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class="vector-toc-text"> <span class="vector-toc-numb">5</span> <span>Correlative Raman imaging</span> </div> </a> <ul id="toc-Correlative_Raman_imaging-sublist" class="vector-toc-list"> </ul> </li> <li id="toc-Biological_Applications" class="vector-toc-list-item vector-toc-level-1 vector-toc-list-item-expanded"> <a class="vector-toc-link" href="#Biological_Applications"> <div class="vector-toc-text"> <span class="vector-toc-numb">6</span> <span>Biological Applications</span> </div> </a> <ul id="toc-Biological_Applications-sublist" class="vector-toc-list"> </ul> </li> <li id="toc-See_also" class="vector-toc-list-item vector-toc-level-1 vector-toc-list-item-expanded"> <a class="vector-toc-link" href="#See_also"> <div class="vector-toc-text"> <span class="vector-toc-numb">7</span> <span>See also</span> </div> </a> <ul id="toc-See_also-sublist" class="vector-toc-list"> </ul> </li> <li id="toc-References" class="vector-toc-list-item vector-toc-level-1 vector-toc-list-item-expanded"> <a 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dir="ltr"><div class="shortdescription nomobile noexcerpt noprint searchaux" style="display:none">Laser microscope used for Raman spectroscopy</div> <figure class="mw-default-size" typeof="mw:File/Thumb"><a href="/wiki/File:Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg" class="mw-file-description"><img alt="Photo of a confocal Raman imaging microscope" src="//upload.wikimedia.org/wikipedia/commons/thumb/9/98/Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg/220px-Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg" decoding="async" width="220" height="147" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/9/98/Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg/330px-Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/9/98/Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg/440px-Confocal_Raman_imaging_microscope_Witec_alpha300_.jpg 2x" data-file-width="5184" data-file-height="3456" /></a><figcaption>Confocal Raman imaging microscope</figcaption></figure> <figure class="mw-default-size" typeof="mw:File/Thumb"><a href="/wiki/File:InVia_Raman_microscope_-_March_2008.jpg" class="mw-file-description"><img alt="Photo of a Raman microscope, with a sample enclosure" src="//upload.wikimedia.org/wikipedia/commons/thumb/e/e3/InVia_Raman_microscope_-_March_2008.jpg/220px-InVia_Raman_microscope_-_March_2008.jpg" decoding="async" width="220" height="165" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/e/e3/InVia_Raman_microscope_-_March_2008.jpg/330px-InVia_Raman_microscope_-_March_2008.jpg 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/e/e3/InVia_Raman_microscope_-_March_2008.jpg/440px-InVia_Raman_microscope_-_March_2008.jpg 2x" data-file-width="3264" data-file-height="2448" /></a><figcaption>Raman microscope</figcaption></figure> <p>The <b>Raman microscope</b> is a laser-based <a href="/wiki/Microscope" title="Microscope">microscopic</a> device used to perform <a href="/wiki/Raman_spectroscopy" title="Raman spectroscopy">Raman spectroscopy</a>.<sup id="cite_ref-Anderson_1-0" class="reference"><a href="#cite_note-Anderson-1"><span class="cite-bracket">[</span>1<span class="cite-bracket">]</span></a></sup> The term <b>MOLE</b> (molecular optics laser examiner) is used to refer to the Raman-based microprobe.<sup id="cite_ref-Anderson_1-1" class="reference"><a href="#cite_note-Anderson-1"><span class="cite-bracket">[</span>1<span class="cite-bracket">]</span></a></sup> The technique used is named after <a href="/wiki/C._V._Raman" title="C. V. Raman">C. V. Raman</a>, who discovered the scattering properties in liquids.<sup id="cite_ref-2" class="reference"><a href="#cite_note-2"><span class="cite-bracket">[</span>2<span class="cite-bracket">]</span></a></sup> </p> <meta property="mw:PageProp/toc" /> <div class="mw-heading mw-heading2"><h2 id="Configuration">Configuration</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=1" title="Edit section: Configuration"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>The Raman microscope begins with a standard <a href="/wiki/Optical_microscope" title="Optical microscope">optical microscope</a>, and adds an <a href="/wiki/Excited_state" title="Excited state">excitation</a> <a href="/wiki/Laser" title="Laser">laser</a>, <a href="/wiki/Optical_filter#longpass" title="Optical filter">laser rejection filters</a>, a <a href="/wiki/Spectrometer" title="Spectrometer">spectrometer</a> or <a href="/wiki/Monochromator" title="Monochromator">monochromator</a>, and an optical sensitive <a href="/wiki/Detector" class="mw-redirect" title="Detector">detector</a> such as a <a href="/wiki/Charge-coupled_device" title="Charge-coupled device">charge-coupled device</a> (CCD), or <a href="/wiki/Photomultiplier_tube" title="Photomultiplier tube">photomultiplier tube</a>, (PMT). Traditionally Raman microscopy was used to measure the Raman spectrum of a point on a sample, more recently the technique has been extended to implement Raman spectroscopy for direct <a href="/wiki/Chemical_imaging" title="Chemical imaging">chemical imaging</a> over the whole field of view on a <a href="/wiki/Three-dimensional_space" title="Three-dimensional space">3D</a> sample. </p> <div class="mw-heading mw-heading2"><h2 id="Imaging_modes">Imaging modes</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=2" title="Edit section: Imaging modes"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>In <i>direct imaging</i>, the whole field of view is examined for scattering over a small range of wavenumbers (Raman shifts). For instance, a wavenumber characteristic for cholesterol could be used to record the distribution of cholesterol within a cell culture. The other approach is <i><a href="/wiki/Hyperspectral_imaging" title="Hyperspectral imaging">hyperspectral imaging</a></i> or <i>chemical imaging</i>, in which thousands of Raman spectra are acquired from all over the field of view. The data can then be used to generate images showing the location and amount of different components. Taking the cell culture example, a hyperspectral image could show the distribution of cholesterol,<sup id="cite_ref-3" class="reference"><a href="#cite_note-3"><span class="cite-bracket">[</span>3<span class="cite-bracket">]</span></a></sup> as well as proteins, nucleic acids, and fatty acids.<sup id="cite_ref-4" class="reference"><a href="#cite_note-4"><span class="cite-bracket">[</span>4<span class="cite-bracket">]</span></a></sup><sup id="cite_ref-5" class="reference"><a href="#cite_note-5"><span class="cite-bracket">[</span>5<span class="cite-bracket">]</span></a></sup><sup id="cite_ref-6" class="reference"><a href="#cite_note-6"><span class="cite-bracket">[</span>6<span class="cite-bracket">]</span></a></sup> Sophisticated signal- and image-processing techniques can be used to ignore the presence of water, culture media, buffers, and other interference. </p> <div class="mw-heading mw-heading2"><h2 id="Resolution">Resolution</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=3" title="Edit section: Resolution"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>Raman microscopy, and in particular <a href="/wiki/Confocal_microscopy" title="Confocal microscopy">confocal microscopy</a>, can reach down to sub-micrometer lateral spatial resolution.<sup id="cite_ref-7" class="reference"><a href="#cite_note-7"><span class="cite-bracket">[</span>7<span class="cite-bracket">]</span></a></sup> Because a Raman microscope is a <a href="/wiki/Diffraction-limited_system" title="Diffraction-limited system">diffraction-limited system</a>, its spatial resolution depends on the wavelength of light and the <a href="/wiki/Numerical_aperture" title="Numerical aperture">numerical aperture</a> of the focusing element. In confocal Raman microscopy, the diameter of the confocal aperture is an additional factor. As a rule of thumb, the lateral spatial resolution can reach approximately the laser wavelength when using air objective lenses, while oil or water immersion objectives can provide lateral resolutions of around half the laser wavelength. This means that when operated in the visible to near-infrared range, a Raman microscope can achieve lateral resolutions of approx. 1 µm down to 250 nm, while the depth resolution (if not limited by the optical penetration depth of the sample) can range from 1-6 µm with the smallest confocal pinhole aperture to tens of micrometers when operated without a confocal pinhole.<sup id="cite_ref-ApplSpectrosc_8-0" class="reference"><a href="#cite_note-ApplSpectrosc-8"><span class="cite-bracket">[</span>8<span class="cite-bracket">]</span></a></sup><sup id="cite_ref-9" class="reference"><a href="#cite_note-9"><span class="cite-bracket">[</span>9<span class="cite-bracket">]</span></a></sup><sup id="cite_ref-10" class="reference"><a href="#cite_note-10"><span class="cite-bracket">[</span>10<span class="cite-bracket">]</span></a></sup> Since the objective lenses of microscopes focus the laser beam down to the micrometer range, the resulting photon flux is much higher than achieved in conventional Raman setups. This has the added effect of increased <a href="/wiki/Photobleaching" title="Photobleaching">photobleaching</a> of molecules emitting interfering fluorescence. However, the high photon flux can also cause sample degradation, and thus, for each type of sample, the laser wavelength and laser power have to be carefully selected. </p> <div class="mw-heading mw-heading2"><h2 id="Raman_imaging">Raman imaging</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=4" title="Edit section: Raman imaging"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <figure class="mw-default-size" typeof="mw:File/Thumb"><a href="/wiki/File:Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png" class="mw-file-description"><img alt="Chemical Imaging of a pharmaceutical emulsion with confocal Raman microscopy." src="//upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png/220px-Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png" decoding="async" width="220" height="220" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png/330px-Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/2/2f/Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png/440px-Confocal_Raman_Image_of_a_pharmaceutical_emulsion..png 2x" data-file-width="2048" data-file-height="2048" /></a><figcaption>Chemical image of a pharmaceutical emulsion acquired by confocal Raman microscopy (alpha300 microscope, WITec; blue: Active pharmaceutical ingredient, green: Oil, red: Silicon impurities).</figcaption></figure> <p>Another tool that is becoming more popular is global Raman imaging. 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 <a href="/wiki/Graphene" title="Graphene">graphene</a> layers,<sup id="cite_ref-11" class="reference"><a href="#cite_note-11"><span class="cite-bracket">[</span>11<span class="cite-bracket">]</span></a></sup> <a href="/wiki/J-aggregate" title="J-aggregate">J-aggregated dyes</a> inside <a href="/wiki/Carbon_nanotube" title="Carbon nanotube">carbon nanotubes</a> and multiple other 2D materials such as <a href="/wiki/Molybdenum_disulfide" title="Molybdenum disulfide">MoS<sub>2</sub></a><sup id="cite_ref-12" class="reference"><a href="#cite_note-12"><span class="cite-bracket">[</span>12<span class="cite-bracket">]</span></a></sup> and <a href="/wiki/Tungsten_diselenide" title="Tungsten diselenide">WSe<sub>2</sub></a>. Since the excitation beam is dispersed over the whole field of view, those measurements can be done without damaging the sample. By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of microscopic regions of samples can be measured. As a result, the fluorescence of water, media, and buffers can be removed. Consequently, it is suitable to examine proteins, cells and organelles. </p><p>Raman microscopy for biological and medical specimens generally uses near-infrared (NIR) lasers (785 nm <a href="/wiki/Diode-pumped_solid-state_laser" title="Diode-pumped solid-state laser">diodes</a> and 1064 nm <a href="/wiki/Nd:YAG_laser" title="Nd:YAG laser">Nd:YAG</a> are especially common). This reduces the risk of damaging the specimen by applying higher energy wavelengths. However, the intensity of NIR Raman scattering is low (owing to the ω<sup>4</sup> dependence of Raman scattering intensity), and most detectors require very long collection times. Recently, more sensitive detectors have become available, making the technique better suited to general use. Raman microscopy of inorganic specimens, such as rocks, ceramics and polymers,<sup id="cite_ref-Schmidt_133–143_13-0" class="reference"><a href="#cite_note-Schmidt_133–143-13"><span class="cite-bracket">[</span>13<span class="cite-bracket">]</span></a></sup> can use a broader range of excitation wavelengths. </p><p>A related technique, <a href="/wiki/Tip-enhanced_Raman_spectroscopy" title="Tip-enhanced Raman spectroscopy">tip-enhanced Raman spectroscopy</a>, can produce high-resolution hyperspectral images of single molecules<sup id="cite_ref-14" class="reference"><a href="#cite_note-14"><span class="cite-bracket">[</span>14<span class="cite-bracket">]</span></a></sup> and DNA.<sup id="cite_ref-15" class="reference"><a href="#cite_note-15"><span class="cite-bracket">[</span>15<span class="cite-bracket">]</span></a></sup> </p> <div class="mw-heading mw-heading2"><h2 id="Correlative_Raman_imaging">Correlative Raman imaging</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=5" title="Edit section: Correlative Raman imaging"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <figure class="mw-default-size" typeof="mw:File/Thumb"><a href="/wiki/File:Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png" class="mw-file-description"><img alt="Correlative Raman-SEM imaging of a hematite." src="//upload.wikimedia.org/wikipedia/commons/thumb/2/2d/Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png/220px-Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png" decoding="async" width="220" height="199" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/2/2d/Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png/330px-Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/2/2d/Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png/440px-Correlative_Raman_SEM_image_of_hematite_acquired_with_RISE_microscope.png 2x" data-file-width="516" data-file-height="466" /></a><figcaption>Correlative Raman-SEM imaging of a hematite (taken with RISE microscope, WITec). The Raman image is overlaid over the SEM image.</figcaption></figure> <p>Confocal Raman microscopy can be combined with numerous other microscopy techniques. By using different methods and correlating the data, the user attains a more comprehensive understanding of the sample. Common examples of correlative microscopy techniques are <a href="/wiki/Atomic_force_microscopy" title="Atomic force microscopy">Raman-AFM</a>,<sup id="cite_ref-16" class="reference"><a href="#cite_note-16"><span class="cite-bracket">[</span>16<span class="cite-bracket">]</span></a></sup><sup id="cite_ref-Schmidt_133–143_13-1" class="reference"><a href="#cite_note-Schmidt_133–143-13"><span class="cite-bracket">[</span>13<span class="cite-bracket">]</span></a></sup> Raman-<a href="/wiki/Near-field_scanning_optical_microscope" title="Near-field scanning optical microscope">SNOM</a>,<sup id="cite_ref-17" class="reference"><a href="#cite_note-17"><span class="cite-bracket">[</span>17<span class="cite-bracket">]</span></a></sup> and Raman-<a href="/wiki/Scanning_electron_microscope" title="Scanning electron microscope">SEM</a>.<sup id="cite_ref-18" class="reference"><a href="#cite_note-18"><span class="cite-bracket">[</span>18<span class="cite-bracket">]</span></a></sup> </p><p>Correlative SEM-Raman imaging is the integration of a confocal Raman microscope into an SEM chamber which allows correlative imaging of several techniques, such as SE, BSE, <a href="/wiki/Energy-dispersive_X-ray_spectroscopy" title="Energy-dispersive X-ray spectroscopy">EDX</a>, <a href="/wiki/Electron_backscatter_diffraction" title="Electron backscatter diffraction">EBSD</a>, <a href="/wiki/Electron_beam-induced_current" title="Electron beam-induced current">EBIC</a>, <a href="/wiki/Cathodoluminescence" title="Cathodoluminescence">CL</a>, <a href="/wiki/Atomic_force_microscopy" title="Atomic force microscopy">AFM</a>.<sup id="cite_ref-19" class="reference"><a href="#cite_note-19"><span class="cite-bracket">[</span>19<span class="cite-bracket">]</span></a></sup> The sample is placed in the vacuum chamber of the electron microscope. Both analysis methods are then performed automatically at the same sample location. The obtained SEM and Raman images can then be superimposed.<sup id="cite_ref-20" class="reference"><a href="#cite_note-20"><span class="cite-bracket">[</span>20<span class="cite-bracket">]</span></a></sup><sup id="cite_ref-21" class="reference"><a href="#cite_note-21"><span class="cite-bracket">[</span>21<span class="cite-bracket">]</span></a></sup> Moreover, adding a <a href="/wiki/Focused_ion_beam" title="Focused ion beam">focused ion beam</a> (FIB) on the chamber allows removal of the material and therefore 3D imaging of the sample. Low-vacuum mode allows analysis on biological and non-conductive samples. </p> <div class="mw-heading mw-heading2"><h2 id="Biological_Applications">Biological Applications</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=6" title="Edit section: Biological Applications"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>By using Raman microspectroscopy, <i>in vivo</i> time- and space-resolved Raman spectra of microscopic regions of samples can be measured. Sampling is non-destructive and water, media, and buffers typically do not interfere with the analysis. Consequently, <i>in vivo</i> time- and space-resolved Raman spectroscopy is suitable to examine <a href="/wiki/Proteins" class="mw-redirect" title="Proteins">proteins</a>, <a href="/wiki/Cell_(biology)" title="Cell (biology)">cells</a> and <a href="/wiki/Organ_(anatomy)" class="mw-redirect" title="Organ (anatomy)">organs</a>. In the field of microbiology, confocal Raman microspectroscopy has been used to map intracellular distributions of macromolecules, such as proteins, polysaccharides, and nucleic acids and polymeric inclusions, such as poly-β-hydroxybutyric acid and polyphosphates in bacteria and sterols in microalgae. Combining stable isotopic probing (SIP) experiments with confocal Raman microspectroscopy has permitted determination of assimilation rates of <sup>13</sup>C and <sup>15</sup>N-substrates as well as D<sub>2</sub>O by individual bacterial cells.<sup id="cite_ref-22" class="reference"><a href="#cite_note-22"><span class="cite-bracket">[</span>22<span class="cite-bracket">]</span></a></sup> </p> <div class="mw-heading mw-heading2"><h2 id="See_also">See also</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=7" title="Edit section: See also"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <ul><li><a href="/wiki/Raman_scattering" title="Raman scattering">Raman scattering</a></li> <li><a href="/wiki/Coherent_Raman_Scattering_Microscopy" class="mw-redirect" title="Coherent Raman Scattering Microscopy">Coherent Raman Scattering Microscopy</a></li> <li><a href="/wiki/Scanning_electron_microscope" title="Scanning electron microscope">Scanning electron microscope</a></li> <li><a href="/wiki/Tip-enhanced_Raman_spectroscopy" title="Tip-enhanced Raman spectroscopy">Tip-enhanced Raman spectroscopy</a></li></ul> <div class="mw-heading mw-heading2"><h2 id="References">References</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=Raman_microscope&action=edit&section=8" title="Edit section: References"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <style data-mw-deduplicate="TemplateStyles:r1239543626">.mw-parser-output .reflist{margin-bottom:0.5em;list-style-type:decimal}@media screen{.mw-parser-output .reflist{font-size:90%}}.mw-parser-output .reflist .references{font-size:100%;margin-bottom:0;list-style-type:inherit}.mw-parser-output .reflist-columns-2{column-width:30em}.mw-parser-output .reflist-columns-3{column-width:25em}.mw-parser-output .reflist-columns{margin-top:0.3em}.mw-parser-output .reflist-columns ol{margin-top:0}.mw-parser-output .reflist-columns li{page-break-inside:avoid;break-inside:avoid-column}.mw-parser-output .reflist-upper-alpha{list-style-type:upper-alpha}.mw-parser-output .reflist-upper-roman{list-style-type:upper-roman}.mw-parser-output .reflist-lower-alpha{list-style-type:lower-alpha}.mw-parser-output .reflist-lower-greek{list-style-type:lower-greek}.mw-parser-output .reflist-lower-roman{list-style-type:lower-roman}</style><div class="reflist"> <div class="mw-references-wrap mw-references-columns"><ol class="references"> <li id="cite_note-Anderson-1"><span class="mw-cite-backlink">^ <a href="#cite_ref-Anderson_1-0"><sup><i><b>a</b></i></sup></a> <a href="#cite_ref-Anderson_1-1"><sup><i><b>b</b></i></sup></a></span> <span class="reference-text"><i>Microscopical techniques in the use of the molecular optics laser examiner Raman microprobe</i>, by M. E. Andersen, R. Z. Muggli, Analytical Chemistry, 1981, 53 (12), pp 1772–1777 <a rel="nofollow" class="external autonumber" href="http://pubs.acs.org/doi/abs/10.1021/ac00235a013">[1]</a></span> </li> <li id="cite_note-2"><span class="mw-cite-backlink"><b><a href="#cite_ref-2">^</a></b></span> <span class="reference-text"><style data-mw-deduplicate="TemplateStyles:r1238218222">.mw-parser-output cite.citation{font-style:inherit;word-wrap:break-word}.mw-parser-output .citation q{quotes:"\"""\"""'""'"}.mw-parser-output .citation:target{background-color:rgba(0,127,255,0.133)}.mw-parser-output .id-lock-free.id-lock-free a{background:url("//upload.wikimedia.org/wikipedia/commons/6/65/Lock-green.svg")right 0.1em center/9px no-repeat}.mw-parser-output .id-lock-limited.id-lock-limited a,.mw-parser-output .id-lock-registration.id-lock-registration a{background:url("//upload.wikimedia.org/wikipedia/commons/d/d6/Lock-gray-alt-2.svg")right 0.1em center/9px no-repeat}.mw-parser-output .id-lock-subscription.id-lock-subscription a{background:url("//upload.wikimedia.org/wikipedia/commons/a/aa/Lock-red-alt-2.svg")right 0.1em center/9px no-repeat}.mw-parser-output .cs1-ws-icon a{background:url("//upload.wikimedia.org/wikipedia/commons/4/4c/Wikisource-logo.svg")right 0.1em center/12px no-repeat}body:not(.skin-timeless):not(.skin-minerva) .mw-parser-output .id-lock-free a,body:not(.skin-timeless):not(.skin-minerva) .mw-parser-output .id-lock-limited a,body:not(.skin-timeless):not(.skin-minerva) .mw-parser-output .id-lock-registration a,body:not(.skin-timeless):not(.skin-minerva) .mw-parser-output .id-lock-subscription a,body:not(.skin-timeless):not(.skin-minerva) .mw-parser-output .cs1-ws-icon a{background-size:contain;padding:0 1em 0 0}.mw-parser-output .cs1-code{color:inherit;background:inherit;border:none;padding:inherit}.mw-parser-output .cs1-hidden-error{display:none;color:var(--color-error,#d33)}.mw-parser-output .cs1-visible-error{color:var(--color-error,#d33)}.mw-parser-output .cs1-maint{display:none;color:#085;margin-left:0.3em}.mw-parser-output .cs1-kern-left{padding-left:0.2em}.mw-parser-output .cs1-kern-right{padding-right:0.2em}.mw-parser-output .citation .mw-selflink{font-weight:inherit}@media screen{.mw-parser-output .cs1-format{font-size:95%}html.skin-theme-clientpref-night .mw-parser-output .cs1-maint{color:#18911f}}@media screen and (prefers-color-scheme:dark){html.skin-theme-clientpref-os .mw-parser-output .cs1-maint{color:#18911f}}</style><cite id="CITEREFKrishnanRaman1928" class="citation journal cs1">Krishnan, K. S.; Raman, C. V. (1928). "A New Type of Secondary Radiation". <i>Nature</i>. <b>121</b> (3048): 501–502. <a href="/wiki/Bibcode_(identifier)" class="mw-redirect" title="Bibcode (identifier)">Bibcode</a>:<a rel="nofollow" class="external text" href="https://ui.adsabs.harvard.edu/abs/1928Natur.121..501R">1928Natur.121..501R</a>. <a href="/wiki/Doi_(identifier)" class="mw-redirect" title="Doi (identifier)">doi</a>:<a rel="nofollow" class="external text" href="https://doi.org/10.1038%2F121501c0">10.1038/121501c0</a>. <a href="/wiki/ISSN_(identifier)" class="mw-redirect" title="ISSN (identifier)">ISSN</a> <a rel="nofollow" class="external text" href="https://search.worldcat.org/issn/1476-4687">1476-4687</a>. <a href="/wiki/S2CID_(identifier)" class="mw-redirect" title="S2CID (identifier)">S2CID</a> <a rel="nofollow" class="external text" href="https://api.semanticscholar.org/CorpusID:4128161">4128161</a>.</cite><span title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.jtitle=Nature&rft.atitle=A+New+Type+of+Secondary+Radiation&rft.volume=121&rft.issue=3048&rft.pages=501-502&rft.date=1928&rft_id=info%3Adoi%2F10.1038%2F121501c0&rft_id=https%3A%2F%2Fapi.semanticscholar.org%2FCorpusID%3A4128161%23id-name%3DS2CID&rft.issn=1476-4687&rft_id=info%3Abibcode%2F1928Natur.121..501R&rft.aulast=Krishnan&rft.aufirst=K.+S.&rft.au=Raman%2C+C.+V.&rfr_id=info%3Asid%2Fen.wikipedia.org%3ARaman+microscope" class="Z3988"></span></span> </li> <li id="cite_note-3"><span class="mw-cite-backlink"><b><a href="#cite_ref-3">^</a></b></span> <span class="reference-text"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1238218222"><cite id="CITEREFMatthäusKrafftDietzekBrehm2012" class="citation journal cs1">Matthäus, Christian; Krafft, Christoph; Dietzek, Benjamin; Brehm, Bernhard R.; Lorkowski, Stefan; Popp, Jürgen (2012-10-16). 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class="navbox-title" colspan="2"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1129693374"><style data-mw-deduplicate="TemplateStyles:r1239400231">.mw-parser-output .navbar{display:inline;font-size:88%;font-weight:normal}.mw-parser-output .navbar-collapse{float:left;text-align:left}.mw-parser-output .navbar-boxtext{word-spacing:0}.mw-parser-output .navbar ul{display:inline-block;white-space:nowrap;line-height:inherit}.mw-parser-output .navbar-brackets::before{margin-right:-0.125em;content:"[ "}.mw-parser-output .navbar-brackets::after{margin-left:-0.125em;content:" ]"}.mw-parser-output .navbar li{word-spacing:-0.125em}.mw-parser-output .navbar a>span,.mw-parser-output .navbar a>abbr{text-decoration:inherit}.mw-parser-output .navbar-mini abbr{font-variant:small-caps;border-bottom:none;text-decoration:none;cursor:inherit}.mw-parser-output .navbar-ct-full{font-size:114%;margin:0 7em}.mw-parser-output .navbar-ct-mini{font-size:114%;margin:0 4em}html.skin-theme-clientpref-night .mw-parser-output .navbar li a abbr{color:var(--color-base)!important}@media(prefers-color-scheme:dark){html.skin-theme-clientpref-os .mw-parser-output .navbar li a abbr{color:var(--color-base)!important}}@media print{.mw-parser-output .navbar{display:none!important}}</style><div class="navbar plainlinks hlist navbar-mini"><ul><li class="nv-view"><a href="/wiki/Template:Raman_spectroscopy" title="Template:Raman spectroscopy"><abbr title="View this template">v</abbr></a></li><li class="nv-talk"><a href="/wiki/Template_talk:Raman_spectroscopy" title="Template talk:Raman spectroscopy"><abbr title="Discuss this template">t</abbr></a></li><li class="nv-edit"><a href="/wiki/Special:EditPage/Template:Raman_spectroscopy" title="Special:EditPage/Template:Raman spectroscopy"><abbr title="Edit this template">e</abbr></a></li></ul></div><div id="Raman_spectroscopy" style="font-size:114%;margin:0 4em"><a href="/wiki/Raman_spectroscopy" title="Raman spectroscopy">Raman spectroscopy</a></div></th></tr><tr><th scope="row" class="navbox-group" style="width:1%;text-align:center">Techniques</th><td class="navbox-list-with-group navbox-list navbox-odd" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><a href="/wiki/Coherent_anti-Stokes_Raman_spectroscopy" title="Coherent anti-Stokes Raman spectroscopy">Coherent anti-Stokes Raman spectroscopy</a></li> <li><a href="/wiki/Raman_optical_activity" title="Raman optical activity">Raman optical activity</a></li> <li><a href="/wiki/Resonance_Raman_spectroscopy" title="Resonance Raman spectroscopy">Resonance Raman spectroscopy</a></li> <li><a href="/wiki/Rotating-polarization_coherent_anti-Stokes_Raman_spectroscopy" title="Rotating-polarization coherent anti-Stokes Raman spectroscopy">Rotating-polarization coherent anti-Stokes Raman spectroscopy</a></li> <li><a href="/wiki/Spatially_offset_Raman_spectroscopy" title="Spatially offset Raman spectroscopy">Spatially offset Raman spectroscopy</a></li> <li><a href="/wiki/Stimulated_Raman_spectroscopy" title="Stimulated Raman spectroscopy">Stimulated Raman spectroscopy</a></li> <li><a href="/wiki/Surface-enhanced_Raman_spectroscopy" title="Surface-enhanced Raman spectroscopy">Surface-enhanced Raman spectroscopy</a></li> <li><a href="/wiki/Tip-enhanced_Raman_spectroscopy" title="Tip-enhanced Raman spectroscopy">Tip-enhanced Raman spectroscopy</a></li> <li><a href="/wiki/Transmission_Raman_spectroscopy" title="Transmission Raman spectroscopy">Transmission Raman spectroscopy</a></li></ul> </div></td></tr><tr><th scope="row" class="navbox-group" style="width:1%;text-align:center">Applications</th><td class="navbox-list-with-group navbox-list navbox-even" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><a href="/wiki/Raman_amplification" title="Raman amplification">Raman amplification</a></li> <li><a href="/wiki/Raman_cooling" title="Raman cooling">Raman cooling</a></li> <li><a href="/wiki/Raman_laser" title="Raman laser">Raman laser</a></li> <li><a class="mw-selflink selflink">Raman microscope</a></li> <li><a href="/wiki/SHERLOC" class="mw-redirect" title="SHERLOC">SHERLOC</a></li> <li><a href="/wiki/Stimulated_Raman_adiabatic_passage" title="Stimulated Raman adiabatic passage">Stimulated Raman adiabatic passage</a></li></ul> </div></td></tr><tr><th scope="row" class="navbox-group" style="width:1%;text-align:center">Theory</th><td class="navbox-list-with-group navbox-list navbox-odd" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><a href="/wiki/Depolarization_ratio" title="Depolarization ratio">Depolarization ratio</a></li> <li><a href="/wiki/Four-wave_mixing" title="Four-wave mixing">Four-wave mixing</a></li> <li><a href="/wiki/Nonlinear_optics" title="Nonlinear optics">Nonlinear optics</a></li> <li><a href="/wiki/Raman_scattering" title="Raman scattering">Raman scattering</a></li> <li><a href="/wiki/Rayleigh_scattering" title="Rayleigh scattering">Rayleigh scattering</a></li> <li><a href="/wiki/Rule_of_mutual_exclusion" title="Rule of mutual exclusion">Rule of mutual exclusion</a></li> <li><a href="/wiki/Stokes_shift" title="Stokes shift">Stokes shift</a></li></ul> </div></td></tr><tr><th scope="row" class="navbox-group" style="width:1%;text-align:center">Journals</th><td class="navbox-list-with-group navbox-list navbox-even" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><i><a href="/wiki/Journal_of_Raman_Spectroscopy" title="Journal of Raman Spectroscopy">Journal of Raman Spectroscopy</a></i></li> <li><i><a href="/wiki/Vibrational_Spectroscopy" title="Vibrational Spectroscopy">Vibrational Spectroscopy</a></i></li></ul> </div></td></tr><tr><td class="navbox-abovebelow" colspan="2"><div> <ul><li><span class="noviewer" typeof="mw:File"><span title="Category"><img alt="" src="//upload.wikimedia.org/wikipedia/en/thumb/9/96/Symbol_category_class.svg/16px-Symbol_category_class.svg.png" 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href="https://commons.wikimedia.org/wiki/Category:Raman_spectroscopy" class="extiw" title="commons:Category:Raman spectroscopy">Commons</a></b></li> <li><b><a href="/wiki/Spectroscopy" title="Spectroscopy">Spectroscopy</a></b></li></ul> </div></td></tr></tbody></table></div> <div class="navbox-styles"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1129693374"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1236075235"></div><div role="navigation" class="navbox" aria-labelledby="Optical_microscopy" style="padding:3px"><table class="nowraplinks hlist mw-collapsible autocollapse navbox-inner" style="border-spacing:0;background:transparent;color:inherit"><tbody><tr><th scope="col" class="navbox-title" colspan="3"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1129693374"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1239400231"><div class="navbar plainlinks hlist navbar-mini"><ul><li class="nv-view"><a href="/wiki/Template:Optical_microscopy" title="Template:Optical microscopy"><abbr title="View this template">v</abbr></a></li><li class="nv-talk"><a href="/wiki/Template_talk:Optical_microscopy" title="Template talk:Optical microscopy"><abbr title="Discuss this template">t</abbr></a></li><li class="nv-edit"><a href="/wiki/Special:EditPage/Template:Optical_microscopy" title="Special:EditPage/Template:Optical microscopy"><abbr title="Edit this template">e</abbr></a></li></ul></div><div id="Optical_microscopy" style="font-size:114%;margin:0 4em"><a href="/wiki/Microscopy" title="Microscopy">Optical microscopy</a></div></th></tr><tr><td class="navbox-abovebelow" colspan="3"><div> <ul><li><b><a href="/wiki/Microscope" title="Microscope">Microscope</a></b></li> <li><b><a href="/wiki/Optical_microscope" title="Optical microscope">Optical microscopy</a></b></li></ul> </div></td></tr><tr><th scope="row" class="navbox-group" style="width:1%">Illumination and<br />contrast methods</th><td class="navbox-list-with-group navbox-list navbox-odd" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><a href="/wiki/Bright-field_microscopy" title="Bright-field microscopy">Bright-field microscopy</a></li> <li><a href="/wiki/K%C3%B6hler_illumination" title="Köhler illumination">Köhler illumination</a></li> <li><a href="/wiki/Dark-field_microscopy" title="Dark-field microscopy">Dark-field microscopy</a></li> <li><a href="/wiki/Phase-contrast_microscopy" title="Phase-contrast microscopy">Phase contrast</a></li> <li><a href="/wiki/Quantitative_phase-contrast_microscopy" title="Quantitative phase-contrast microscopy">Quantitative phase-contrast microscopy</a></li> <li><a href="/wiki/Differential_interference_contrast_microscopy" title="Differential interference contrast microscopy">Differential interference contrast (DIC)</a></li> <li><a href="/wiki/Dispersion_staining" title="Dispersion staining">Dispersion staining</a></li> <li><a href="/wiki/Second-harmonic_imaging_microscopy" title="Second-harmonic imaging microscopy">Second harmonic imaging (SHIM)</a></li> <li><a href="/wiki/4Pi_microscope" title="4Pi microscope">4Pi microscope</a></li> <li><a href="/wiki/Microscopy#Structured_illumination" title="Microscopy">Structured illumination</a></li> <li><a href="/wiki/Sarfus" class="mw-redirect" title="Sarfus">Sarfus</a></li> <li><a href="/wiki/Interference_reflection_microscopy" title="Interference reflection microscopy">Interference reflection microscopy (IRM/RICM)</a></li> <li><a class="mw-selflink selflink">Raman</a></li></ul> </div></td><td class="noviewer navbox-image" rowspan="3" style="width:1px;padding:0 0 0 2px"><div><span typeof="mw:File"><a href="/wiki/File:Loupe-binoculaire-p1030891.jpg" class="mw-file-description"><img src="//upload.wikimedia.org/wikipedia/commons/thumb/c/c3/Loupe-binoculaire-p1030891.jpg/150px-Loupe-binoculaire-p1030891.jpg" decoding="async" width="150" height="111" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/c/c3/Loupe-binoculaire-p1030891.jpg/225px-Loupe-binoculaire-p1030891.jpg 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/c/c3/Loupe-binoculaire-p1030891.jpg/300px-Loupe-binoculaire-p1030891.jpg 2x" data-file-width="2535" data-file-height="1882" /></a></span></div></td></tr><tr><th scope="row" class="navbox-group" style="width:1%">Fluorescence methods</th><td class="navbox-list-with-group navbox-list navbox-even" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><a href="/wiki/Fluorescence_microscope" title="Fluorescence microscope">Fluorescence microscopy</a></li> <li><a href="/wiki/Confocal_microscopy" title="Confocal microscopy">Confocal microscopy</a></li> <li><a href="/wiki/Two-photon_excitation_microscopy" title="Two-photon excitation microscopy">Multiphoton microscopy</a> (<a href="/wiki/Two-photon_excitation_microscopy" title="Two-photon excitation microscopy">Two-photon</a>, <a href="/wiki/Three_photon_microscopy" class="mw-redirect" title="Three photon microscopy">Three-photon</a>)</li> <li><a href="/wiki/Deconvolution#Optics_and_other_imaging" title="Deconvolution">Image deconvolution</a></li> <li><a href="/wiki/Total_internal_reflection_fluorescence_microscope" title="Total internal reflection fluorescence microscope">Total internal reflection fluorescence microscopy (TIRF)</a></li> <li><a href="/wiki/Light_sheet_fluorescence_microscopy" title="Light sheet fluorescence microscopy">Lightsheet microscopy (LSFM/SPIM)</a></li> <li><a href="/wiki/Lattice_light-sheet_microscopy" title="Lattice light-sheet microscopy">Lattice light-sheet microscopy</a></li></ul> </div></td></tr><tr><th scope="row" class="navbox-group" style="width:1%">Sub-diffraction<br />limit techniques</th><td class="navbox-list-with-group navbox-list navbox-odd" style="width:100%;padding:0"><div style="padding:0 0.25em"> <ul><li><a href="/wiki/Diffraction-limited_system" title="Diffraction-limited system">Diffraction limit</a></li> <li><a href="/wiki/STED_microscopy" title="STED microscopy">Stimulated emission depletion (STED)</a></li> <li><a href="/wiki/Photoactivated_localization_microscopy" title="Photoactivated localization microscopy">Photo-activated localization microscopy (PALM/STORM)</a></li> <li><a href="/wiki/Near-field_scanning_optical_microscope" title="Near-field scanning optical microscope">Near-field (NSOM/SNOM)</a></li></ul> </div></td></tr><tr><td class="navbox-abovebelow hlist" colspan="3"><div> <ul><li><span class="noviewer" typeof="mw:File"><span title="Category"><img alt="" src="//upload.wikimedia.org/wikipedia/en/thumb/9/96/Symbol_category_class.svg/16px-Symbol_category_class.svg.png" decoding="async" width="16" height="16" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/en/thumb/9/96/Symbol_category_class.svg/23px-Symbol_category_class.svg.png 1.5x, //upload.wikimedia.org/wikipedia/en/thumb/9/96/Symbol_category_class.svg/31px-Symbol_category_class.svg.png 2x" data-file-width="180" data-file-height="185" /></span></span> <b><a href="/wiki/Category:Optical_microscopy" title="Category:Optical microscopy">Category</a></b></li> <li><span class="noviewer" typeof="mw:File"><span title="Commons page"><img alt="" src="//upload.wikimedia.org/wikipedia/en/thumb/4/4a/Commons-logo.svg/12px-Commons-logo.svg.png" decoding="async" width="12" height="16" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/en/thumb/4/4a/Commons-logo.svg/18px-Commons-logo.svg.png 1.5x, //upload.wikimedia.org/wikipedia/en/thumb/4/4a/Commons-logo.svg/24px-Commons-logo.svg.png 2x" data-file-width="1024" data-file-height="1376" /></span></span> <b><a href="https://commons.wikimedia.org/wiki/Category:Optical_microscopy" class="extiw" title="commons:Category:Optical microscopy">Commons</a></b></li></ul> </div></td></tr></tbody></table></div>    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