Super-Resolution Spatial Proximity Detection with Proximity-PAINT.

Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single-molecule sensitivity. Here, we introduce Proximity-PAINT (pPAINT), a variation of the super-resolution microscopy technique DNA-PAINT. pPAINT uses a split-docking-site configuration to detect spatial proximity with high sensitivity, low false-positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha- and beta-tubulin in microtubules using super-resolution detection.

Environmental fluctuations and their effects on microbial communities, populations and individuals.

From the homeostasis of human health to the cycling of Earth's elements, microbial activities underlie environmental, medical and industrial processes. These activities occur in chemical and physical landscapes that are highly dynamic and experienced by bacteria as fluctuations. In this review, we first discuss how bacteria can experience both spatial and temporal heterogeneity in their environments as temporal fluctuations of various timescales (seconds to seasons) and types (nutrient, sunlight, fluid flow, etc.). We then focus primarily on nutrient fluctuations to discuss how bacterial communities, populations and single cells respond to environmental fluctuations. Overall, we find that environmental fluctuations are ubiquitous and diverse, and strongly shape microbial behavior, ecology and evolution when compared with environments in which conditions remain constant over time. We hope this review may serve as a guide toward understanding the significance of environmental fluctuations in microbial life, such that their contributions and implications can be better assessed and exploited.

Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT.

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.