Images were acquired using an Olympus BX61 widefield epi-fluorescence microscope outfitted with a 100X/1.35 NA UPlanApo objective. Samples were visualized using the Chroma 41007 filter (Cy3), Chroma 31000 filter (DAPI), and DIC. Metamorph (Molecular Devices) was used as the acquisition software in combination with a CoolSNAP HQ CCD camera (Photometrics). Z-sections were acquired at 200 nm intervals over an optical range of 4.0 um. Exposure times for each z-section include 1200 ms (Cy3), 200 ms (Cy5) and 25 ms (DAPI). Cell volumes are then assembled by maximum intensity projections of z-sections using Metamorph. Localize, developed by Dr. Daniel Larson, was used to detect, locate and quantify intensities for fluorescent spots based on a two-dimensional Gaussian mask algorithm detailed previously5 (link)26 (link). Cell-segmentation software was used to identify cell and nuclear boundaries, and nascent mRNA counts were determined by dividing the transcription site intensity by the mean single transcript intensities, and rounding up or down to the nearest whole number5 (link). The mean initiation interval = ((lenth of the gene in Kb/mean # of nascent mRNA)/1.5 Kb per minute)). The maximum initiation interval = ((lenth of the gene in Kb/maximun # of nascent mRNA)/1.5 Kb per minute)).
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