Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that {beta}-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 {my}m away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization

Linking complex forest fuel structure and fire behaviour at fine scales

Abstract. Improved fire management of savannas and open woodlands requires better understanding of the fundamental connection between fuel heterogeneity, variation in fire behaviour and the influence of fire variation on vegetation feedbacks. In this study, we introduce a novel approach to predicting fire behaviour at the submetre scale, including measurements of forest understorey fuels using ground-based LIDAR (light detection and ranging) coupled with infrared thermography for recording precise fire temperatures. We used ensemble classification and regression trees to examine the relationships between fuel characteristics and fire temperature dynamics. Fire behaviour was best predicted by characterising fuelbed heterogeneity and continuity across multiple plots of similar fire intensity, where impacts from plot-to-plot variation in fuel, fire and weather did not overwhelm the effects of fuels. The individual plot-level results revealed the significance of specific fuel types (e.g. bare soil, pine leaf litter) as well as the spatial configuration of fire. This was the first known study to link the importance of fuelbed continuity and the heterogeneity associated with fuel types to fire behaviour at metre to submetre scales and provides the next step in understanding the complex responses of vegetation to fire behaviour

Nuclear accessibility of beta-actin mRNA is measured by 3D single-molecule real-time tracking

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that beta-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 microm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization