Shigeki Iwanaga

Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.

Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels beyond the Optical Diffraction Limit

A desire to better understand the role of voltage-gated sodium channels (Na(V)s) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select Na(V) isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to Na(V)s in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective Na(V) labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal Na(V) distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.

Super resolution imaging of HER2 gene amplification

HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

Three-dimensional fluorescence imaging of particles in a glass capillary using tilted light-sheet illumination

We have attempted to image three-dimensional distribution of fluorescence beads embedded within gels in a glass capillary using light-sheet illumination, as a primary step towards developing a three-dimensional molecular imaging flow cytometer. An illumination and a detection path were arranged orthogonal to the longer axis of the capillary. The light-sheet illumination was tilted with respect to the illumination axis to image a projection of a section of the sample by a CCD. Different sections of the sample were imaged through scanning the capillary itself, along its length. By stacking the images after inverse transformation, the three-dimensional distribution of the fluorescence beads was imaged.

Gold thin layer-assisted DNA immobilization for photoelectrochemical DNA sensor

Electrochemical biosensors have been developed due to its potential to be a compact medical diagnostic devise with high sensitivity. So far we have developed a photoelectrochemical DNA sensor using transparent semiconductor films such as tin-doped indium oxide (ITO), in which probe DNAs that captures fluorescence-labeled target DNAs were immobilized on semiconductor via silane coupling reagent such as aminopropyl triethoxy silane (APTES). Here we aimed to provide an effective DNA immobilization technique using gold thin layer in order to obtain higher photocurrents to noise ratio. Gold thin film (1nm thickness) deposited over semiconductor electrode serves as a substrate to immobilize a thiol-modified DNA (24bases) at its end that can capture fluorescence-labeled target DNA by hybridization. The sensitivity in this method was approximately 4times higher than that in APTES.