We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (∼10-20 nm standard deviation or ∼20-40 nm full-width-at-halfmaximum) and temporal resolution (<1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on <20 nm spatial scales. The trapping is cholesterolassisted and much more pronounced for a sphingo-than for a phosphoglycero-lipid, with average trapping times of ∼15 ms and <4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization. M any open questions in biology can be tackled only if the dynamics of individual molecules can be observed noninvasively in vivo and at the appropriate spatial and temporal scale (1-5). Over the years, specific labeling of the cell's constituent parts with fluorescent markers has enabled deeper understanding in many areas of cell biology and allowed, for example, the spatiotemporal tracking of single particles (6, 7). However, to reach the desired spatial and temporal accuracy, single-particle tracking often applies bright but large and clumsy signal markers, which potentially influence the system under study. One notable example is the dynamics of proteins and lipids in cellular membranes and their organization into nanodomains, so-called "lipid rafts" (8-12). Since their proposal, the question of the existence and functional role of such cholesterol-mediated lipid assemblies has caused much controversy (13-16). Owing to the lack of suitable noninvasive techniques to detect these nanodomains in living cells, their spatial extent has been estimated at somewhere in the range of 5-200 nm (17), that is between molecular dimensions and the resolution limit of conventional fluorescence microscopy. Camera-based tracking of single lipids could provide more detailed insight (18)(19)(20). However, so far it either lacked temporal resolution or it made use of rather large gold beads as lipid labels, implying that the measurements may not give comprehensive answers to the open questions. Recently, stimulated-emission-depletion (STED) nanoscopy (21, 22), delivering subdiffraction resolution in live cells, provided direct evidence that certain lipids are transiently trapped in cholesterol-assisted molecular complexes (23, 24). In those experiments, which are evaluate...

Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.

In this work, we present the two-photon excitation spectrum and two-photon-sensitized fluorescence spectra of photosystem I (PS I) of Thermosynechococcus elongatus. The two-photon excitation spectrum of PS I agrees well with known spectra of the carotenoid (Car) S 1 state of -carotene in solution. Only a small intensity of the PS I two-photon spectrum around its 0-0 transition indicates hot-state Car S 1 f chlorophyll (Chl) energy transfer. The two-photon-sensitized fluorescence spectrum of PS I shows no major difference of the intensities of bulk-and red-chlorophyll fluorescence in comparison to the fluorescence spectrum observed after nonselective one-photon excitation of the PS I chlorophylls. Fluorescence spectra measured after selective excitation of red chlorophylls show a decreased fluorescence of bulk chlorophylls around 685 nm, indicating that the thermal equilibration of the excitations between bulk and red chlorophylls is not complete prior to emission. Taking these results together, there seems to be almost no preference for Car S 1 f red chlorophyll energy transfer, even though this could have been energetically favorable. We conclude that the small number of red chlorophylls is not sufficient to act as major acceptors for Car S 1 energy. The fact that no increased red-chlorophyll emission is observed after two-photon excitation also provides a strong indication that no two-photon-allowed excitonic states can be formed from chlorophyll-chlorophyll or carotenoid-chlorophyll interactions. A comparison of the measured two-photon data with experimental and theoretical chlorophyll energies yields a fraction of ∼40% carotenoids that are potential Car S 1 donors, neglecting any hot-state energy transfer. Spectral overlap calculations yield estimated vibrational ground-state Car S 1 f Chl energytransfer time constants of 3.1-4.4 ps.

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON-and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of singlemolecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range. Microsc. Res. Tech. 70:1003-1009, 2007