High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.DOI: http://dx.doi.org/10.7554/eLife.21660.001

Difficulty in financing and low financing efficiency is one of the bottlenecks that restrict the high-quality development of China’s energy-saving and environmental protection industry and economy. The key to improving financing efficiency is to understand its influencing factors. This paper uses data envelopment analysis (DEA) and the Malmquist index to measure the overall financing efficiency and the efficiency of different financing methods of 205 Chinese energy-saving and environmental protection industries from 2015 to 2020 from static and dynamic perspectives, respectively, as well as the Tobit model to estimate the impact of factors such as the digital transformation and green technological innovation of enterprises on financing efficiency. The study shows the following: (1) Static analysis shows that: the financing efficiency of the comprehensive technical efficiency of China’s energy conservation and environmental protection industry is less than one, 5.8% to 23.41% of enterprises have very effective comprehensive technical financing efficiency, and fewer than 9% enterprises have very effective scale efficiency levels. Enterprises may have more room for improving their financing efficiency in the future. The four types of financing are, namely, internal financing, equity financing, fiscal financing, and debt financing, in descending order of efficiency. (2) Dynamic analysis shows that the financing efficiencies of debt financing and fiscal financing are both on an upward trend, while internal and equity financing efficiencies are on a downward trend. Additionally, the technological progress change index and scale efficiency are two key factors affecting the financing efficiency of different financing methods. (3) In terms of financing methods, the comprehensive technical efficiency and scale efficiency of endogenous financing and equity financing are high, while the comprehensive technical efficiency and scale efficiency of debt financing and fiscal financing are low and flat. (4) Digital transformation, green technology innovation, the asset–liability ratio, profitability, and operational capability have a significant positive impact on the financing efficiency of energy-saving and environmental protection enterprises. This paper studies the financing efficiency of China’s energy conservation and environmental protection industry under different financing methods and the mechanism through which key factors affect the financing efficiency of enterprises. It aims to provide a theoretical basis for managing financing methods scientifically and rationally and improving the financing efficiency of the energy conservation and environmental protection industry, as well as to provide practical reference for the implementation of digital transformation, green technology innovation and diversified financing in China and other developing economies.

This Letter proposes a method referred to as distorted grating (DG) and double-helix point spread function (DH-PSF) combination microscopy (DDCM), which is capable of multiparticle parallel localization and tracking in a transparent sample thicker than 10 μm, the thickness of cells. A special phase mask, combining the field depth extension capabilities of DG with the three-dimensional (3D) nanolocalization capabilities of the DH-PSF, is designed for multiparticle parallel localization. Time-lapse tracking of one particle moving along the z axis and parallel tracking of two particles are simulated. Results demonstrate that, with only a single snapshot, particles can be localized, tracking with 3D nanoresolution wherever they are. The theoretical localization precisions of DDCM, DH-PSF, and multifocus microscopy are compared. DDCM results in almost constant localization precisions in all three dimensions for a depth of field larger than 10 μm. DDCM is expected to become a tool in investigations of important dynamic events in living cells.

High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled superresolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used threedimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29-34 individual MB probes, we observed 3D finescale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.

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