Lsm 710 axioobserver

Multichannel images were obtained using a ZEISS LSM 710 AxioObserver equipped with a ZEISS Plan Apochromat 63× oil DIC objective lens (NA 1.40). Images were acquired using 512 × 512 pixels, at 440 nm pixel sizes. Samples were illuminated with the following lasers (fiber launching): λ = 405 nm, λ = 488 nm, λ = 561, and λ = 633 nm, excitation filters MBS 488/561/633 and MBS 405. Fluorescence was collated using the corresponding diffraction grating and bandwidth slit settings for emission 416 to 485 nm (CH1), 494 to 554 nm (CH2), 572 to 632 nm (CH3), and 641 to 730 nm (CH4).
The following combinations of lasers (excitation) and bandwidth slit settings were used for our 10-channel recordings:
Validation images of the fluorescent protein constructs stained with primary and secondary antibodies (see S2 Fig and S2 Table) were obtained using an Olympus IX71 microscope equipped with an Olympus UPlanSApo 60× oil objective (1.35 NA). These validation images were only obtained to determine whether the proteins behaved as expected. Rab5a images of S2 Fig and all work relating to the deep neural network was performed on the ZEISS LSM 710 AxioObserver.

SKOVip-kat cells were seeded in 35 mm glass bottom Petri dishes, 10⁵ cells per dish, and grown overnight. Cells were then visualized using an inverted laser scanning confocal fluorescence microscope AxioObserver LSM 710 (Carl Zeiss, Oberkochen, Germany). The images were obtained with a C-Apochromat 63×/1.2 water immersion objective. Fluorescence of TurboFP635 protein was excited by the HeNe laser at 594 nm and collected in the range of 611–740 nm.