Development of Super-Depth and Super-Resolution Microscopy technology
Development of Super-Depth and Super-Resolution Microscopy technology
Research results obtained by Professor Choi Won-Shik’s and Shim Sang-Hee’s groups were published in the prestigious journal Nature Communications.
The research groups led by Professor Choi Won-Shik in the Department of Physics of the College of Science (Deputy director of the Center for Molecular Spectroscopy and Dynamics at the Institute of Basic Science (IBS; President Noh Do-Young)) and Professor Shim Sang-Hee in the Department of Chemistry successfully developed a super-depth and super-resolution microscopy technology.
The emergence of super-resolution fluorescence microscopy has allowed for the direct observation of individual biomolecules using visible light, overcoming the diffraction limit resolution. In particular, single-molecule localization microscopy, a representative super-resolution fluorescent microscopy, enables researchers to achieve high resolutions by means of a simple optical setup. Thanks to these advantages, the method has been applied to varied biological studies, such as the imaging of synapse structures and the imaging of molecular composites, leading to wide-ranging advances including the discovery of new biological structures. As these contributions were highly regarded, a Nobel Prize in Chemistry was granted in this field in 2014.
However, single-molecule localization microscopy has the drawback that image quality is easily lowered by sample aberration. This problem is fundamentally caused by the fluorescent signals released from a single molecule being extremely weak. This complicates measurement as the image is easily distorted by scattering or aberration inside the sample. As a result, the maximum imaging depth accessible by single-molecule localization microscopy is just a few micrometers, limiting the imaging method to extremely thin samples, such as cultured cells and thin brain tissue slices.
The research team employed CLASS (closed-loop accumulation of single-scattering) microscopy, which is a label-free technology of adaptive optics, to measure and correct the aberration caused by biological tissues. This enabled the single-molecule localization microscope to achieve optimal performance when imaging a deep-seated section of a biological tissue sample.
The operating method is as follows. First, an interferometer-based reflecting microscope is used to record the reflection images of a sample at a specific depth, while varying the incidence angle of the light. The CLASS algorithm is applied to the acquired reflection images to determine the aberration. In the next step, a correction pattern for the aberration is displayed on a spatial light modulator installed in the fluorescent path. This allows the fluorescent beam reflected on the spatial light modulator to arrive on the camera with the aberration corrected. The aberration-corrected single-molecule fluorescent images collected in this way are analyzed to finally generate a super-resolution fluorescent image.
Various methods have already been developed to increase the performance of super-resolution microscopes through the correction of aberration. However, the conventional methods all improve image quality by collecting single-molecule fluorescent images. Therefore, when the single-molecule fluorescent images are distorted beyond a certain threshold due to a severe aberration, the fluorescent images become unobtainable, limiting the measurement of aberration. The research group resolved this problem by measuring the aberration using the reflection images rather than the fluorescent images of biological tissues. Taking advantage of this method, the research team successfully acquired super-resolution images of various structures of whole intact zebrafish without preparing thin tissue slices. Through their aberration correction method, the research team increased the number of localized fluorescent molecules by up to 37 times through recovering the images of fluorescent molecules that were previously too distorted to be localized. Based on this powerful improved performance, the research group successfully performed imaging to a depth of 100 micrometers at a resolution of about 30 nanometers.
논문의 Park Sang-Hyeon, the first author of the article, commented, “Our research overcomes the chronic problem of single-molecule localization microscopy, the limited imaging depth, through adaptive optics. Now we are able to apply super-resolution fluorescent imaging, which used to be applicable to only tissue slices, to animal models, such as zebrafish.” Professor Shim Sang-Hee in the Department of Chemistry described the research, “We have extended the scope of the application of super-resolution imaging, which is expected to be a great help to many research fields, including genetics, developmental biology, and neurobiology.” Professor Choi commented on the significance of their research, “The development of super-depth and super-resolution microscopy will be a milestone in microscopy research.”
The research was supported by the Institute of Basic Science of the Ministry of Science and ICT and the National Research Foundation of Korea. The results of the research were published in the prestigious journal Nature Communications (IF = 17.694) on July 13.