Höchstauflösende und ultrasensitive Lichtmikroskopie zur Untersuchung zellulärer Prozesse

High-Resolution Imaging of Specific Celular Structures Through Partial Photoconversion of EosFP

4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. The high resolution allows investigation of organelles and structures too small for the resolution limits of conventional widefield and confocal microscopes.

We apply 4Pi microscopy to cellular imaging with EosFP, a photoconvertable autofluorescent protein, the fluorescence emission wavelength of which can be irreversibly switched from green (516 nm) to red (581 nm) by irradiation with 400 nm light (fig. 1).

fig. 1

Photoswitchable proteins like EosFP enable the combination of 3D optical marking of subcellular regions with subsequent high resolution 4Pi confocal microscopy. For efficient two-photon excitation in 4Pi microscopy and two-photon conversion in 3D-localized optical marking, a thorough characterization of the respective nonlinear properties of the marker protein is required. Therefore, we have measured the spectral dependencies of two-photon excitation (fig. 2a) and two-photon conversion (fig. 2b) of the green form of EosFP as well as the two-photon excitation cross-sections for both the green and the red forms.

fig. 2a		fig. 2b

The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. This knowledge allows us to select optimal imaging parameters for cellular applications of EosFP. Shown as an example is the optical highlighting of small subcellular compartments in HeLa cells expressing EosFP fused to a mitochondrial targeting signal (fig. 3).

Confocal Excitation at 488 nm		4Pi deconvoluted TPE at 970 nm
fig. 3

After two-photon conversion of a small subregion within the mitochondrial network of HeLa cells, the photoactivated volume was resolved in a 3D reconstruction from a dual-color 4Pi image stack (fig. 4).

	Conversion at 810nm, Excitation at 970nm
fig. 4