Eric Bergles

Raman measurement on microscopic inclusions in fluorescent materials requires the ability to measure in small volumes, excellent throughput, and long wavelength excitation such as 1064 nm for fluorescence reduction. Carotenoids and keratin can be measured in epoxy and fossil amber by BaySpec's dispersive 1064 confocal Raman microscope. 

Inclusions, small particles less than 1 mm in diameter embedded in other materials, are of interest in a number of different areas. In some manufactured products they result in cosmetic, structural, or chemical resistance defects. On the other hand, entrapped inclusions in paleology, or paints and stains in artefacts are a time-capsule from our past. This article demonstrates a few examples of fast, nondestructive identification of microscopic fluorescent samples using Raman spectroscopy, which were previously unattainable.  

The challenge for Raman spectroscopy in the past was that it was susceptible to fluorescence. Working with 1064 nm wavelength lasers avoids fluorescence, by avoiding exciting the electronic transitions. Traditionally, due to grating and detector limitations, 1064 nm Raman is often obtained by an FT-Raman system, which is very slow because each spectrum is composed by constantly moving interferometers. Sample mapping using an FT-Raman system is almost impossible. With recent advances in gratings and InGaAs detectors, volume mapping, depth and surface profiling can be accomplished easily with BaySpec's Raman system. For instance, a 1064 nm Raman microscope in dispersive configuration with f2 high-throughput Raman spectrometer is able to create a chemical image as fast as a traditional Raman microscope with visible lasers such as 532 nm. Furthermore, such dispersive configuration allows great flexibility of the system, such as a high-magnification video probe being added to the system to measure the area of interest quickly on samples that do not fit under a microscope. 

Figure 1: 1064 nm Raman spectra of feathers from an extinct pink-headed duck and from a flamingo wing. Courtesy of Dr. Daniel Thomas, Smithsonian Institution. Refer to reference 1 for Raman spectra of feathers in fossil amber. Raman Data were taken by BaySpec’s Nomadicâ¢ 1064 nm Raman microscope.			

Microscopic feathers can occasionally be found embedded in fossil amber, which hold clues about the prehistoric animals. The technique was successfully applied to fossil feathers by using BaySpec's Nomadic™ 1064 nm Raman microscope (Figure 1) (1). The versatility of the video probe in being able to make measurements of areas and depth of a painting is unmatched (Figure 2). In a quick summary, Raman imaging using 1064 nm excitation provides a unique and rapid ability to chemically profile inclusions with fluorescence. 

Figure 2: Painting’s cross-section and spectra of the different layers using a Raman video microprobe attached to a BaySpec RamSpecâ¢ 1064 nm Raman spectrometer.			

 (1) D.B. Thomas, P.C. Nascimbene, C.J. Dove, D.A. Grimaldi, and H.F. James, 

Articles

Raman measurement on microscopic inclusions in fluorescent materials requires the ability to measure in small volumes, excellent throughput, and long wavelength excitation such as 1064 nm for fluorescence reduction.

Chemical Analysis of Microscopic Fluorescent Materials by Dispersive 1064 Raman System

Owing to technological improvements spurred on by the telecommunications boom of the last decade, Raman spectroscopy has become much more accessible to users in all application areas, including agricultural, forensic, pharmaceutical, biomedical, and others.  However, there remains a struggle to extract useful Raman spectra from fluorescent and luminescent samples. 

Most users with fluorescent samples switched to near-infrared wavelengths such as 785 nm and 830 nm to avoid fluorescence. But now, BaySpec's new dispersive 1064 nm Raman spectrometer family offers a turn-key solution combines the speed, sensitivity, and ruggedness of the dispersive Raman instruments with superior fluorescence avoidance of traditional FT-Raman.  

Any new method for routine determination of chemical composition in beverage manufacturing processes should be non-invasive, non-destructive, and rapid. Raman spectroscopy offers the advantages of minimal sample preparation and fast spectral collection. However, the pigments in red wines are usually too fluorescent to allow Raman to be measured, even at 785 nm. But at 1064 nm, a clear Raman spectrum is generated while florescence background is almost totally avoided. 

Figure 1: Identification of methanol in red wine based on its unique Raman features different from ethanol.

Further, glass is mostly transparent to NIR wavelengths, compared to 532 nm and 785 nm, which makes 1064 nm Raman even more suitable for non-destructive measurement of red wine through the bottle. As in Figure 1, comparing the red wine spectrum in a secondary vial with that collected through the bottle, despite a small attenuation in the signal intensity, the Raman features are maintained and distinguishable for further identification and quantification. 

Further component analysis was performed by adding methanol into the wine and measuring as before. As seen in Figure 2, most of the Raman features from pure red wine could be easily identified as ethanol. For the Raman spectrum of red wine with methanol added, a Raman peak centered around 1020 cm

  is apparent, and this peak can be utilized for the quantification of the methanol concentration profile in different red wine samples as seen in Figure 3. Slight variances in spectral intensity on different measuring spots were found due the heterogeneity of the bottle glass. 

Figure 2: Identification of methanol in red wine based on its unique Raman features different from ethanol.			

Based on these experiments, 1064 nm dispersive Raman is demonstrated as a viable new option to determine wine composition and contamination in the bottle. Chemometrics tools such as principal component analysis (PCA) and partial least squares regression (PLS) can significantly increase the accuracy and precision. It can be expected that future development of such applications may provide the wine industry a fast and non-destructive quality control and assurance tool for composition monitoring. This methodology can be easily applied to distilled spirits, beer, and others. 

Figure 3: Quantification of methanol concentrations in red wine based on the intensity of one major Raman feature.			

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Owing to technological improvements spurred on by the telecommunications boom of the last decade, Raman spectroscopy has become much more accessible to users in all application areas, including agricultural, forensic, pharmaceutical, biomedical, and others.

Non-Destructive Red Wine Measurement with Dispersive 1064 nm Raman Spectroscopy

Biological tissues and other materials often autofluoresce at near-infrared wavelengths, prohibiting Raman acquisition. New, dispersive Raman systems at 1064 nm allow fluorescence-free measurement in similar integration times.

Fluorescence is much more likely and intense at short wavelengths where energies are more apt to cause an electronic transition. Yet even at near-IR wavelengths like 785 or 830 nm, many substances still fluoresce, sometimes prohibiting Raman spectral acquisition. For those users who require longer wavelengths such as 1064 nm, the only available option has been FT-Raman, which is typically noisier and slower than dispersive  Raman systems. But now, BaySpec's new dispersive 1064 nm Raman spectrometer family of instruments offers a turn-key solution that combines the speed, sensitivity, and rugged design of traditional dispersive Raman instruments with the fluorescence avoidance of traditional FT-Raman instruments. 

A variety of animal tissues obtained from a local market were interrogated using two BaySpec benchtop systems: the RamSpec™ 785 and the RamSpec™ 1064. Both systems utilized filtered fiber probes designed for each respective wavelength. Acquisition times were 30 s for both systems. Power was set at 50 mW for the 785 nm measurements, and 150 mW for the 1064 nm measurements. 

Pigmented and porphyrin-rich tissues such as kidney are often too fluorescent to be measured, even at 785 nm; see Figure 1. But using 1064 nm, a clear Raman spectrum relatively devoid of fluorescence background is generated using the same integration time. Additionally, because of the extended quantum efficiency of the InGaAs detector, high wavenumber features (C–H, O–H, and N–H stretching modes) are also simultaneously captured with the same laser.  

Figure 1: Kidney (porcine) is highly fluorescent at 785 nm, preventing extraction of a usable Raman signal. At 1064 nm, however, this fluorescence interference is largely avoided and clear Raman bands are evident. 			

In addition to allowing users the ability to measure Raman spectra from highly fluorescent samples, the dispersive 1064 nm Raman systems also reduce the stringent sampling conditions necessary at shorter wavelengths. For example, 785-nm Raman measurement through vials, cuvettes, or under cover slips often requires that these materials be made of fused silica, quartz, or calcium fluoride for their reduced fluorescence, all of which cost considerably more than bulk glass materials. However, even inexpensive glass sample vials can be used for 1064-nm measurement of weak Raman scatterers such as tissue, owing to the fluorescence avoidance of the longer wavelength approach. 

One of the lingering concerns about the use of 1064-nm Raman is the reduced Raman scattering cross-section. As compared to 785-nm Raman, this cross-section, indeed, reduces approximately 3.4×. However, according to permissible standards for human tissue (skin) exposure, the maximum power level that can be tolerated at 1064 nm is approximately 3.4× higher than the power permissible at 785 nm (1). So, even in photosensitive samples such as biological tissue, the physical reduction in Raman efficiency can be totally compensated by increased laser power.  

Based on these experiments, 1064-nm dispersive Raman will provide a viable new option for those users who are studying highly fluorescent tissues, desire to measure multiple samples in simple glass containers, or those users who are interested in simultaneous fingerprint and high wavenumber spectral acquisition. Future studies will certainly evidence further advantages of this approach, as compared to shorter wavelength (785 or 830 nm) Raman approaches or FT-Raman systems. 

(1) National Institute of Standards and Technology, Z136.1, 76–77, (2007). 

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Tissue Raman Measurement at 1064 nm

BaySpec, Inc. has developed a complete line of 1064 nm excitation, dispersive Raman systems that offer maximum reduction in fluorescence interference from biological samples and thus making them very useful tools for biofuel research. 

Biofuels are a current focus of government funding (1) and research efforts from academia, as well as industry, as we look to reduce green house gas emissions and secure our energy future away from fossil fuels. Of these materials being evaluated for biofuel production, algae offers the single greatest promise of becoming a sustainable source as our transportation fuel and at the same time does not produce net green house gas emissions in its life cycle. Algae are unique organisms that can store energy in the form of lipids which can comprise up to 60% of their body mass. These lipids can be readily converted to transportation fuel, such as biodiesel.  As different micro and macro algae are evaluated, timely quantitation of their lipid content becomes increasingly important. 

Traditional, wet chemistry-based methods are time consuming, require large amounts of sample, and are tedious to implement.  Fluorescence based methods allow in-situ lipid content measurement, however, the algae samples have to be stained first with fluorescence labeling molecules (2). Raman spectroscopy is a non-destructive technique that measures the vibrational "fingerprint" and requires essentially no sample preparation and, thus, would be ideal for these applications. Unfortunately, strong background fluorescence from algae tends to overwhelm the Raman spectral features, even with 785 nm laser excitation. 

BaySpec's line of 1064 nm Raman analyzers offer the means to minimize interference from fluorescence and unmask much of the algae Raman spectral "fingerprint." In Figure 1, a sample of micro-algae has been analyzed using the benchtop RamSpec™ 1064 nm system and the handheld Xantus™-1G 785 nm system. While the trace derived from the 785 nm system is mostly from background fluorescence, Raman peaks clearly dominate the 1064 nm trace even without any baseline treatment. The integration time is less than 1 min and the laser power at the sample is about 200 mW.

 Figure 1: Raman spectra of micro-algae sample using the benchtop RamSpecâ¢ 1064nm system (red trace) and the Xantusâ¢-1G 785 nm system.			

BaySpec's RamSpec™ 1064 nm system features high-efficiency Volume Phase Grating (VPG®) Technology and a patented deep cooled InGaAs camera to allow high optical throughput and minimized detector noise from thermal sources. The 1064 nm, narrow bandwidth fiber laser is stabilized for Raman applications and has a power output of 500 mW, with optional power outputs up to 1.5 W, providing the flexibility necessary to ensure peak distinction in samples with reduced Raman scattering. 

In addition to the RamSpec™ benchtop system, BaySpec's 1064 nm dispersive Raman platform also includes a portable system Xantus™-2 and a high performance system interfaced to a research grade microscope, the Nomad™. These systems offer researchers in the biofuels community an unprecedented choice of tools, not only to characterize micro algae but also the potential to quantify lipid content in-situ, with no interruption to their growth process. 

http://www.eere.energy.gov/biomass/pdfs/biodiesel_from_algae.pdf