Using Multi-Focus Microscopy to Capture Transcriptional Dynamics at the Second Timescale

Abstract

Transcriptional dynamics occur at multiple timescales. Transcript initiation can occur from seconds to hours apart. Transcriptional activators have been reported to bind promoters at the hour, minute, second and sub-second timescales. Regulatory pertinent nucleosome dynamics also occur at time scales orders of magnitude apart. To gain a better mechanistic understanding of transcription we must observe the dynamics at all relevant timescales. However, practical limitations in acquisition speed prevent the investigation of single molecule dynamics at the second or sub-second timescales. Single molecules maintain freedom of movement in three dimensions; analysis of single molecules requires image acquisition at multiple focal planes to ensure consistent particle tracking and intensity quantification. However, the necessity of time series z-stacks hinders temporal resolution. Moving and capturing the specimen is difficult and results in time delay between each image in the stack. Beyond timescale, multiple image acquisition requires multiple light exposures per z-stack resulting in enhanced photobleaching and phototoxicity which sets a maximum duration on image acquisition. The Multifocus Microscope (MFM) solves many of these challenges simultaneously by capturing multiple focal planes with only one exposure. We employ the MFM toward the analysis of “fast” transcriptional dynamics at the Pho5 locus in Saccharomyces cerevisiae.