The low-light poison probe “lights up” the mitochondrial.

Researchers from Peking University and Westlake University have teamed up to develop two new probes, PK Mito Red (PKMR) and PK Mito Deep Red (PKMDR), that for the first time have significantly reduced phototoxicity at the level of living cells, according to a study published online in Chemical Science.

Author: Weibo Tang Lin.

The 21st century is considered the age of life sciences. To further demystell the mystery of cellular biology, scientists have developed a variety of bioimaging techniques. Among them, fluorescence ultra-resolution imaging stands out because of simple imaging conditions and compatibleness with biological samples.

However, fluorescence ultra-resolution imaging often requires more photons, as live cell ultra-resolution imaging requires fluorescent probes to emit more photons before bleaching. In addition to focusing on how to improve the light stability of fluorescent probes, scientists are more concerned with the phototoxicity produced by the bleaching process.

Using the newly invented low-phototoxic PKM series of probes to process cells, the team conducted mitochondrial imaging experiments on the self-developed Hessian-SIM instrument. Photo from the Institute of Molecular Medicine, Peking University.

Break through the problem of ultra-resolution imaging “card neck”.

As early as 2018, Hessian SIM, developed by Chen Liangyu of Peking University’s Institute of Molecular Medicine, reveals for the first time the fine inner structure of mitochondrials in living cells and their dynamic changes.

In order to meet the needs of the new optical microscope, many scientific research teams at home and abroad have developed a variety of light stability of the new mitochondrial probe, used to observe the structure of mitochondrials.

However, the high excitation light intensity required for ultra-resolution imaging tends to make mitochondrial fluorescent probes more susceptible to reactive oxygen species (ROS) and does not disappear with increased sampling time intervals.

Such ultra-resolution imaging, even if the fluorescent probe itself is not completely bleached, will quickly destroy the mitochondrial body, so that mitochondrial swelling, rounding.

“Although phototoxicity has no effect on optical resolution, it can have a significant impact on cellular structure, and cells can be injured or even killed, in which case what is seen is completely different from the physiological response.”

Chen Zhi line, a researcher at Peking University’s Institute of Molecular Medicine and one of the authors of the work, told China Science Daily.

In other words, the main problem that live cell ultra-resolution imaging needs to address is actually improving phototoxicity, not just photobleaching.

In response to the new pain points in this field, Chen Zhi line team used the strategy of molecular in-molecular acrylic, successfully synthesized two new low-phototoxic mitochondrial fluorescent probes, PKMR and PKMDR, which for the first time achieved a significant reduction in phototoxicity at the level of living cells.

In addition, the Chen Zhihang team, in collaboration with Chen Liangyi’s team and the Rekai team at Westlake University, conducted further experiments to prove that the phototoxicity of the new dye to the cells was significantly lower than that of the MitoTracker series of commercial dyes, and that 1000 frames could be achieved. The above, mitochondrial body crucible shape basically unchanged ultra-resolution imaging, the first time proved that the probe can be used for stem cell flow selection of vortex insects, without affecting the erraticity of stem cells, can really be used to study the physiological process such as stem cell reprogramming mitochondrial function.

Low-light poison is gentler.

Chen Zhi line introduced that the phototoxicity of fluorescent probes generally comes from the first excitation of single-heavy state (S1) in the process of excitation through the inter-system jump produced by the first excitation triple state (T1), T1 state and oxygen action can produce reactive oxygen species such as single-line oxygen, and thus destroy biological molecules.

To minimize this process and thus reduce phototoxicity, the team used a strategy previously used to reduce photobleaching in the molecular in-molecular tri-line quenching agent (self-healing dye Self-Healing Dye) previously used in areas such as dye lasers and single-molecule imaging.

Light survival experiments on HeLa cells have found that only cyclocytryene (COT) in common three-line quenching groups can effectively reduce phototoxicity in fluorescence imaging.

More importantly, the team also observed that the group (nitrobenzene) that reduces photobleaching does not necessarily reduce phototoxicity.

Because of this, “the phototoxicity and photobleaching properties of fluorescent probes should be tested separately, but in the past fluorescent probe designs often ignored the detailed characteration of the properties of the probe phototoxicity.” Chen explained.

On this basis, the team used COT as a three-line quenching group and synthesized two fluorescent probes PKMR and PKMDR with Cyanine as the master core.

Light survival experiments on HeLa cells have shown that the new probe is less phototoxic than one-half of the corresponding spectral commercial probe (MitoTrackers) and can provide the same or even higher brightness under the same conditions.

“It’s fair to say that this work has found a new direction for the field, with the hope of reducing the phototoxicity of the probe to a lower level or even eliminating it al completely in the future.” Chen said.

Collaborative innovation to the extreme.

In June 2018, after returning to China, Chen Zhi line joined the Institute of Molecular Medicine of Peking University.

At that time, Hessian SIM, developed by Chen Liangyu’s team, not only took the performance of optical microscopes to a whole new level, but also greatly improved the phototoxicity of ultra-resolution imaging technology in both mathematical and physical terms.

To achieve the ultimate in ultra-resolution imaging technology, collaborative innovation must be made from the direction of the chemical probe.

And the chemical research background of Chen Zhi line’s addition, no doubt for the team injected a strong momentum.

From the beginning of probe research in June 2018 to today’s major breakthrough, the team took just over a year to iterate through three generations of design, and finally found an effective solution.

After testing the application of the new probe in the field of vortex stem cell sub-selection, the Rekai team, which has been engaged in the research of vortex stem cell regeneration for a long time, found that the new probe-labeled mitochondrials can help to sub-select stem cell groups, and that the new probe-labeled stem cells can maintain good activity and dryness outside the body, demonstrating its excellent biosystosis in fluid fluorescence analysis.

Even more valuable is that the new probe also offers superior advantages in long-range ultra-resolution imaging.

Hessian SIM imaging results show that the new probe provides a resolution close to the theoretical value of the 2D Hessian SIM, showing a clear image of the mitochondrial crucible structure, while maximizing the original form of mitochondrial health.

The mitochondrial marked with commercial dye MitoTracker Red CMXRos (MTR) swells and deforms significantly at about 140 frames, and normal physiological structures such as crucibles are no visible at more than 200 frames, while PKMR-labeled mitochondrials can provide more than 1000 frames of healthy mitochondrial images, and the deformation of mitochondrials around 2000 frames is still less than 200 frames of commercial needles.

“Next, we will continue to work together in math, physics, and chemistry to push technology to the extreme. At the same time, strive for the future transformation, break the monopoly of foreign companies in the direction of high-end imaging instruments / probes, and truly realize the provision of biomedical researchers beyond foreign products of domestic high-end imaging tools. Chen said.