High-speed Two-photon Microscopy-based 3-D Multiple-particle Tracking- Principles and Applications

顯微影像技術的進步，已使細胞的結構得以被解析至次奈米的尺度；然而關於細胞作用之各項機制，迄今卻仍存在許多待解的謎團。藉由追蹤微粒的運動，我們可以觀察物質在細胞間的傳輸，細胞在遷徙、分生或重新組織骨架時黏彈特性的變化，和許多相應之生理現象，從而為細胞功能性活動的探索提供一可行之途徑。 由於這些現象的發生多半牽涉至三維空間的活動，欲完整地分析細胞內各項作用的機制，端賴具備三維解析能力之追跡技術；此外，因為細胞間存在的歧異性，要獲致具統計意義之結果，往往需要大量的資料分析，亦導致了對多微粒追跡的需求。然而，目前除了全內反射螢光顯微術(Total Internal Reflection Fluorescent Microscopy)外；就我們所知，尚無可達視頻之三維之多微粒追跡技術。而受限於漸逝波的穿透範圍，全內反射螢光顯微術僅能應用於探索玻片下約250 nm深度內之細胞動態。 在這篇論文中，我們以高速雙光子顯微術為基礎，提出並實現了具三維解析能力的多微粒追跡系統，更近一步將其應用在細胞力學之探討。 由於雙光子激發的效率與光子流量(單位面積單位時間內通過的光子數目)成平方關係，因此激發範圍可被限定在焦點附近約1微米立方的空間，除了提供微米級軸向空間解析力外，亦大幅減少焦點外高強度光源激發所帶來的光傷害及光漂白效應。此外，由於所使用的近紅外線光源在生物樣本內的衰減遠低於用於單光子顯微術的紫外或可見光，使得雙光子顯微術在生物造影上有相當廣泛的應用。但是，在細胞動態的觀察上，以掃描為基礎的雙光子顯微術卻面臨了速度上的瓶頸；以目前的商用雙光子顯微系統而言，掃描速度約落在0.3~2 Hz之間，僅能用以觀察緩慢之細胞作用。 針對掃描速度的限制，我們利用微透鏡矩陣、讓激發光在進入物鏡前先分為多道射束，俟進入物鏡後，在樣本上產生雙光子激發之焦點陣列。配合高速掃瞄鏡系統，達成每秒30 幅影像之掃描速度，使我們可以肉眼直視即時之雙光子影像。另一方面，為了達成多微粒的三維追跡，我們在偵測光路中導入一長焦距之圓柱透鏡，利用x與y軸焦距差異所產生之像差，將微粒之空間資訊編碼於其影像中。透過校正，即可由其影像還原其三維空間資訊。在每秒10張影像的擷取速率下，徑向的定位精確度(標準誤差)優於10 nm，而軸向精確度則在20 nm左右。藉由追蹤螢光小球於預定軌跡、及在甘油溶液中自由擴散的運動，我們亦示範了此系統追蹤動態物體之響應。 此外，我們以此三維追跡系統為基礎，進行細胞力學特性之研究。透過細胞膜上附著的磁性小球，我們以磁鑷子對細胞施予應力；並藉由附著於細胞表面之螢光小球之軌跡，觀察細胞骨架在施受外力時所產生的對應變化。實驗結果顯示，細胞除在徑向上有可見之位移外，其軸向的位移亦甚為顯著；當評估細胞受力之反應時，當將各軸向應力納入整體考慮，以期建構適恰之細胞力學模型。Nowadays, the advancement in microscopy has enabled the structure of cells to be resolved at sub-nanometer scale. However, the mechanisms of cellular process still leave a lot to be understood. Tracking the movement of particles in cells is one promising way to uncover the mystery of cell mechanics. Particle tracking techniques allows us to monitor the intra-cellular transportation of materials, viscoelasticity changes during cell differentiation and migration, and other related physiological phenomena. Since these phenomena involved three-dimensional activities, three-dimensional particle tracking is required to understand the complete mechanism of cell’s response. Moreover, due to the large variance among cells, numerous data should be collected to give a statistically reliable conclusion, thus motivates the development for multiple- particle tracking techniques. So far, to our knowledge, total internal reflection fluorescent microscopy is the only technique that can achieve three-dimensional multiple particle tracking at video-rate. However, because of the penetration depth of evanescent wave, total internal reflection fluorescent microscopy can only be applied to probe the cell dynamics within 250 nm under the coverslip. In this thesis, based on high-speed two-photon microscopy, we proposed and developed a three-dimensional multiple particle tracking system, and used it to study cell mechanics. Because the probability of two-photon process is proportional to the square of the photon flux, the excitation can be localized within a 1 μm3 volume. This feature of two-photon excitation not only provides the optical sectioning capability, but also minimizes the photodamage and photobleach away from the focal plane. Furthermore, the near infrared light employed in two-photon excitation has a much less attenuation in biological specimens than the UV light used in one-photon techniques. However, a practical limitation of two-photon microscopy is the slow imaging speed. The imaging rate of commercial two-photon systems ranges from 0.3 to 2 Hz, which are unable to provide us the observation of speedy cell dynamics. To satisfy the requirement of high imaging speed, we employed a microlens array in our system. The microlens array splits the incident beam into several beamlets; the objective then focuses these beamlets into a matrix of two-photon foci on the specimens. As multiple foci are scanned over the sample simultaneously, the total scanning time can be greatly reduced. Accompanied by a high-speed galvo-mirror scanner, the system achieved a frame rate of 30 Hz, which allows us to directly view the two-photon images. Additionally, to achieve the three-dimensional multiple particle tracking, we inserted a long-focal-length cylindrical lens in the detection beampath. The aberration caused by the focal length difference between x and y axes encodes the position information of particles into the two-photon image. With a calibration process, we can recover the three-dimensional spatial information from the images. The radial precision (standard deviation) is better than 10 nm, and the axial precision is around 20 nm at a frame rate of 10 frames per second. We also demonstrated the tracking of beads moving along defined trajectories and diffusing in glycerol solutions. In addition, we applied this three-dimensional tracking system to study the cell mechanics. Via the magnetic beads attached to the cell surface, we used a magnetic tweezer to apply force on the cell. By tracking the fluorescent beads attached to the cell surface near the magnetic bead, we are able to observe the creep response of the cell. The results showed noticeable movements in both radial and axial directions, implying that the force in the axial direction, as well as in the radial direction, should be considered in building a proper model for the cellular process.Chapter 1 Introduction 1 1.1 Particle tracking 1 1.2 Two-photon microscopy 4 1.3 Scope of this thesis 6 Chapter 2 Basic Principles 11 2.1 Basic concepts of microscopy 11 2.1.1 Diffraction and resolution 11 2.1.2 Optical aberrations 17 2.2 Two-photon microscopy 23 2.2.1 Two-photon excitation 23 2.2.2 Optical properties of two-photon microscopy 25 2.2.3 Multifocal multi-photon microscopy 31 2.3 3-D particle tracking 34 Chapter 3 Development of a multi-foci two-photon microscope- based 3-D tracking system ] 40 3.1 Experimental setup 40 3.1.1 Multifocal multi-photon excitation 40 3.1.2 High-speed scanning galvo-mirror system 42 3.1.3 3-D positioning using cylindrical optics- induced aberration 43 3.1.4 Selection of imaging device 45 3.1.5 A whole view of the system design 48 3.2 Results & discussions 51 3.2.1 GM curve measurement 51 3.2.2 Precision Test 56 3.2.3 Particle tracking for beads along defined trajectories 57 3.2.4 Particle tracking for beads diffusing in glycerol 59 3.3 Conclusion 63 Chapter 4 Application in cell mechanics study 65 4.1 Introduction 66 4.2 Methods and material 68 4.2.1 Magnetic trap 68 4.2.2 Bead protocol 70 4.2.3 Cell culture 71 4.3 Results and discussions 73 4.4 Conclusion 76 Chapter 5 Summary 8