Miysaka Lab. Keisuke Yamada Alexandros Pertsinidis, Yunxiang Zhang & Steven Chu

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 ・ Disadvantage diffraction-limited optics ( 回折限界 ) limiting the resolution of the microscopy (~200 nm) ・ Advantage nondestructive, noncontact observe inside of a sample NA : numerical aperture λ : Wavelength : resolution

Super-Resolving Microscopy ・ 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. Example of the method The resolution power is higer than the optical microscope ( ≦ 100 nm)

STED + = DM Excitation laser STED laser The diameter fluorescence spot is decreased and get better resolution power detector Excitation spot STED spot

SNOM ~100 nm SNOM is a kind of Scanning Probe Microscope using optical fiber as probe.

PALM/STORM 生物物理 50 （ 4 ）， 藤田克昌 Super-resolution with wide-field microscope

Motivation To know about the structure of multisubunit biological complexes in biologically relevant environment To improve the spatial resolution utilizing closed-loop feedback system The spatial resolution is limited by noise and systematic effects that include drift and vibration of the measurement apparatus

Sample glass silan glass silan PEG glass PEG silan biotin coated glass BSAStreptavidinbiotin-PEG beadsDNA silanize coated with PEG coated with biotin 100  m

Setup Closed-Loop feedback system

Result 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 320 nm 3.2 nm

Sample Laser Optical Tweezers Polystyrene bead measurement Locked by feedback system Measuring the position of the Alexa 647 and using the mapping calibration

Result 0.77 nm spatial resolution is achieved a b c

Conclusion ・ The spatial resolution of 0.77 nm is attained for the optical microscope. ・ 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 closed-loop control and registration could become essential concepts in design of future sub-10-nm optical lithography tools