In this study, several strains of yeast cells, both wild-type (WT) and engineered, were analyzed by Raman imaging microscopy. Two biologically active compounds produced within the engineered yeast cells were readily identified by their characteristic Raman spectral peaks. The locales and the relative abundance of those biologically active compounds were visualized from the Raman microscopic images of the individual cells. Raman imaging microscopy further reveals the differences in efficiencies with which the biologically active compounds were produced by different yeast cell strains.

In recent years, bioengineering of yeast cells has made them a “biological cell factory” for producing a range of chemicals, including biologically active compounds through fermentation processes. This “biological cell factory” can efficiently convert starch, sucrose, or lignocellulose-based raw materials into precursor metabolites, which can further be converted into the products of interest (1). Bulk chemical analyses, such as gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR), can be used for screening fermentation cultures, assessing product yield, and delivering process optimization. These methods, however, are destructive, time-consuming, and insufficient, because they fail to provide specificity at the cellular level (2). Fluorescence microscopy-based imaging is capable of visualizing large or small molecules at the cellular level, but the success of this approach largely hinges on the photostability and brightness of fluorescent labels, their possible perturbations to the biological system, and the methods by which the labels are attached (3). Confocal Raman microscopy-based imaging has emerged as a great alternative (3,4). Raman spectroscopy provides chemical annotations to the constituents within the cell, and the high spatial resolution intrinsic to confocal microscopy provides cellular level information. In contrast to the fluorescence microscopy-based imaging approach, Raman imaging is nondestructive and requires minimal sample preparation.

In this study, wild-type (WT) and several strains of engineered yeast cells were analyzed by Raman imaging. Two biologically active compounds produced within the engineered yeast strains were readily identified. Furthermore, it was revealed that different strains produced the two compounds with different efficiencies. Raman images of the individual cells show the locales as well as the relative abundance of those compounds. The information extracted provides valuable information that helps optimize the fermentation process.

A 2 μL sample of the WT and each engineered yeast cell strain sample was transferred to an aluminum-coated, 12-positions slide, and then it was air dried prior to the Raman imaging experiments. The two biologically active reference compounds are denoted as “compound 1” and “compound 2”, respectively. Compound 1 is a terpene and compound 2 is a sterol. For the two reference compounds in solid powder form, a small amount of each was transferred to a glass slide, and the Raman spectra were collected.

The Raman spectra and the Raman area images (for an area containing one or two cells within each sample) were acquired using a Thermo Scientific DXR3xi Raman Imaging Microscope. The key experimental parameters for Raman imaging are listed below:

Raman images were obtained by the correlation profiling option of the OM-NICxi software, using a representative Raman spectrum from within a cell as the reference. The correlation profile shows the correlation between the spectrum at each pixel (sampling point) in the Raman image and the specified reference spectrum. Each color in the rainbow-colored intensity scale represents a degree of correlation with the reference spectrum. The higher the intensity value, the greater similarity to the reference spectrum.

Figure 1 shows the representative Raman spectra of the two biologically active compounds, compound 1 (pink trace, top) and compound 2 (red trace, bottom). Both compounds have prominent features in the C-H stretching region (~2900 cm

). The two strong peaks attributed to the C=C stretch- ing vibrations are located at 1667 cm

, respectively, for compound 1 and compound 2. The downshift of the C=C stretching peak in compound 2 (1605 cm

) is because of a ring structure. There are no strong interfering Raman peaks from the cellular matrices in this frequency range. Hence, these peaks are chosen as the Raman markers to distinguish the two biologically active compounds in the yeast cell samples by Raman imaging.

Figure 2 shows the Raman image of a WT yeast cell (Figure 2a), the representative Raman spectra from the different locations of the imaged region (Figure 2b), and the representative Raman spectra of compound 1 and compound 2. As can be seen in Figure 2a, the cells (in red) can be spectroscopically differentiated from the surrounding media (in blue), with a clearly delineated contour in green color. As expected, the cells are devoid of either compound 1 or compound 2 (Figure 2c). Raman image of the strain B yeast cells is shown in Figure 3. Both compound 1 and compound 2 are produced in the cell, as evidenced by Figures 3b and 3c. Interestingly, for the same strain B, there are cell-to-cell variations in the level of produced compounds. Based on the relative peak intensities, the cell in the upper- left corner appears to produce more compound 1 than compound 2, whereas the cell in the center produces similar amounts of compound 1 and compound 2.

Figure 4 summarizes the Raman imaging analyses of the three different strains of yeast cells—strain A, strain C, and strain D—in terms of their yield in producing compound 1 and compound 2. At a quick glance, strain A appears to be a low producer of compound 1 and does not produce compound 2. Strains C and D, on the other hand, appear to produce both compound 1 and compound 2, but strain D has a higher yield for compound 1 than for compound 2.

Figure 5 summarizes the yield with which compound 1 and compound 2 are produced by the four different engineered strains of yeast cells (strains A–D). Strain A (blue) appears to be a low producer of compound 1 and does not produce compound 2. Strains B, C, and D all produce both compound 1 and compound 2. Strain B displays nonuniform levels of produced compounds from cell-to-cell, some cells (pink) producing higher level of compound 1 than the others (cyan). Strain C (green) seems to produce higher level of compound 2, and strain D (red) has the highest yield for compound 1. Caution should be exercised when assessing the product yields from the Raman peak intensity. A more rigorous, quantitative analysis requires the respective Raman cross sections for the compounds 1 and 2 be properly accounted for.

In this study, Raman imaging was successfully applied to analyze different strains of yeast cells, both WT and engineered. The target bioactive compounds produced by the engineered yeast cells can be readily identified and differentiated by their respective Raman spectra, allowing for assessment of the product yields of different yeast strains by Raman imaging. Raman images of the individual cells from the four engineered yeast cell strains indicate that these strains produce compounds 1 and 2 with different efficiencies. Raman imaging can also be used to estimate the relative yield of the bioactive compounds. A quantitative analysis of product yield, however, requires a more systematic study on the Raman cross-sections of those compounds. The information obtained from the Raman imaging will be beneficial for a deeper understanding of the fermentation process and for optimization of the process for higher product yield.

The authors would like to acknowledge Dr. Michael Leavell and his colleagues at Amyris for providing the samples and many insightful discussions.

(1) Y. Chen, L. Daviet, M. Schalk, V. Siewers, and J. Nielsen, 

(2) R. Miyaoka, M. Hosokawa, M. Ando, T. Mori, H.O. Hamaguchi, and H. Takeyama, 

(4) L. Mikoliunaite, R.D. Rodriguez, E. Sheremet, V. Kolchuzhin, J. Mehner, A. Ramanavicius, and D.R.T. Zahn, 

 are with Thermo Fisher Scientific in Madison, Wisconsin. 

 was formerly a scientist with Amyris in Emeryville, California, when this work was being conducted. Direct correspondence to: 

Articles

Using Raman imaging, wild-type and engineered yeast cells were compared for their ability to produce bioactive compounds. Raman imaging microscopy is able to visualize locales, relative abundance, and production efficiencies of biologically active compounds for the individual yeast cells.

Tracking Bioactive Compounds Produced by Genetically Engineered Yeast Cells Using Raman Imaging

Surface enhanced Raman spectroscopy (SERS), although popular in research, has not yet been adopted in industrial applications. This is largely because of the challenge of manufacturing cost-effective, reproducible SERS substrates in volume that can be read by a compact reader for practical deployment in the field. Here we demonstrate and characterize a new dual-nanostructured SERS substrate approach that could be amenable to large-scale production. This dual-nanostructured surface combines long-range ordering of silver nanowires (AgNWs) with dense non-ordered decoration via silver nanoparticles (AgNPs) to improve on the SERS response characteristics of the individual structures, using fabrication methods that could be scaled for deposition via roll-to-roll Mayer rod coating. Characterization of the substrates’ SERS response using 638 nm Raman spectroscopy and ATP as the reporter molecule found the dual-nanostructured SERS substrate to offer both greater spatial uniformity and a consistently higher SERS enhancement factor (EF) than the nanowires alone.

Surface-enhanced Raman spectroscopy (SERS) is a technique whereby the Raman effect is enhanced by a factor of 10

 or more through interaction of the analyte with noble metal nanostructures (1). This increases the limit of detection of Raman significantly for both quantitative and qualitative applications, and reduces performance demands on the associated instrumentation to allow greater deployment in the field. In SERS, the Raman signal enhancement originates from a combination of electromagnetic (EM) field enhancement because of the interaction of light with the nanostructures and the chemical enhancement (CE) that occurs when the analyte is chemisorbed on a SERS-active surface.

Common SERS-active structures include nanoparticles, nanowires, and nanostructured fabricated surfaces, with the greatest EM enhancement occurring in SERS hotspots, junctions between two nanostructures (1). Although both colloidal and solid SERS structures have been explored, solid substrates offer the greatest potential for commercial use, provided that they possess a sufficient number of SERS hotspots uniformly distributed within the spot size of the reader, in order to produce a reproducible, concentration-dependent signal while retaining high sensitivity. SERS substrates also allow convenient solution sampling, with the analyte becoming concentrated on the surface as the solvent evaporates to further increase interaction between the analyte and signal-enhancing substrate within the laser focus.

Both silver nanoparticles (AgNPs) and silver nanowires (AgNWs) are known to produce significant SERS signals, and can be synthesized relatively easily in the laboratory. However, the current fabrication methods utilize reaction conditions that pose challenges for large-scale production, from the need for meticulous controls over synthesis parameters to slow reaction times. This study demonstrates a SERS substrate where AgNWs and AgNPs are used as complementary nanostructures to improve on the SERS response characteristics of AgNWs alone, and where both structures are fabricated via easy, scalable synthetic methods.

For the AgNWs, a simple one-pot synthesis with a fast reaction rate and low temperature is used, based on the popular “polyol” method. The polyol method utilizes hot ethylene glycol (EG) to reduce silver ions in the presence of polyvinylpyrrolidone (PVP, Figure 1a). The method is popular due to the use of environmentally-friendly reactants and relatively mild reaction conditions compared to other thermal solvolysis methods. Here, we employ FeCl

 to catalyze the reduction of silver and create shorter, thicker wires (2), as well as halide ions to promote wire formation.

For the AgNPs, a silver ion molecular complex is used as an alternative to traditional AgNP fabrication, in which a solid silver surface is rapidly produced when a diamminesilver(I) complex is exposed to an aldehyde functional group. The silver complex used here is μ-oxalatobis(ethylenediamine)silver(I), shown in Figure 1b. By utilizing a carboxylic acid as a ligand, gaseous CO

 is produced upon decomposition, and the decomposition reaction is slower. Because the reaction produces all gaseous organic species, a clean silver surface is produced absent of background interference from the capping agents often used to ensure colloidal stability (3), or additives required to avoid the coffee stain effect common in SERS substrates (uneven distribution of AgNPs resulting in concentrated rings on the surface) (4). Though the silver complex alone has previously failed to yield the morphology required for SERS response (5), the consistent formation of nanoparticles and known shelf stability (6) makes it a promising choice for incorporation with AgNWs into a dual-nanostructured SERS substrate.

Additionally, by drawing on established industrial processing techniques like use of a whirlpool tank to isolate the AgNWs and roll-to-roll Mayer rod coating to deposit the SERS-active materials, a pathway is proposed for a SERS substrate which could be both manufacturable and cost-effective in volume.

Both AgNW substrates and AgNW substrates coated with AgNPs were fabricated using a variety of reaction parameters and deposition methods. These were evaluated to explore whether the easily produced AgNW coating could be utilized as a substrate to produce a uniform AgNP surface through the decomposition of an aqueous ink of the Ag complex on the AgNW substrate without the need of any additives, thus creating a dual-nanostructured surface. Results presented herein represent the optimized conditions relevant to demonstrating the dual-nanostructured surface; full details of the optimization process are presented elsewhere (7).

A synthetic method was developed to produce AgNWs based on the “polyol” method, incorporating a transition metal cation catalyst, and utilizing a selective precipitation purification method.

To synthesize the AgNWs, PVP (~150 mg) with a molecular weight of ~55,000 was dissolved in EG (20 mL ) with vigorous stirring, protected from light exposure. Silver nitrate was dissolved directly into the same reaction vessel, with a mole ratio of PVP monomer to silver of 2.0 to help direct one-dimensional growth of nanowires. An 11 mM stock solution of FeCl3 in EG (100 μL) was added with gentle swirling. The solution was heated to 100 °C in an oven for 45 min before increasing the temperature to 140 °C for 70 min, then cooled to room temperature.

The reaction products were dissolved in water (40 mL ) to create a homogenous dispersion. Acetone was then added slowly, dropwise with swirling, until a visible granularity was observed, and the mixture was allowed to settle for 15 min. The supernatant was removed, and water (~20 mL ) was added to resuspend the AgNWs. The selective precipitation was repeated 3–5 times, as necessary. Residual acetone and water were removed by centrifuging the solution at 3000 rpm for 3 min, removing the supernatant, redispersing in ethanol, and repeating the procedure four times before suspending the purified wires in a stock solution of ethanol (~5 mL ).

Qualitative analysis of the wire size distribution and purity was determined by bright- field optical microscopy using an Olympus BX60 microscope equipped with a DP73 color camera and polarization filters. SEM images were taken using a Hitachi SU8000 UHR FE-SEM. Composition and structure were characterized using UV-vis spectroscopy, thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM), reported elsewhere (7).

The silver complex μ-oxalato-bis (ethylenediamine) silver(I) was synthesized as reported in literature (6). First, 0.5 M aqueous solutions of silver nitrate and oxalic acid were combined in an Ag

:oxalate volume ratio of 5:3. The silver oxalate precipitate was collected by filtration, washed with water, and dried at room temperature under vacuum. The silver oxalate was then dissolved in small increments, while stirring, in an aqueous solution of ethylene diamine (20% v/v), followed by precipitation with an excess of isopropanol, and subsequently filtered. The white precipitate was washed with cold isopropanol and vacuum dried at room temperature for 6 h prior to storage at -20 °C. The resulting silver complex was prepared as an ink for substrate fabrication by dissolving the silver complex in water (11 mg/mL). Care was taken to avoid light exposure during all synthetic steps and storage. The structure of the silver complex was confirmed via infrared (IR) and 

C nuclear magnetic resonance (NMR) spectroscopy, as well as TGA reported elsewhere (7).

Nanowire and Dual-Nanostructure Substrate Fabrication

Mayer rod coating was used to produce AgNW surfaces for direct use as a SERS substrate, herein referred to as a nanowire substrate (NWS), and for the fabrication of dual-nanostructured surfaces (DNS). To produce a NWS, the stock AgNW solution was pipetted (25 μL) in a line on glass slides and spread by hand using a #10 Mayer rod. The slides were dried at room temperature for ~30 min.

SERS quality DNS were produced by first preheating a NWS slide to 125 °C, then 25 μL of the Ag complex ink was drop-cast in the center of one half of the slide, and allowed to decompose for 5 min before cooling to room temperature, when it was then gently rinsed with ethanol and allowed to dry.

The uniformity of the fabricated surfaces was examined using an optical microscope. The morphology of the nanostructures on the surfaces were characterized using a Veeco Dimension 3100 AFM, operating in tapping mode.

SERS Characterization and Instrumentation

Aminothiolphenol (ATP) was used as the SERS reporter molecule for characterization of the substrates, drop-casted in small aliquots onto both the NWS and DNS sides of the SERS substrates, and air dried at room temperature for 2 min. Raman spectra were acquired with a fully integrated Wasatch Photonics 638 nm Raman spectroscopy system, utilizing a backscattering sampling geometry and spot size of ~60 μm, with detection via a fixed holographic transmission grating and a thermal electric-cooled (TEC) charge-coupled device (CCD) detector. SERS measurements were acquired using a laser power of 5 mW, taking 1 s per scan. Slide positioning was achieved using a Thorlabs MT3 XYZ stage controller equipped with micrometers to control translation (XY) and focus (Z) of the laser on the slide surfaces.

Nanowire synthesis produced a narrow distribution of diameters of 80–120 nm, with lengths of 10–30 μm, as verified by optical microscopy, yielding an aspect ratio of ~200.

Notably, the reaction conditions employed required no control over stirring, injection rate, or other factors that would be difficult or expensive to incorporate into industrial scale reactions. The mechanical strength of the ~100 nm wires is enough to remain mostly straight with only a small amount of curvature, which bodes well for processability of the wires for substrate manufacture. The selective precipitation successfully isolates the desired AgNWs in a high purity and is readily adaptable to large scale execution in a whirlpool tank.

Deposition of AgNWs onto a slide via Mayer rod coating resulted in uniform coating and areas of linear structure with a high density of wire intersections to generate SERS-active hotspots, as imaged using microscopy (Figure 2a). To assess the uniformity of the SERS response in relation to spatial variations in sampling, SERS spectra of a 10 μL sample of 0.5 μM ATP solution were taken every 500 μm along a 1 cm linear path on the NWS, averaging four scans per location (Figure 2b). The SERS response of the NWS was found to be highly dependent on the position sampled, dropping to zero at some locations. The inconsistent spatial response of the NWS is a consequence of the AgNWs’ length being comparable to the spot size of the laser, which limits the number of SERS hotspots, as well as to gaps in deposition, that could be significantly improved in an industrial setting through the use of process automation.

Because the AgNWs retain a surface layer of PVP as a byproduct of synthesis, it was hypothesized that the NWS could provide a guiding template for the deposition of a silver complex to populate the NWS with AgNPs in the interstitial space between and on the AgNWs, thus creating a dual-nanostructured surface with improved SERS characteristics, without the use of additives.

The NWS was then utilized as a substrate to produce a uniform AgNP surface via dropcasting and subsequent thermal decomposition of the aqueous ink of the Ag complex as the solvent evaporated. The outer edge of the deposited ink spot experienced a concentration of silver deposition due to the coffee stain effect, and thus all characterization was performed on the central area. While dropcasting was chosen to optimize SERS response for proof of concept, a pathway exists to adapt this step to Mayer rod coating for volume production (7).

Optical microscopy showed craters interspersed with the AgNWs due to the elimination of the gaseous decomposition products (Figure 3a), while AFM revealed clusters of AgNPs at wire intersections, confirming the dual-nanostructured surface (Figure 3a, inset). The particles exhibit extremely small sizes and exist on a large range of Z heights. The same spatial uniformity assessment was applied to the DNS substrate, sampling SERS spectra of ATP every 500 μm along a 1 cm linear path for comparison to the NWS, averaging four scans per location (Figure 3b). The SERS response of the DNS retains some spatial fluctuations in the intensity of the acquired spectra, as expected due to variation in the underlying AgNW layer, but provides a minimum SERS response at every sampled location, as well as a consistently higher SERS response overall when compared to the NWS.

Augmenting the NWS with dense, nonordered AgNPs produced from a silver complex with good shelf stability (Figure 1b) resulted in an increase in the number of SERS hotspots. This approach improved the magnitude of the SERS response, while decreasing the spatial variability of the signal obtained from the AgNWs alone.

To quantify and compare enhancement factors (EF), the SERS spectra of a 5 μL sample of 423.5 μM ATP solution were taken at multiple locations of both a NWS and DNS fabricated on the same slide. Due to the spatial variability in the NWS, a total of 15 positions with local SERS signal max- ima were sampled, a process which took roughly 30 s per location. No optimization was needed for the DNS, and thus 10 random locations were sampled twice, at both 

 s, to capture the initial response, and to approximate the laser exposure time seen in the NWS sampling. Spectra are shown in Figure 4.

 are due to vibrations of the aromatic ring of ATP, with contribution from the dimerization of ATP to 4,4’-dimercaptoazobenzene (DMAB) at 1084 cm

 (8). The effects of ATP concentration and laser exposure on dimerization for these substrates were explored in more detail elsewhere (7). The calculated EFs for these peaks are shown in Table I. Peaks at 1150 cm

 were also observed, largely attributed to the selective enhancement of the 

 vibrational modes of ATP during SERS analysis (9,10).

Although the contribution from DMAB to the spectrum makes it difficult to reliably quantify the increase in EF for the DNS vs. the NWS (7), all substrates demonstrate an EF of 105 or greater, with the dual-nanostructured substrate showing a clear improvement in EF over the nanowire substrate.

A nanowire substrate utilizing AgNWs and a dual-nanostructured SERS surface that also incorporated AgNPs were fabricated using synthesis methods and production processes amenable to cost-effective volume production. The AgNWs synthesized were of high purity and narrow size distribution, ~10 μm in length and ~100 nm in diameter, and were deposited using Mayer rod coating to form a SERS-active substrate. Although high spatial variation in the number of SERS hotspots relative to the spot size of the laser limits its applicability as a quantitative SERS device, the AgNWs were shown to be an effective substrate to facilitate subsequent dropcasting with an aqueous solution of a silver complex to populate the interstitial spaces between the AgNWs and improve the SERS response using an additive-free process.

The resulting DNS exhibited both improved spatial uniformity in the SERS response compared to the NWS alone, as well as a significant increase in intensity. Through further process development and adaptation of AgNP deposition to a Mayer rod coating method, the dual-nanostructured SERS approach offers a pathway to industrial fabrication of a low-cost, SERS-active substrate.

(1) C.L. Haynes, A.D. McFarland, and R.P. Van Duyne, 

(2) K. Zhan, R. Su, S. Bai, Z. Yu, N. Cheng, C. Wang, S. Xu, W. Liu, S. Guo, and X.-Z. Zhao, 

(4) A.T. Fafarman, S.-H. Hong, S.J. Oh, H. Caglayan, X. Ye, B.T. Diroll, N. Engheta, C.B. Murray, and C.R. Kagan, 

(5) X. Liu, C. Zong, K. Ai, W. He, and L. Lu, 

(6) K.R. Zope, D. Cormier, and S.A. Williams, 

(7) K.B. Castro, M.Sc. dissertation, Old Dominion University, Norfolk, Virginia (2019). 

(8) Y.-F. Huang, H.-P. Zhu, G.-K. Liu, D.-Y. Wu, B. Ren, and Z.-Q. Tian, 

(9) K. Uetsuki, P. Verma, T.-a. Yano, Y. Saito, T. Ichimura, and S. Kawata, 

(10) Y.-C. Chen, J.-H. Hsu, and Y.-K. Hsu, 

 are with the Department of Chemistry at Old Dominion University, in Norfolk, Virginia. 

 is Vice President of Marketing at Wasatch Photonics, in Morrisville, North Carolina. Direct correspondence to: 

SERS can amplify Raman signals, but to make the technique practical for industrial use, large quantities of substrate are needed. The approach described here could enable cost-effective, reproducible manufacturing of SERS substrates at large scale.

A Dual Nanostructured Approach to SERS Substrates Amenable to Large-Scale Production

Perovskites are ubiquitous in science and technology, from solar cells to high-temperature and exotic superconductivity. Exciting representatives of the perovskite family, such as metal-doped oxy-chalcogen calcium ruthenates, recently attracted the attention of researchers. Rapid modification of their properties is a key for research competitiveness. Crystalline structure plays an important role in the electronic properties of these materials, in particular superconductivity. Flux-assisted growth may deliver an advanced performance of these perovskites due to a higher level of crystallinity. In this article, we demonstrate that Raman spectroscopy enables a fast evaluation of improvement of the crystalline quality of Ca

 by using chlorine as a flux. In particular, ~0.6 wt.% of chlorine significantly modifies the properties of polycrystalline material. While standard energy dispersive X-ray analysis (EDX) diagnostics could not resolve the presence of chlorine and distinguish between samples with and without it, Raman spectroscopy resolved the chlorine-related features with very high accuracy at initial synthesis. Moreover, after specially performed high-temperature treatment, which sublimated chlorine from the specimen, Raman analysis confirmed the absence of chlorine and presence of required crystalline phase. The feedback received in Raman analysis is very informative, and faster than provided by any other method.

Contemporary solid-state physics is hard to imagine without ubiquitous perovskites. Perovskites gained attention after the discovery of high-temperature superconductivity in copper oxides by Bednorz and Müller (1). Later, triplet superconductivity was discovered in strontium ruthenates (2), and, very recently, high-temperature superconductivity was reported in calcium ruthenates (3). However, perovskites are not related only to the world of superconductivity. They are interesting objects for solar cells; see, for example, (4) and (5), and other applications. Interestingly, they constitute a major part of the earth’s mantle (6). Recently, possible superconductivity was reported in reference (7), which may have a relationship with the findings of reference (2). These discoveries initiated our studies in calcium ruthenates, which was facilitated by Raman diagnostics. By this report we would like to share our excitement concerning perovskites with a broader audience.

Our ceramic samples were prepared similarly to the case of doped strontium ruthenates described in (7,8). Here, we will briefly summarize the various exploratory procedures.

In total, five batches of samples were studied. For the first batch, the dried precursors, RuO

, were powderized and mixed initially in stoichiometric proportions Ca

 composition (214 hereafter). A hand grinding and mixing process in an agate mortar was applied. The powder mixture was calcined at 1070 °C (this temperature was reported in [2]) for 20 min, with 10 h linear heating time and 5 h linear cooling time. The calcined powder was again ground and separated into two parts. To one of these parts, as a chlorine flux (see, for example, [8]), 1 wt.% of NaCl was added, and hand mixed in the agate mortar. Both powders were then pelletized and annealed in the regime similar to that described above (8). A second batch was prepared in a similar manner (also with and without flux); however, pellets were first annealed for a longer time (1070 °C for 3 h, with 10 h linear heating and cooling, respectively). A third batch was prepared similarly with two differences: 20 at.% excess ruthenium in initial ingredients, and the pellets were annealed by much longer time (1070 °C for 50 h). This temperature and lengthier annealing time is recommended in (2), with 10 h linear heating followed by 10 h of cooling. Excess initial amount of ruthenium was applied to compensate its deficit (see the EDX data (in Table I) when preparing with stoichiometric composition (9). The fourth batch was also prepared with 20 at.% excess ruthenium. The powder was calcined at 1070 °C for 20 min, with 10 h linear heating time and 3 h linear cooling time. The first annealing of pellets was made at 1070 °C in air for 3 h with linear temperature increases and decreases each at the 10 h interval. The second annealing was made at 1370 °C in air for 30 h with linear temperature increase and decrease during 6 h and 4 h, respectively. The final, fifth batch was also prepared with 20 at.% of excess ruthenium. It was calcined at 1000 °C in air for 20 min with linear temperature increase and decrease at 3 h each. Ground and pelletized samples were annealed at 1370 °C in air for 30 h, with linear temperature increases and decreases at the rates 10 h and 4 h respectively. At the annealing, the pellets were arranged on top of each other pairwise. This worked better than using powder underlayment (9) for exclusion of the interaction of pellet material with the supporting alumina plates. Long-time and high-temperature annealing was performed in KSL-1700TX Zirconia Sintering Furnace (Hansunthermal).

The Raman spectra were taken at ambient temperature using a Thermo Fisher DXR Raman microscope equipped with a 532 nm (green) unpolarized excitation laser and a 5 cm

resolution grating. The calibration was performed automatically, using an internally attached calibration toolkit that included a standard polystyrene film. We used the usual double exposure protocol where half of the total scans were dedicated to the background collection. Throughout this process, the Raman spectra were measured using a 10x objective, 5 mW laser power at the sample, a 50 micron pinhole, and a total exposure time between 2 to 10 min.

An X-ray diffraction (XRD) experiment was performed on polycrystalline calcium ruthenate sample pellets using CuKα radiation on a powder diffractometer (Siemens D-500), the angle interval 2θ = 10–80° with the step 0.04° and scanning rate 0.01°/s. Match! 3.3 Crystal Impact software was used for phase analysis.

Surface morphology and composition of polycrystalline samples were analyzed using a Hitachi SU3500 scanning electron microscope (SEM) with Oxford Instrument energy dispersive X-ray analysis (EDX) unit operated using AZtec software.

Figure 1 demonstrates major results obtained by our Raman diagnostics in combination with XRD and SEM analysis. As follows from this figure, achievement of the required crystalline phase 214 is far from being reached. There is also direct evidence that influence of a minor amount of chlorine during synthesis yields changes in the substance which are not visible via XRD or EDX (see Table I above), while their presence is consistently revealed via Raman diagnostics.

As follows from this table, the Ca to Ru ratio is about 2:1. However, both Raman and XRD data contradict this observation. The puzzle is most likely because of the existence of parasitic (possibly amorphous) phases existing in the material that are responsible for integral composition close to 214, while the crystalline phase is mainly 113 (that is, CaRuO

Since the regimes used for obtaining the results shown in Figure 1 have not delivered the required phase 214, but rather the phase 113, further Raman-guided studies were undertaken.

Figure 2 contains the results of this next stage of research that included high-temperature annealing.

While annealing at 1070 °C for 50 h yields a sample with the majority 113-phase (blue and green), identified by their fingerprint region (including the 439 cm

 peak of the B2g-mode [10]), a two-step annealing at 1070 °C and 1370 °C (orange) reveals a mix of both CaRuO

). However, a prominent background between 580 and 800 cm

, suggests the presence of unknown phases. At the same time, the direct annealing at 1370 °C improves representation of 214 phase where these features are suppressed (red curve). We should also note that while XRD diffraction is an integral characteristic over the sample surface, Raman microscopy allowed us to explore the selected small areas and confirm the homogeneity of the material. Table II below shows compositional data obtained by EDX. These data support Raman and XRD observations.

By using Raman spectroscopy, a powerful tool for phase identification, we were able to narrow down the optimal synthesis routes to our target material, Ca

. While it is best to combine the experimental results with other characterization tools such as XRD and SEM/EDX, having the capabilities and appropriate library database of Raman spectra can significantly accelerate material exploration work in condensed matter physics.

We are grateful to A. Rzhevskii for help with Raman measurements, and to Thermo Fisher Scientific for providing us access to the Raman microscope. This work was supported by ONR grants N00014-16-1-2269, N00014-17-1-2972, N00014-18-1-2636, N00014-19-1-2265, and N00014-20-1-2442.

(2) Y. Maeno, H. Hashimoto, K. Yoshida, S. Nishizaki, T. Fujita, J.G. Bednorz, and F. Lichtenberg, 

(3) H. Nobukane, K. Yanagihara, Y. Kunisada, Y. Ogasawara, K. Isono, K. Nomura, K. Tanahashi, T. Nomura, T. Akiyama, and S. Tanda, 

(4) M.I.H. Ansari, A. Qurashi, and M.K. Nazeeruddin, 

(5) P. Roy, N.K. Sinha, S. Tiwari, and A. Khare, 

(6) K. Hirose, R. Sinmyo, and J. Hernlund, 

(7) Y. Aleshchenko, B. Gorshunov, E. Zhukova, A. Muratov, A. Dudka, R. Dulal, S. Teknowijoyo, S. Chahid, V. Nikoghosyan, and A. Gulian, 

Quantum Studies: Mathematics and Foundations

(9) S. Xu, Y. Hu, Y. Liang, C. Shi, Y. Su, J. Guo, Q. Gao, M. Chao, and E. Liang, 

(10) N. Kolev, C.L. Chen, M. Gospodinov, R.P. Bontchev, V.N. Popov, A.P. Litvinchuk, M.V. Abrashev, V.G. Hadjiev, and M.N. Iliev, 

(12) S. Jiao, K-N.P. Kumar, K.T. Kilby, and D.J. Fray, 

(13) D. Fobes, E. Vehstedt, J. Peng, G. C. Wang, T. J. Liu, and Z. Q. Mao, 

 are with the Advanced Physics Laboratory at Chapman University, in Burtonsville, Maryland. Direct correspondence to: 

Perovskites are known to be useful for fabrication of solar cells, and their crystalline structure plays an important role in their electronic properties. Here, we show how Raman analysis is able to confirm the presence of the required crystalline phase for solar cell production.

Raman Technology for Today's Spectroscopists

Tracking Bioactive Compounds Produced by Genetically Engineered Yeast Cells Using Raman Imaging

A Dual Nanostructured Approach to SERS Substrates Amenable to Large-Scale Production

Raman Spectroscopy as a Tool for Rapid Feedback of Perovskite Growth Crystallinity and Composition