The retinal photoreceptor ribbon synapse is a chemical synapse structurally and functionally specialized for the tonic release of neurotransmitter. It is characterized by the presynaptic ribbon, an electron-dense organelle at the active zone covered by hundreds of synaptic vesicles. In conventional synapses, dense-core transport vesicles carrying a set of active zone proteins are implicated in early steps of synapse formation. In photoreceptor ribbon synapses, synaptic spheres are suggested to be involved in ribbon synapse assembly, but nothing is known about the molecular composition of these organelles. With light, electron, and stimulated emission depletion microscopy and immunocytochemistry, we investigated a series of presynaptic proteins during photoreceptor synaptogenesis. The cytomatrix proteins Bassoon, Piccolo, RIBEYE, and RIM1 appear early in synaptogenesis. They are transported in nonmembranous, electron-dense, spherical transport units, which we called precursor spheres, to the future presynaptic site. Other presynaptic proteins, i.e., Munc13, CAST1, RIM2, and an L-type Ca(2+) channel alpha1 subunit are not associated with the precursor spheres. They cluster directly at the active zone some time after the first set of cytomatrix proteins has arrived. By quantitative electron microscopy, we found an inverse correlation between the numbers of spheres and synaptic ribbons in the postnatally developing photoreceptor synaptic terminals. From these results, we suggest that the precursor spheres are the transport units for proteins of the photoreceptor ribbon compartment and are involved in the assembly of mature synaptic ribbons.

New photostable rhodamine dyes represented by the compounds 1 a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N',N-bis(2,2,2-trifluoroethyl) derivatives 1 e, 1 i, 1 j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4' and 5'. Two fluorine atoms were introduced into the positions 2' and 7' of the 3',6'-diaminoxanthene fragment in compounds 1 a-d, 1 i-l and 1 m-r. The new rhodamine dyes may be excited with λ=488 or 514 nm light; most of them emit light at λ=512-554 nm (compounds 1 q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited-state lifetimes (>3 ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments.

Stimulated emission depletion (STED) fluorescence microscopy images protein distribution in cells in two‐color channels at a resolution of ≈30 nm in the focal plane (see image), corresponding to a seven‐to eightfold improvement over confocal microscopy resolution.

LSM- and OCT-measurements are efficient non-invasive tools for the characterization of morphological structures of the skin. On the one hand, the optical methods have a clear advantage in the case of kinetic measurements. On the other hand, histological investigations are characterized by a high information density and a high resolution, also in deep tissue layers. The selection of the best method for the analysis of the skin morphology depends on the target and the task of the investigation.

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