DNA probes without the damage
Image: SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy [Johnsson/EPFL]
Switzerland-based researchers have developed a DNA stain for safely studying live mammalian cells for days, under demanding imaging conditions.
Fluorescent stains that light up a cell’s DNA are popular in live-cell imaging as they allow researchers to track key biological processes such as cell division.
However, currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy.
As Professor Kai Johnsson from EPFL points out: "A popular stain - the DNA minor groove-binder Hoechst 33342 - is non-toxic when used at low concentrations but requires cell-damaging blue light illumination and is not compatible with super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy."
"In contrast, the anthraquinone-based intercalator DRAQ5 is a far-red DNA stain but is toxic at the concentrations used for live-cell microscopy," he adds.
With these limitations in mind, Johnsson and colleagues synthesised a new far-red DNA stain, SiR–Hoechst, that displays minimal toxicity.
Based on the far-red fluorescent silicon rhodamine molecule and a well-established DNA stain, Hoechst, the new stain works by binding to a part of the DNA helix known as the "minor groove", and only then emitting a fluorescent red light.
Laser scanning confocal imaging of HeLa cells using a customized Zeiss LSM780 microscope equipped with a × 40 1.4 NA oil DIC Plan-Apochromat objective, and controlled by ZEN 2011 software and an autofocus macro, AutofocusScreen. [Johnsson et al. Nature Communications, 6, 8497]
According to the researchers, the stain binds to DNA without affecting its biological function in the cell, can be used across many cells and tissues, and is compatible with super-resolution microscopy.
What's more, SiR–Hoechst can safely maintain high-quality staining in live cells for over 24 hours, allowing biologists to identify individual cells in tissue or culture, or track delicate processes, such as cell division, in real time.
To demonstrate their new DNA stain, the researchers imaged Hela cells using high resolution time-lapse confocal microscopy. These cells were stained with 200 nM SiR-Hoechst before imaging.
As Kai Johnsson says: “The introduction of SiR-Hoechst brings bioimaging closer to one of its main goals: observing the wonders of nature without disturbing them.”
The team is now preparing to commercialize SiR-Hoechst through their EPFL startup company, Spirochrome.
Research is published in Nature Communications.