細菌可以藉由旋轉鞭毛來游向營養物質或避免毒素，其中鞭毛由稱為細菌鞭毛馬達的蛋白質複合物所驅動。細菌鞭毛馬達是一種分子機器，它在黏性環境中為游泳運動提供推進力。如同許多旋轉馬達一樣，細菌鞭毛馬達的結構也可分為轉子和定子。細菌鞭毛馬達上的定子利用儲存橫跨在細胞膜上的電化學梯度中的能量與轉子交互作用藉以產生機械扭矩。然而，鞭毛運動能量轉換的機制仍然被了解甚少。我們對離子流入與鞭毛馬達旋轉之間的關係很感興趣。有許多實驗結果提出細菌鞭毛馬達的旋轉可能與通過鞭毛馬達的離子通量緊密耦合。然而，沒有任何證據已被直接觀測到。 為了驗證細菌鞭毛馬達的緊密耦合模型，我們設計了一種新的相關顯微鏡來研究細菌鞭毛馬達的機制。相關顯微鏡結合了單細胞測量的三個重要功能，即活體單細胞鈉離子濃度的測量、即時細菌鞭毛馬達轉速的測量和馬達運動的控制。我們藉由附著在馬達萬象軸上的磁珠子來使用磁力鑷子操縱細菌鞭毛馬達的運動。背焦面偵測器可以在磁鑷施加外部扭矩時同時測量鞭毛馬達的旋轉速度。最後，細胞體內的鈉離子濃度可以通過鈉離子螢光指示劑和螢光顯微鏡觀察。 細胞內鈉離子濃度是鈉離子流進與流出的平衡。大腸桿菌具有部份鈉離子調控的能力。我們測量了當磁力鑷子加快或減慢鞭毛馬達旋轉時細胞體內鈉離子濃度的變化。結果表明在加速操作中鈉離子濃度增加了大約 20%。但是，減速操作中並未顯示鈉離子濃度發生顯著變化。我們推測鈉離子的變化可能被固定子單元的組裝所抵消了。在我們的實驗中觀察到的證據揭示了鈉離子流入與鞭毛馬達旋轉之間的緊密耦合關係。未來還需要利用相關顯微鏡來研究，用以完成理解細菌鞭毛馬達的能量使用。;Bacteria can swim to the nutrient or avoid toxin by rotating their flagella, where the flagella are driven by a protein complexes called bacterial flagellar motor. Bacterial flagellar motor is a molecular machine that produces propulsive force for swimming motility in the viscous environments. Like many rotary motors, the structure of flagellar motor can also be divided into rotor and stator. The stator of flagellar motor utilizes the energy stored in the electrochemical gradient across the cytoplasmic membrane interacting with rotor to generate mechanical torque. However, the mechanism of flagellar motor energy conversion remains poorly understood. We are interested in the relationship between the ion influx to the motor rotation. There are experimental evidences supporting that the motor rotation tightly couples with the ion flux through the motor. However, there is no direct test has been made. To verify the tight-coupling model of bacterial flagellar motors, we designed a new correlative microscope to investigate the mechanism of bacterial flagellar motor. The correlative microscope combines three important functions for single-cell measurements, which are the live single-cell sodium-ion concentration measurement, instant bacterial flagellar motor rotational speed measurement, and the motion control of motor. We used the magnetic tweezers to manipulate the motion of flagellar motor via a magnetic bead attached to its hook. The back-focal-plane detection can simultaneously measure the rotational speed of motor when an external torque is applied by the magnetic tweezers. At the same time, the intracellular sodium-ion concentration is monitored via ion fluorescence indicator using fluorescence microscope. The intracellular sodium-ion concentration is the balance of sodium influx and efflux. Escherichia coli has partial homeostasis of internal sodium-ion concentration. We measured the change of sodium-ion concentration inside a cell when the magnetic tweezers speed up or slow down the rotation of flagellar motor. The results showed an increase of approximately 20% of the sodium concentration in the accelerated operations. However, there was no significant change in intracellular sodium-ion concentration during the deceleration operations. We speculated that the change might be offset by the assembly of extra stator-units. The evidences observed in our experiment reveal the tight-coupling relationship between influx of ion to the rotation of flagellar motor. Further experiments using the correlative microscope are required for the complete understanding of bacterial flagellar motor energy transduction.
