Deltavision wide field fluorescent microscope

Deconvolution three-dimensional imaging was performed as described previously [19 (link)]. In brief, z-stack images were collected (from bottom to top, 20 sections of 0.5 µm) using identical acquisition parameters with a DeltaVision wide-field fluorescent microscope (Applied Precision, GE) equipped with a digital camera (CoolSNAP HQ; Photometrics), using a 1.4-numerical aperture 100× objective lens. Excitation light was generated using the Insight SSI solid-state illumination module (Applied Precision, GE), and images were deconvolved with the SoftWoRx deconvolution software (Applied Precision, GE). Following deconvolution, images were quantified by Imaris (Bitplane) software using the Surfaces feature function, generating surfaces around red puncta. Three-dimensional views of images were generated using the Surpass mode of Imaris software.

Z-stack images were collected with a DeltaVision wide field fluorescent microscope (Applied Precision, GE) equipped with a digital camera (CoolSNAP HQ; Photometrics), using a 1.4-numerical aperture 100× objective lens. Excitation light was generated with an Insight SSI solid state illumination module (Applied Precision, GE) and were deconvolved with SoftWoRx deconvolution software (Applied Precision, GE). In any experiment, identical acquisition conditions were used to acquire all images. Following deconvolution, images were analyzed by Imaris 7.6.4 (Bitplane). An algorithm was designed using the surface feature function in Imaris to generate surfaces around signal of interest and the maximum fluorescence intensity associated within these surfaces were measured. The algorithm was applied to all images within the same experiment. For live cell experiments, cells were plated in delta DPG dishes (Thermo Fisher Scientific). Cells were maintained at 5% CO₂ at 37°C in an environmental chamber on a DeltaVision microscope, and images were captured in a z series on an electron multiplied charge coupled device digital camera (EMCCDCascade 2; Photometrics) and deconvolved using SoftWoRx deconvolution software. Images were acquired every 15 seconds for 10 minutes.