Editing Raman spectroscopy (section)

== Microspectroscopy ==
{{Main|Raman microscope}}
[[File:Raman cement clinker remnant FigTOC Thomas Schmid and Petra Dariz in Heritage 2(2) (2019) 1662-1683 landscape.png|400px|thumb|Hyperspectral Raman imaging can provide distribution maps of chemical compounds and material properties: Example of an unhydrated [[clinker (cement)|clinker]] remnant in a 19th-century [[cement]] mortar (cement chemist's nomenclature: C ≙ CaO, A ≙ Al<sub>2</sub>O<sub>3</sub>, S ≙ SiO<sub>2</sub>, F ≙ Fe<sub>2</sub>O<sub>3</sub>).<ref name="Heritage" />]]
Raman spectroscopy offers several advantages for [[microscopy|microscopic]] analysis. Since it is a light scattering technique, specimens do not need to be fixed or sectioned. Raman spectra can be collected from a very small volume (< 1&nbsp;μm in diameter, < 10&nbsp;μm in depth); these spectra allow the identification of species present in that volume.<ref name="AnnuRevAnalChem">{{cite journal |author=Lothar Opilik |author2=Thomas Schmid |author3=Renato Zenobi |title=Modern Raman Imaging: Vibrational Spectroscopy on the Micrometer and Nanometer Scales |journal=Annual Review of Analytical Chemistry |volume=6  |pages=379–398 |date=2013 |doi=10.1146/annurev-anchem-062012-092646 |pmid=23772660 |issn=1936-1335  |bibcode=2013ARAC....6..379O }}</ref> Water does not generally interfere with Raman spectral analysis. Thus, Raman spectroscopy is suitable for the microscopic examination of [[mineral]]s, materials such as polymers and ceramics, [[cell (biology)|cell]]s, [[proteins]] and forensic trace evidence. A [[Raman microscope]] begins with a standard optical microscope, and adds an excitation laser, a [[monochromator]] or [[polychromator]], and a sensitive detector (such as a [[charge-coupled device]] (CCD), or [[photomultiplier]] tube (PMT)). [[Fourier transform spectroscopy|FT-Raman]] has also been used with microscopes, typically in combination with [[near-infrared]] (NIR) laser excitation.  [[microscopy#Ultraviolet microscopy|Ultraviolet microscopes]] and UV enhanced optics must be used when a UV laser source is used for Raman microspectroscopy.

In ''direct imaging'' (also termed ''global imaging''<ref>{{cite journal|title=Raman Spectroscopy hyperspectral imager based on Bragg Tunable Filters |journal=SPIE Photonics North |volume=8412 |pages=84121J |date=2012 | doi=10.1117/12.2000479 |series=Photonics North 2012 |last1=Marcet |first1=S. |last2=Verhaegen |first2=M. |last3=Blais-Ouellette |first3=S. |last4=Martel |first4=R. |editor1-first=Jean-Claude |editor1-last=Kieffer |bibcode=2012SPIE.8412E..1JM |s2cid=119859405 }}</ref> or ''wide-field illumination''), the whole field of view is examined for light scattering integrated over a small range of wavenumbers (Raman shifts).<ref name="AnalChem">{{cite journal |author=Sebastian Schlücker |author2=Michael D. Schaeberle |author3=Scott W. Huffman |author4=Ira W. Levin |title=Raman Microspectroscopy: A Comparison of Point, Line, and Wide-Field Imaging Methodologies |journal=Analytical Chemistry |volume=75  |issue=16 |pages=4312–4318 |date=2003 |doi=10.1021/ac034169h |pmid=14632151 |issn=1520-6882  }}</ref> For instance, a wavenumber characteristic for cholesterol could be used to record the distribution of cholesterol within a cell culture. This technique is being used for the characterization of large-scale devices, mapping of different compounds and dynamics study. It has already been used for the characterization of [[graphene]] layers,<ref>{{cite journal |author=Robin W. Havener |display-authors=etal  |title=High-Throughput Graphene Imaging on Arbitrary Substrates with Widefield Raman Spectroscopy |journal=ACS Nano |volume=6 |issue=1 |pages=373–80 |date=December 2011 | doi=10.1021/nn2037169 |pmid=22206260  }}</ref> J-aggregated dyes inside [[carbon nanotubes]]<ref>{{cite journal |title=Giant Raman scattering from J-aggregated dyes inside carbon nanotubes for multispectral imaging |journal=Nature Photonics |pages=72–78 |date=2014 | doi=10.1038/nphoton.2013.309 |volume=8|issue=1 |last1=Gaufrès |first1=E. |last2=Tang |first2=N. Y.-Wa |last3=Lapointe |first3=F. |last4=Cabana |first4=J. |last5=Nadon |first5=M.-A. |last6=Cottenye |first6=N. |last7=Raymond |first7=F. |last8=Szkopek |first8=T. |last9=Martel |first9=R.  |bibcode=2014NaPho...8...72G |s2cid=120426939 |url=https://zenodo.org/record/918289 }}</ref> and multiple other 2D materials such as [[Molybdenum disulfide|MoS<sub>2</sub>]] and [[Tungsten diselenide|WSe<sub>2</sub>]]. Since the excitation beam is dispersed over the whole field of view, those measurements can be done without damaging the sample.

The most common approach is ''[[hyperspectral imaging]]'' or ''[[chemical imaging]]'', in which thousands of Raman spectra are acquired from all over the field of view by, for example, raster scanning of a focused laser beam through a sample.<ref name="AnalChem" /> The data can be used to generate images showing the location and amount of different components. Having the full spectroscopic information available in every measurement spot has the advantage that several components can be mapped at the same time, including chemically similar and even [[polymorphism (materials science)|polymorphic]] forms, which cannot be distinguished by detecting only one single wavenumber. Furthermore, material properties such as [[stress (mechanics)|stress]] and [[strain (mechanics)|strain]], [[orientation imaging microscopy|crystal orientation]], [[crystallinity]] and incorporation of foreign ions into crystal lattices (e.g., [[doping (semiconductor)|doping]], [[solid solution|solid solution series]]) can be determined from hyperspectral maps.<ref name="Heritage" /> Taking the cell culture example, a hyperspectral image could show the distribution of cholesterol, as well as proteins, nucleic acids, and fatty acids. Sophisticated signal- and image-processing techniques can be used to ignore the presence of water, culture media, buffers, and other interferences.

Because a Raman microscope is a [[diffraction-limited system]], its spatial resolution depends on the wavelength of light, the [[numerical aperture]] of the focusing element, and — in the case of [[confocal microscopy]] — on the diameter of the confocal aperture. When operated in the visible to near-infrared range, a Raman microscope can achieve lateral resolutions of approx. 1&nbsp;μm down to 250&nbsp;nm, depending on the wavelength and type of objective lens (e.g., air ''vs.'' water or oil immersion lenses). The depth resolution (if not limited by the optical penetration depth of the sample) can range from 1–6&nbsp;μm with the smallest confocal pinhole aperture to tens of micrometers when operated without a confocal pinhole.<ref>{{Cite book|date=2018|editor-last=Toporski|editor-first=Jan|editor2-last=Dieing|editor2-first=Thomas|editor3-last=Hollricher|editor3-first=Olaf|title=Confocal Raman Microscopy|series=Springer Series in Surface Sciences|volume=66|doi=10.1007/978-3-319-75380-5|issn=0931-5195|isbn=978-3-319-75378-2|url=https://cds.cern.ch/record/1339422|publisher=Springer|bibcode=2018crm..book.....T }}</ref><ref name="ApplSpectrosc">{{cite journal |author=Neil J. Everall |title=Confocal Raman Microscopy: Performance, Pitfalls, and Best Practice |journal=Applied Spectroscopy |volume=63  |issue=9 |pages=245A–262A |date=2009 |doi=10.1366/000370209789379196 |pmid=19796478 |issn=1943-3530  |bibcode=2009ApSpe..63..245E |doi-access=free }}</ref><ref>[https://media.nature.com/original/nature-assets/srep/2015/151217/srep18410/extref/srep18410-s1.pdf Supporting Information] {{Webarchive|url=https://web.archive.org/web/20190703105747/https://media.nature.com/original/nature-assets/srep/2015/151217/srep18410/extref/srep18410-s1.pdf |date=2019-07-03 }} of {{cite journal |author=T. Schmid |author2=N. Schäfer |author3=S. Levcenko |author4=T. Rissom |author5=D. Abou-Ras |title=Orientation-distribution mapping of polycrystalline materials by Raman microspectroscopy |journal=Scientific Reports |volume=5  |pages=18410 |date=2015 |doi=10.1038/srep18410 |pmid=26673970 |issn=2045-2322  |pmc=4682063 |bibcode=2015NatSR...518410S }}</ref><ref name="AnnuRevAnalChem" /> Depending on the sample, the high laser power density due to microscopic focussing can have the benefit of enhanced [[photobleaching]] of molecules emitting interfering fluorescence. However, the laser wavelength and laser power have to be carefully selected for each type of sample to avoid its degradation.

Applications of Raman imaging range from materials sciences to biological studies.<ref name="AnnuRevAnalChem" /><ref>{{cite journal |author=Ellis DI |author2=Goodacre R |title=Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy |journal=Analyst |volume=131 |issue=8 |pages=875–85 |date=August 2006 |pmid=17028718 |doi=10.1039/b602376m|bibcode = 2006Ana...131..875E |s2cid=9748788 }}</ref> For each type of sample, the measurement parameters have to be individually optimized. For that reason, modern Raman microscopes are often equipped with several lasers offering different wavelengths, a set of objective lenses, and neutral density filters for tuning of the laser power reaching the sample. Selection of the laser wavelength mainly depends on optical properties of the sample and on the aim of the investigation.<ref>{{cite journal |author=David Tuschel |title=Selecting an Excitation Wavelength for Raman Spectroscopy |journal=Spectroscopy Online |volume=31 |issue=3 |pages=14–23 |date=2016 |url=http://www.spectroscopyonline.com/selecting-excitation-wavelength-raman-spectroscopy  }}</ref> For example, Raman microscopy of biological and medical specimens is often performed using red to near-infrared excitation (e.g., 785&nbsp;nm, or 1,064&nbsp;nm wavelength). Due to typically low [[absorbance]]s of biological samples in this spectral range, the risk of damaging the specimen as well as [[autofluorescence]] emission are reduced, and high penetration depths into tissues can be achieved.<ref>{{cite journal |author=K. Christian Schuster |author2=Ingo Reese |author3=Eva Urlaub |author4=J. Richard Gapes |author5=Bernhard Lendl |title=Multidimensional Information on the Chemical Composition of Single Bacterial Cells by Confocal Raman Microspectroscopy |journal=Analytical Chemistry |volume=72  |issue=22 |pages=5529–5534 |date=2000 |doi=10.1021/ac000718x |pmid=11101227 |issn=1520-6882  }}</ref><ref>{{cite journal |author=Shan Yang |author2=Ozan Akkus |author3=David Creasey |title=1064-nm Raman: The Right Choice for Biological Samples? |journal=Spectroscopy Online |volume=32 |issue=6 |pages=46–54 |date=2017 |url=http://www.spectroscopyonline.com/1064-nm-raman-right-choice-biological-samples  }}</ref><ref>{{cite journal |author=Zanyar Movasaghi |author2=Shazza Rehman |author3=Ihtesham U. Rehman |title=Raman Spectroscopy of Biological Tissues |journal=Applied Spectroscopy Reviews |volume=42 |issue=5 |pages=493–541 |date=2007 |doi=10.1080/05704920701551530 |issn= 1520-569X  |bibcode=2007ApSRv..42..493M |s2cid=218638985 }}</ref><ref>{{cite journal |author=Peter J.Caspers |author2=Hajo A.Bruining |author3=Gerwin J.Puppels |author4=Gerald W.Lucassen |author5=Elizabeth A.Carter |title=''In Vivo'' Confocal Raman Microspectroscopy of the Skin: Noninvasive Determination of Molecular Concentration Profiles |journal=Journal of Investigative Dermatology |volume=116  |issue=3 |pages=434–442 |date=2001 |doi=10.1046/j.1523-1747.2001.01258.x |pmid=11231318 |issn=0022-202X  |url=http://repub.eur.nl/pub/10881 |hdl=1765/10881 |hdl-access=free }}</ref> However, the intensity of Raman scattering at long wavelengths is low (owing to the ω<sup>4</sup> dependence of Raman scattering intensity), leading to long acquisition times. On the other hand, [[resonance raman spectroscopy|resonance Raman]] imaging of single-cell [[algae]] at 532&nbsp;nm (green) can specifically probe the [[carotenoid]] distribution within a cell by a using low laser power of ~5&nbsp;μW and only 100&nbsp;ms acquisition time.<ref>{{cite journal |author=Pawel L. Urban |author2=Thomas Schmid |author3=Andrea Amantonico |author4=Renato Zenobi |title=Multidimensional Analysis of Single Algal Cells by Integrating Microspectroscopy with Mass Spectrometry |journal=Analytical Chemistry |volume=83  |issue=5 |pages=1843–1849 |date=2011 |doi=10.1021/ac102702m |pmid=21299196 |issn=1520-6882  }}</ref>

Raman scattering, specifically tip-enhanced Raman spectroscopy, produces high resolution hyperspectral images of single molecules,<ref>{{Cite journal|last1=Apkarian|first1=V. Ara|last2=Nicholas Tallarida|last3=Crampton|first3=Kevin T.|last4=Lee|first4=Joonhee|date=April 2019|title=Visualizing vibrational normal modes of a single molecule with atomically confined light|journal=Nature|volume=568|issue=7750|pages=78–82|doi=10.1038/s41586-019-1059-9|pmid=30944493|issn=1476-4687|bibcode=2019Natur.568...78L|s2cid=92998248}}</ref> atoms,<ref>{{Cite journal|last1=Crampton|first1=Kevin T.|last2=Lee|first2=Joonhee|last3=Apkarian|first3=V. Ara|date=2019-06-25|title=Ion-Selective, Atom-Resolved Imaging of a 2D Cu2N Insulator: Field and Current Driven Tip-Enhanced Raman Spectromicroscopy Using a Molecule-Terminated Tip|journal=ACS Nano|volume=13|issue=6|pages=6363–6371|doi=10.1021/acsnano.9b02744|pmid=31046235|s2cid=143433439 |issn=1936-0851}}</ref> and DNA.<ref name="He 753–757">{{Cite journal|last1=He|first1=Zhe|last2=Han|first2=Zehua|last3=Kizer|first3=Megan|last4=Linhardt|first4=Robert J.|last5=Wang|first5=Xing|last6=Sinyukov|first6=Alexander M.|last7=Wang|first7=Jizhou|last8=Deckert|first8=Volker|last9=Sokolov|first9=Alexei V.|date=2019-01-16|title=Tip-Enhanced Raman Imaging of Single-Stranded DNA with Single Base Resolution|journal=Journal of the American Chemical Society|volume=141|issue=2|pages=753–757|doi=10.1021/jacs.8b11506|pmid=30586988|bibcode=2019JAChS.141..753H |s2cid=58552541 |issn=0002-7863}}</ref>