"As a large-scale and high-level scientific research platform, Zhejiang University has great attraction. After I came back, my School of Physics gave me great support, so that I could do scientific research with peace of mind. By the way, I grew up in Shaanxi, but my ancestral home is in Shaoxing. At that time, due to the war, the ancestors had to flee to the west. Now that I have returned to Zhejiang University and my hometown, I am also in line with the spirit of Zhejiang University's westward migration. Zhang Delong, a researcher at the Hundred Talents Program at the School of Physics of Zhejiang University, said.
Figure | Zhang Delong (Source: Zhang Delong)
In 2019, after completing his PhD in the United States and completing his postdoctoral research, Zhang Delong returned to China to join Zhejiang University, mainly focusing on molecular spectroscopic imaging and cross-cutting frontier technologies.
Recently, he and his team developed a photothermal relaxation localization technique (PEARL) based on photothermal relaxation localization. In the fields of biology, medicine and materials science, these technologies can play the label-free positioning advantages that cannot be replaced by traditional fluorescence super-resolution technology, and have a wide range of application prospects.
PEARL: Compatible with existing fluorescence techniques and able to track specific proteins
In short, Zhang proposed a new microscopy technique that can achieve super-resolution imaging of non-fluorescent molecules through photothermal relaxation. It gets rid of the dependence of traditional super-resolution imaging technology on fluorescent labeling and can directly perform super-resolution imaging.
In recent years, super-resolution technology has developed into a hot technology. In 2014, American applied physicist Eric Betzig, German physicist Stefan W. Hell, and American chemist William Esco Moerner were jointly awarded the Nobel Prize in Chemistry for their contributions to super-resolution technology.
Optical technology belongs to the field of physics, and they should be awarded a physics prize. The Nobel Prize Committee's answer is: the previous resolution depended on the quality of the lens grind, and today's breakthrough in resolution is due to the discovery and synthesis of magical fluorescent molecules.
Fluorescent labeling technology is very mature today and is undoubtedly one of the most widely used optical technologies. However, no technology is perfect. The so-called "success and defeat", fluorescence technology is still limited by the dependence on fluorescent molecules, and there are problems such as photobleaching, low labeling efficiency and poor selectivity.
Although super-resolution fluorescence technology has become more and more advanced over the past decade, there is still a lot of room for the development of non-fluorescence super-resolution microscopy. For a long time, scholars in the field of optical imaging have been looking for label-free super-resolution technology, but have suffered from the lack of a widely applicable technology.
Among common imaging modalities, photothermal microscopy is a powerful technique with high sensitivity and versatility, compatible with both electron and vibration absorption.
The super-resolution imaging technology based on photothermal relaxation positioning developed by Zhang Delong this time realizes label-free imaging that breaks through the diffraction limit by detecting the temporal characteristics of photothermal effects. In the study, he and his team used position-dependent photothermal dissipation to break the resolution limits of photothermal microscopy.
The technology also has a wide applicability, that is, molecules or structures that are optically absorbed can basically be imaged beyond the diffraction limit.
In contrast, the application of existing label-free super-resolution technologies is mostly limited to specific systems or molecular structures, and is not widely applicable.
In the paper, Zhang Delong's group also demonstrated the imaging ability of molecular vibration spectroscopy in the mid-infrared region, the characteristic peaks of lipids and proteins, and also demonstrated the imaging ability of electron absorption spectroscopy represented by gold nanoparticles in the visible light region.
These two modes cover the spectral range from visible light to mid-infrared (about 400nm~10000nm), which are representative spectroscopic application cases and fully prove the wide applicability of super-resolution imaging technology based on photothermal relaxation localization.
At the same time, taking the yeast model as an example, the team also demonstrated the research potential of this technology in biology and other aspects.
(Source: Nature Photonics)
Through infrared absorption characteristic peaks, they selectively super-resolved imaging of spatially aggregated fat and protein molecules. In traditional methods, fluorescent labeling of small metabolites such as fat is often difficult, usually indirectly through specific proteins, but even then difficult to quantify.
The super-resolution imaging technology based on photothermal relaxation positioning can directly perform label-free super-resolution imaging of fat, which can make unprecedented and accurate observation of biological metabolism and other processes, which has irreplaceable value for deciphering biological processes.
In addition, this technology is compatible with existing fluorescence techniques, allowing simultaneous immunofluorescence tracking of specific proteins on the basis of molecular imaging.
It is worth mentioning that it is generally difficult to label more than 3 simultaneous labels in fluorescence imaging. The width of molecular spectral peaks is generally very narrow, and it has the ability to track multiple molecules at the same time.
Previously, other teams tracked dozens of different molecules at the same time [1], and the super-resolution imaging technology based on photothermal relaxation localization developed by Zhang Delong's group also inherited this high-throughput spectral multiplexing advantage.
Let's start with the "cat" you use when surfing the Internet
According to reports, this work is carried out on the basis of Zhang Delong's previous results [3], that is, through the ultra-sensitive detection technology derived from the photothermal effect, optical absorption is observed and applied to infrared imaging, which breaks through the limitations of traditional infrared imaging and realizes three-dimensional, sub-micron resolution live-cell imaging.
Zhang Delong said: "For PEARL's work, it was quite accidental at the beginning. "This has to mention modulation. In engineering, modulation technology is extremely widely used, from radio to 5G communications.
For example, the "cat" we use when surfing the Internet is the common name of modem, which is the abbreviation of modulator-demodulator (modem). The core of modulation technology is actually very simple, like we use flashing lights when driving, so that the car "jumps" out of the complex surrounding environment.
Modulation is also often used in scientific research, that is, the excitation light is "flickered" at frequency f (that is, modulated), and then this "flicker" (that is, demodulation) is detected by the detector at frequency f, which can improve the signal-to-noise ratio.
Zhang Delong said: "The academic community's understanding of modulation technology has always continued in this way, and no one has explored more application potential. But is it right to always be like this? So after I returned to China, I began to explore this area. ”
For the discovery in the exploration, Zhang Delong gave an example: When we were children, we played the "red and white machine" game, the music sound was very strange, the same C major, but far from the sound of the piano.
This is because synthetic music can produce pure sinusoidal vibration, while piano strings cannot produce pure sine vibration, which is accompanied by many "imperfect" vibrations, which physically produce many frequency multiplier vibrations, this phenomenon is generally called harmonics. And for such sounds, the human ear will feel more natural.
Realizing this, Zhang and his team began to observe signals at harmonic frequencies, and initially they wanted to add them all together to further improve the signal-to-noise ratio (by using this idea, another research group made independent research results).
However, what Zhang Delong did not expect was that the imaging results on the harmonic frequency were not completely consistent with the original picture. This made him feel very unexpected, and driven by curiosity, this project began.
After the official establishment of the project, he and his team took the microsphere system as the standard and deeply studied the relationship between scanning imaging resolution and harmonic order. After obtaining reproducible results, the study of live-cell imaging is gradually carried out boldly.
During this time, they noticed that the physics of PEARL also apply to general photothermal phenomena. Therefore, the photothermal imaging of electron absorption began to be studied, and the most representative material such as nanostructures was selected for imaging, so that the improvement of PEARL resolution was verified.
(Source: Nature Photonics)
However, Zhang Delong described the entire research process as "twists and turns, and bad fate". He said: "Needless to say, our experiments have been interrupted more than once. And in the experiment, after one week of installation and operation, one of the core lasers suddenly broke down without warning and had to be returned to the factory for repair. ”
In this case, they used this "half-bad" laser and finally completed the experiment a week before the repair pickup.
In addition, some experiments are to be completed in the infrared femtosecond system. At that time, the condition of the laser was very poor, and engineers had to visit the house for maintenance about every three months, and the research progress was also seriously hampered.
And the most difficult thing is that when they did the experiment of visible light pumped imaging, they did not have a light source at hand. Later, Zhang Delong searched up and down in the institute, and finally borrowed the idle red laser and green pulse laser to ensure the completion of the experimental verification.
He continued: "It is very fortunate that the first author of this paper, Fu Pengcheng, is the first doctoral student to join our group. Peng Cheng has also grown from a 'little white' whose clothes were burned out of holes by lasers during experiments, to a researcher who can operate the system independently. One day he told me with a solemn expression that he had ruined our scanning platform of hundreds of thousands. Later, after carefully investigating the reason, it was found that after the new version of the driver, the definition of the pin was modified, which turned out to be a false alarm. These big and small things have exercised students' 'debug' ability, and also allowed us to create a set of scientific research methods. ”
Finally, the related paper was published in Nature Photonics under the title "Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy", with Fu Pengcheng as the first author. Zhang Delong served as the corresponding author [2].
Figure | Related papers (Source: Nature Photonics)
In fact, PEARL is also one of the emerging frontier directions in recent years, so Zhang Delong will continue to explore deeply. On the one hand, they hope to further optimize system performance, such as increasing acquisition bandwidth and throughput, and improving resolution. On the other hand, they will also carry out more external cooperation in application expansion, and will develop more novel imaging technologies in the field of infrared super-resolution imaging.
Resources:
1.Nature 544, 465–470 (2017)
2.Fu, P., Cao, W., Chen, T.et al. Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy. Nat. Photon. (2023). https://doi.org/10.1038/s41566-022-01143-3
3.Science Advances 2, e1600521 (2016)