Resonance Raman Spectroscopy Measurements Research Articles

Resonance Raman spectra and excited state properties of methyl viologen and its radical cation from time-dependent density functional theory.

Time-dependent density functional theory (TDDFT) was applied to gain insights into the electronic and vibrational spectroscopic properties of an important electron transport mediator, methyl viologen (MV2+ ). An organic dication, MV2+ has numerous applications in electrochemistry that include energy conversion and storage, environmental remediation, and chemical sensing and electrosynthesis. MV2+ is easily reduced by a single electron transfer to form a radical cation species (MV•+ ), which has an intense UV-visible absorption near 600 nm. The redox properties of the MV2+ /MV•+ couple and light-sensitivity of MV•+ have made the system appealing for photo-electrochemical energy conversion (e.g., solar hydrogen generation from water) and the study of photo-induced charge transfer processes through electronic absorption and resonance Raman spectroscopic measurements. The reported work applies leading TDDFT approaches to investigate the electronic and vibrational spectroscopic properties of MV2+ and MV•+ . Using a conventional hybrid exchange functional (B3-LYP) and a long-range corrected hybrid exchange functional (ωB97X-D3), including with a conductor-like polarizable continuum model to account for solvation, the electronic absorption and resonance Raman spectra predicted are in good agreement with experiment. Also analyzed are the charge transfer character and natural transition orbitals derived from the TDDFT vertical excitations calculated. The findings and models developed further the understanding of the electronic properties of viologens and related organic redox mediators important in renewable energy applications and serve as a reference for guiding the interpretation of electronic absorption and Raman spectra of the ions.

Probing Enhanced Electron-Phonon Coupling in Graphene by Infrared Resonance Raman Spectroscopy.

We report on resonance Raman spectroscopy measurements with excitation photon energy down to 1.16eV on graphene, to study how low-energy carriers interact with lattice vibrations. Thanks to the excitation energy close to the Dirac point at K, we unveil a giant increase of the intensity ratio between the double-resonant 2D and 2D^{'} peaks with respect to that measured in graphite. Comparing with fully abinitio theoretical calculations, we conclude that the observation is explained by an enhanced, momentum-dependent coupling between electrons and Brillouin zone-boundary optical phonons. This finding applies to two-dimensional Dirac systems and has important consequences for the modeling of transport in graphene devices operating at room temperature.

6,6'-Ditriphenylamine-2,2'-bipyridine: Coordination Chemistry and Electrochemical and Photophysical Properties.

A 2,2'-bipyridine with bulky triphenylamine substituents in the 6 and 6' positions of the ligand (6,6'-ditriphenylamine-2,2'-bipyridine, 6,6'-diTPAbpy) was generated. Despite the steric bulk, the ligand readily formed bis(homoleptic) complexes with copper(I) and silver(I) ions. Unfortunately, efforts to use the 6,6'-diTPAbpy system to generate heteroleptic [Cu(6,6'-diTPAbpy)(bpy)]+ complexes were unsuccessful with only the [Cu(6,6'-diTPAbpy)2](PF6) complex observed. The 6,6'-diTPAbpy ligand could also be reacted with 6-coordinate metal ions that featured small ancillary ligands, namely, the [Re(CO)3Cl] and [Ru(CO)2Cl2] fragments. While the complexes could be formed in good yields, the steric bulk of the TPA units does alter the coordination geometry. This is most readily seen in the [(6,6'-diTPAbpy)Re(CO)3Cl] complex where the Re(I) ion is forced to sit 23° out of the plane formed by the bpy unit. The electrochemical and photophysical properties of the family of compounds were also examined. 6,6'-diTPAbpy exhibits a strong ILCT absorption band (356 nm, 50 mM-1 cm-1) which displays a small increase in intensity for the homoleptic complexes ([Cu(6,6'-diTPAbpy)2]+; 353 nm, 72 mM-1 cm-1, [Ag(6,6'-diTPAbpy)2]+; 353 nm, 75 mM-1 cm-1), despite containing 2 equiv of the ligand, attributed to an increased dihedral angle between the TPA and bpy moieties. For the 6-coordinate complexes the ILCT band is further decreased in intensity and overlaps with MLCT bands, consistent with a further increased TPA-bpy dihedral angle. Emission from the 1ILCT state is observed at 436 nm (τ = 4.4 ns) for 6,6'-diTPAbpy and does not shift for the Cu, Ag, and Re complexes, although an additional 3MLCT emission is observed for [Re(6,6'-diTPAbpy)(CO)3Cl] (640 nm, τ = 13.8 ns). No emission was observed for [Ru(6,6'-diTPAbpy)(CO)2Cl2]. Transient absorption measurements revealed the population of a 3ILCT state for the Cu and Ag complexes (τ = 80 ns). All assignments were supported by TD-DFT calculations and resonance Raman spectroscopic measurements.

Fabrication of vertical van der Waals gap array using single-and multi-layer graphene

Arrays of van der Waals gaps were manufactured by synthesizing the vertically aligned graphene layer stacked between two copper (Cu) catalytic films. The Cu–graphene–Cu laminated structure was obtained by directly synthesizing graphene on a patterned Cu film followed by depositing a second copper layer for optical measurements. The synthesis of graphene on the Cu surface was optimized by adjusting the synthesis temperatures and pre-annealing time using plasma enhanced chemical vapor deposition (PECVD). Resonant Raman spectroscopy measurements reveal that graphene can be synthesized on both bulk Cu foil and relatively thin Cu film under the same growth mechanism using PECVD. Structural and optical characterizations of the array of graphene van der Waals gaps were implemented by the transmission electron microscope and terahertz-time domain spectroscopy (THz–TDS). In THz–TDS, the measured THz amplitude transmitted through the graphene van der Waals gap slit array was constant regardless of the gap width determined by the number of graphene layers between the Cu thin films in a single slit. These results imply that the optical dielectric constant of graphene at THz frequencies in the out-of-plane direction is linearly proportional to the gap width. Our results of the manufacturing method can be adopted to investigate mechanical, electrical, and optical properties of other 2D materials such as h-BN, MoS2, and others. Furthermore, metal-graphene-metal structures with vertical orientations can be used in many electronic, optic, and optoelectronic applications.

Front Cover: Assessment of trans‐scleral iontophoresis delivery of lutein to the human retina (J. Biophotonics 3/2018)

Trans‐scleral iontophoresis device was shown to be effective for in‐situ delivery of lutein to the retina of human donor eyes. After treatment, Resonance Raman Spectroscopy measurements demonstrated that lutein greatly enriched the inner sclera, choroid and retina. Clinical studies are going to prove if the methodology would be a valuable approach to enrich the human macular pigment and prevent local oxidative damage in patients at risk of AMD progression.Further details can be found in the article by Marco Lombardo et al. (e201700095). image

Correlations Between Macular, Skin, and Serum Carotenoids.

PurposeOcular and systemic measurement and imaging of the macular carotenoids lutein and zeaxanthin have been employed extensively as potential biomarkers of AMD risk. In this study, we systematically compare dual wavelength retinal autofluorescence imaging (AFI) of macular pigment with skin resonance Raman spectroscopy (RRS) and serum carotenoid levels in a clinic-based population.MethodsEighty-eight patients were recruited from retina and general ophthalmology practices from a tertiary referral center and excluded only if they did not have all three modalities tested, had a diagnosis of macular telangiectasia (MacTel) or Stargardt disease, or had poor AFI image quality. Skin, macular, and serum carotenoid levels were measured by RRS, AFI, and HPLC, respectively.ResultsSkin RRS measurements and serum zeaxanthin concentrations correlated most strongly with AFI macular pigment volume under the curve (MPVUC) measurements up to 9° eccentricity relative to MPVUC or rotationally averaged macular pigment optical density (MPOD) measurements at smaller eccentricities. These measurements were reproducible and not significantly affected by cataracts. We also found that these techniques could readily identify subjects taking oral carotenoid-containing supplements.ConclusionsLarger macular pigment volume AFI and skin RRS measurements are noninvasive, objective, and reliable methods to assess ocular and systemic carotenoid levels. They are an attractive alternative to psychophysical and optical methods that measure MPOD at a limited number of eccentricities. Consequently, skin RRS and MPVUC at 9° are both reasonable biomarkers of macular carotenoid status that could be readily adapted to research and clinical settings.

Skin Carotenoids as Biomarker for Vegetable and Fruit Intake: Validation of the Reflection‐Spectroscopy Based “Veggie Meter”

Skin is a relatively stable storage medium for carotenoids; non‐invasive optical measurements of carotenoids in this tissue via Resonance Raman spectroscopy (RRS) serve as a non‐invasive biomarker for fruit and vegetable (F/V) intake. The RRS method has been validated with HPLC‐based measurements of carotenoid concentrations in serum and excised skin tissue samples and has been shown in feeding studies to respond to changes in F/V intake.An attractive alternative to RRS is based on reflection spectroscopy (RS), which requires less complex instrumentation and has an advantage in also measuring skin melanin and hemoglobin absorptions. Using a suitable algorithm, the latter can be taken into account in the RS‐based derivation of skin carotenoid levels. Furthermore, these levels can be quantified in optical density units, which directly correlate with the carotenoid concentrations encountered in the measured tissue volume. The objective of this study was to validate the RS method via comparison with the RRS method.We compared RS and RRS measurements in 5 diverse human subject cohorts of different age groups and ethnicities in community and clinical settings. These measurements included inter‐subject comparisons of carotenoid levels in 146 adults and children, tracking of carotenoid changes in response to dietary interventions in 29 adults, and an ongoing exploration of a potential correlation between skin and retinal carotenoid levels in adult patients with vision disorders. Depending on the particular study, correlation coefficients varied between R ~ 0.80 and 0.96, showing that the RS method provides a valid substitute for RRS in skin carotenoid measurements of all age groups. A compact, portable instrument configuration will be presented, which permits rapid non‐invasive measurement and tracking of F/V derived carotenoid levels. In its capacity as “Veggie Meter” it should be attractive for screening of large populations and the assessment of the effectiveness of F/V intake interventions.Support or Funding InformationSupport for this research was provided in part by the Paso del Norte Health Foundation and the USDA‐ARS.

Using Skin Carotenoids to Assess Potential Dietary Changes After One Academic Year in the Shaping Healthy Choices Program

Reported dietary intake is often used in community interventions to assess intake of fruits and vegetables (F/V); however, dietary assessment methods are inaccurate, and time and labor intensive. Skin carotenoids are a potential biomarker to assess F/V intake given that carotenoids are predominately found in F/V and their concentration in the skin can reflect dietary intakes of F/V. The Resonance Raman Spectroscopy (RRS) method is a quick and validated method to assess skin carotenoids. The objective of this study was to determine whether skin carotenoids measured by RRS can be used to confirm changes in dietary intake of F/V in 4th‐grade students participating in the Shaping Healthy Choices Program (SHCP). The SHCP is a multi‐component nutrition intervention and one of its aims is to increase F/V intake. Dietary intake was assessed using the Block Food Frequency Questionnaire in a subset of students (n=30) and skin carotenoids were measured using RRS (n=82) before and after the SHCP intervention. Pre‐post changes in F/V intake measured by each method were tested using Student's t‐tests. After 9 months, reported intake of carotenoids decreased by 19% (1469mcg, P=0.05) and skin carotenoids decreased by 9% (2882 RRS intensity units, P<0.001). Change in reported intake correlated with change in skin carotenoids (r=0.43, P=0.02). While the reported decrease in F/V intake in this population was unanticipated given that the intervention aimed to increase intake, the RRS measurements confirmed this change. In conclusion, optically measured skin carotenoids can be used to help evaluate changes after 9 months of F/V intake in 4th‐grade students.Support or Funding InformationFunding was provided by UCANR competitive grant #11‐1018, USDA 2011‐38420‐20082, and USDA NIFA, Hatch project 221082.

Quantitative Comparison of Organic Photovoltaic Bulk Heterojunction Photostability Under Laser Illumination

The photostability of bulk heterojunction organic photovoltaic films containing a polymer donor and a fullerene-derivative acceptor was examined using resonance Raman spectroscopy and controlled laser power densities. The polymer donors were poly(3-hexylthiophene-2,5-diyl) (P3HT), poly[[9-(1-octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophenediyl] (PCDTBT), or poly({4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b′]dithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl}) (PTB7). Four sample preparation methods were studied: (i) thin or (ii) thick films with fast solvent evaporation under nitrogen, (iii) thick films with slow solvent evaporation under nitrogen, and (iv) thin films dried under nitrogen followed by thermal annealing. Polymer order was assessed by monitoring a Raman peak’s full width at half-maximum and location as a function of illumination time and laser power densities from 2.5 × 103 to 2.5 × 105 W cm–2. Resonance Raman spectroscopy measurements show that before prolonged illumination, PCDTBT and PTB7 have the same initial order for all preparation conditions, while P3HT order improves with slow solvent drying or thermal annealing. All films exhibited changes to bulk heterojunction structure with 2.5 × 105 Wcm–2 laser illumination as measured by resonance Raman spectroscopy, and atomic force microscopy images show evidence of sample heating that affects the polymer over an area greater than the illumination profile. Photostability data are important for proper characterization by techniques involving illumination and the development of devices suitable for real-world applications.

Resonance Raman spectroscopic measurements delineate the structural changes that occur during tau fibril formation.

The aggregation of the microtubule-associated protein, tau, into amyloid fibrils is a hallmark of neurodegenerative diseases such as the tauopathies and Alzheimer's disease. Since monomeric tau is an intrinsically disordered protein and the polymeric fibrils possess an ordered cross-β core, the aggregation process is known to involve substantial conformational conversion besides growth. The aggregation mechanism of tau in the presence of inducers such as heparin, deciphered using probes such as thioflavin T/S fluorescence, light scattering, and electron microscopy assays, has been shown to conform to ligand-induced nucleation-dependent polymerization. These probes do not, however, distinguish between the processes of conformational conversion and fibril growth. In this study, UV resonance Raman spectroscopy is employed to look directly at signatures of changes in secondary structure and side-chain packing during fibril formation by the four repeat functional domain of tau in the presence of the inducer heparin, at pH 7 and at 37 °C. Changes in the positions and intensities of the amide Raman bands are shown to occur in two distinct stages during the fibril formation process. The first stage of UVRR spectral changes corresponds to the transformation of monomer into early fibrillar aggregates. The second stage corresponds to the transformation of these early fibrillar aggregates into the final, ordered, mature fibrils and during this stage; the processes of conformational conversion and the consolidation of the fibril core occur simultaneously. Delineation of these secondary structural changes accompanying the formation of tau fibrils holds significance for the understanding of generic and tau-specific principles of amyloid assembly.

Effects of Proton Motive Force on the Structure and Dynamics of Bovine Cytochrome c Oxidase in Phospholipid Vesicles

A conventional method for reconstituting cytochrome c oxidase (CcO) into phospholipid vesicles (COV) has been modified to permit resonance Raman (RR) analysis in the presence and absence of proton motive force (ΔμH(+)). The COV has an average diameter of 20 nm and contains one CcO molecule within a unified orientation with CuA located outside the COV. The process of generation of ΔμH(+) across the membrane was monitored spectrophotometrically with rhodamine123 dye. The COV exhibits a respiratory control ratio (RCR) value of >30 and is tolerant to RR measurements with 10 mW laser illumination for 60 min at 441.6 nm. Structural perturbations at the heme sites caused by incorporation into vesicles were clarified by spectral comparisons between solubilized CcO and COV. Absorption spectroscopy revealed that the rate of electron transfer from cytochrome c to O2 is reduced significantly more in the presence of ΔμH(+) than in its absence. RR spectroscopic measurements indicate that CcO in COV in the "respiratory-controlled" state adopts a mixed-valence state (heme a(2+) and heme a3(3+)). This study establishes a supramolecular model system for experimentally examining the energy conversion protein machinery in the presence of ΔμH(+).

Effect of age and other factors on macular pigment optical density measured with resonance Raman spectroscopy

Macular pigment is a defense system against phototoxic damage of the retina by visible light. It is still under debate whether or not macular pigment optical density (MPOD) levels decline with age, because the age effect varied depending on the technique used to measure MPOD levels. Resonance Raman spectroscopy (RRS) is an objective method to measure MPOD, and studies using RRS showed a drastic age-related decline of MPOD levels; however, since RRS measurements are influenced by cataracts, it has been argued that the age-related decline of RRS measurements is an artifact from lens changes in aged subjects. In the present study, MPOD levels were measured with RRS in pseudophakic eyes, and the effects of age and other factors on MPOD levels were investigated. The subjects included 144 patients with no fundus disorders who received cataract surgery with untinted intraocular lens implantation. MPOD levels were measured in 144 eyes using integral RRS 1day post surgery. Factors potentially associated with MPOD levels such as age, gender, smoking habits, body mass index, diabetes, glaucoma, axial length, pupil diameter, spherical equivalent refractive error, and foveal thickness were examined by multiple regression analysis. The macular pigment RRS levels ranged from 776 to 11,815 Raman counts, with an average level of 4,375 ± 1,917 (standard deviation [SD]) Raman counts. Multiple regression analysis revealed that age and axial length were significantly correlated with low MPOD values (regression coefficient of -59 for age and -404 for axial length, respectively). No significant correlations were observed for other factors. After removing the potentially confounding effect of age-related lens yellowing on the RRS measurements, age remained a significant patient parameter for lowered MPOD levels. MPOD levels were found to decline by more than 10% each decade. Axial length was also a negative predictor of MPOD levels. Since the present study included only patients aged 50years and older, the effects of age and other factors on MPOD levels for younger subjects remain unknown.

Tunable Electric Field Enhancement and Redox Chemistry on TiO2 Island Films via Covalent Attachment to Ag or Au Nanostructures

Ag–TiO2 and Au–TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b5 (Cyt b5) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b5 were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s–1) of Cyt b5 to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film.

The influence of endurance exercise on the antioxidative status of human skin

Oxidative stress is supposed to be responsible for a diversity of diseases. For protection purposes, the human organism exhibits a line-up of antioxidant substances functioning as radical catchers. As a result of neutralization of free radicals, antioxidants are destroyed. Therefore, the degradation of the antioxidants can be utilized as an indirect parameter for the measurement of free radical formation. As physical exercise may also induce oxidative stress, the aim of the present study was to determine the antioxidant substances, and more precisely, the carotenoid concentration in the skin of male volunteers during different sportive exposures (cycling and running with two different exercise intensities) with resonance Raman spectroscopic measurements. The results revealed that moderate and high intensity cycling and running decrease the carotenoid concentration of the skin, whereas both sport disciplines and both exercise intensities revealed similar results. It can be concluded that above a certain threshold, physical exercise leads to oxidative stress also in the skin associated with the decrease in the antioxidant concentration. This gives rise to the impairment of the first defence line of the skin and means an increase in the risk of sun exposure-induced damage, e.g., when exercise training is performed outside. Nevertheless, it has to be emphasized that sport in general applied at moderate loads has predominantly positive effects on the health of humans especially concerning cardiovascular and metabolic diseases.

Raman spectroscopic analysis of the carotenoid concentration in egg yolks depending on the feeding and housing conditions of the laying hens

Resonance Raman spectroscopic measurements are suited to analyze the concentration of carotenoid antioxidants in biological samples. Previously, it has been shown that the carotenoid concentration of nutritional egg yolks has a direct influence on the carotenoid content of human skin in vivo. In the present study, resonance Raman spectroscopy was used to analyze the carotenoid concentration in the yolks of hen eggs, which were housed in battery cages or alternatively on free-range grassland. The egg yolks of hens, which had access to grassland, contained approximately double the amount of carotenoid concentration than the egg yolks of hens housed in battery cages (p < 0.001). The kinetics of the carotenoid concentration in the egg yolks, depending on fodder, housing and weather conditions, were investigated.

Expression and Spectroscopic Characterization of Melanopsin and Squid Rhodopsin

Melanopsin, found in intrinsically photosensitive retinal ganglion cells, drives circadian rhythm and pupillary response. A variety of studies indicate that melanopsin shares many properties with invertebrate rhodopsin such as found in the eyes of squid including activation by an initial photon absorption and regeneration to the original state with a second red-shifted photon. We report the expression and initial spectroscopic characterization of recombinant expressed melanopsin. Mouse melanopsin, carrying a C-terminal deca-his-tag, was expressed in Sf9 insect cells, using recombinant baculovrius. Good expression levels of intact melanopsin protein were achieved (2-4 mg/l). To regenerate the active photopigment 11-cis retinal was added. However, solubilization efficiency was quite low for most detergents. Best results were obtained with dodecylphosphocholine (DPC). Efficient single-step purification was achieved with affinity chromatography over immobilized Ni-2+ ions. So far only a small percentage of the purified melanopsin was photoactive, showing a maximum absorbance at 480-500 nm. Preliminary low-temperature FTIR difference and resonance Raman spectroscopic measurements are reported for melanopsin and squid rhodopsin in order to compare structural features and conformational changes upon light activation including retinal isomerization and protein backbone changes. Supported by a grants from the NIH-NEI to KJR and from NWO-CW to WJdG.

Single-Walled Carbon Nanotubes Probing the Denaturation of Lysozyme.

Resonance Raman spectroscopy measurements of lysozyme-bound single-walled carbon nanotubes have been made during different stages of the chemically and thermally induced misfolding and of the denaturation process of nanotube-bound lysozymes. Changes to the Raman intensity of single-walled carbon nanotubes (SWNTs) have been observed during the denaturation of lysozyme. The Raman intensity changes are attributed to excitonic transition energy (Eii ) shifts of the SWNTs during the denaturation of lysozyme. The Eii shift of SWNTs was confirmed by photoluminescence measurements.

Radical Production by Infrared A Irradiation in Human Tissue

The influence of the ultraviolet (UV) irradiation of the sun on the formation of free radicals in human skin is well investigated. Up to now, only small amounts of data are available stating that infrared (IR) irradiation can produce free radicals in the skin. In the present study, the formation of free radicals in human skin, subsequent to IRA irradiation (600–1,500 nm), has been demonstrated by means of two different methods. Firstly, the radical formation was detected indirectly by the degradation of the cutaneous carotenoid antioxidants β-carotene and lycopene, which was investigated in vivo by resonance Raman spectroscopic measurements. Secondly, the direct observation of produced radicals subsequent to IRA irradiation of the skin was performed in vitro by electron paramagnetic resonance spectroscopy. Taking into account the results of the present study and previous UV light studies, it can be expected that also solar irradiation in the visible spectral range will produce free radicals in the human skin. Therefore, the current sun protection strategies should be reconsidered. Furthermore, it was shown in the present study that the side effect in the form of radical formation could be significantly reduced by increasing the protection system of the human organism in form of the antioxidant network.

Resonant Raman-active localized vibrational modes inAlyGa1−yNxAs1−xalloys: Experiment and first-principles calculations

The localized vibrational modes associated with substitutional aluminium and nitrogen atoms in ${\mathrm{Al}}_{y}{\mathrm{Ga}}_{1\ensuremath{-}y}{\mathrm{N}}_{x}{\mathrm{As}}_{1\ensuremath{-}x}$ have been studied within first-principles density functional theory using a supercell approach. Localized vibrational modes related to $\mathrm{N}\text{\ensuremath{-}}{\mathrm{Al}}_{m}{\mathrm{Ga}}_{4\ensuremath{-}m}$ $(1\ensuremath{\leqslant}m\ensuremath{\leqslant}4)$ complexes have been identified, which reveal the formation of $\mathrm{N}\text{\ensuremath{-}}{\mathrm{Al}}_{4}$ units well above random abundance, in qualitative agreement with a large calculated value $(391\phantom{\rule{0.3em}{0ex}}\mathrm{meV})$ of the Al-N bond formation energy. We determine the resonant Raman-active modes from the selection rule obtained by calculating the electron-phonon coupling strength and optical transition matrix elements and compare them with resonant Raman spectroscopy measurements. The localized modes from Raman scattering measurements with frequencies around 325, 385, 400, 450, 500, and $540\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$ are found to be in good agreement with the calculated modes (326, 364, 384, 410, 456, 507, and $556\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$). The modes are classified as follows: the two modes at 326 and $556\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$ belong to the $\mathrm{N}\text{\ensuremath{-}}\mathrm{Al}{\mathrm{Ga}}_{3}$ configuration; there are three modes which belong to $\mathrm{N}\text{\ensuremath{-}}{\mathrm{Al}}_{2}{\mathrm{Ga}}_{2}$ with frequencies at 326, 364, and $507\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$; the $\mathrm{N}\text{\ensuremath{-}}{\mathrm{Al}}_{3}\mathrm{Ga}$ configuration gives rise to modes whose frequencies are 384 and $456\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$; and the mode at a frequency of $410\phantom{\rule{0.3em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$ belongs to the $\mathrm{N}\text{\ensuremath{-}}{\mathrm{Al}}_{4}$ complex. The comparison of line intensities from samples before and after rapid thermal annealing allows us to experimentally distinguish vibrational modes associated with different clusters, in agreement with the theoretical assignments.

Detailed evaluation of the performance of microfluidic T mixers using fluorescence and ultraviolet resonance Raman spectroscopy

A reliable device that produces efficient mixing with a short dead time has enormous utility in the kinetic analysis of biochemical and chemical processes. We have designed two different T mixers that use moderate flow rates (0.2–0.4ml∕s), can monitor reactions up to several milliseconds, and achieve mixing times as low as 20μs. The two mixers are easy to build and dismantle, reliable, and can perform hundreds of experiments without blocking. The first mixer comprises a stainless steel block, containing a microchannel, glued to a quartz cuvette, containing a 200×200μm2 observation channel defining a conventional T mixer. The reactions are monitored by imaging the length of the observation channel onto a charge-coupled device camera. In the second mixer the entire T (200×200μm2 internal cross section) is contained within a 40-mm-long quartz cuvette. We have adopted a novel approach to controlling the entrance channel bore by inserting a stainless steel wire in order to increase the linear speed of the impinging fluids. Using a dye to visualize the flow profile inside the second T mixer, it was shown that in this T geometry segregation of the reactants is observed in the junction between the inlet channels and the observation channel (T junction) and mixing occurs entirely in the observation channel. We thoroughly tested the two mixers through several kinetic reactions using both fluorescence and ultraviolet resonance Raman spectroscopy measurements. We show that both mixers provide efficient mixing with nominal dead times (using 1:10 v∕v dilution), calculated using the quenching of the fluorescence of N-acetyl-L-tryptophanamide by N-bromosuccinimide, of 200±20 and 100±10μs, for each mixer, respectively. However, the ability to monitor within the inlet channels and the entire observation channel of the second mixer shows that this standard approach to estimating the dead time is artifactual, since it relies on assuming a constant flow speed throughout the observation channel, a feature that we show is not adhered to at short distances from the T junction. Using both mixers the refolding of the A state of cytochrome c to the native state was followed by fluorescence and ultraviolet resonance Raman spectroscopy, revealing the ability of these instruments to provide insights into the early stages of protein folding using only milligrams of sample.