Super-Resolution Microscopy Imaging Protocol

Cells were imaged using an Elyra PS.1 AxioObserver Z1 motorized inverted microscope with a EMCCD camera and Plan-Apochromat 63×/1.4 Oil DIC (differential interference contrast) M27 Elyra objective (Zeiss) with solid-state lasers of 488 nm and 561 nm as light sources. ZEN lite software was used for both acquisition of z stacks (5 phases and 3 rotations grating) and reconstruction. Quality of raw and reconstructed data was determined using the ImageJ plugin SIM check (Ball et al., 2015 (link)). Cells were prepared as for confocal imaging.

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Sposini S., Jean-Alphonse F.G., Ayoub M.A., Oqua A., West C., Lavery S., Brosens J.J., Reiter E, & Hanyaloglu A.C. (2017). Integration of GPCR Signaling and Sorting from Very Early Endosomes via Opposing APPL1 Mechanisms. Cell Reports, 21(10), 2855-2867.

Publication 2017

PS.1 AxioObserver Z1 Plan-Apochromat 63×/1.4 Oil DIC

Cells Light Microscope Reconstruction

Corresponding Organization :

Other organizations : Imperial College London, University of Pittsburgh, Physiologie de la Reproduction et des Comportements, Hammersmith Hospital, University of Warwick, Coventry University

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Variable analysis

independent variables

Imaging using Elyra PS.1 AxioObserver Z1 motorized inverted microscope
Solid-state lasers of 488 nm and 561 nm as light sources

dependent variables

Quality of raw and reconstructed data

control variables

EMCCD camera
Plan-Apochromat 63×/1.4 Oil DIC (differential interference contrast) M27 Elyra objective (Zeiss)
ZEN lite software for acquisition of z stacks (5 phases and 3 rotations grating) and reconstruction
Cells prepared as for confocal imaging

controls

No positive or negative controls explicitly mentioned

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