Flatbed scanner

Cells bearing LOV2GIVe constructs growing exponentially in SDGalactose media were pelleted as described above (‘Yeast Strains and Manipulations’), and resuspended at equal densities. Equal volumes of each strain were spotted on agar plates in four identical sets. Two of the sets were seeded on SDGalactose-TRP-URA plates with histidine and the other two sets were seeded on SDGalactose-TRP-URA-HIS (supplemented with 5 mM 3-amino-1,2,4-triazole). From each one of the two pairs of sets, one of the plates was exposed to a homemade array of blue LED strips positioned approximately 12 cm above the plates (~2,000 Lux as determined by a Trendbox Digital Light Meter HS1010A) whereas the other one was incubated side by side under the same light but tightly wrapped in aluminum foil. Plates were incubated simultaneously under these conditions for 4 days at 30°C and then imaged using an Epson flatbed scanner.

Pulse-chase labeling of log phase yeast with vacuolar fluorescent dye FM4-64 and fluorescence microscopy imaging were performed as described (Nickerson et al. 2012 (link)), except that cells were grown in nonselective YPD prior to labeling and imaging. DNA in agarose gels was stained using ethidium bromide and visualized using ultraviolet light in a BioRad Chemi-doc system with digital camera and Quantity One imaging software (BioRad, Hercules, CA, USA). All gel images were exported from Quantity One as .TIFF images, except Figure 2B, which was printed to photographic paper and later scanned in .TIFF format using an Epson flatbed scanner. Images were cropped using Adobe Photoshop CS6 (Adobe, San Jose, CA, USA). Fluorescence microscopy images were overlaid using ImageJ (NIH, https://imagej.nih.gov/ij/). Images were arranged as annotated figures using Canvas Draw 4 vector graphics software (Canvas GFX, Boston, MA, USA).

0.5 ml of cell suspension in 0.36% agarose containing culture media was plated into each well on the top of existing 0.6% bottom agarose in 24-well tissue culture plates. 0.5 ml of culture media containing compound or compound combinations at various doses was then loaded into each well. Plates are incubated in a 37°C, 5% CO₂ incubator for a period of 2 – 4 weeks to allow cell colonies to grow large enough to be visualized through MTT tetrazolium dye staining. At the assay endpoint, 0.04 ml of 5 mg/ml MTT solution (Sigma) were added into each well and further incubated at 37°C for 3 h. Cell colony images of each well were acquired using a flatbed scanner (Epson). The number of colonies formed in each well was then quantified using ImageJ software. Percent cell colony formation was calculated relative to DMSO control treated cells.

After full germination, root growth was captured by scanning plates with an Epson flatbed scanner and a resolution set to 600 ppi. Primary root length of individual seedlings was then measured using Fiji [99 (link)]. Average root elongation rate (mm/d) was calculated as an average of daily root elongation rates following the protocol described in [100 ].
Primary root apical meristems were analyzed to measure meristematic cortical cell length (μm), meristem size (μm), and meristem cell number by performing a propidium iodide (PI) stain following the protocol described in [101 (link)]. Root tips were examined using a Leica SP2 confocal microscope with a 40× oil immersion objective. The resulting image data was processed with semi-automatic image analysis software, Cell-O-Tape [102 (link)].

Imaging experiments were performed using a Bruker Autoflex Speed MALDI/TOF-TOF mass spectrometer. Before matrix deposition, tissues were scanned using an Epson flatbed scanner and collected at 300 dpi for acquisition of a digital baseline image. The mass spectrometer method was set to collect 5000 spectra/spot and images. Intact protein images were collected using linear mode, and trypsin peptide images were collected using reflectron mode. Calibration was conducted using a mixture of angiotensin II, bovine insulin, PEG 600, and ephrinA1-His standard. All control and acute MI samples were imaged at 100 μm spatial resolution. Data was collected in positive ionization mode and a Nd:YAG laser repetition rate of 2000 Hz was used (50 μm laser size) during image collection.