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研究生:德士柏
研究生(外文):Subir Das
論文名稱:Signal Processing in Stimulated Emission Based Pump-Probe Microscopy
論文名稱(外文):Signal Processing in Stimulated Emission Based Pump-Probe Microscopy
指導教授:高甫仁
指導教授(外文):Fu-Jen Kao
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生醫光電研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:108
語文別:英文
論文頁數:70
中文關鍵詞:N/A
外文關鍵詞:Pump-probe microscopyStimulated emissionSignal processingLock-in detectionFluorescence
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Fluorescence imaging has been a powerful technique due to its high sensitivity and specificity, especially for biomedical applications. Fluorescence emission is a spontaneous process and the photons are emitted incoherently and isotropically, filling full solid angle (4π). Therefore, high numerical aperture (NA) optics and highly sensitive detectors are required for fluorescence imaging, to achieve both higher spatial resolution and efficient collection of the emitted photons.
In comparison, stimulated emission (SE) exhibits a very different way of emitting and propagation, attributing to its coherence. In SE based signal detection, two beams at pump-probe configuration are usually used, one at wavelength λ_pu (pump) and the other at λ_pr, (probe or SE). The pump beam is modulated and the modulation is transferred to the probe beam via stimulated emission of the excited fluorophores. Recently, stimulated emission (SE) has presented as an advantageous method in fluorescence imaging due to its unique properties, including the detection of dark fluorophores and the integration with interferometric techniques.
This doctoral thesis work focuses on three themes: first, the fundamental and physical (or optical) limits of SE and its signal processing (Chapter 2). Second, the implement of high modulation frequency (subharmonic) for SE microscopy (Chapter 3) and third, demonstration of spontaneous loss (SL) detection (Chapter 4). In the first part, the development and the implement of SE microscopy is realized with two synchronized semiconductor diode lasers. The SE signal is extracted by two different approaches of modulation, single and double modulation. The double modulation can extract SE signal by significantly improving the imaging contrast and removing spontaneous background. Rigorous theoretical estimation is also conducted by photon statistics observing Poisson distribution.
The second part elucidates the implement of subharmonic fast gating synchronization for SE microscopy, to achieve much higher modulation frequency and better signal-to-noise ratio. The high modulation frequency (~38 MHz) of the pump laser is achieved with the frequency divider (FD) circuit on the probe laser’s repetition rate, which synchronously triggers the pump laser and provides the reference for the lock-in amplifier. The greatly improved signal-to-noise ratio allows close to shot noise limited sensitivity and reduced time constant (TC)~ 0.1 ms for the lock-in detection and pixel dwell time. The subharmonic modulation is also demonstrated on a labeled biological sample.
The third part is aimed to demonstrate new methods. Note that dynamic range and detector saturation are the limiting factors for SE detection, as well as for other pump-probe contrasts. We have implemented spontaneous loss (SL) detection as a proof of concept in overcoming the above limiting factors. In this way, a high gain detector, such as a photomultiplier tube (PMT), can be selected for SE contrast. All exemplification is conducted through ATTO 647N fluorescent dye.
Table of Contents

Abstract i
Acknowledgements iii
List of Figures iv
List of Tables viii
Chapter 1 Introduction 1
1 Introduction 1
1.1 Non-linear optical imaging 2
1.2 Pump-probe microscopy 3
1.2.1 Pump-probe theory 5
1.2.2 Stimulated emission (SE) 6
Chapter 2 Dual frequency modulation for background free SE imaging 12
2 Introduction 12
2.1 Operation principle 14
2.2 Experimental design 15
2.2.1 Double modulation SE microscope setup 15
2.2.2 Sample preparation 17
2. 3 Results and discussions 17
2.3.1 Single and double modulation SE images 17
2.3.2 Theoretical SNRs estimation 20
2.4 Summary 26
Chapter 3 SE microscopy with subharmonic synchronized modulation 27
3 Introduction 27
3.1 Working principle 28
3.2 Experimental 30
3.2.1 FD and PD circuits description 30
3.2.2 Bandwidth of PD circuit 34
3.2.3 Pulse stretching 34
3.2.4 Microscope setup 36
3.2.5 Biological sample preparation 38
3.3 Results and discussions 39
3.3.1 Characteristics of SE signal 39
3.3.2 Subharmonic frequency versus SE signal 40
3.3.3 Evaluation of shot noise limited sensitivity 41
3.3.4 Subharmonic SE image 43
3.4 Summary 44
Chapter 4 Spontaneous loss detection 45
4 Introduction 45
4.1 Working principle 47
4.2 Experimental 48
4.2.1 Spectral detection scheme 48
4.2.2 Optical microscope setup 49
4.3 Results and discussion 50
4.3.1 SL signal detection 50
4.3.2 Time-resolved spontaneous loss imaging 52
4.3.3 Noise level 53
4.3.4 Signal-to-noise ratio 54
4.4 Summary 56
Chapter 5 Conclusions and future prospective 57
Bibliography 59
Appendix 66
Abbreviations 69
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