Super-resolution imaging of living tissues achieved by multi-confocal image scanning microscopy

Editors' notes

This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility:

fact-checked

trusted source

proofread

There is a growing demand for non-invasive insights into the complex three-dimensional subcellular dynamics within living tissues at the frontier of biological research. Professor Xi Peng's group at Peking University has developed a novel imaging technique known as multi-confocal image scanning microscopy (MC-ISM), which enhances spatial resolution, imaging depth, and minimizes phototoxicity in biological imaging.

Their paper is published in National Science Review.

Recent advancements have demonstrated that MC-ISM can significantly transform biological imaging techniques. By integrating confocal scanning with structural illumination super-resolution, this approach allows researchers to achieve superior imaging capabilities essential for studying living tissues.

Current imaging techniques struggle to balance spatial resolution and phototoxicity while capturing rapid biological processes in living tissues. The traditional confocal laser scanning microscopy (CLSM) is limited by the trade-off between pinhole size and signal-to-noise ratio (SNR).

MC-ISM addresses these challenges by integrating confocal scanning imaging with structural illumination super-resolution, allowing for enhanced three-dimensional imaging capabilities in biological specimens. This advancement enables researchers to capture the intricate dynamics of living organisms with unprecedented clarity and minimal damage.

The research team utilized MC-ISM technology to perform three-color 3D super-resolution imaging on sections of the mouse kidney. The technique operates within a volume of 66.5 μm × 66.5 μm × 12 μm, achieving an axial interval of 150 nm.

By optimizing pinhole diameter and spacing, applying optical lock-in detection (OLID) to suppress out-of-focus signals, and utilizing a frame reduction reconstruction algorithm, MC-ISM significantly enhances imaging speed and quality. The imaging was also tested on DAPI-stained zebrafish heads and plant cells, showcasing its versatility and capability in deep tissue imaging.

Key findings

MC-ISM improved lateral and axial resolutions to 131 nm and 336 nm, respectively, effectively suppressing out-of-focus signals and demonstrating optical sectioning comparable to spinning disk confocal microscopy.

The technique exhibited enhanced photon collection efficiency and lower photobleaching rates compared to existing methods, maintaining 67% fluorescence intensity after 1,000 frames of imaging.

In live U2OS cells, MC-ISM imaged mitochondria at 7.5 frames per second without any swelling, highlighting its low phototoxicity.

The technology successfully overcame tissue scattering and autofluorescence in plant cells, allowing for in situ super-resolution imaging of mitochondria in Arabidopsis hypocotyls.

More information: Wei Ren et al, Expanding super-resolution imaging versatility in organisms with multi-confocal image scanning microscopy, National Science Review (2024). DOI: 10.1093/nsr/nwae303

Provided by Peking University