Nis elements 4.60 lambda software

Yeast strains were grown as detailed above and concentrated by centrifuging for 30 s at 3,800 x g at RT, and 1.6 μL of cells were placed on a glass slide and covered with a #1.5 coverslip. Images were acquired with an Axioskop 2 microscope equipped with a 100x/1.4 Plan-Apochromat objective (Zeiss) and an Orca-ER cooled CCD camera (Hamamatsu) and a pE-4000 LED illumination system (coolLED) controlled by NIS Elements 4.60 Lambda software (Nikon). GFP and mCherry were excited using a 470 nm LED with a ET470/40x filter, and a 561 nm LED with a ET572/35x filter respectively (Chroma). Emission was collected through a dual eGFP/mCherry cube (Chroma, 59,222). GFP and mCherry images were deconvolved using a constrained iterative restoration algorithm assuming 507 nm and 610 nm excitation wavelength, respectively, with 100% confidence limit and 60 iterations using Volocity 6.3.

Fluorescence microscopy was performed with one of the following imaging systems: (1) a Zeiss Axioskop 2 Plus upright fluorescence microscope with a 100×/1.4 Numerical Aperture (NA) Zeiss Plan-Apochromat objective lens (Carl Zeiss Inc., Thornwood, NY), Light-Emitting Diode (LED) module (CoolLED pE-4000, Andover, UK) and an Orca ER cooled charge-coupled device (CCD) camera (Hamamatsu Photonics, Hamamatsu City, Japan), and (2) a Zeiss AxioObserver.Z1 inverted fluorescence microscope with a 100x/1.3 oil EC Plan-Neofluar objective lens, a metal-halide lamp and an LED Colibri system (Carl Zeiss Inc., Thornwood, NY), and Orca ER cooled CCD. The first system was controlled by NIS Elements 4.60 Lambda software (Nikon, Melville, NY), and the second system was controlled by Zen Blue (Carl Zeiss Inc., Thornwood, NY). Details about the imaging conditions are given within each experimental section below.