Postdoc in the group of Ernst H. K. Stelzer,
Buchmann Institute for Molecular Life Science (BMLS), Goethe University, Frankfurt am Main, Germany
Postdoc in the group of Su-Yu Chiang,
Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Taiwan, R.O.C.
Ph.D. student in the group of Long Hsu,
Institute of Electro-Optical Engineering, National Chiao Tung University, Taiwan, R.O.C.
Thesis: “Design and study of an optical tweezers system for measuring adhesion and extension properties of biological materials”
Graduate student in the group of Long Hsu,
Department of ElectroPhysics, National Chiao Tung University, Taiwan, R.O.C.
|1995–1999||Student in Department of ElectroPhysics, National Chiao Tung University, Taiwan, R.O.C.|
Development of coherent structured illumination light sheet based-fluorescence microscope (csiLSFM)
Modern fluorescence microscope requires high resolution, low photo-damage, live imaging, and in vivo observation. Nevertheless, a compromise has always to be made in between these requirements. In this project, we combine the structured illumination microscopy and light sheet microscopy.
Structured illumination microscope (SIM) has always been an attractive option for high-resolution imaging with a moderate excitation intensity as well as higher acquisition speed as compared to other super-resolution techniques, e.g. STED, GSD, d/STORM, and PALM. Nevertheless, photo-damage is still accumulated when recording a 3D image stack. In SIM, the photo-damage grows dramatically with the desired number of planes because it requires at least 9 to 15 raw images on each plane. For instance, the last plane of an image stack containing 100 planes has been illuminated by 1485 (99×15) times before it is actually imaged. This is because the optical arrangement of a conventional microscope that the illumination and detection are collinear thus, whenever a single plane is imaged the entire specimen is illuminated. In contrast, Light sheet-based fluorescence microscope (LSFM) uses a light sheet to illuminate the specimen from one side and observes the fluorescence at an angle of 90°. The light sheet overlaps the detection focal plane thus only the observed plane is illuminated. Consequently, the photo-damage is dramatically reduced when axially scanning the specimen through the focal plane to form a 3D image stack. In addition, LSFM has the intrinsic optical sectioning ability by taking the advantage of no out of focus fluorescence is excited.
The combination of SIM and LSFM is inspired because they have lots in common. They are both based on the wide-field illumination and detection schemes. Both techniques use a CCD camera to record the image, pursue for low photo-damage, and require no special fluorophores thus multiple colors and in vivo imaging are possible. By taking the advantages of both techniques proposes a new microscope features with high-resolution imaging, as well as low photo-damage, and 3D image observation. Additionally, the specimen is held in a 3D environment so a biological specimen can behaves closer to its physiological (natural) condition, as suggested with a conventional LSFM. The intrinsic optical sectioning ability of LSFM also makes it possible for rapid frame rate of 2D live imaging without the disturbance of out of focus fluorescence, i.e. improved image contrast and quality.
We believe this csiLSFM features super resolution, fast imaging, low photo-toxicity and photo-bleaching, and true optical sectioning; as well as being able to take images deep inside physiological relevant 3D biological specimens. This microscope will also bring an impact for both structured illumination and light sheet based techniques.
B.-J. Chang, Y.-J. Huang, C.-H. Chan, L. Hsu, H.-L. Peng, H.-Y. Chang, T.-R. Yew, C.-H. Liu, and S. Chi, "Measurement of the adhesive force between a single Klebsiella pneumoniae type 3 fimbria and collagen IV using optical tweezers", Biochem. Biophys. Res. Commun. 350, 33-38 (2006).
C.-F. Hsieh, B.-J. Chang, C.-H. Pai, H.-Y. Chen, J.-W. Tsai, Y.-H. Yi, Y.-T. Chiang, D.-W. Wang, S. Chi, L. Hsu, and C.-H. Lin, "Stepped Changes of Monovalent Ligand-binding Force during Ligand-induced Clustering of Integrin αIIBβ3", J. Biol. Chem. 281, 25466-25474 (2006).