Supplementary Methods - 4Pi microscopy
The focal spot size of any optical microscope directly depends on the
objective lense’s aperture angle. Therefore the axial resolution can be
improved 3-7 fold in the 4Pi-microscope by coherently adding the focused
wavefronts of two opposing lenses1. When coherently adding two wavefronts,
the lack of a 90° semiaperture angle spawns off at least two major satellite
spots of the point spread function, called axial sidelobes. While the ‘ghost
images’ produced by these axial sidelobes have to be taken into account
when analyzing the raw data they are well suitable for measuring the axial
extension of small objects2. A thin ribbon will produce a strongly modulated
axial profile featuring deep minima between the ‘ghost images’ while a thicker
ribbon will ‘smear out’ this modulation. For a typical two-photon excitation
wavelength of 870 nm in a 4Pi-confocal microscope, the modulation of a
ribbon with an axial extend of D = 150 nm is substantially blurred and for D
=550 nm, the modulation almost disappears. Assuming ellipsoidal or spherical
ribbons, D can be readily extracted in the range of  6n -  n , i.e. 100-650
nm.
In order to determine the average ribbon dimensions we compared the
measured size distribution with the predictions of two different models:
For spherical ribbons we would only expect a single peak, as the extension
along the optical axes is independent of the orientation in space. For an
ellipsoid the medial diameter dominates but the other two principal axes lead
to shoulders on either sides of the distribution (Fig. 1k, Supplementary Table
1). Spectral un-mixing of Alexa488 and Alexa546 fluorescence was performed
essentially as described in reference 3.
1.
2.
3.
Hell, S. & Stelzer, E. H. K. Properties of a 4Pi-confocal fluorescence
microscope. J. Opt. Soc. Am. A 9, 2159-2166 (1992).
Egner, A., Jakobs, S. & Hell, S. W. Fast 100-nm resolution 3D-microscope
reveals structural plasticity of mitochondria in live yeast. Proc. Natl. Acad.
Sci. USA 99, 3370-3375 (2002).
Zimmermann, T., Rietdorf, J., Girod, A., Georget, V. & Pepperkok, R.
Spectral imaging and linear un-mixing enables improved FRET efficiency
with a novel GFP2-YFP FRET pair. FEBS Lett 531, 245-9 (2002).