DGIST Scholar: Investigation of unique background noise in STED optical nanoscopy and a novel method for suppressing the noise

Department of New Biology Theses Master

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Investigation of unique background noise in STED optical nanoscopy and a novel method for suppressing the noise

Title: Investigation of unique background noise in STED optical nanoscopy and a novel method for suppressing the noise

Author(s): Jaeyong Kim

DGIST Authors: Jaeyong Kim ; Jong-Chan Lee ; Cheol Song

Advisor: 이종찬

Co-Advisor(s): Cheol Song

Issued Date: 2022

Awarded Date: 2022/02

Type: Thesis

Subject: Optical microscopy, Super-resolution microscopy, STED nanoscopy, Background noise, Fluorescence Correlation Spectroscopy

Description: Optical microscopy, Super-resolution microscopy, STED nanoscopy, Background noise, Fluorescence Correlation Spectroscopy

Table Of Contents: Ⅰ. Introduction 1
1.1 Emergence of super-resolution techniques 1
1.1.1 Widefield fluorescence microscopy 1
1.1.2 Confocal scanning microscopy 1
1.1.3 Optical diffraction limit and point-spread-function 3
1.1.4 Development of super-resolution microscopy 4
1.2 Stimulated emission depletion microscopy 5
1.2.1 Stimulated emission 5
1.2.2 Principle of STED nanoscopy 6
1.2.3 3D STED 8
1.3 STED-specific background noise 9
1.3.1 Direct excitation background noise 9
1.3.2 Incomplete depletion background noise 10
1.3.3 Characteristics of the STED-specific background noise 10
1.4 Background noise suppressing STED imaging techniques 11
1.4.1 STED-only subtraction method 11
1.4.2 Time-gating STED 12
1.4.3 Stimulated emission double depletion 13
1.4.4 Polarization switching STED 14
1.5 Limitation of the current background suppressing techniques 15
1.5.1 Insufficient background suppression effect 15
1.5.2 Setup difficulty and cost increase caused by additional components 16
1.5.3 The strategy to overcome conventional limitation 16
Ⅱ. Theoretical Development 17
2.1 Basic idea for subtracting STED-specific background noise 17
2.2 Double subtraction STED scheme 17
2.3 Strategy to find the optimal STED beam power, and subtraction factor 18
Ⅲ. Methods 20
3.1 STED nanoscopy simulation 20
3.1.1 Jablonski diagram model and fluorescence detection with STED 20
3.1.2 STED point spread function calculation and image formation 22
3.1.3 Simulation parameters 23
3.2 Home-built STED nanoscope 24
Ⅳ. Results 26
4.1 Accurate selection of optimal STED beam power, and subtraction factor 26
4.2 Comparison between point spread functions of dsSTED and other techniques 28
4.3 Simulated cytoskeleton structure images of dsSTED and other techniques 29
4.4 Resolution comparison between dsSTED and other techniques 30
Ⅴ. Conclusion 34
Supplementary information 36
S.1 Direct excitation background in sub-STED PSF 36
S.2 SBR calculation on 3D PSF 37
S.3 Cytoskeleton structure images in different sample densities 38
S.4 Home-built STED setup 39
S.4.1 Photonic crystal fiber setup 39
S.4.2 Polarization maintaining single-mode fiber coupling 41
S.4.3 Piezo actuator scanning system 42
S.4.4 Image scanning and acquisition software 43
S.4.5 Pinhole alignment 43
S.4.6 Laser pulse delay setting 44
S.4.7 Excitation beam shape fitting 45
S.4.8 Confocal imaging test 46
S.4.9 Phase plate alignment to generate XY STED beam 48
S.5 Fluorescence correlation spectroscopy experiments for live-cell membrane lipids 50
References 54
국문요약 62