Eclipse ti e inverted microscope

3D SIM was performed with a Nikon N-SIM based on an Eclipse TiE inverted microscope using an SR Apo-TIRF 100x/1.49 oil-immersion objective and an Andor iXon 3 EMCCD camera. Images were acquired in 3D-SIM mode using excitation at 405, 488, and 561 nm and the following emission bandpasses: 425-475 nm for DAPI; 500-550 nm for the green channel; 570-630 nm for the red channel. Image z-stacks were collected with a z interval of 125 nm. Focus stabilization (Perfect Focus System, Nikon) was used to maintain focus during image acquisition. SIM image reconstruction, channel alignment and 3D reconstruction were performed using NIS-Elements AR software.

Time-series images were generated by TIRF and widefield epifluorescence microscopy on a Nikon Eclipse Ti-E inverted microscope equipped with an Andor Zyla 4.2 PLUS sCMOS camera and viewed with an Apo TIRF 60×/1.49 NA oil objective. Excitation at 488 nm was sourced by a Nikon LU-NV laser unit. YFP-DAT and IDT307 emissions were collected with a 525 ± 18 nm emission filter. SavQD-605 and SavQD-655 signals were collected with 603 ± 15 nm and 655 ±15 nm emission filters, respectively. For SPT experiments, time series were produced at a 10 Hz frame rate. For clustering experiments, time series were produced at a 17 Hz frame rate.

Epifluorescence images of Sup35 yTRAP sensors were taken at 100× magnification (Plan Apo 100× oil objective, NA 1.4) using an Eclipse Ti-E inverted microscope (Nikon Instruments, Inc.). Images were acquired in DIC and in fluorescent (GFP) channels. Filters and light sources were automatically controlled by the supplier’s software (NIS-Elements Advanced Research).
For microscopy on induced / uninduced RBPs the following protocol was used: overnight cultures were diluted into CSM media at OD = 0.1 and grown for 2 hours at 30°C prior to induction with 100 nM estradiol. Induction was continued for at least 4 hours before imaging. Cells were imaged live within ten minutes of their preparation on glass slides. Epifluorescence images were taken at 100× magnification (Plan Apo Lambda 100× oil objective, NA 1.45) using an Eclipse Ti-E inverted microscope and a CCD camera (Andor technology). Bright field (BF) images were collected along with images in green and red fluorescence channels. Images were processed with ImageJ, and linear intensity transformations were set identically for induced and uninduced samples.

DFs or VSMCs were plated at subconfluent density. Once cells had reached 100% confluence, a P200 pipette tip was used to create the scratch in the bottom of the tissue culture well. The tissue culture plate was immediately placed on a Nikon Eclipse Ti-E inverted microscope equipped with a TID-NA stage adapter and incubation chamber and an Andor Zyla camera (VSC-05009) and subjected to automated live imaging every 4 hours for 24 hours. The cell-free area was measured using ImageJ software (NIH).

For fluorescent imaging, all strains were grown to saturation in synthetic complete media with 2% glucose, diluted to OD₆₀₀ = 0.2, and induced for 6 hours with estradiol. All images were acquired with a Nikon Plan Apo 100x oil objective (NA 1.4) using a Nikon Eclipse Ti-E inverted microscope and a CCD camera (Andor technology). The fluorescent reporter strains were identical to those used in for the high content microscopy. Specifically, the Kar2_SS-msfGFP-HDEL gene was driven by the TDH3 promoter and integrated in single copy at the TRP1 locus. Both Kar2 and Scj1 were C-terminally fused to mNG at their endogenous locus. Total cellular GFP fluorescence was quantified manually using ImageJ. The fraction of cells with mislocalized GFP (i.e. GFP in the cytoplasm instead of the ER) was determined visually. For the experiments demonstrating STE24 rescue of the IAPP-induced phenotypes, STE24 was inserted with a single-copy integrating plasmid at the URA3 locus under the control of the pZ promoter. Due to the additional estradiol-inducible promoter binding site introduced by integrating STE24 in this manner, for the low toxicity experiments the concentration of estradiol used was double that of the screening experiments. For each data point, the fluorescence intensity of at least 100 cells was quantified using ImageJ. Experiment were performed in biological triplicate.