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-half-maximum) 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 cholesterol-assisted 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.

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.

We present results of a two‐photon fluorescence‐correlation study carried out with glycosylated and untreated 20 nm fluorescing spheres that interacted with the carbohydrate‐binding proteins soybean agglutinin (SBA) and concanavalin A (Con A). The assay principle allows protein–carbohydrate binding interactions to be determined without protein labeling. This assay might serve as a simple model system for studying physical and chemical interactions between proteins and carbohydrates, for example, at cell or virus surfaces. In experiments with galactosylated 20 nm beads and SBA, several stages of protein–carbohydrate interactions could be clearly distinguished. Initially, only a few lectins bound to the nanospheres. At higher lectin concentrations polymerization occurred, and aggregates consisting of about 2.6×10 5 glycosylated nanospheres were formed. At very high lectin concentrations, the degree of polymerization dropped, and the size of single SBA‐covered nanospheres increased to ∼40 nm. When Con A was used instead of SBA, a significantly smaller degree of aggregation (4×10 4 spheres) was obtained. Treatment of unglycosylated 20 nm beads with SBA as a negative control sample resulted in a much lower unspecific aggregation (5×10 3 spheres). The assay principle can thus help to elucidate relative binding affinities.

We present a method for label-free microscopic analysis of nonmetallic nanoparticles such as biopolymers or technical polymers diffusing freely in an aqueous environment. We demonstrate the principal feasibility of the approach with first measurements of 20-200 nm sized polystyrene spheres and of the approximately 10 nm protein complex Photosystem I (PS I) of Thermosynechococcus elongatus. The approach is based on the combination of a microscope setup with a deep-nulling interferometer for measuring minute refractive index changes or absorptions in the focal area of the microscope. It is possible to obtain transient nulls in a microscope setup on the order of 10(-5), corresponding to optical pathway differences of less than 0.6 nm and to stabilize the nulls to about 5.10(-4). With this level of stabilization it is possible to perform a fast (1 s) correlational analysis of aqueous solutions containing the protein complex PS I or 20 nm spheres and to detect in real time single diffusional transits of larger nanospheres through the focal area of the microscope. A modulated heating of the samples in the microscope focus is not necessary for detection. The interferometer correlation data correspond well to conventional two-photon excited fluorescence correlation (FCS) data measured in the same setup, providing evidence that the detection volumes are of a similar size (approximately 200 nm). We also conducted first nulling experiments using coherent near-field sources of about 30 nm diameter. Theoretical considerations indicate that this combination with nanometric near-field sources will even allow label-free single-protein analysis.

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 β-carotene in solution. Only a small intensity of the PS 1 two-photon spectrum around its 0-0 transition indicates hot-state Car S 1 → 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 → 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 Si donors, neglecting any hot-state energy transfer. Spectral overlap calculations yield estimated vibrational ground-state Car S 1 → Chl energy-transfer time constants of 3.1-4.4 ps.