Development of super-resolution microscopy towards unlimited resolution via selective deactivation of molecular transition

다국어 초록 (Multilingual Abstract)

Understanding how cells function at the subcellular level has become important. The phrase “seeing is believing” can exactly represent the scientist’s desire. They want to see the smaller and faster moving of micro-scale molecules. Microscopy is a right that technique to visualize objects and areas of objects that cannot be seen with the naked eyes. Advances in microscopy technique open the new window not only for biologist but also various experts who deal with micro-scale systems. However, the resolution of conventional optical microscopy is limited by the diffraction limit of scattered light. This limit also called the point spread function (PSF) is 200~300 nm in the x and y direction, and 450~700 nm in the axial direction. This scale is larger or similar to subcellular structures that can’t be seen by using a diffraction limited conventional microscope.
To overcome the diffraction limit, super-resolution techniques have been developed in recent 20 years to visualize the biomolecular structure under few nanometer scales. Although the detailed strategies of super-resolution techniques are different from each other, they are based on the same mechanism, which uses the switching between on and off states of the target molecule’s signal.
Stimulated emission depletion (STED) microscopy is one of these super-resolution techniques that impose the switching mechanism of fluorophores. STED reengineers the effective PSF by suppressing fluorescence with stimulated emission at the periphery of the confocal fluorescence PSF using donut-shaped beam. The STED microscopy can provide unlimited resolution theoretically. However, when it comes to real experimental application, STED microscopy has been reported that tends to give lower spatial resolution even when compared to STORM. The photobleaching and quenching of fluorophore induced by intense depletion beam is a severe problem that limits the resolution of the STED microscopy. Additionally, the fluorscent super-resolution microscopy methods require endogenous (e.g., fluorescence proteins) or exogenous (e.g., immunostaining) fluorophore tags. Since these tags typically have a bulky size (~ 25 nm if primary and secondary antibodies are used), they could give significant uncertainty to the localization of a target molecule. Also, exogenous tags can influence the function of biomolecules.
We demonstrated the novel method to unlimited resolution, using selective deactivation of molecular transition. With the scheme of signal switch-off, that based on the idea using donut beam, we introduced super-resolution optical microscopy technique that could solve the two fundamental problems in STED microscopy: first, increasing the spatial resolution in fluorescence microscopy by combining expansion microscopy (ExM) with STED microscopy (Chapter 1-2); second, developing label-free super-resolution nonlinear vibrational microscopy technique with stimulated Raman scattering (SRS) microscopy (Chapter 3-4).
Chapter 1 outlines the theoretical background for fluorescence and a brief introduction of fluorescence microscopy and conventional super-resolution microscopy techniques. Chapter 2 describes the amplified expansion stimulated emission depletion microscopy method. we developed extremely bright expansion nanoscopy by using biotin–avidin signal amplification to increase the labeling density. Our method provides up to sevenfold increases in fluorescence signal intensity in expanded samples, thus enabling the use of STED imaging with maximum depletion intensities of a commercial microscope in the order of GWcm-2. We demonstrated the method by using biotinylated antibodies and genetic incorporation approaches that allow localization of biotin in a specific molecule or organelle. Chapter 3 depicts the theoretical background and introduction for coherent nonlinear microscopy. In chapter 4, we introduce a new method for sub-diffraction Raman imaging based on depletion of stimulated Raman scattering (SRS) with label-free and theoretically unlimited depletion efficiency. The idea is based on donut-shaped depletion light as in STED microscopy. To reduce the effective focal spot of the SRS signal, it is required to find a spectroscopic process that efficiently depletes SRS signal at the periphery of the focal spot. Here, we use the three-beam femtosecond SRS scheme formulated and demonstrated to simultaneously induce two different SRS processes associated with Raman-active modes in the same molecule. Two SR gains involving a common pump pulse are coupled and compete: As one of the Stokes beam intensities increases, the other SRS is selectively suppressed. We provide a theoretical description and experimental evidence that the selective suppression behavior is due to the limited number of pump photons used for both of the two SRS processes when an intense depletion beam induces one SRS process. The maximum suppression efficiency was ∼60% with our experimental setup, where the SR gain of the ring breathing mode of benzene is the target SRS signal, which is allowed to compete with another SRS process, induced by an intense depletion beam, of the CH stretching mode. With these experimental and theoretically derived data, we expect practical biological imaging based on Raman scattering with sub-100-nm resolution.

목차 (Table of Contents)

• Abstract
• 1. Fluorescence Microscopy
• 1.1 Introduction 8
• 1.2 Fluorescence and confocal microscopy 9
• 1.3 Diffraction limit in optical microscopy 13
• 1.4 Super-resolution fluorescence microscopy 14
• 1.4.1 Patterned illumination based method 15
• 1.4.2 Single molecule localization based method 19
• 1.4.3 Expansion microscopy 23
• 1.5 References 25
• 2. Amplified Expansion Stimulated Emission Depletion Microscopy
• 2.1 Introduction 28
• 2.2 Experimental method 31
• 2.3 Result and discussion 35
• 2.4 Conclusion 46
• 2.5 References 48
• 3. Coherent Nonlinear Microscopy
• 3.1 Introduction 51
• 3.2 Pump probe microscopy 53
• 3.3 Coherent Raman scattering microscopy 55
• 3.3.1 Classical derivation of Raman effect 56
• 3.3.2 The spontaneous Raman scattering 58
• 3.3.3 Stimulated Raman scattering (SRS) microscopy 60
• 3.3.4 Coherent anti-Stokes Raman scattering (CARS) microscopy 67
• 3.4 References 70
• 4. Breaking the Diffraction Limit of Stimulated Raman Scattering (SRS) Microscopy
• 4.1 Introduction 74
• 4.2 Selective suppression of stimulated Raman scattering with another competing stimulated Raman scattering 78
• 4.2.1 Theoretical description of three-beam SRS processes 78
• 4.2.2 Experimental method 89
• 4.2.3 Results and discussion 94
• 4.2.4 Summary and a few concluding remarks 107
• 4.3 Stimulated Raman scattering depletion microscopy 109
• 4.3.1 Experimental setup 111
• 4.3.2 Results and discussion 114
• 4.4 References 125
• Acknowledgement 128