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

• Main Authors: Yen-Yu Chen, 陳彥宇
• Other Authors: Hsiao-Wen Chung
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/04051669688739329783

博士 === 國立臺灣大學 === 電機工程學研究所 === 93 === 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.