4Pi-SMS microscopy

In the simplest case, an SMS image is a histogram of sufficiently many marker positions, each being calculated from the diffraction-limited fluorescence image of a single emitter. Here, the localization in the dimension along the optic axis poses a challenge. The 4Pi-SMS microscope solves this problem by using two opposing objective lenses coherently for fluorescence detection [Aquino et al., 2011]. The analysis of at least three interference patterns for each marker, each exhibiting a different relative phase, allows precise axial localization.

The 4Pi-SMS microscope can localize markers with <10 nm precision in three dimensions in an extended layer of 650 nm thickness. It can also distinguish between different species of markers, e.g. markers of different color.

	Left: xy-SMS image of microtubules (red) and peroxisomes (blue) in an intact PtK2 cell. Right: same region with color-coded axial position. (Image from [Aquino et al., 2011])

Further information:

Aquino, D., A. Schönle, C. Geisler, C. v. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, A. Egner (2011):
"Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores"
Nature Methods, 8 (4), 353 - 359

Hell, S. W., R. Schmidt, A. Egner (2009):
"Diffraction-unlimited three-dimensional optical nanoscopy with opposing lenses"
Nature Photonics 3, 381 - 387

Middendorff, C. v, A. Egner, C. Geisler, S. W. Hell, A. Schönle (2008):
"Isotropic 3D Nanoscopy based on single emitter switching"
Opt. Expr. 16 (25), 20774-20788