Nature 466 , 647Ğ651 (29 July 2010) Alexandros Pertsinidis, Yunxiang Zhang & Steven Chu Miysaka Lab. Keisuke Yamada Contents I. Introduction II. Experiment Sample & Setup III. Results & Discussion The separation between differently coloured fluorescent molecules IV. Coclusion Optical detection Optical microscope A type of microscope which uses visible light and a system of lenses to magnify images of small samples ・ Advantage nondestructive, noncontact observe inside of a sample ・ Disadvantage diffraction-limited optics (回折限界) limiting the resolution of the microscopy (~200 nm) : resolution 0.61 NA NA : numerical aperture λ : Wavelength QuickTimeý Dz TIFFÅiîÒà• èkÅ j êLí£Év ÉçÉOÉâÉÄ Ç™Ç±ÇÃÉsÉN É`ÉÉÇ ¾å©ÇÈǞǽDžÇÕïKóvÇ Ç• ÅB Super-Resolving Microscopy The resolution power is higer than the optical microscope (≦100 nm) Example of the method ・ STimulated Emission Depletion (STED) Microscopy ・ Scanning Near Field Optical Microscopy (SNOM) ・ Photoactivated Localization Microscopy (PALM) ・ STochastic Optical Reconstruction Microscopy(STORM) ・ To identify the structure of a biological molecule ・ Nanoscale property in materials can be evaluated. STED DM DM detector Excitation laser STED laser + = Excitation spot STED spot The diameter fluorescence spot is decreased and get better resolution power SNOM ~100 nm SNOM is a kind of Scanning Probe Microscope using optical fiber as probe. PALM/STORM 生物物理50（4），174-179 藤田克昌 Super-resolution with wide-field microscope Motivation To know about the structure of multisubunit biological complexes in biologically relevant environment The spatial resolution is limited by noise and systematic effects that include drift and vibration of the measurement apparatus To improve the spatial resolution utilizing closed-loop feedback system Sample PEG silan glass silan glass glass silanize 100 m BSA coated glass biotin PEG silan glass coated with PEG coated with biotin Streptavidin biotin-PEG beads DNA Setup Closed-Loop feedback system Result 320 nm 3.2 nm The feedback systems controll the position ・ By locking the pinhole position in each image, they avoid the effect of the apparatus’ drift ・ By locking a Cy3 molecule and kept same the position, they demonstrate 0.7 nm molecule to molecule reproducibitliy Sample Polystyrene bead measurement Locked by feedback system Laser Optical Tweezers Measuring the position of the Alexa 647 and using the mapping calibration a Result c b 0.77 nm spatial resolution is achieved Conclusion ・ The Closed-roop feedback system could avoid vibration of the measurement apparatus and the combined effects of the relay imaging optics and CCD array. ・ The spatial resolution of 0.77 nm is attained for the optical microscope. ・ The closed-loop control and registration could become essential concepts in design of future sub-10-nm optical lithography tools
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