近年來，越來越多的研究員透過模式生物來學習腦部的運作方式並將其對應到人類的神經網路。在果蠅腦中，已經發現有非常多的控制基因跟功能是和人類大腦類似的。為了要研究果蠅腦的構造和功能，目前常用共軛焦顯微鏡來擷取腦部螢光切片影像並重建回立體的腦模型。因為有光學穿透深度以及會阻擋腦部光學訊號的高光學吸收係數黑色腦殼的限制，在使用共軛焦顯微鏡時需要將腦殼剝除。但這種侵入式的手術可能會對腦部的功能造成影響以及無法對同一隻果蠅的腦進行長期的觀察。在此研究當中，我們嘗試證實光學解析度光聲顯微術於表皮完整活體果蠅腦造影的可行性。不同於激發光與偵測光會被腦殼阻擋的純光學成像，我們假設果蠅表皮的聲波阻抗與水及腦組織相似，讓因受雷射激發的螢光蛋白所產生的光聲波可以穿透過腦殼，而能在表皮完整的果蠅上取得光學解析度光聲顯微腦影像。在此研究中，我們藉由量測了因果蠅表皮造成的入射光以及傳出的光聲波強度衰減還有解析度變化，以及進行活體實驗來驗證我們的假設。此外，我們亦量測了所使用的螢光蛋白在多次實驗下的光吸收衰減，以了解同一隻果蠅同一群螢光蛋白可重覆實驗的次數。整體而言，我們證實了光學解析度光聲顯微造影擁有不用進行侵入式手術就能得到光學對比且能得到共軛焦顯微術相近解析度的果蠅全腦成像潛力。

Recently, more and more researchers learn how the brain works through model organisms and map the learned knowledge to the neural network of human beings. In the Drosophila brain, it is discovered that several controlling genes of brain are highly similar to human ones, and so as how they function. To study the structure and functions of the Drosophila brain, confocal microscopy is commonly used to capture fluorescent brain slices and reconstruct individual 3-D brain models. Since the limitation of optical penetration and high optical absorption of the black head cuticle which blocks the optical signal from the brain, surgical removal of the head cuticle of Drosophila is required for confocal microscopy. However, such invasive surgery may influence brain’s functions and hinders long term monitoring. In this study, we attempt to verify the feasibility of non-invasive optical-resolution photoacoustic microscopy (OR-PAM) of cuticle intact Drosophila brain in vivo. Unlike pure optical imaging where the cuticle hinders both the optical excitation and detection, we hypothesize that the laser induced photoacoustic wave generated by the fluorescence proteins, which server as optical absorbers for OR-PAM, inside the Drosophila brain can pass through the intact cuticle because of the similar acoustic impedance of the cuticle to those of water and brain tissues. This hypothesis is verified through the measurement of the acoustic and optical attenuation of the cuticles and degradation of spatial resolution caused by the cuticles. In vivo OR-PAM of cuticle intact Drosophila brain is also performed. In addition, the optical absorption bleaching of the imaged fluorescence protein is also measured which tells the repetition number of the same experimental procedure on the same Drosophila brain. Overall, we demonstrate that OR-PAM owns a great potential in imaging the whole Drosophila brain without invasive surgery while providing optical absorption contrast and comparable resolution to confocal microscopy.

摘要.............................................................I
Abstract........................................................II
Table of Contents...............................................IV
List of Figures .............................................VI
Chapter 1 Introduction...........................................1
1.1 Drosophila and Brain Research.............................1
1.1.1 Advantages..............................................1
1.1.2 Drosophila Brain Imaging................................4
1.2 Optical-resolution Photoacoustic Microscopy...............5
1.2.1 Principles of Photoacoustic Imaging.....................5
1.2.2 Laser-scanning OR-PAM...................................7
1.2.3 Hypothesis of Non-invasive Imaging Procedure............8
1.3 Motivations...............................................9
1.4 Thesis Organization.......................................9
Chapter 2 Materials and Methods..............................11
2.1 Reflection-mode Laser-scanning OR-PAM (LSOR-PAM).........11
2.1.1 System Setup...........................................11
2.1.2 Scanning Mechanism.....................................13
2.2 Verification of Hypothesis...............................14
2.2.1 Design of the Testing Procedure........................14
2.2.2 Optical and Acoustic Attenuation of the Cuticle........16
2.2.3 Degradation in Spatial Resolution......................19
2.3 Drosophila...............................................20
2.3.1 Drosophila Preparation and Holder Design...............20
2.3.2 Selection of Fluorescent Protein.......................21
2.3.3 Experimental Procedure.................................23
2.3.4 Anatomical Plane.......................................24
Chapter 3 Experimental Results and Discussions...............25
3.1 Results of Hypothesis Test...............................25
3.1.1 Acoustic and Optical Attenuation of the Cuticle........25
3.1.2 Degradation in Spatial Resolution......................28
3.2 Drosophila Brain Imaging.................................29
3.2.1 In-vivo experiments....................................29
3.2.2 Comparison to Confocal Microscopy......................31
3.2.3 Super-resolution and 3-D Model.........................33
3.3 Discussions..............................................35
3.3.1 Distance from the Cuticle to the Air Force Target......35
3.3.2 Air Sac in Drosophila Brain............................37
3.3.3 Optical Absorption Bleaching Measurement...............39
Chapter 4 Conclusions and Future Work........................41
4.1 Conclusions..............................................41
4.2 Future work..............................................41
References......................................................44

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