KENBIKYO Vol.52▶No.2 2017
■Feature Articles: The Forefront of Advances in Super-Resolution Microscopy

Super-Resolution Imaging by SPoD-ExPAN

Tetsuichi Wazawa, Yoshiyuki Arai and Takeharu Nagai

Abstract: SPoD-ExPAN is a super-resolution technique that uses a fluorescence microscope with illumination of patterned polarization and a reconstruction calculation of super-resolved images. In this technique the whole field from the microscope is acquired by a camera device at fast frame rates of 1–10 frames/s, and the spatial resolution is better than 100 nm. Many of conventional super-resolution microscopy techniques use illumination lights at extremely high power densities of 0.1 kW/cm2–1 GW/cm2, which unfortunately leads to serious phototoxicity to live cells and thereby difficulty in their time-lapse super-resolution observation. Here we have developed a novel super-resolution imaging technique using our recently-developed photoswithcable fluorescent protein Kohinoor and SPoD-ExPAN so that super-resolution observation can be achieved at an illumination power density of as low as ~1 W/cm2. This article presents the outline of the principle and the implementation of SPoD-ExPAN microscopy as well as the practical example of SPoD-ExPAN imaging using Kohinoor that we have performed.

Key words: fluorescence modulation, fluorescence polarization, biocompatible imaging, photoswitchable fluorescent protein, regularized maximum likelihood