Plasmonics nanorod biosensor for in situ intracellular detection of gene expression biomarkers in intact plant systems.

The intracellular developmental processes in plants, particularly concerning lignin polymer formation and biomass production are regulated by microRNAs (miRNAs). MiRNAs including miR397b are important for developing efficient and cost-effective biofuels. However, traditional methods of monitoring miRNA expression, like PCR, are time-consuming, require sample extraction, and lack spatial and temporal resolution, especially in real-world conditions. We present a novel approach using plasmonics nanosensing to monitor miRNA activity within living plant cells without sample extraction. Plasmonic biosensors using surface-enhanced Raman scattering (SERS) detection offer high sensitivity and precise molecular information. We used the Inverse Molecular Sentinel (iMS) biosensor on unique silver-coated gold nanorods (AuNR@Ag) with a high-aspect ratio to penetrate plant cell walls for detecting miR397b within intact living plant cells. MiR397b overexpression has shown promise in reducing lignin content. Thus, monitoring miR397b is essential for cost-effective biofuel generation. This study demonstrates the infiltration of nanorod iMS biosensors and detection of non-native miRNA 397b within plant cells for the first time. The investigation successfully demonstrates the localization of nanorod iMS biosensors through TEM and XRF-based elemental mapping for miRNA detection within plant cells of Nicotiana benthamiana. The study integrates shifted-excitation Raman difference spectroscopy (SERDS) to decrease background interference and enhance target signal extraction. In vivo SERDS testing confirms the dynamic detection of miR397b in Arabidopsis thaliana leaves after infiltration with iMS nanorods and miR397b target. This proof-of-concept study is an important stepping stone towards spatially resolved, intracellular miRNA mapping to monitor biomarkers and biological pathways for developing efficient renewable biofuel sources.

Evaluation of Modern Approaches for the Assessment of Dietary Carotenoids as Markers for Fruit and Vegetable Consumption.

The assessment of dietary carotenoids via blood measurements has been widely used as a marker for fruit and vegetable consumption. In the present study, modern, non-invasive approaches to assess dietary carotenoids, such as skin measurements and an app-based short dietary record (ASDR), were compared with conventional methods such as plasma status and handwritten 3-day dietary records. In an 8-week observational study, 21 healthy participants aged 50-65 years recorded their daily consumption of carotenoid-rich fruits and vegetables via a specially developed ASDR. Anthropometry, blood samplings and assessment of skin carotenoids via Raman and reflection spectroscopy were performed at baseline, after four weeks and at the end of the study. App-based intake data showed good correlations with plasma α-carotene (r = 0.74, p < 0.0001), ß-carotene (r = 0.71, p < 0.0001), and total plasma carotenoids (r = 0.65, p < 0.0001); weak correlations with plasma lutein/zeaxanthin and ß-cryptoxanthin (both r = 0.34, p < 0.05); and no correlation with plasma lycopene. Skin measurements via reflection and Raman spectroscopy correlated well with total plasma carotenoids (r = 0.81 and 0.72, respectively; both p < 0.0001), α-carotene (r = 0.75-0.62, p < 0.0001), and ß-carotene (r = 0.79-0.71, p < 0.0001); moderately with plasma lutein/zeaxanthin (both r = 0.51, p < 0.0001); weakly with plasma ß-cryptoxanthin (r = 0.40-0.31, p < 0.05); and showed no correlation with plasma lycopene. Skin measurements could provide a more convenient and noninvasive approach of estimating a person's fruit and vegetable consumption compared to traditional methods, especially in studies that do not intend blood sampling. ASDR records might function as a suitable, convenient tool for dietary assessment in nutritional intervention studies.

Comparison of individual and common wavelength-operation for 785 nm Y-branch DBR ridge waveguide diode lasers with adjustable spectral distance.

An experimental comparison between individual and common wavelength-operation of a Y-branch distributed Bragg reflector (DBR) ridge waveguide (RW) laser at 785 nm with an electrically adjustable spectral distance is presented. The dual-wavelength Y-branch laser combines two laser cavities via a Y-section to a common output section. DBR gratings with different grating periods are associated with the two cavities, which set the emission wavelengths of the two branches. Implemented resistive heater elements allow separate wavelength tuning of the two branches, which can be operated individually for alternating emission wavelengths in applications such as differential absorption spectroscopy or shifted excitation Raman difference spectroscopy. Common wavelength operation simultaneously generates two emission lines suitable for the generation of THz radiation using difference frequency mixing. Hereby, the devices could potentially be used as single-chip light sources for a combination of Raman and THz applications. For the wavelength-operation comparison presented, the devices were operated up to optical output powers of about 105 and 185 mW in individual and common wavelength-operation mode, respectively. In individual operation mode, the devices show spectral single-mode emission over the whole operation range. In common operation mode, the spectral emission is predominantly single mode up to an optical output power of 65 mW. In both operation modes, mode hops typical for DBR lasers occur. At an optical output power of 50 mW, tuning of the spectral distance between the two wavelengths using the implemented resistor heaters is demonstrated. In both modes of wavelength operation, a flexible frequency difference between 0 and 0.8 THz (0 and 1.6 nm) with predominantly single-mode spectral emission is obtained.

Determination of Soil Constituents Using Shifted Excitation Raman Difference Spectroscopy.

Soil analysis to estimate soil fertility parameters is of great importance for precision agriculture but nowadays it still relies mainly on complex and time-consuming laboratory methods. Optical measurement techniques can provide a suitable alternative. Raman spectroscopy is of particular interest due to its ability to provide a molecular fingerprint of individual soil components. To overcome the major issue of strong fluorescence interference inherent to soil, we applied shifted excitation Raman difference spectroscopy (SERDS) using an in-house-developed dual-wavelength diode laser emitting at 785.2 and 784.6 nm. To account for the intrinsic heterogeneity of soil components at the millimeter scale, a raster scan with 100 individual measurement positions has been applied. Characteristic Raman signals of inorganic (quartz, feldspar, anatase, and calcite) and organic (amorphous carbon) constituents within the soil could be recovered from intense background interference. For the first time, the molecule-specific information derived by SERDS combined with partial least squares regression was demonstrated for the prediction of the soil organic matter content (coefficient of determination R2 = 0.82 and root mean square error of cross validation RMSECV = 0.41%) as important soil fertility parameter within a set of 33 soil specimens collected from an agricultural field in northeast Germany.

Mode-locked Cr:LiSAF laser far off the gain peak: tunable sub-200-fs pulses near 1 µm.

We report, to the best of our knowledge, the first mode-locking results of a Cr:LiSAF laser near the 1 µm region. The system is pumped only by a single 1.1 W high-brightness tapered diode laser at 675 nm. A semiconductor saturable absorber mirror (SESAM) with a modulation depth of 1.5% and non-saturable losses below 0.5% was used for mode-locking. Once mode-locked, the Cr:LiSAF laser produced almost-transform-limited sub-200-fs pulses with up to 12.5 mW of average power at a repetition rate of 150 MHz. Using an intracavity birefringent filter, the central wavelength of the pulses could be smoothly tuned in the 1000-1020 nm range. Via careful dispersion optimization, pulse widths could be reduced down to the 110-fs level. The performance in this initial study was limited by the design parameters of the SESAM used, especially its passive losses and could be improved with an optimized SESAM design.

783 nm wavelength stabilized DBR tapered diode lasers with a 7 W output power.

Wavelength stabilized distributed Bragg reflector (DBR) tapered diode lasers at 783 nm will be presented. The devices are based on GaAsP single quantum wells embedded in a large optical cavity leading to a vertical far field angle of about 29° (full width at half maximum). The 3-inch (7.62 cm) wafers are grown using metalorganic vapor phase epitaxy. In a full wafer process, 4 mm long DBR tapered lasers are manufactured. The devices consist of a 500 µm long 10th order surface DBR grating that acts as rear side mirror. After that, a 1 mm long ridge waveguide section is realized for lateral confinement, which is connected to a 2.5 mm long flared section having a full taper angle of 6°. At an injection current of 8 A, a maximum output power of about 7 W is measured. At output powers up to 6 W, the measured emission width limited by the resolution of the spectrometer is smaller than 19 pm. Measured at 1/e2 level at this output power, the lateral beam waist width is 11.5 µm, the lateral far field angle 12.5°, and the lateral beam parameter M2 2.5. The respective parameters measured using the second moments are 31 µm, 15.2°, and 8.3. 70% of the emitted power is originated from the central lobe.

Accurate in vivo tumor detection using plasmonic-enhanced shifted-excitation Raman difference spectroscopy (SERDS).

For the majority of cancer patients, surgery is the primary method of treatment. In these cases, accurately removing the entire tumor without harming surrounding tissue is critical; however, due to the lack of intraoperative imaging techniques, surgeons rely on visual and physical inspection to identify tumors. Surface-enhanced Raman scattering (SERS) is emerging as a non-invasive optical alternative for intraoperative tumor identification, with high accuracy and stability. However, Raman detection requires dark rooms to work, which is not consistent with surgical settings. Methods: Herein, we used SERS nanoprobes combined with shifted-excitation Raman difference spectroscopy (SERDS) detection, to accurately detect tumors in xenograft murine model. Results: We demonstrate for the first time the use of SERDS for in vivo tumor detection in a murine model under ambient light conditions. We compare traditional Raman detection with SERDS, showing that our method can improve sensitivity and accuracy for this task. Conclusion: Our results show that this method can be used to improve the accuracy and robustness of in vivo Raman/SERS biomedical application, aiding the process of clinical translation of these technologies.

Wide Field Spectral Imaging with Shifted Excitation Raman Difference Spectroscopy Using the Nod and Shuffle Technique.

Wide field Raman imaging using the integral field spectroscopy approach was used as a fast, one shot imaging method for the simultaneous collection of all spectra composing a Raman image. For the suppression of autofluorescence and background signals such as room light, shifted excitation Raman difference spectroscopy (SERDS) was applied to remove background artifacts in Raman spectra. To reduce acquisition times in wide field SERDS imaging, we adapted the nod and shuffle technique from astrophysics and implemented it into a wide field SERDS imaging setup. In our adapted version, the nod corresponds to the change in excitation wavelength, whereas the shuffle corresponds to the shifting of charges up and down on a Charge-Coupled Device (CCD) chip synchronous to the change in excitation wavelength. We coupled this improved wide field SERDS imaging setup to diode lasers with 784.4/785.5 and 457.7/458.9 nm excitation and applied it to samples such as paracetamol and aspirin tablets, polystyrene and polymethyl methacrylate beads, as well as pork meat using multiple accumulations with acquisition times in the range of 50 to 200 ms. The results tackle two main challenges of SERDS imaging: gradual photobleaching changes the autofluorescence background, and multiple readouts of CCD detector prolong the acquisition time.

Microsecond pulse-mode operation of a micro-integrated high-power external-cavity tapered diode laser at 808 nm.

We investigate microsecond pulse-mode operation of a micro-integrated high-power diode laser based on volume Bragg grating external-cavity feedback around 808 nm. The laser system contains a tapered amplifier consisting of a ridge-waveguide section and a tapered section with separated electrical contacts. Thus, the diode laser system can be pulsed by modulating the injected current either to the ridge waveguide section (IRW) or to the tapered amplifier section (ITA). With a trigger signal of a 50 µs pulse width and a 10 kHz repetition rate, comparing the modulation depth, peak output power, beam propagation factor, and spectral bandwidth, we conclude that the pulse-mode operation achieved by modulating the ITA gives better results than by modulating the IRW due to the decreased thermal effect. At a constant IRW of 0.2 A and a modulated ITA of 6.0 A, 4.3 W of peak output power is obtained with an emission spectral bandwidth with an upper bound of 0.2 nm, and a beam propagation factor in the slow axis, Mslow2, of 2.6 (1/e2). The modulation depth is almost 100%. The results show that the tapered diode laser system may be a good candidate for microsecond pulse-mode solid-state lasers.

Micro-integrated high-power narrow-linewidth external-cavity tapered diode laser at 808 nm.

A novel compact micro-integrated high-power narrow-linewidth external-cavity diode laser around 808 nm is demonstrated. The laser system contains a tapered amplifier consisting of a ridge-waveguide section and a tapered section with separated electrical contacts. Thus, the injection currents to both sections can be controlled independently. An external volume Bragg grating is utilized for spectral narrowing and stabilization. The diode laser system is integrated on a 5mm×13mm aluminum nitride micro-optical bench on a conduction cooled package mount with a footprint of 25mm×25mm. The diode laser system is characterized by measuring the output power and spectrum with the injection currents to the ridge-waveguide section (IRW) and tapered amplifier section (ITA) changed in steps of 25 and 50 mA, respectively. At IRW=200mA and ITA=6.0A, 3.5 W of output power is obtained with an emission spectral linewidth with an upper bound of 6 pm, and a beam propagation factor in the slow axis, M2, of 2.6 (1/e2). The characterization of the temperature stabilization of the laser system shows an increase of the wavelength at a rate of 6.5 pm/K, typical for the applied volume Bragg grating.

Direct SERDS sensing of molecular biomarkers in plants under field conditions.

Molecular biomarkers such as microRNAs (miRNAs) play important roles in regulating various developmental processes in plants. Understanding these pathways will help bioengineer designing organisms for efficient biomass accumulation. Current methods for RNA analysis require sample extraction and multi-step sample analysis, hindering work in field studies. Recent work in the incorporation of nanomaterials for plant bioengineering research is leading the way of an agri-tech revolution. As an example, surface-enhanced Raman scattering (SERS)-based sensors can be used to monitor RNA in vivo. However, the use of SERS in the field has been limited due to issues with observing Raman signal over complex background. To this end, shifted-excitation Raman difference spectroscopy (SERDS) offers an effective solution to extract the SERS signal from high background based on a physical approach. In this manuscript, we report the first application of SERDS on SERS sensors. We investigated this technique on SERS sensor developed for the detection of a microRNA biomarker, miR858. We tested the technique on in vitro samples and validated the technique by detecting the presence of exogenous miR858 in plants directly under ambient light in a growth chamber. The possibility of moving the detection of nucleic acid targets outside the constraints of laboratory setting enables numerous important bioengineering applications. Such applications can revolutionize biofuel production and agri-tech through the use of nanotechnology-based monitoring of plant growth, plant health, and exposure to pollution and pathogens.

In vivo detection of changes in cutaneous carotenoids after chemotherapy using shifted excitation resonance Raman difference and fluorescence spectroscopy.

BACKGROUND: Various cutaneous toxicities under chemotherapy indicate a local effect of chemotherapy by secretion after systemic application. Here, changes in the fluorescence and Raman spectral properties of the stratum corneum subsequent to intravenous chemotherapy were assessed. METHODS: Twenty healthy subjects and 20 cancer patients undergoing chemotherapy were included. Measurement time points in cancer patients were before the first cycle of chemotherapy (Tbase ) and immediately after intravenous application of the chemotherapy (T1 ). Healthy subjects were measured once without any further intervention. Measurements were conducted using an individually manufactured system consisting of a handheld probe and a wavelength-tunable diode laser-based 488 nm SHG light source. Hereby, changes in both skin fluorescence and shifted excitation resonance Raman difference spectroscopy (SERRDS) carotenoid signals were assessed. RESULTS: Healthy subjects showed significantly (P < .001) higher mean concentrations of carotenoids compared to cancer subjects at Tbase . An increase in fluorescence intensity was detected in almost all patients after chemotherapy, especially after doxorubicin infusion. Furthermore, a decrease in the carotenoid concentration in the skin after chemotherapy was found. CONCLUSION: The SERRDS based noninvasive detection can be used as an indirect quantitative assessment of fluorescent chemotherapeutics. The lower carotenoid SERRDS intensities at Tbase might be due to cancerous diseases and co-medication.

Shifted Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device (CCD) Lock-In Detection.

Shifted excitation Raman difference spectroscopy (SERDS) can provide effective, chemically specific information on fluorescent samples. However, the restricted ability for fast alternating detection (usually < 10 Hz) of spectra excited at two shifted laser wavelengths can limit its effectiveness when rapidly varying emission backgrounds are present. This paper presents a novel charge-shifting lock-in approach permitting fast SERDS operation (exemplarily demonstrated at 1000 Hz) using a specialized dual-wavelength diode laser (emitting at 829.40 nm and 828.85 nm) and a custom-built charge-coupled device (CCD) enabling charge retention and shifting back and forth on the CCD chip. For six selected mineral samples (moved irregularly during spectral acquisition), results demonstrate superior reproducibility of the fast charge-shifting read-out over the conventional read-out (operated at 5.4 Hz). Partial least squares discriminant analysis revealed improved classification performance of charge-shifting (four latent variables, sensitivity: 99%, specificity: 94%) versus conventional read-out (six latent variables, sensitivity: 90%, specificity: 92%). The charge-shifting concept was also successfully translated to sub-surface analysis using spatially offset Raman spectroscopy (SORS). Charge-shifting SERDS-SORS spectra recorded from a polytetrafluoroethylene layer, concealed behind a 0.25 mm thick, opaque, heterogeneous layer, matched reference spectra much more closely and exhibited a signal-to-background-noise (S/NB) ratio two times higher than that achieved with conventional CCD read-out SERDS-SORS. The novel approach overcomes fundamental limitations of conventional CCDs. In conjunction with the inherent capability of the charge-shifting lock-in technique to suppress rapidly varying ambient light interference demonstrated by us earlier it is expected to be particularly beneficial with heterogeneous fluorescent samples in field applications.

Efficient Tm:YAG and Tm:LuAG lasers pumped by 681 nm tapered diodes.

In this paper, we present highly efficient continuous-wave (cw) laser operation of Tm:YAG and Tm:LuAG lasers pumped by high-brightness red tapered diodes. The single-emitter tapered diode lasers (TDLs) provide up to 1 W of pump power around 680 nm. By adjusting the operation temperature of the TDL, the pump central wavelength could be matched to the strong absorption peak of Tm3+ ions in this region (H63→F33 excitation). This absorption peak is around threefold stronger than the usually employed 785 nm transition (H63→H34). In the cw laser experiments, we have achieved slope efficiencies exceeding 55% at room temperature, which is far above the Stokes limited slope efficiency (34%), indicating presence of a strong two-for-one cross-relaxation process. Pumping with high-brightness tapered diode lasers further facilitated usage of smaller pump spots (enabling quite low lasing thresholds) and generation of near-diffraction limited output beam profiles from standard z-type cavities. To the best of our knowledge, this is the first report of diode pumping of Tm-doped solid-state lasers around 680 nm as well as the first usage of TDLs as pump sources in Tm-doped laser systems.

Extended 9.7 nm tuning range in a MOPA system with a tunable dual grating Y-branch laser.

In this Letter, we present a tunable Y-branch hybrid master oscillator power amplifier (MOPA) with 5.5 W output power, emitting between 973.7 and 983.4 nm. The MO is a monolithic Y-branch distributed Bragg reflector (DBR) diode laser, which is collimated and coupled into a tapered amplifier using cylindrical microlenses in a compact 25 mm×25 mm conduction cooled laser package. The wavelength spacing between the two laser branches is 2.2 nm. Each branch can be electrically tuned by up to 7.5 nm of quasi-continuous wavelength tuning, which in combination covers up to 9.7 nm of spectral range. Over this range, an output power variation of 0.5% and 0.2% is observed for the left and right arms, respectively, while maintaining a spectral width of less than 17 pm. In addition, the MOPA system can be operated as a dual-wavelength system by operating both branches simultaneously. The presented device is suitable for nonlinear frequency conversion applications.

Dual-wavelength diode laser with electrically adjustable wavelength distance at 785 nm.

A spectrally adjustable monolithic dual-wavelength diode laser at 785 nm as an excitation light source for shifted excitation Raman difference spectroscopy (SERDS) is presented. The spectral distance between the two excitation wavelengths can be electrically adjusted between 0 and 2.0 nm using implemented heater elements above the distributed Bragg reflector (DBR) gratings. Output powers up to 180 mW at a temperature of 25°C were measured. The spectral width is smaller than 13 pm, limited by the spectrum analyzer. The device is well-suited for Raman spectroscopy, and the flexible spectral distance allows a target-specific adjustment of the excitation light source for shifted excitation Raman difference spectroscopy (SERDS).

Highly efficient single-pass sum frequency generation by cascaded nonlinear crystals.

The cascading of nonlinear crystals has been established as a simple method to greatly increase the conversion efficiency of single-pass second-harmonic generation compared to a single-crystal scheme. Here, we show for the first time that the technique can be extended to sum frequency generation, despite differences in the phase relations of the involved fields. An unprecedented 5.5 W of continuous-wave diffraction-limited green light is generated from the single-pass sum frequency mixing of two diode lasers in two periodically poled nonlinear crystals (conversion efficiency 50%). The technique is generally applicable and can be applied to any combination of fundamental wavelengths and nonlinear crystals.

Compact Handheld Probe for Shifted Excitation Raman Difference Spectroscopy with Implemented Dual-Wavelength Diode Laser at 785 Nanometers.

A compact handheld probe for shifted-excitation Raman difference spectroscopy (SERDS) with an implemented dual-wavelength diode laser with an emission at 785 nm is presented. The probe is milled from aluminum and has dimensions 100 × 28 × 12 mm. The diode laser provides two excitation lines with a spectral distance of 10 cm(-1) (0.62 nm), has a spectral width smaller than 11 pm, and reaches an optical power of 120 mW ex probe. Raman experiments were carried out using polystyrene (PS) as the test sample. During a measurement time of over 1 h, a stable spectral center position of the Raman line at 999 cm(-1) of PS was achieved within a spectral window of 0.1 cm(-1). Here, the Raman intensity of this line was observed with a peak-to-peak variation smaller than ±2%, dominated by shot noise interference. A deviation of the center position of a Raman line with <±1 cm(-1) was observed over the whole excitation power range. Raman investigations of the quartz glass window of the SERDS probe showed minor interference. The results demonstrate the suitability of the developed handheld probe for Raman investigations and the application of in situ SERDS experiments to fields such as food safety control, medical diagnostics, and process control.

Capability of shifted excitation Raman difference spectroscopy under ambient daylight.

We present the capability of shifted excitation Raman difference spectroscopy (SERDS) under ambient daylight. A dual-wavelength diode laser emitting at 785 nm is used as the excitation light source. The monolithic diode laser provides more than 110 mW in cw operation. Both excitation lines show an emission width ≤0.2 cm(-1) and a spectral distance of 10 cm(-1) as targeted for SERDS. Polystyrene (PS) is used as the test sample and ambient daylight to generate real-world background interference. Here, a broadband background signal with narrowband absorption lines from water vapor and Fraunhofer lines from singly ionized calcium (Ca II) obscure the Raman lines of PS. SERDS clearly separates the Raman signals from the background signals with a 13-fold improvement in signal-to-background noise.

Tunable 975 nm nanosecond diode-laser-based master-oscillator power-amplifier system with 16.3 W peak power and narrow spectral linewidth below 10 pm.

A spectrally tunable, narrow linewidth master oscillator power amplifier system emitting ns pulses with high peak power is presented. The master oscillator is a distributed feedback ridge waveguide (DFB-RW) laser, which is operated in continuous wave (CW) mode and emits at about 975 nm with a spectral line width below 10 pm. The oscillator can be tuned over a range of 0.9 nm by varying the injection current. The tapered amplifier (TA) consists of an RW section and a flared gain-guided section. The RW section of the amplifier acts as an optical gate and converts the CW input beam emitted by the DFB-RW laser into a train of short optical pulses, which are subsequently amplified by the tapered section. The width of the pulses is 8 ns at a repetition rate of 25 kHz. The peak power is 16.3 W. The TA preserves the spectral properties of the emission of the DBR-RW laser. The amplified spontaneous emission is suppressed by about 40 dB.