Generation of nanopores down to 10 nm for use in deep-nulling interferometry.

Scanning electron microscope images show that it is easy to generate nanopores on polycarbonate membranes with well-defined pore diameters by ion-track perforation and subsequent magnetron sputtering with metal. The size reduction of the nanopores during sputtering with gold is a linear function of time. Images of different angles and from the bottom side of the membrane show that the channels are the smallest very close to the surface of the metal layer, have a conelike shape, and reach about half as much into the polymer membranes as the metal-layer thickness. This topographical pore shape is ideal for use as optically coherent near-field sources in deep-nulling microscopy. We present the first results of significantly improved nulling stabilization in the presence (<2 nm optical pathway difference) and the absence (<0.6 nm optical pathway difference) of the nanoapertures in the focal region of a deep-nulling microscope.

A bioassay based on the ultrafast response of a reporter molecule.

The capability of using ultrafast detection technologies for a fast analysis of biomolecular reactions has been explored. As an example, the ultrafast response of tetramethylrhodamine (TMR)-labeled bovine serum albumin (BSA) as a function of different extents in proteolytic cleavage was investigated. The authors compared 4 samples of masses differing over several orders of magnitude: untreated, TMR-labeled BSA (66 kDa), TMR-labeled BSA treated with elastase (6-33 kDa) and with subtilisin (< 3 kDa), and the pure label TMR (0.4 kDa). A direct comparison with gel electrophoresis revealed that various ultrafast parameters give robust information about the progress of the proteolytic cleavage. The authors found the ratio of the transient absorption signal observed at 0 psec and 50 psec after excitation (lambda(Pump) = 540 nm, lambda(Probe) = 570 nm) to be the most precise parameter for determining the cleavage. This parameter allowed determining the mass accurately within 1 sec (Z' factor of 0.83) or 600 msec (Z' factor of 0.64), measuring time per sample. This indicates that many of the known ultrafast detection technologies might be used for monitoring biochemical reactions, probably even without any labeling procedure. The authors also discuss briefly which ultrafast processes contribute to the signals and how they are affected by changes in the biomolecular environment.

Time-resolved two-photon spectroscopy of photosystem I determines hidden carotenoid dark-state dynamics.

We present time-resolved fs two-photon pump-probe data measured with photosystem I (PS I) of Thermosynechococcus elongatus. Two-photon excitation (lambda(exc)/2 = 575 nm) in the spectral region of the optically forbidden first excited singlet state of the carotenoids, Car S1, gives rise to a 800 fs and a 9 ps decay component of the Car S1 --> S(n) excited-state absorption with an amplitude of about 47 +/- 16% and 53 +/- 10%, respectively. By measuring a solution of pure beta-carotene under exactly the same conditions, only a 9 ps decay component can be observed. Exciting PS I at exactly the same spectral region via one-photon excitation (lambda(exc) = 575 nm) also does not show any sub-ps component. We ascribe the observed constant of 800 fs to a portion of about 47 +/- 16% beta-carotene states that can potentially transfer their energy efficiently to chlorophyll pigments via the optically dark Car S1 state. We compared these data with conventional one-photon pump-probe data, exciting the optically allowed second excited state, Car S2. This comparison demonstrates that the fast dynamics of the optically forbidden state can hardly be unravelled via conventional one-photon excitation only because the corresponding Car S1 populations are too small after Car S2 --> Car S1 internal conversion. A direct comparison of the amplitudes of the Car S1 --> S(n) excited-state absorption of PS I and beta-carotene observed after Car S2 excitation allows determination of a quantum yield for the Car S1 formation in PS I of 44 +/- 5%. In conclusion, an overall Car S2 --> Chl energy-transfer efficiency of approximately 69 +/- 5% is observed at room temperature with 56 +/- 5% being transferred via Car S2 and probably very hot Car S1 states and 13 +/- 5% being transferred via hot and "cold" Car S1 states.

Correlational analysis of proteins and nonmetallic nanoparticles in a deep-nulling microscope.

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.

A two-photon excitation study on the role of carotenoid dark states in the regulation of plant photosynthesis.

Plants are exposed to sun light intensities that vary rapidly over several orders of magnitude during a typical day. It is known that the regulation of photosynthetic activity under these circumstances is essential for the survival and fitness of natural and gene modified plants. A quick balancing between utilization and dissipation of absorbed light energy ensures optimized levels of CO(2) fixation and protection from photo damage by excessive light-irradiation. Despite intensive investigations the biophysical mechanisms of these regulation processes are still poorly understood. Potentially involved singlet states of carotenoids are optically "dark" and so far it was impossible to investigate their role directly in living plants by conventional absorption or fluorescence spectroscopy. Here, we show by selective two-photon excitation of the carotenoid dark states in plant that a dominant part of the regulation is correlated with a substantial change in the energy transfer between these states and the chlorophylls (Chl). The results support a considerable role of the molecular gear shift model in which a reversible and step-wise enzymatic modification of the electronic structure of xanthophyll carotenoids enables a switching between carotenoid-to-Chl light-harvesting and Chl-to-carotenoid quenching. The shifting can be observed in real time in any plant. Treatment with the xanthophyll cycle inhibitor dithiothreitol slowed down both the light adaptation and the carotenoid-Chl energy flow changes to the same extent. Based on these results, we propose a biophysical quenching model in which both carotenoid dark states and radical cations contribute to the dissipation of excessive excitation energy.