Correlating structural and photochemical heterogeneity in cyanobacteriochrome NpR6012g4.

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from Nostoc punctiforme In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequences of structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily.

Noncanonical Photodynamics of the Orange/Green Cyanobacteriochrome Power Sensor NpF2164g7 from the PtxD Phototaxis Regulator of Nostoc punctiforme.

Forward and reverse primary (<10 ns) and secondary (>10 ns) photodynamics of cyanobacteriochrome (CBCR) NpF2164g7 were characterized by global analysis of ultrafast broadband transient absorption measurements. NpF2164g7 is the most C-terminal bilin-binding GAF domain in the Nostoc punctiforme phototaxis sensor PtxD (locus Npun_F2164). Although a member of the canonical red/green CBCR subfamily phylogenetically, NpF2164g7 exhibits an orange-absorbing 15ZPo dark-adapted state instead of the typical red-absorbing 15ZPr dark-adapted state characteristic of this subfamily. The green-absorbing 15EPg photoproduct of NpF2164g7 is unstable, allowing this CBCR domain to function as a power sensor. Photoexcitation of the 15ZPo state triggers inhomogeneous excited-state dynamics with three spectrally and temporally distinguishable pathways to generate the light-adapted 15EPg state in high yield (estimated at 25-30%). Although observed in other CBCR domains, the inhomogeneity in NpF2164g7 extends far into secondary relaxation dynamics (10 ns -1 ms) through to formation of 15EPg. In the reverse direction, the primary dynamics after photoexcitation of 15EPg are qualitatively similar to those of other red/green CBCRs, but secondary dynamics involve a "pre-equilibrium" step before regenerating 15ZPo. The anomalous photodynamics of NpF2164g7 may reflect an evolutionary adaptation of CBCR sensors that function as broadband light intensity sensors.

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics.

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

Characterization of femtosecond laser-induced breakdown spectroscopy (fsLIBS) and applications for biological samples.

We characterize the femtosecond laser-induced breakdown spectroscopy (fsLIBS) signal for biological tissues as a function of different excitation parameters with femtosecond laser systems. These parameters include laser energy, depth of focus, and number of pulses per focal volume. We used femtosecond laser pulses of 800 nm and energy between 25 and 123 µJ to generate LIBS signals in biological tissues. As expected, we observed a linear increase in the fsLIBS intensity as a function of the laser energy. In addition, we show that moving the beam out of focus and the presence of overlapping pulses on the same focal area leads to a decrease in fsLIBS intensity due to changes in focal spot size. We also demonstrate that fsLIBS can distinguish between different biological tissue samples.

Primary and secondary photodynamics of the violet/orange dual-cysteine NpF2164g3 cyanobacteriochrome domain from Nostoc punctiforme.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Like phytochromes, CBCRs photointerconvert between two photostates that accompany photoisomerization of their bilin chromophores. While phytochromes typically exhibit red/far-red photocycles, CBCR photocycles are much more diverse, spanning the near-ultraviolet and the entire visible region. All CBCRs described to date have a conserved Cys residue covalently attached to the linear tetrapyrrole (bilin) chromophore; two CBCR subfamilies also exploit a second thioether linkage to the chromophore for detection of near-ultraviolet to blue light. Here, we present the photodynamic analysis of the insert-Cys CBCR NpF2164g3, a representative of the second class of two-cysteine CBCRs. Using broadband transient absorption pump-probe spectroscopy, we characterize the primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics in both directions, examining photodynamics over nine decades of time. Primary isomerization dynamics occur on a ~10 ps time scale for both forward and reverse reactions. In contrast to previous studies on Tlr0924, a representative of the other class of two-cysteine CBCRs, formation and elimination of the second linkage are slower than the 1 ms experimental range probed here. These results extend our understanding of dual-cysteine CBCR photocycles in the phytochrome superfamily.

Primary photochemistry of the dark- and light-adapted states of the YtvA protein from Bacillus subtilis.

The primary (100 fs to 10 ns) and secondary (10 ns to 100 µs) photodynamics in the type II light-oxygen-voltage (LOV) domain from the blue light YtvA photoreceptor extracted from Bacillus subtilis were explored with transient absorption spectroscopy. The photodynamics of full-length YtvA were characterized after femtosecond 400 nm excitation of both the dark-adapted D447 state and the light-adapted S390 state. The S390 state relaxes on a 43 min time scale at room temperature back into D447, which is weakly accelerated by the introduction of imidazole. This is ascribed to an obstructed cavity in YtvA that hinders access to the embedded FMN chromophore and is more open in type I LOV domains. The primary photochemistry of dark-adapted YtvA is qualitatively similar to that of the type I LOV domains, including AsLOV2 from Avena sativa, but exhibits an appreciably higher (60% greater) terminal triplet yield, estimated near the maximal ΦISC value of ≈78%; the other 22% decays via non-triplet-generating fluorescence. The subsequent secondary dynamics are inhomogeneous, with three triplet populations co-evolving: the faster-decaying (I)T* population (38% occupancy) with a 200 ns decay time is nonproductive in generating the S390 adduct state, a slower (II)T* population (57% occupancy) exhibits a high yield (Φadduct ≈ 100%) in generating S390 and a third (5%) (III)T*population persists (>100 µs) with unresolved photoactivity. The ultrafast photoswitching dynamics of the S390 state appreciably differ from those previously resolved for the type I AcLOV2 domain from Adiantum capillus-veneris [Kennis, J. T., et al. (2004) J. Am. Chem. Soc. 126, 4512], with a low-yield dissociation (Φdis ≈ 2.5%) reaction, which is due to an ultrafast recombination reaction, following photodissociation, and is absent in AcLOV2, which results in the increased photoswitching activity of the latter domain.

Note: a flexible light emitting diode-based broadband transient-absorption spectrometer.

This Note presents a simple and flexible ns-to-ms transient absorption spectrometer based on pulsed light emitting diode (LED) technology that can be incorporated into existing ultrafast transient absorption spectrometers or operate as a stand-alone instrument with fixed-wavelength laser sources. The LED probe pulses from this instrument exhibit excellent stability (∼0.5%) and are capable of producing high signal-to-noise long-time (>100 ns) transient absorption signals either in a broadband multiplexed (spanning 250 nm) or in tunable narrowband (20 ns) operation. The utility of the instrument is demonstrated by measuring the photoinduced ns-to-ms photodynamics of the red/green absorbing fourth GMP phosphodiesterase/adenylyl cyclase/FhlA domain of the NpR6012 locus of the nitrogen-fixing cyanobacterium Nostoc punctiforme.

Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches.

The aim of this article is to review the basic principles of two-photon excitation fluorescence (2PEF) microscopy and to compare the advantages and disadvantages of 2PEF imaging to other microscopy methodologies. 2PEF imaging is a nonlinear approach that generates images of optical sections and that is particularly well suited for deep-tissue and in vivo imaging of live animals. The nonlinear excitation used for 2PEF offers the advantage, too, of being able to generate contrast from second or third harmonic generation as well as coherent anti-Stokes Raman scattering. We also review the recent use of nonlinear excitation to provide image resolution beyond the diffraction limit and discuss the progress in non-scanning (planar) 2PEF microscopy, an approach that holds great potential for large-scale quantitative imaging and plate reading, e.g., in screening applications.

Versatile method for manipulating and contacting nanowires.

We present a simple, non-lithographic method for electrically connecting nanowires with electrodes on planar as well as non-planar microsystems. A rigid nanowire is used as a local shadow mask during deposition of metal contacts, which we use to contact structures of widely different conductances: Multiwalled carbon nanotubes, para-hexaphenylene nanofibers, as well as indium arsenide and indium phosphide nanowires. Finally we demonstrate how the method can be extended to different electrode materials on each side of the electrode gap, as well as for investigation of the electromechanical properties of a nanowire integrated in a cantilever.

A simple electron-beam lithography system.

A large number of applications of electron-beam lithography (EBL) systems in nanotechnology have been demonstrated in recent years. In this paper we present a simple and general-purpose EBL system constructed by insertion of an electrostatic deflector plate system at the electron-beam exit of the column of a scanning electron microscope (SEM). The system can easily be mounted on most standard SEM systems. The tested setup allows an area of up to about 50 x 50 microm to be scanned, if the upper limit for acceptable reduction of the SEM resolution is set to 10 nm. We demonstrate how the EBL system can be used to write three-dimensional nanostructures by electron-beam deposition.

Towards pick-and-place assembly of nanostructures.

We examine an approach to three-dimensional pick-and-place assembly of wire-like nanoscale components, such as carbon nanotubes and silicon nanowires, on microstructures inside a scanning electron microscope. In this article we demonstrate that microfabricated electrostatically acutuated tweezers can pick up silicon nanowires and show how electron beam deposition of carbon residues can be used to assemble carbon nanotubes on microelectrodes.

The primary photodynamics of aqueous nitrate: formation of peroxynitrite.

We have examined the photochemical reactions occurring after irradiation at 200 nm of the aqueous nitrate ion, NO3(-)(aq). Using femtosecond transient absorption spectroscopy over the range 194-388 nm, we have characterized the formation and subsequent relaxation of the primary photoproducts of nitrate photolysis. The dominant photoproduct is the cis-isomer of peroxynitrite, which accounts for 48% of the excited state molecules initially produced. A slightly smaller fraction, 44%, of the excited molecules return to the electronic ground state of NO3(-) and relax to the vibrational ground state in 2 ps. The remaining 8% of the molecules initially excited react via the *NO + *O2(-) or the NO- + O2 dissociation channels. Formation of NO2(-) and *NO2 is not observed, suggesting that the previous observations of these species in steady-state photolysis are caused by reactions occurring on a longer time scale.