| 摘要: | 許多細菌的運動來自於細菌鞭毛馬達。細菌鞭毛馬達是由轉子、定子和鞭毛透過自我組裝機制所構成的動力機械裝置。細菌鞭毛馬達藉由離子電動勢產生能量而進一步推動細菌鞭毛馬達旋轉，旋轉的細菌鞭毛馬達帶動細菌鞭毛在流體中產生剪力形成推進力。此外，鞭毛馬達的旋轉方向具有可切換性。當細菌體內的趨化蛋白和轉子產生鍵結，轉子的結構會產生形變，使細菌鞭毛馬達可以順時鐘旋轉或是逆時鐘旋轉。細菌鞭毛馬達藉由雙旋轉態便可以更有效率的克服熱擾動產生長距離的移動和空間探索。
本論文主要探索細菌的鞭毛運動行為和化學趨向性的研究。因此，在研究工具上必須建立「避免邊界效應干擾」的三維立體空間追跡和「細菌鞭毛運動狀態」的視覺化方法。首先，我們引入球面像差在一般的相位差顯微鏡上，使得點擴散函數在縱向產生不對稱性。因此，擷取到的影像便可以藉由已知的系統點擴散函數計算二維相關函數而重建立體軌跡資訊。然而，寬度約為20奈米的細菌鞭毛過於纖細導致對比度不足，使得一般光學顯微鏡無法輕易的觀察。因此，我們巧妙地選用帶有「鞘」的細菌鞭毛；「鞘」為外膜延伸覆蓋到細菌鞭毛上，所以其成份類似細胞膜的脂質。基於這種先天的性質，這類的細菌鞭毛便可以使用厭水性的螢光染料FM1-43標定細菌鞭毛。但是，細菌馬達的轉速非常快（大約幾百赫茲），所以大部分的科研相機都必須透過縮小觀察視野才能提升相機的幀率，而「觀察視野不足」對於研究細菌的游泳是個致命的問題。於是，我們開發了一套「頻閃照明方法」：使用資料擷取系統產生脈寬調變訊號，然後同步控制相機拍照跟激發光源的曝光時間長度。這種拍攝方式的最大優點在於：可以在相機幀率不高（33-100赫茲）的情況下，保持最大的觀察視野，並且清楚地記錄此刻（150-200微秒）的鞭毛運動組態。
目前已知的細菌游泳模式有大腸桿菌的「前進和翻滾」、溶藻弧菌的「前進、後退和偏折」以及有些菌會「纏繞」。這些不同的細菌使用不同的運動策略更有效率地達成生物上的目標。本論文選用費氏弧菌作為研究的生物系統。它具有多條鞭毛，鞭毛的出現位置不像大腸桿菌分散在身體周圍，而是像溶藻弧菌集中在身體的其中一端。透過我們建立的分析方法，我們發現費氏弧菌：（1.）前進和後退的游泳速率是不一樣的；（2.）在不同黏稠程度的環境採取不同的運動策略，低黏稠環境是採用「前進和後退」模式，而高黏稠環境採用「前進和纏繞」模式；（3.）在高黏稠環境並且所有鞭毛馬達同步旋轉方向時，發現全新的「纏繞」游泳模式：在「纏繞」狀態使細菌鞭毛構型可以直接左右旋構型轉換，而大幅延長倒退的時間。最後，我們完整地建立費氏弧菌的運動模式轉換關係，並且透過數值模擬的方式揭露出：在高黏滯的環境，費氏弧菌大幅度延長倒退時間的行為是為了更有效率的增加化學趨向的能力。
;Many motile bacteria are driven by the bacterial flagellar motor (BFM). The BFM consists of a rotor, a stator, and a filament through a self-assembly mechanism. The BFM is powered by a specific ion motive force (IMF) that generates torque and propulsion for the bacteria. Also, a BFM is a rotational switchable two-state motor. The rotor switches between counterclockwise/clockwise (CCW/CW) rotation when intracellular chemotaxis signaling proteins bind/unbind to the rotor of the BFM (C-ring). As a result, bacteria can overcome thermal fluctuations and navigate a wide range of environments.
In this thesis, we aim to study the motility and chemotaxis of lophotrichous bacteria. We have developed a 3-dimensional tracking method and real-time visualization of flagella during swimming. In phase-contrast 3D tracking, we introduce spherical distortion into phase-contrast microscopy to induce the point spread function (PSF) asymmetry on the z-axis. The z-position of bacteria can be mapped by computing the 2D cross-correlation between the target image and the pre-labeled depth database. Therefore, we can reconstruct spatial trajectory information. However, standard optical microscopes cannot easily observe these 20 nm thin flagellar filaments due to the low scattering cross-section. Therefore, we take the advantage of sheathed flagella that is contiguous with the outer membrane for rapid fluorescence labeling by the lipophilic dye. To capture the flagellar filament configuration during the swimming at several hundred Hz rotations in the wild field of view, we developed the strobe lighting method. We integrate and synchronize cameras and lights through NI-DAQ. Send a series of pulse-width modulation (PWM) signals through NI-DAQ to trigger the devices simultaneously. Thus, we can achieve the imaging of a large field of view (> 1024 pixels) at low frame rates (< 100 Hz) with clear rotating flagellar filament configuration within 200 microseconds (> 5000 Hz).
Known swimming patterns are run-and-tumble in Escherichia coli, run-reverse in Vibrio alginolyticus, and push-wrap in some bacteria. Different swimming patterns allow bacteria to efficiently navigate various environments. We chose A. fischeri, which is a lophotrichous system, as our study system. We discovered two alternative speeds for forward and backward motion through phase-contrast 3D tracking. Then, A. fischeri faced low- and high-viscosity environments using push-pull and push-wrap modes. In addition, A. fischeri synchronizes motor switching and polymorphic changes to prolong backward duration. Finally, we constructed a complete swimming pattern of A. fischeri by fluorescence strobe illumination microscopy. Our simulations then showed that prolonged backward run time enables efficient chemotactic navigation at high viscosity. |
