Compact and versatile laser system for polarization-sensitive stimulated Raman spectroscopy.

We demonstrate a compact and versatile laser system for stimulated Raman spectroscopy (SRS). The system is based on a tunable continuous wave (CW) probe laser combined with a home-built semi-monolithic nanosecond pulsed pump Nd:YVO4 laser at 1064 nm. The CW operation of the probe laser offers narrow linewidth, low noise and the advantage that temporal synchronization with the pump is not required. The laser system enables polarization-sensitive stimulated Raman spectroscopy (PS-SRS) with fast high resolution measurement of the depolarization ratio by simultaneous detection of Raman scattered light in orthogonal polarizations, thus providing information about the symmetry of the Raman-active vibrational modes. Measurements of the depolarization ratios of the carbon-hydrogen (CH) stretching modes in two different polymer samples in the spectral range of 2825-3025 cm-1 were performed. Raman spectra are obtained at a sweep rate of 20 nm/s (84 cm-1/s) with a resolution of 0.65 cm-1. A normalization method is introduced for the direct comparison of the simultaneously acquired orthogonal polarized Raman spectra.

In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers.

Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.

Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe.

In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped "nanoprobe" with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these '"DNA tightropes" can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2's binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level.

The effect of point mutation on the equilibrium structural fluctuations of ferric Myoglobin.

The ultrafast equilibrium fluctuations of the Fe(III)-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.

ULTRA: A Unique Instrument for Time-Resolved Spectroscopy.

We report the development of a high-sensitivity time-resolved infrared and Raman spectrometer with exceptional experimental flexibility based on a 10-kHz synchronized dual-arm femtosecond and picosecond laser system. Ultrafast high-average-power titanium sapphire lasers and optical parametric amplifiers provide wavelength tuning from the ultraviolet (UV) to the mid-infrared region. Customized silicon, indium gallium arsenide, and mercury cadmium telluride linear array detectors are provided to monitor the probe laser intensity in the UV to mid-infrared wavelength range capable of measuring changes in sample absorbance of ΔOD ~ 10(-5) in 1 second. The system performance is demonstrated for the time-resolved infrared, two-dimensional (2D) infrared, and femtosecond stimulated Raman spectroscopy techniques with organometallic intermediates, organic excited states, and the dynamics of the tertiary structure of DNA.