S-Space College of Natural Sciences (자연과학대학) Dept. of Physics and Astronomy (물리·천문학부) Physics (물리학전공) Theses (Ph.D. / Sc.D._물리학전공)
Accelerated super-resolution imaging with FRET-PAINT
FRET-PAINT를 이용한 초고속 초고해상도 이미징
- 자연과학대학 물리·천문학부(물리학전공)
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 물리·천문학부(물리학전공), 2018. 8. 홍성철.
- Optical microscopy, especially fluorescence microscopy, is one of the most widely used tools for biological studies. Due to several methods to label biological samples with fluorophores such as biological fluorescent stain, immunofluorescence, and fluorescent protein expression, high sensitivity and specificity can be obtained. However, its resolution is limited by the diffraction. Therefore molecules and structures smaller than few hundred nanometers cannot be resolved with the conventional fluorescence microscopy.
Several decades ago, super-resolution fluorescence microscopy techniques were developed and they opened a way to resolve ultra-fine structures without being limited by the optical diffraction. The achievement, however, was not obtained without sacrifice. Compared to the conventional fluorescence microscopy, the super-resolution fluorescence microscopy techniques usually suffer from the aggravated photobleaching and the slowed-down imaging speed. Due to these problems, the super-resolution fluorescence microscopy in the current form is hard to be directly used to image large volume samples.
Recently developed DNA-PAINT microscopy has overcome the photobleaching problem by using transient binding of fluorescently labeled short DNA strands to docking DNA strands conjugated to target molecules. Since photobleached probes are continuously replaced with the other probes in the imaging buffer, fluorescence imaging can be performed without being limited by photobleaching. Furthermore, DNA-PAINT technique can acquire more photon numbers from a fluorophore than other single-molecule localization techniques because its imaging time is not limited by photobleaching. The imaging speed of DNA-PAINT (1-3 frames per hour), however, is extremely slow compared to those of other super-resolution fluorescence microscopy techniques. The slow imaging speed of DNA-PAINT is due to slow binding of the imager strand. Since the binding rate of the imager strand to a docking strand is proportional to the imager concentration, an obvious solution to this problem is to use higher imager concentration. In current DNA-PAINT technology, however, the imager concentration cannot be increased more than a few nM because the background noise also proportionally increases with the imager concentration.
I developed a novel super-resolution imaging technique which is based on both the DNA-PAINT technique and the FRET technique to accelerate imaging speed of DNA-PAINT without compromising its unique advantages, such as the photobleaching-resistance, high localization precision, and high multiplexing capability. In this technique that is named FRET-PAINT, the docking strand has two DNA binding sites: one for a donor strand and the other for an acceptor strand. For single-molecule localization, the FRET signal of the acceptor is used. Since the acceptor is not directly excited by an illumination beam but by the FRET, several hundred times higher imager (donor and acceptor) concentrations could be used. As a demonstration, microtubules were imaged with 300 nM donor strands and 300 nM acceptor strands. As a result, the imaging speed of the FRET-PAINT was accelerated 240 times faster than that of the DNA-PAINT. Since the donor and the acceptor strands bind to docking strand transiently as the imager strand does in the DNA-PAINT, the FRET-PAINT is also resistant to the photobleaching.
Another advantage of the DNA-PAINT technique over other super-resolution techniques is high multiplexing capability. By labeling a certain antibody with a certain docking strand whose DNA sequence is different from other docking strands, specific target molecules can be imaged orthogonally because the only complementary imager strands can bind to that docking strand. The imager strands are 7 to 10 nucleotides long, thus 16384 to 1048576 combinations are possible. Practically, every biomolecule can be specifically imaged with the DNA-PAINT.
Since the FRET-PAINT uses the same complementary base pairing of the donor and the acceptor strands to the docking strand, the FRET-PAINT can also possess high multiplexing capability. As a demonstration, microtubules and mitochondria were imaged. The merged image showed no cross-talk between these two structures.
Due to the high imaging speed together with the other advantages such as the photobleaching resistance and the high multiplexing capability, the FRET-PAINT technique will be a useful addition to the advancement of super-resolution fluorescence microscopy.
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