CoolSNAP HQ2 CCD camera and an automatized stage for both chip (CFI Plan Apo VC 60XWI, NIKON) and glass slide (CFI Plan Fluor 40X). Image acquisition was performed by using NIKON software. Z-stack images were first treated with Fiji, using background subtraction, gamma and maximum intensity projection functions. Then, PLA signals were quantified using CellProfiler (2.2.0, rev ac0529e) in the following modules: Identify primary objects/identify objects manually for defining nuclei using DAPI staining; identify secondary object for defining cytoplasm using cytokeratin (CK) staining or DAPI staining with N-distance method; enhance speckles features to enhance PLA signals and then identify primary objects for defining PLA signals. For each cell, the presence of cytokeratin staining was determined using the thresholding based on fluorescence signal intensity on the background. Object enhancing and filtering size were adjusted depending on the objective used for control experiments on chip and on glass slides, which also varied between patient samples.