The authors would like to thank Andreas Hesselschwerdt and Jouni Rautio from the design office and workshop at JRC-Karlsruhe for the design and the manufacture of the radioactive sample holder for Raman analysis. Patrick Lajarge, Daniel Freis (JRC-Karlsruhe), and Mark Sarsfield (NNL, UK) are acknowledged for providing the AmO2 samples investigated with the present technique. The authors also would like to thank Boris Burakov (Khlopin Radium Institute) for providing the sample of the Chernobyl lava and Philipp P&#246;ml and Ralf Gretter (both at JRC-ITU) for the sample preparation.

1. Experiment Planning
<ol>
	<li>Make sure that the Raman spectrometer to be used is equipped with an X-Y stage below the slide holder, with a central aperture at least 60 mm in diameter.</li>
	<li>Make sure to have at least 150 mm of free room below the stage to introduce the capsule and that this room is easily accessible (from the top or bottom).</li>
	<li>Ensure that the Raman spectrometer is equipped with an objective with at least a 10-mm working distance.</li>
	<li>Ensure that the sample to analyze is stored and transferrable to the capsule in the proper atmosphere (refer to the local procedure to transfer samples from glove boxes).</li>
	<li>Make sure that the sample can be handled with the tweezers, small chemical spoons, or spatulas needed to load the sample into the capsule.</li>
	<li>High-pressure (HP) option: Make sure that the confinement system is equipped with a system to pump and fill the high-pressure capsule.</li>
	<li>Ask the local radioprotection officer for radioprotection measures to implement for the complete procedure.</li>
</ol>
2. Preparation of the Sample Holder
<ol>
	<li>Collect all of the parts composing the capsule or, optionally, the high-pressure capsule.</li>
	<li>Fixing the window on the capsule body
	<ol>
		<li>Evenly apply a small amount of epoxy resin directly with the glue applicator on the outer part of the groove fitting the window. Note that different types of epoxy resins have been tried. The glue indicated in the Materials List was the best adapted for this application because of its specific viscosity.</li>
		<li>To make sure that the installed window is optically clean, wear clean gloves and unpack the window from its original packaging. Place it in the groove on the acrylic glass capsule, moving it with a finger in order to disperse the glue between the window and the groove.</li>
		<li>Carefully check through the window to see if the glue is evenly applied between the window and the acrylic glass.</li>
		<li>Let the glue cure as long as indicated in the glue instruction sheet.</li>
		<li>Check again through the glass to see whether the window and the acrylic glass are properly glued; no bubbles should be visible.</li>
	</ol>
	</li>
	<li>HP option: Fixing the window on the high pressure capsule body
	<ol>
		<li>Check with a magnifying glass whether the surfaces in contact with the capsule O-ring are clean and evenly machined, which ensures a good tightness.</li>
		<li>Place a capsule O-ring in the dedicated grove on the window side of the high-pressure capsule body.</li>
		<li>Place the high-pressure capsule window on the high-pressure capsule body over the capsule O-ring.</li>
		<li>Place the polyoxymethylene flat ring over the high-pressure capsule window.</li>
		<li>Place the closing flange at the top of the windows side of the high-pressure capsule window and affix it with the 6 top-sink screws.</li>
	</ol>
	</li>
	<li>Fixing the bag on the capsule
	<ol>
		<li>Insert the capsule, windows first, from the wide side of the funnel-shaped bag into the narrow part of the funnel-shaped bag, ending up to the point where the cylinder cannot slide further because of the jut.</li>
		<li>If necessary, adjust the bag position so that the cylinder sticks out of the funnel-shaped bag by about 1.5 cm.</li>
		<li>Place the tightening O-ring over the bag in the groove of the cylinder.</li>
		<li>Tape the bag with flexible electrical tape onto the cylinder in order to leave about 8 mm of the cylinder&#39;s upper part uncovered. This part will be used to fix the cylinder in the Raman microscope.</li>
	</ol>
	</li>
	<li>Capsule assembly tightness test
	<ol>
		<li>Bring the funnel-shaped bag equipped with the capsule (bag assembly) to the installation dedicated to the tightness testing of glove box gloves and sacs, usually available in nuclear facilities (in this case, an Ar-H2 over-pressurized device with an H2 detector).</li>
		<li>Fix the bag assembly in the test flange.</li>
		<li>Tape it to the flange using electrical tape.</li>
		<li>Fill up to 500 mbar with an Ar + 5% H2 gas mixture.</li>
		<li>Move the portable H2 detector all around the capsule and the bag, taking special care around the area where the window is glued.</li>
		<li>If H2 is detected, repeat the procedure from step 2.5.2, as the bag assembly is not tight enough.</li>
	</ol>
	</li>
	<li>Preparation of the plunger
	<ol>
		<li>Install the sliding O-ring in the plunger groove.</li>
		<li>Install the pin stub mount on the plunger.</li>
		<li>Stick the double-sided adhesive tab on the pin stub mount, keeping the protective layer on the surface towards the outside.</li>
		<li>Screw the pull screw in the other side of the plunger.</li>
		<li>If the sample is powder or has parts smaller than 1 mm, install the external circlip with circlip pliers in the last groove of the plunger (toward the screw). For samples larger than 1 mm, adjust the position of the external circlip on the plunger groove to the thickness of the sample.</li>
	</ol>
	</li>
	<li>Preparation of the high-pressure plunger
	<ol>
		<li>Check with a magnifying glass whether both surfaces in contact with the capsule O ring are cleaned and evenly machined, ensuring good tightness.</li>
		<li>Install a capsule O-ring in the dedicated plunger groove on the sample side.</li>
		<li>Screw the sample holder at the top of the plunger.</li>
		<li>Stick a piece of a double-sided adhesive tab, of a size corresponding to the sample holder, on the sample holder, keeping the protective layer on the surface towards the outside.</li>
		<li>Check with a magnifying glass whether both surfaces in contact with the inlet O-ring are cleaned and evenly machined, ensuring good tightness.</li>
		<li>Install the inlet O-ring in the dedicated groove on the gas inlet side of the plunger.</li>
		<li>Screw the adapter in the plunger.</li>
		<li>Install the ball valve on the adapter according its installation procedure.</li>
	</ol>
	</li>
</ol>
3. Installation of the Sample in the Sample Holder
<ol>
	<li>Install the sample holder on the confinement system according the local procedure and at the same time, install the plunger in the confinement system. (HP option): Install the 6 bottom-sink screws as well.</li>
	<li>Remove the protective layer from the double-sided adhesive tab.</li>
	<li>Hold the plunger and place the sample on the adhesive tab. If the sample is a single piece, press a little bit on the sample with the tweezers or the chemical spoon, if possible. If the sample is powder, spread it very gently on the sample holder. HP option: Place a sample smaller than 1 mm in height on the high-pressure sample holder.</li>
	<li>Insert the plunger (HP option: plunger flange) in the capsule. Push it in until it cannot go any further in, while taking care to keep the capsule in a vertical position. From this point, make sure that the capsule is held as much as possible in the vertical position.</li>
	<li>HP option: Tighten the 6 bottom-sink screws to fix the plunger flange at the bottom of the high-pressure capsule body assembly.</li>
	<li>HP option: Pressurization of the high-pressure capsule.
	<ol>
		<li>Connect the ball valve to the vacuum/gas supply line in the confinement.</li>
		<li>Open the valve and evacuate the capsule.</li>
		<li>Pressurize the capsule with the chosen gas, taking care that it does not exceed 20 bars and that the gas is inert with respect to the capsule material.</li>
		<li>Close the valve.</li>
	</ol>
	</li>
	<li>Separate the capsule from the confinement according the local procedure, and take care that the valve does not damage the plastic bag. Reduce the volume of the bag by taping it together, in order to fit it below the microscope stage (see step 4.4). Note that the procedure may require taping a second bag over the first one.</li>
</ol>
4. Installation of the Capsule under the Raman Microscope
<ol>
	<li>Fix the metal ring slide with a blocking screw (see Figure 3) on the tape-free upper part of the capsule. Tighten the side screw to block it.</li>
	<li>Insert the capsule from either the top or the bottom of the microscope stage.</li>
	<li>Mount the metal ring slide on the stage slide holder (see Figure 3). Secure it with the slide-holder springs.</li>
	<li>Check if the bag below the stage can freely move within any needed X, Y, and Z movements of the stage. If not, tape the bag together to reduce its volume.</li>
</ol>
5. Raman Spectra Measurement
<ol>
	<li>Calibration of the frequency of the Raman spectrometer.
	<ol>
		<li>Place a single silicon crystal on the window of the capsule.</li>
		<li>Choose the objective to use and focus the microscope.</li>
		<li>Select the laser wavelength for the measurement and determine the T2g excitation of the single silicon crystal, for which the reference band is at 520.5 cm-1 26. Using the software, adjust the frequency scale accordingly.</li>
	</ol>
	</li>
	<li>Calibration of the intensity of the Raman spectrometer.
	<ol>
		<li>Adjust the optical path, laser, inlet slits, polarization configuration, confocality spatial filter, and the CCD opening using the strong Si crystal peak in order to maximize the peak intensity while keeping the slits as closed as possible without decreasing the maximal intensity. Compare this intensity to the value obtained after &#34;factory&#34; alignment in the same conditions.</li>
		<li>Close the slits and spatial filter to reach the required spectral and z-axis spatial resolutions, respectively.</li>
	</ol>
	</li>
	<li>Measurement of the sample.
	<ol>
		<li>Measure the sample in a similar manner as the unencapsulated samples. Note that this step depends strongly on the type of Raman spectrometer used, as well as on the type of measurement. Refer to the manual of the Raman spectrometer. For very small samples (for example when measuring one grain of the size of the laser spot), fluorescence signal might appear in the Raman spectrum mainly because of illuminating the double adhesive tab. In this case make sure to refocus the microscope in order to illuminate the maximum of the surface of the small sample and reduce the entrance slit aperture of the spectrometer to analyze the central part of the illuminated spot only. Make sure as well to not illuminate the double-sided adhesive directly with the laser. The laser power could burn the adhesive and release volatile organic molecules adding fluorescence in the measured spectrum.</li>
		<li>Check that, in the measured spectrum, the spectral lines of the fused silica27 of the window do not appear. This may happen when using a Raman spectrometer with poor confocality.</li>
	</ol>
	</li>
</ol>

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<li>Böhler, R. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0925838814016454">The solidification behaviour of the UO2-ThO2 system in a laser heating study.</a> J. Alloys Compd. 616, 5-13 (2014).</li>
<li>Böhler, R. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0022311514000919">Recent advances in the study of the UO2-PuO2 phase diagram at high temperatures.</a> J. Nucl. Mater. 448 (1-3), 330-339 (2014).</li>
<li>Begun, G. M., Haire, R. G., Wilmarth, W. R., Peterson, J. R. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/002250889090465V">Raman spectra of some actinide dioxides and of EuF2.</a> J. Less-Common MET. 162 (1), 129-133 (1990).</li>
<li>Hobart, D. E., Begun, G. M., Haire, R. G., Hellwege, H. E. Characterization of transplutonium orthophosphates and trimetaphosphates by Raman spectrophotometry. J. Less-Common MET. 93, 359(1983).</li>
<li>Hobart, D. E., Begun, G. M., Haire, R. G., Hellwege, H. E. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jrs.1250140114/abstract">Raman spectra of the transplutonium orthophosphates and trimetaphosphates.</a> J. Raman Spectrosc. 14 (1), 59-62 (1983).</li>
<li>Nguyen Trung, C., Begun, G. M., Palmer, D. A. <a target="_blank" href="http://pubs.acs.org/doi/abs/10.1021/ic00051a021">Aqueous uranium complexes. 2. Raman spectroscopic study of the complex formation of the dioxouranium(VI) ion with a variety of inorganic and organic ligands.</a> Inorg. Chem. 31 (25), 5280-5287 (1992).</li>
<li>Guimbretière, G. <a target="_blank" href="http://aip.scitation.org/doi/abs/10.1063/1.4816285">In situ Raman monitoring of He2+ irradiation induced damage in a UO2 ceramic.</a> Appl. Phys. Lett. 103 (4), (2013).</li>
<li>Canizarès, A. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jrs.4088/full">In situ Raman monitoring of materials under irradiation: study of uranium dioxide alteration by water radiolysis.</a> J. Raman Spectrosc. 43 (10), 1492-1497 (2012).</li>
<li>Johnston, A. H. Radiation Damage of Electronic and Optoelectronic Devices in Space. Proceedings of the 4th International Workshop on Radiation Effects on Semiconductor Devices for Space Application. 2000 Oct 11-13, Tsukuba, Japan, , (2000).</li>
<li>Nucleonica Nuclear Science Portal v.3.0.49. , Nucleonica GmbH. Karlsruhe, Germany. Available from:
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<li>Strahlenschutzverordnung. Bundesministerium für & Naturschutz und Reaktorsicherheit Umwelt. , 54-55 (2013).</li>
<li>Prieur, D., et al. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0022311512006319">Accommodation of multivalent cations in fluorite-type solid solutions: Case of Am-bearing UO2.</a> J. Nucl. Mater. 434 (1-3), 7-16 (2013).</li>
<li>Lebreton, F., Belin, R. C., Prieur, D., Delahaye, T., Blanchart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Situ+Study+of+the+Solid-State+Formation+of+U1-xAmxO2%C2%B1%CE%B4+Solid+Solution%5BTitle%5D)+AND+%22Inorg.+Chem%22%5BJournal%5D)">In Situ Study of the Solid-State Formation of U1-xAmxO2±δ Solid Solution.</a> Inorg. Chem. 51 (17), 9369-9375 (2012).</li>
<li>Prieur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+Structure+and+Charge+Distribution+in+Mixed+Uranium-Americium+Oxides%3A+Effects+of+Oxygen+Potential+and+Am+Content%5BTitle%5D)+AND+%22Inorg.+Chem%22%5BJournal%5D)">Local Structure and Charge Distribution in Mixed Uranium-Americium Oxides: Effects of Oxygen Potential and Am Content.</a> Inorg. Chem. 50 (24), 12437-12445 (2011).</li>
<li>Prieur, D. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0022311511000109">Self-irradiation effects in dense and tailored porosity U1−yAmyO2−x (y = 0.10; 0.15) compounds.</a> J. Nucl. Mater. 411 (1-3), 15-19 (2011).</li>
<li>Wiss, T. <a target="_blank" href="https://www.cambridge.org/core/journals/journal-of-materials-research/article/tem-study-of-alpha-damaged-plutonium-and-americium-dioxides/C9CAF6A2E3DB7A42EE2F0686014CCC76">TEM study of alpha-damaged plutonium and americium dioxides.</a> Journal of Materials Research. 30 (9), 1544-1554 (2015).</li>
<li>Parker, J. H., Feldman, D. W., Ashkin, M. <a target="_blank" href="http://journals.aps.org/pr/abstract/10.1103/PhysRev.155.712">Raman Scattering by Silicon and Germanium.</a> Phys. Rev. 155, 712-714 (1967).</li>
<li>Hass, M. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/0022369770900818">Raman spectra of vitreous silica, germania and sodium silicate glasses.</a> J. Phys. Chem. Solids. 31 (3), 415-422 (1970).</li>
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<li>Naji, M. <a target="_blank" href="http://pubs.acs.org/doi/abs/10.1021/acs.jpcc.5b12068">Raman Scattering from Decoupled Phonon and Electron States in NpO2.</a> J Phys Chem C. 120 (9), 4799-4805 (2016).</li>
</ol>

The authors have nothing to disclose.

The present experimental approach relies on an original capsule, which can be easily designed and manufactured in a workshop equipped with a good turning machine. Except for the external diameter, which should fit with a commercially available funnel-shaped bag, the other dimensions of the capsule are not strictly necessary. However, for the high-pressure capsule, the surface exposed to high pressure should be minimized, particularly the surface perpendicular to the capsule axis. Here, for example, the maximum surface is the 5 mm-radius (r) window, which corresponds to an area A of about 127 mm&#178; (A = &#960;r&#178;). A pressure P of 20 bars exposed to this surface develops a force F of 254 N on the window (P = F*A), P in Pa, F in N, and A in m&#178;. This force, distributed on the 6 screws, results in about 42 N/screw. This should be taken into consideration when designing the capsule and the plunger side. A second point should be taken in account: the tightness of the plunger, as well as the volume of gas at high pressure. As the plunger is placed inside the confinement bag, the gas expands inside the confinement bag in case of a leak, possibly compromising the tightness of the confinement. The design should ensure that the volume of the gas expanding in case of a leak is negligible when compared to the bag capacity. The design should also ensure that the surfaces in contact with the O-ring are well-manufactured, assuring the proper level of tightness. Quality control of those surfaces, as well as of the O-ring, should be carried out. Note that very radioactive samples may end up damaging the capsule materials over time. Therefore, capsules should not be used to store radioactive samples for long periods. Also note that this system is a nuclear material confinement system and may require the approval of local safety authorities.
The advantages of this technique are numerous in comparison with a partial or complete Raman spectrometer confinement2,3,4,5,6,16,17. No special confinement (glove box and hot cells) is required, and thus, no extra material is generated that must be treated as nuclear waste at the end of life. There is no customization of the Raman spectrometer (needed in case of confinement). There is no limitation on the measurement possibilities in terms of wavelength, polarization, mode of measurement, or the easy setting of the atmosphere in which the measurement is done.
In comparison with the method used at ORNL &#8211; USA12,13,14,15, microscopy can be applied in proper optical conditions (single optical window instead of tubes), reducing the sample quantity needed, as well as the requirements for laser power.
Some limitations to the system should be pointed out. The distance between the sample and the microscope objective due to the presence of the capsule window imposes the use of a long focal objective, which may reduce the sensitivity of a wide-aperture Raman spectrometer. The insertion of a non-coated fused silica window between the sample and the objective may also reduce the imaging quality. Moreover, the current encapsulation system is also not reusable due to the fact that the funnel-shaped bag is definitively fixed on the capsule. However, this could be solved if the small side of the funnel-shaped bag was equipped with an integrated O-ring, allowing the possibility of applying the transfer bag technique to the capsule as well. This would make the use of a more complex capsule possible. For example, a mechanism to allow gas flow; a temperature-measurement device; or a mechanical pressure-controlled stage for the analysis of solids as well as liquids, or for the in situ measurement of kinetic effects, would be possible. A point to pay attention to is that the Raman spectra of highly radioactive samples like americium should be measured very quickly (sometimes in less than a week) because of an additional fluorescence signal that adds to the Raman spectrum with time. That phenomenon may be due to the degradation of the double sided adhesive tab after a few days of exposure to radiation, resulting to the production of volatile organic molecules that condense on the sample surface.
The present system is particularly well adapted to the study of radioactive nuclear materials. It can also be applied to the study of any other kind of material that the user should be protected from (hazardous samples) or of samples that must be protected from the atmospheric environment.

Raman spectroscopy is widely used as a nondestructive analytical method in fields like pharmaceuticals, cosmetics, geology, mineralogy, nanotechnology, environmental science, archeology, forensics, and art identification1. It is used for the analysis of vibrational, rotational, and other low-frequency modes in crystals or molecules. This technique is sensitive to crystal structure, composition, crystalline state, temperature, electronic state, stress, pressure, grain size (especially in the case of nano-structured crystallites), inclusions, and defects. For single molecules (gas- or matrix-isolated molecules), Raman is sensitive to chemical composition, local coordination, and electronic structure. The fact that it can be used as an electronic resonant or surface-enhanced spectroscopic technique makes it extremely sensitive for the detection and measurement of compounds at very low concentrations.
With its ease of use, limited sample preparation, and the possibility for remote measurement, Raman spectroscopy is of particular interest in the nuclear field. It has been used recently for applied studies of radiation damage (defect) in spent nuclear fuel2,3,4,5, as well as for fundamental studies on actinide compound systems6,7,8,9,10,11,12,13,14,15. The major challenge for Raman measurement of nuclear materials is the inherent risk of radiation exposure and incorporation. Those risks can be managed: for radiation by shielding, and for incorporation by confinement. Typically, a confinement system like an acrylic glass glove box is sufficient to confine and shield alpha emitters. Beta and gamma may require additional high-density shielding material, like lead or lead-doped glass. Neutron emitters will need shielding composed of a material that is able to easily capture neutrons and is rich in hydrogen, such as water or paraffin. Up to now, most Raman spectroscopic measurements of nuclear materials have been performed in shielded cells in remote configurations, e.g., with the help of a remote head connected with glass fibers2,3,4,5,6,16,17. This technique is even suited for the direct analysis of spent nuclear fuel2. Unfortunately, this approach has some important limitations: the first being that all remote Raman spectrometer parts in the cell are in direct contact with the radioactive material rapidly damaging them18 and converting them into radioactive waste. Further limitations are inherent to the remote technique. For example, the use of fiber optics limits the possibility of employing different excitation wavelengths, confocality, polarization, etc.
Another experimental approach was developed in the 1990&#39;s at the Oak Ridge National Laboratory (ORNL - USA)12,13,14,15. The radioactive sample was sealed in a double-quartz capillary, itself placed in a third confinement consisting of a borosilicate glass tube. This allowed the first Raman measurement of actinide-containing species. However, the measurement had to be carried out through a few layers of curved quartz and borosilicate glass, yielding a too-low signal. In this way, it was not possible to obtain, for example, a quality spectrum of AmO212. Moreover, Begun et al.12 had to use a relatively high laser power (few hundred mW) that may have affected the sample by local heating.
It should be possible to employ all the Raman spectrometer features (excitation wavelength, spectrometer mode, polarization, etc.) in order to obtain sound reference Raman spectra of actinide compounds. In view of this, we have developed a new technique of local encapsulation of radioactive samples. It permits the use of a standard non-contaminated or customized micro-Raman spectrometer for the measurement of nuclear materials. The use of a microscope for Raman analysis (micro-Raman spectroscopy, or &#181;RS) presents an important advantage in that it requires only a very small amount of sample to be observed and measured properly. Basically, a sample size ranging from the order of few tens of micrometers is sufficient for &#181;RS, thanks to the few-micrometer spatial resolution of the microscope equipped with a 10X or 50X objective. The volume of a sample exposing 2,500 &#181;m2 (of a 50 x 50 &#181;m size) to the microscope, depending on the shape, is about 0.1 mm3, which corresponds to a weight of about 1 mg, considering a density of 12 g/cm3 (typical for actinide oxides). A 1 mg sample of the highly radioactive 241Am exposes the user to about 50 &#181;Sv/h at 10 cm or 0.5 &#181;Sv/h at 1 m19. These levels remain easily within the legal dose limits, typically on the order of mSv/day for the hands and tens of &#181;Sv/day for the body20. In addition, this system also isolates the sample from the atmospheric environment, including high humidity levels or the presence of oxygen. Depending on the needs of the measurement, from vacuum, the user can even choose the best atmosphere up to 20 bars, reactive or protective. This is particularly important while studying materials chemically reactive to their atmospheric environment, like actinide oxides, fluoride salts, metals (oxidation, reduction, and reaction with water). The intense laser irradiation of the sample, usually needed for Raman measurement, enhances the kinetics of those reactions because the sample may be heated by the laser. Those reactions can be compensated for by choosing the right atmosphere. This type of procedure could also be helpful to any optical measurement on hazardous specimens, like chemicals or infectious biological substances.
The alpha-radiation and atmospheric tight Raman sample holder consist of an acrylic glass cylinder, 44 mm in diameter and 60 mm long, in the axis of which is drilled a 15 mm-deep hole (Figure 1). This part, the capsule, is closed on one side with a 2 mm-thick, 20 mm-diameter, one-wave optically polished fused silica window. A 14.9 mm-diameter acrylic glass rod, the plunger, holding the sample is inserted in the capsule up to the point that the sample comes right below the window. The samples (powder or small fragments of a disk) are fixed with the help of a double-sided adhesive tab on a standard aluminum pin stub mount 12.7 mm in diameter, itself fixed at the end of the acrylic glass rod (the plunger). The plunger is equipped with an external circlip to avoid the risk of pushing the sample and its holder too far into the fused silica window, which might lead to the rupture of the capsule and dispersion of radioactivity in the laboratory. Moreover, the external circlip can be set at different positions, in one of the grooves made for that purpose in the plunger, in order to adjust the distance between the sample and the window. The plunger is also equipped with an O-ring for a smooth sliding of the rod in the cylinder. To avoid compressing the gas or atmosphere in the cylinder while inserting the rod, a groove in the inner surface of the cylinder allows the evacuation of the gas during the mounting procedure. A screw can be fixed in a thread bored at the bottom of the plunger in order to pull the rod out of the cylinder. The samples can thus be removed after the generally non-destructive Raman analysis.
A second sample holder has been developed in order to perform the Raman analysis under a chosen atmosphere up to 20 bars (Figure 2). This high-pressure-resistant alpha-radiation and gas-tight Raman sample holder consists of a polyether ether ketone (PEEK) cylinder body 44 mm in diameter and 65 mm long, in which a 16-mm hole its drilled in the axis. This part, the capsule body, is closed on one side with a 3 mm-thick, 12.7 mm-diameter, one-wave optically polished fused silica uncoated window maintained by a metal flange fixed on the capsule body by 6 screws. To achieve tightness, the window rests on an O-ring placed in a groove made in the body. To protect the window from direct contact with the metal flange, a fluoropolymer elastomer flat joint is placed between both of them. The other side of the capsule is closed by another metal flange (plunger flange) also fixed to the body with screws. The plunger flange is equipped with a plunger, at the end of which a sample holder is screwed (next to the window). Just below the sample holder, the plunger is equipped with an O-ring placed in a groove, assuring the high pressure tightness of the capsule. The plunger is drilled over the whole length by a capillary ending just after the O-ring, ensuring the tightness. It is intended to pump the vacuum or put the sample chamber under pressure. The sample is fixed on the sample holder the same way as explained previously. The plunger flange is equipped with an adaptor to a 6-mm, stainless-steel gas tube in order to couple to a valve for the gas inlet or vacuum pumping.
In order to interface the outer part of the capsules and the confinement system where the sample is stored without having to break the confinement, a well-established transfer bag technique is used. This technique is commonly used, especially in the nuclear industry, to securely transfer samples between two separated confinements. The funnel-shaped bag used here is specially designed for the use of this technique. On the sample holder side, the bag end is funnel-shaped, the smallest diameter fitting with the external diameter of the capsule. A groove and a jut are realized on the outer surface of the cylinder in order to install a tight O-ring around the bag, keeping it in place, and to prevent the cylinder from sliding too far into the bag, respectively.
This paper provides details on the experimental approach, as well as three representative example applications of the technique. One example concerns the Raman study of highly radioactive americium dioxide. This is of particular interest in the study of Am transmutation in special nuclear fuels aiming at the radioactivity reduction of long-lived nuclear waste21,22,23,24, but also as a replacement of 238Pu in radioisotope generators for the powering of deep-space exploration spacecraft25. The measurement of this highly radioactive material specimen demonstrates the strength of the developed technique. The second example also deals with a material planned for transmutation. It reports a more fundamental study of the Raman features of NpO2, including the influence of 17O doping, using three different excitations wavelengths and various laser power levels. The resulting sample temperature was estimated here by measuring the ratio between Stokes and anti-Stokes line intensities, with the help of the triple spectrometer configuration. This successful test demonstrates the instrumental flexibility that is offered by this technique and helps to identify vibronic Raman bands that can be used as NpO2 fingerprints. In the last example, the present approach was used to Raman-map a sample taken from the Chernobyl lava formed in 1986 after the reactor core meltdown. This aims at the identification of the different phases present in the material.

<table><tbody><tr><td>(standard) acrylic glass &lt;strong&gt;capsule body&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(standard) &lt;strong&gt;UV fused silicate Window&lt;/strong&gt; 20 mm x 2 mm</td><td>Edmund Optics GmbH, Karlsruhe (Germany)</td><td>45464</td><td></td></tr><tr><td>(standard) acrylic glass &lt;strong&gt;Plunger&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(standard) fluoropolymer elastomer &lt;strong&gt;sliding O ring&lt;/strong&gt; 10 x 2 mm</td><td></td><td></td><td></td></tr><tr><td>(standard) &lt;strong&gt;Epoxi resin:&lt;/strong&gt; uhu schnellfest 2k epoxit kleber&amp;nbsp;</td><td>UHU (germany)</td><td>45725</td><td></td></tr><tr><td>(standard) &lt;strong&gt;External circlip&lt;/strong&gt; DIN 471 40 mm</td><td></td><td></td><td></td></tr><tr><td>(standard) hexagon socket head cap &lt;strong&gt;pull screw&lt;/strong&gt; DIN 912 M4 x 30 mm</td><td></td><td></td><td></td></tr><tr><td>(standard) aluminum SEM &lt;strong&gt;pin stub mount&lt;/strong&gt;</td><td>Plano GmbH, Wetzlar (Germany)</td><td>G301</td><td></td></tr><tr><td>(standard + high pressure) 1.4301 stainless steal &lt;strong&gt;metal ring slide&lt;/strong&gt; with blocking screw</td><td>home made</td><td></td><td></td></tr><tr><td>(standard + high pressure) &lt;strong&gt;Electrician tape&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(standard + high pressure) fluoropolymer elastomer &lt;strong&gt;tightening O ring&lt;/strong&gt; 40 x 4 mm</td><td></td><td></td><td></td></tr><tr><td>(standard + high pressure) &lt;strong&gt;double-sided adhesives tabs&lt;/strong&gt;</td><td>Plano GmbH, Wetzlar (Germany)</td><td>G3347</td><td></td></tr><tr><td>(standard + high pressure) &lt;strong&gt;Funnel-shaped bag&lt;/strong&gt;; Sac PVC 300 &amp;micro; TA Diam 40/185 x 540 mm Tronc conique</td><td>Plastunion, Bondy (France)</td><td>4.123</td><td></td></tr><tr><td>(High pressure) polyether ether ketone &lt;strong&gt;high pressure capsule body&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(High pressure) &lt;strong&gt;High pressure capsule window&lt;/strong&gt;: &amp;Oslash;12.7 x 3 mm UVFS Broadband Precision Window, Uncoated</td><td>THORLABS GMBH, Dachau (Germany)</td><td>WG40530</td><td></td></tr><tr><td>(High pressure) &lt;strong&gt;High pressure ball valve&lt;/strong&gt;: K&amp;uuml;kenhahn, Edelstahl, 6 mm Rohrverschraubung, Cv 1,6</td><td>Swagelok, Forst(Germany)</td><td>SS-6P4T-MM</td><td></td></tr><tr><td>(High pressure) 1.4301 stainless steel &lt;strong&gt;sample holder&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(High pressure) 1.4301 stainless steel &lt;strong&gt;high pressure plunger&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(High pressure) 1.4301 stainless steel &lt;strong&gt;adapter&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(High pressure) 1.4301 stainless steel &lt;strong&gt;closing flange&lt;/strong&gt;</td><td>home made</td><td></td><td></td></tr><tr><td>(High pressure) 2 x fluoropolymer elastomer &lt;strong&gt;capsule O ring&lt;/strong&gt; 10*1 mm</td><td></td><td></td><td></td></tr><tr><td>(High pressure) fluoropolymer elastomer &lt;strong&gt;inlet O Ring&lt;/strong&gt; 6*1 mm</td><td></td><td></td><td></td></tr><tr><td>(High pressure) 6 x DIN 7991 M4 * 25 mm &lt;strong&gt;bottom sink screw&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(High pressure) 6 x DIN 7991 M4 * 18 mm &lt;strong&gt;top sink screw&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(High pressure) &lt;strong&gt;Polyoxymethylen flat ring&lt;/strong&gt; 13/10*1 mm</td><td>home made</td><td></td><td></td></tr></tbody></table>

a novel technique for raman analysis of highly radioactive samples using any standard micro-raman spectrometer

A novel approach for the Raman measurement of nuclear materials is reported in this paper. It consists of the enclosure of the radioactive sample in a tight capsule that isolates the material from the atmosphere. The capsule can optionally be filled with a chosen gas pressurized up to 20 bars. The micro-Raman measurement is performed through an optical-grade quartz window. This technique permits accurate Raman measurements with no need for the spectrometer to be enclosed in an alpha-tight containment. It therefore allows the use of all options of the Raman spectrometer, like multi-wavelength laser excitation, different polarizations, and single or triple spectrometer modes. Some examples of measurements are shown and discussed. First, some spectral features of a highly radioactive americium oxide sample (AmO2) are presented. Then, we report the Raman spectra of neptunium oxide (NpO2) samples, the interpretation of which is greatly improved by employing three different excitation wavelengths, 17O doping, and a triple mode configuration to measure the anti-stokes Raman lines. This last feature also allows the estimation of the sample surface temperature. Finally, data that were measured on a sample from Chernobyl lava, where phases are identified by Raman mapping, are shown.

Three unique and representative results demonstrating the potential of this system are reported in this section.
These measurements have been recorded with a Raman spectrometer equipped with an 1,800 groove-per-mm grating; a low-noise, LN2-cooled symphony CCD detector, a subtractive pre-monochromator (in triple mode), which allows access to low wavenumbers (up to 10 cm-1); and anti-Stokes lines or edge filters (in single mode), blocking the elastic laser scattering coming from the sample. The incident light is focused using a long working distance (10.6 mm) objective, which offers a 0.5 numerical aperture with a 5x104 magnification factor. The microscope system is equipped with a Z-motor, piezo-based positioner providing fast focus and long-time stability. Switching between parallel and cross-polarization configurations can be done using a λ/2 plate for the incident beam, with a combination of λ/4 and 90° polarizers for the back-scattered light. The back-scattered light passes through an adjustable spatial filter, permitting it to work in confocal conditions. The stage is motorized in both the X and Y axis in order to allow automatic area mapping. Excitation sources are Ar+ continuous wave (CW) lasers with main wavelengths at 488 nm and 514.5 nm, or Kr+ CW lasers with main wavelengths at 647 nm and 752 nm. The nominal output power of both lasers can be digitally adjusted from a few mW up to a few W, depending on the wavelength. A monochromator or band-pass filters are used to block background plasma and secondary emission lines. The power impinging the sample surface is measured at the microscope objective output using a coherent power meter. Using the long focal 50X objective and the single spectrometer mode permits a good spectral resolution (± 1 cm-1), independent of the surface shape, with a spatial resolution of 2 µm x 2 µm on the sample surface.
The Raman Spectrum of AmO2
The Raman spectrum of pure americium dioxide was measured with a lower-energy excitation source compared with previous research28. It was measured in the capsule in atmospheric air. For AmO2 with a defect-free fluorite structure, group theory predicts only one Raman active mode (T2g)28, which corresponds to the vibration of the Am−O bond of an Am cation surrounded by eight oxygen anions in a cubic environment. Although the position of T2g in UO2 is precisely known to be about 445 cm−1 (despite small variations between authors), that of AmO2 has never been clearly identified. Figure 4 shows a typical Raman spectrum of AmO2 acquired with an excitation wavelength of 647 nm. The same spectrum was recorded by Naji et al.28 and Horlait et al.29 in previous studies of americium oxides. It is dominated by a broad, asymmetric band centered at ~380 cm-1 and tentatively attributed to an oxygen-stretching motion in the fluorite structure.
The reason why this mode is observed at rather low frequencies compared to the other actinide dioxides is still a matter of debate. It is possible that this shift is due to the photo-reduction of AmO2 to Am2O3+z by the excitation laser phonons, as recently proposed by Naji et al.27. Such an effect would be consistent with the very high oxygen potential of AmO2. In order to clarify this point, further Raman measurements are being prepared under high oxygen pressure in the modified setup shown in Figure 2.
The Raman Spectrum of NpO2
The current technique has also been used to investigate the Raman features of neptunium dioxide (NpO2) using three different excitations sources, with incident photon energies of 647 nm, 514 nm, and 488 nm, respectively30. The asymmetric profile of the T2g band in NpO2 has been probed. In addition, a 17O-enriched NpO2 sample (30% enrichment) has been analyzed in order to distinguish vibrational and electronic contributions to the Raman spectrum30. The temperature at the sample surface was determined by measuring the Stokes and anti-Stokes T2g line intensity ratio of NpO2 using Bose-Einstein statistics (Figure 6, top and middle). Our results clearly demonstrate, for the first time, the existence of a secondary mode at ~431 cm−1 (Figures 5b, 5d and Figure 6, bottom), inducing a low-wavenumber asymmetry in the T2g peak (Figure 5c), which is a signature of the NpO2 lattice. From the isotopic shift (Figure 5d), laser energy dependence, and temperature behavior of the Raman intensity (Figure 6, bottom), we demonstrated that this mode arises from a band with an electronic origin. We suggest that it corresponds to the bare, decoupled crystal-field level predicted by the theoretical bound state.
Raman Analysis of Chernobyl Lava
During the Chernobyl accident, the interaction between the high temperature fuel (up to 2,600 °C), the destroyed fuel cladding, and the silicate materials (concrete, sand, and serpentinite) dropped into the reactor resulted in the formation of a kind of lava called corium. Liquid corium flew into the basement of the plant through discharge corridors. The first samples of this lava were collected in 1987 with help of an AK47 machine gun because of the high activity and the strong mechanical properties of the corium. In 1990, hydrothermal alteration and disintegration of the lava was observed with the formation of uranyl-like secondary phases. Some of those Chernobyl lava samples were provided to JRC-Karlsruhe by the Khlopin Radium Institute for multiple analyses. Several Raman measurements were performed on various cut and polished surfaces of Chernobyl corium samples. All these measurements were coupled with SEM-EDX data to identify the elemental composition and the phases present on the surface. Figure 7 shows an optical picture of a representative sample having different-phase "spots" that were analyzed using Raman spectroscopy.
Figure 8 shows the Raman spectra obtained from spots 1, 2, and 3 in Figure 7. The spectra correspond to SiO2 glasses: (U,Zr)SiO4, USiOx, and (U,Zr)Ox, respectively. These phases were either formed from direct interaction of the cladding with the nuclear fuel and then transported as xenocrysts in the melt or were crystallized from the silicate melt during transport and cooling.
<img alt="Exploded diagram of a pressure sensor assembly with labeled components for structural analysis." class="xfigimg" src="/files/ftp_upload/54889/54889fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Schematic of the standard capsule. The capsule essentially consists of the closed acrylic glass capsule body with a fused silica window on one side and, on the opposite side, the plunger holding the pin stub mount on which the sample is fixed with the help of the adhesive tab. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="High-pressure capsule assembly diagram showing components: flange, O-Ring, sample holder, valve." class="xfigimg" src="/files/ftp_upload/54889/54889fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Schematic of the high-pressure capsule. The capsule essentially consists of a PEEK capsule body closed on one side with a fused silica window with the help of a screwed flange. On the other side, the plunger flange supports the sample holder, on which the sample is fixed with the help of the adhesive tape. This capsule is equipped with a ball valve in order to evacuate or fill the capsule with gas. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy setup for optical excitation, labeled parts; stage slide holder, diagram." class="xfigimg" src="/files/ftp_upload/54889/54889fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Photo of the capsule placed on the microscope stage. The capsule is fixed on the stage with the help of a metal-ring adaptor. In this example, a 647-nm (red) laser beam is used as excitation source through a 50X long focal-distance objective for the measurement of low-activity (U,Np)O2 samples (some fragments can be seen through the capsule window). <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Raman spectroscopy graph showing intensity vs. wavenumber for molecular vibration analysis." class="xfigimg" src="/files/ftp_upload/54889/54889fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Raman spectrums of AmO2 measured in the standard capsule. The Raman spectrums of AmO2 measured in the standard- and high-pressure capsules under N2 at atmospheric pressure and 15 bar of O2, respectively. The Raman spectrum shows the intense mode at ~380 cm-1, which is a signature of reduced AmO2. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Raman spectroscopy graphs showing phonon modes and wavenumber shifts; spectral fitting analysis." class="xfigimg" src="/files/ftp_upload/54889/54889fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: The Raman spectrum of NpO2 measured at different energies and submitted to peak-deconvolution analysis. a) Raman spectrum of Np16O2 at different energies. b) The T2g band of Np16O2 measured at 2.41 eV. Peak fit and deconvolution show the presence of an additional peak at 441 cm-1. c) Comparison of the T2g of Np16O2 measured at different energies. d) Comparison of the T2g of Np16O2 and Np17O2 measured at 2.41 eV. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Raman spectroscopy graphs showing intensity vs wavenumber, temperature vs laser power, and mode intensity." class="xfigimg" src="/files/ftp_upload/54889/54889fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Summary of the NpO2 temperature measurements and results. Top: Stokes and anti-Stokes spectrums of Np16O2 measured at different laser irradiation power levels. Middle: Sample surface temperature determined by applying Bose-Einstein statistics to the Stokes/anti-Stokes T2g peak ratios as a function of the laser irradiation power. Bottom: Intensity of the peak at 431 cm-1 as a function of the calculated temperature. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopic view of mineral surface texture analysis; numbered zones indicate specific points of interest." class="xfigimg" src="/files/ftp_upload/54889/54889fig7.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Picture of the Chernobyl lava sample with the positions of the Raman measurement points. The sample consists of a 1 mm-thick slide of approximately 10 mm x 5 mm cut in a piece of lava. Different inclusions in the dark mass of the glass can be seen; three of them have been chosen as examples. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Raman spectroscopy graph, (U,Zr)SiO₄ phases, showing spectral peaks, Raman shift/cm⁻¹." class="xfigimg" src="/files/ftp_upload/54889/54889fig8.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Raman spectrum of the different phases identified in Figure 7. The Raman spectra are obtained from spots 1, 2, and 3 of Figure 7. They correspond to silicate glasses: (U,Zr)SiO4, USiOx, and (U,Zr)Ox, respectively. <a href="http://cloudflare.jove.com/files/ftp_upload/54889/54889fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Technique for Raman Analysis of Highly Radioactive Samples Using Any Standard Micro-Raman Spectrometer at JoVE.com

A Novel Technique for Raman Analysis of Highly Radioactive Samples Using Any Standard Micro-Raman Spectrometer

We present a technique for Raman spectroscopic analysis of highly radioactive samples compatible with any standard micro-Raman spectrometer, without any radioactive contamination of the instrument. We also show some applications using actinide compounds and irradiated fuel materials.

A novel approach for the Raman measurement of nuclear materials is reported in this paper. It consists of the enclosure of the radioactive sample in a ...

a-novel-technique-for-raman-analysis-highly-radioactive-samples-using

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Molecular Vibrational Spectroscopy

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12.8K Views.  Joint Research Centre (JRC).  We presenteren een techniek voor het Raman spectroscopische analyse van sterk radioactieve monsters compatibel met elke standaard micro-Raman spectrometer, zonder enige radioactieve besmetting van het instrument. We tonen ook een aantal applicaties met behulp van actinide verbindingen en bestraalde splijtstof materialen. 

Video: A Novel Technique for Raman Analysis of Highly Radioactive Samples Using Any Standard Micro-Raman Spectrometer

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

Bioengineering

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Engineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the presence of carbon nanotubes. These nanowires are extreme in the sense that they are the limit of miniaturization of nanowires and their behavior is not always a simple extrapolation of the behavior of larger nanowires as their diameter decreases. The paper then describes the methods required to obtain Raman spectra from extreme nanowires and the fact that due to the van Hove singularities that 1D systems exhibit in their optical density of states, that determining the correct choice of photon excitation energy is critical. It describes the techniques required to determine the photon energy dependence of the resonances observed in Raman spectroscopy of 1D systems and in particular how to obtain measurements of Raman cross-sections with better than 8% noise and measure the variation in the resonance as a function of sample temperature. The paper describes the importance of ensuring that the Raman scattering is linearly proportional to the intensity of the laser excitation intensity. It also describes how to use the polarization dependence of the Raman scattering to separate Raman scattering of the encapsulated 1D systems from those of other extraneous components in any sample.

The paper describes a method for producing extreme nanowires by melt infiltration into carbon nanotubes and how 1D systems may be characterized and investigated using Resonance Raman Spectroscopy to determine vibrational and optical excitation energies.

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Biochemistry

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Raman Spectroscopy for Chemical Analysis

Source: Laboratory of Dr. Ryoichi Ishihara &#8212; Delft University of Technology
Raman spectroscopy is a technique for analyzing vibrational and other low frequency modes in a system. In chemistry it is used to identify molecules by their Raman fingerprint. In solid-state physics it is used to characterize materials, and more specifically to investigate their crystal structure or crystallinity. Compared to other techniques for investigating the crystal structure (e.g. transmission electron microscope and x-ray diffraction) Raman micro-spectroscopy is non-destructive, generally requires no sample preparation, and can be performed on small sample volumes.
For performing Raman spectroscopy a monochromatic laser is shone on a sample. If required the sample can be coated by a transparent layer which is not Raman active (e.g., SiO2) or placed in DI water. The electromagnetic radiation (typically in the near infrared, visible, or near ultraviolet range) emitted from the sample is collected, the laser wavelength is filtered out (e.g., by a notch or bandpass filter), and the resulting light is sent through a monochromator (e.g., a grating) to a CCD detector. Using this, the inelastic scattered light, originating from Raman scattering, can be captured and used to construct the Raman spectrum of the sample.
In the case of Raman micro-spectroscopy the light passes through a microscope before reaching the sample, allowing it to be focused on an area as small as 1 &#181;m2. This allows accurate mapping of a sample, or confocal microscopy in order to investigate stacks of layers. Care has to be taken, however, that the small and intense laser spot does not damage the sample.
In this video we will briefly explain the procedure for obtaining a Raman spectra, and an example of a Raman spectrum captured from carbon nanotubes will be given.

Source: Laboratory of Dr. Ryoichi Ishihara &#8212; Delft University of Technology
Raman spectroscopy is a technique for analyzing vibrational and other ...

JoVE Science Education

A Raman Spectroscopy-Based Direct Immunoassay to Detect a Specific Antigen

Source: Hanson, C., et al. <a href="https://app.jove.com/v/54795">Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.</a> J. Vis. Exp.(2016).
In this video, we describe the Raman spectroscopy-based direct immunoassay method for detecting specific target antigens. The antibodies, which are adsorbed onto the surface of gold nanoparticles attached to Raman probes, interact with target antigens and produce a specific Raman spectrum, which is subsequently detected.

Source: Hanson, C., et al. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform. J. Vis. Exp.(2016).
In this video, we describe the Raman ...

JoVE Encyclopedia of Experiments

Biological Techniques

Spectroscopy Techniques

Raman Spectroscopy: Overview

The underlying principle of Raman spectroscopy is based on the interaction between light and matter, specifically molecules&#39; inelastic scattering of photons. When a monochromatic beam of light, typically from a laser source, interacts with a sample, most scattered light has the same frequency as the incident light. This is known as Rayleigh scattering.
However, a small fraction of the scattered light exhibits a frequency shift due to the exchange of energy between the incident photons and the vibrational energy levels of the molecules in the sample. This phenomenon is called Raman Scattering.
To obtain Raman spectra, the sample is irradiated with a monochromatic beam of laser light. The scattered radiation is collected at a suitable angle (usually 180&#176;) using a spectrometer equipped with a sensitive detector. The measurement of the frequency-shifted scattered light as a function of wavelength or wavenumber provides the sample&#39;s Raman spectrum.
Resonance Raman scattering occurs when the excitation wavelength is close to an electronic transition of the molecule. This enhances the Raman scattering intensity, making it easier to detect even weak signals. However, choosing an appropriate excitation wavelength is crucial to avoid fluorescence interference, which can overwhelm the Raman signal. In many cases, near-infrared or visible lasers are used to minimize fluorescence.
When a molecule absorbs a photon, it is temporarily elevated to a virtual energy level. This virtual state is short-lived, and the molecule quickly returns to a vibrational energy level by emitting a photon. The difference in energy between the incident and emitted photons corresponds to the molecule&#39;s vibrational energy levels.
There are two types of Raman scattering: Stokes and anti-Stokes. In Stokes scattering, the emitted photon has lower energy (longer wavelength) than the incident photon, while in anti-Stokes scattering, the emitted photon has higher energy (shorter wavelength).
The frequency shifts of the inelastically scattered radiation correspond to the vibrational frequency of the molecule. By analyzing these frequency shifts, valuable information about the molecular structure and chemical composition of the sample can be obtained.
Raman spectra are typically presented as plots of intensity versus wavenumber (reciprocal of wavelength). The peaks in the spectrum represent specific vibrational modes of the molecules in the sample, providing a unique &#34;fingerprint&#34; for identification.
Consider a sample of carbon tetrachloride. Both Stokes and anti-Stokes scattering will occur when irradiated with a monochromatic laser. The intensity of the Stokes signal will be stronger than the anti-Stokes signal, as the population of molecules in the ground vibrational state is higher than those in the excited state at room temperature.
Vibrational modes can be identified by analyzing the Raman spectrum of carbon tetrachloride. The frequency shifts associated with these vibrational modes provide valuable information about the molecule&#39;s chemical composition and bonding.

The underlying principle of Raman spectroscopy is based on the interaction between light and matter, specifically molecules&#39; inelastic scattering of ...

A Novel Technique for Raman Analysis of Highly Radioactive Samples Using Any Standard Micro-Raman Spectrometer

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