MINSTED tracking of single biomolecules.

Here we show that MINSTED localization, a method whereby the position of a fluorophore is identified with precisely controlled beams of a STED microscope, tracks fluorophores and hence labeled biomolecules with nanometer/millisecond spatiotemporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within <250 µs using about 13 photons. The power of MINSTED tracking is demonstrated by resolving the stepping of the motor protein kinesin-1 walking on microtubules and switching protofilaments.

Near index matching enables solid diffractive optical element fabrication via additive manufacturing.

Diffractive optical elements (DOEs) have a wide range of applications in optics and photonics, thanks to their capability to perform complex wavefront shaping in a compact form. However, widespread applicability of DOEs is still limited, because existing fabrication methods are cumbersome and expensive. Here, we present a simple and cost-effective fabrication approach for solid, high-performance DOEs. The method is based on conjugating two nearly refractive index-matched solidifiable transparent materials. The index matching allows for extreme scaling up of the elements in the axial dimension, which enables simple fabrication of a template using commercially available 3D printing at tens-of-micrometer resolution. We demonstrated the approach by fabricating and using DOEs serving as microlens arrays, vortex plates, including for highly sensitive applications such as vector beam generation and super-resolution microscopy using MINSTED, and phase-masks for three-dimensional single-molecule localization microscopy. Beyond the advantage of making DOEs widely accessible by drastically simplifying their production, the method also overcomes difficulties faced by existing methods in fabricating highly complex elements, such as high-order vortex plates, and spectrum-encoding phase masks for microscopy.

MINSTED nanoscopy enters the Ångström localization range.

Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power. By blue-shifting the STED beam and separating fluorophores by on/off switching, individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Because our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up new areas of application in the study of macromolecular complexes in cells.

Transcriptionally active enhancers in human cancer cells.

The growth of human cancer cells is driven by aberrant enhancer and gene transcription activity. Here, we use transient transcriptome sequencing (TT-seq) to map thousands of transcriptionally active putative enhancers in fourteen human cancer cell lines covering seven types of cancer. These enhancers were associated with cell type-specific gene expression, enriched for genetic variants that predispose to cancer, and included functionally verified enhancers. Enhancer-promoter (E-P) pairing by correlation of transcription activity revealed ~ 40,000 putative E-P pairs, which were depleted for housekeeping genes and enriched for transcription factors, cancer-associated genes, and 3D conformational proximity. The cell type specificity and transcription activity of target genes increased with the number of paired putative enhancers. Our results represent a rich resource for future studies of gene regulation by enhancers and their role in driving cancerous cell growth.