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searchaux" style="display:none">Method of specimen illumination used in optical microscopy</div> <p><b>Köhler illumination</b> is a method of specimen <a href="/wiki/Illumination_(lighting)" class="mw-redirect" title="Illumination (lighting)">illumination</a> used for transmitted and reflected light (trans- and epi-illuminated) <a href="/wiki/Optical_microscopy" class="mw-redirect" title="Optical microscopy">optical microscopy</a>. Köhler illumination acts to generate an even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp <a href="/wiki/Electrical_filament" class="mw-redirect" title="Electrical filament">filament</a>) is not visible in the resulting image. Köhler illumination is the predominant technique for sample illumination in modern scientific light microscopy. It requires additional optical elements which are more expensive and may not be present in more basic light microscopes. </p> <meta property="mw:PageProp/toc" /> <div class="mw-heading mw-heading2"><h2 id="History_and_motivation">History and motivation</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=K%C3%B6hler_illumination&action=edit&section=1" title="Edit section: History and motivation"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>Prior to Köhler illumination <a href="/wiki/Critical_illumination" title="Critical illumination">critical illumination</a> was the predominant technique for sample illumination. Critical illumination has the major limitation that the image of the light source (typically a <a href="/wiki/Light_bulb" class="mw-redirect" title="Light bulb">light bulb</a>) falls in the same plane as the image of the specimen, i.e., the bulb filament is visible in the final image. The image of the light source is often referred to as the <b>filament image</b>. Critical illumination therefore gives uneven illumination of the sample; bright regions in the filament image illuminate those regions of the sample more strongly. Uneven illumination is undesirable as it can introduce artifacts such as <a href="/wiki/Glare_(vision)" title="Glare (vision)">glare</a> and shadowing in the image. </p><p>Various methods can be used to diffuse the filament image, including reducing power to the light source or using an opal glass bulb or an opal glass diffuser between the bulb and the sample. These methods are all, to some extent, functional at reducing the unevenness of illumination however they all reduce intensity of illumination and alter the range of wavelengths of light which reach the sample. </p><p>To address these limitations <a href="/wiki/August_K%C3%B6hler" title="August Köhler">August Köhler</a> designed a method of illumination which uses a perfectly defocused image of the light source to illuminate the sample. This work was published in 1893 in the <i><a href="/w/index.php?title=Zeitschrift_f%C3%BCr_wissenschaftliche_Mikroskopie&action=edit&redlink=1" class="new" title="Zeitschrift für wissenschaftliche Mikroskopie (page does not exist)">Zeitschrift für wissenschaftliche Mikroskopie</a></i><sup id="cite_ref-wissenschaft_1893_1-0" class="reference"><a href="#cite_note-wissenschaft_1893-1"><span class="cite-bracket">[</span>1<span class="cite-bracket">]</span></a></sup> and was soon followed by publication of an English translation in the <i><a href="/wiki/Journal_of_the_Royal_Microscopical_Society" class="mw-redirect" title="Journal of the Royal Microscopical Society">Journal of the Royal Microscopical Society</a></i>.<sup id="cite_ref-Royal_Society_1894_2-0" class="reference"><a href="#cite_note-Royal_Society_1894-2"><span class="cite-bracket">[</span>2<span class="cite-bracket">]</span></a></sup> </p><p>Köhler illumination has also been developed in the context of <a href="/wiki/Nonimaging_optics" title="Nonimaging optics">nonimaging optics</a>.<sup id="cite_ref-IntroNio2e_3-0" class="reference"><a href="#cite_note-IntroNio2e-3"><span class="cite-bracket">[</span>3<span class="cite-bracket">]</span></a></sup> </p> <div class="mw-heading mw-heading2"><h2 id="Optical_principles">Optical principles</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=K%C3%B6hler_illumination&action=edit&section=2" title="Edit section: Optical principles"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>The primary limitation of critical illumination is the formation of an image of the light source in the specimen image plane. Köhler illumination addresses this by ensuring the image of the light source is perfectly <a href="/wiki/Defocus" class="mw-redirect" title="Defocus">defocused</a> in the sample plane and its <a href="/wiki/Conjugate_focal_plane" title="Conjugate focal plane">conjugate image planes</a>. In a ray diagram of the illumination light path, this can be seen as the image-forming rays passing parallel through the sample. </p><p>Köhler illumination requires several optical components to function: </p> <ol><li>Collector lens and/or field lens</li> <li>Field diaphragm</li> <li>Condenser diaphragm</li> <li><a href="/wiki/Condenser_lens" class="mw-redirect" title="Condenser lens">Condenser lens</a></li></ol> <figure class="mw-default-size mw-halign-center" typeof="mw:File/Thumb"><a href="/wiki/File:Kohler_Illumination_en.svg" class="mw-file-description"><img src="//upload.wikimedia.org/wikipedia/commons/thumb/7/73/Kohler_Illumination_en.svg/660px-Kohler_Illumination_en.svg.png" decoding="async" width="660" height="282" class="mw-file-element" srcset="//upload.wikimedia.org/wikipedia/commons/thumb/7/73/Kohler_Illumination_en.svg/990px-Kohler_Illumination_en.svg.png 1.5x, //upload.wikimedia.org/wikipedia/commons/thumb/7/73/Kohler_Illumination_en.svg/1320px-Kohler_Illumination_en.svg.png 2x" data-file-width="769" data-file-height="329" /></a><figcaption>Schematics of Köhler illumination. Top: Illumination beam path, conjugated planes marked with the light green bar. Bottom: Imaging beam path, conjugated planes marked with the light blue bar. See text for further details.</figcaption></figure> <p>These components lie in this order between the light source and the specimen and control the illumination of the specimen. The collector/field lenses act to collect light from the light source and focus it at the plane of the condenser diaphragm. The condenser lens acts to project this light, without focusing it, through the sample. This illumination scheme creates two sets of conjugate image planes, one with the light source and its images and one with the specimen and its images. These two sets of image planes are found at the following points (see image for numbers and letters): </p> <dl><dd>Light source image planes (labeled with the light green bar in the above image): <ul><li>Lamp filament (1)</li> <li>Condenser diaphragm (2)</li> <li>Back focal plane of the objective (3)</li> <li>The eyepoint (4)</li></ul></dd> <dd>Specimen image planes (labeled with the light blue bar in the above image): <ul><li>Field diaphragm (A)</li> <li>Specimen (B)</li> <li>Intermediate image plane (the <a href="/wiki/Eyepiece" title="Eyepiece">eyepiece</a> diaphragm) (C)</li> <li>The eye retina or camera sensor (D)</li></ul></dd></dl> <div class="mw-heading mw-heading2"><h2 id="Advantages">Advantages</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=K%C3%B6hler_illumination&action=edit&section=3" title="Edit section: Advantages"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>The primary advantage of Köhler illumination is the uniform illumination of the sample. This reduces image artifacts and provides high sample contrast. Uniform illumination of the sample is also critical for advanced illumination techniques such as <a href="/wiki/Phase_contrast" class="mw-redirect" title="Phase contrast">phase contrast</a> and <a href="/wiki/Differential_interference_contrast" class="mw-redirect" title="Differential interference contrast">differential interference contrast</a> microscopy. </p><p>Adjusting the condenser diaphragm alters sample <a href="/wiki/Contrast_(vision)" title="Contrast (vision)">contrast</a>. Furthermore, altering the size of the condenser diaphragm allows adjustment of sample <a href="/wiki/Depth_of_field" title="Depth of field">depth of field</a> by altering the effective <a href="/wiki/Numerical_aperture" title="Numerical aperture">numerical aperture</a> of the microscope. The role of the condenser diaphragm is analogous to the <a href="/wiki/Aperture" title="Aperture">aperture</a> in <a href="/wiki/Photography" title="Photography">photography</a> although the condenser diaphragm of a microscope functions by controlling illumination of the specimen, while the aperture of a camera functions by controlling illumination of the detector. </p><p>Altering the condenser diaphragm allows the amount of light entering the sample to be freely adjusted without altering the wavelengths of light present, in contrast to reducing power to the light source with critical illumination (which changes the <a href="/wiki/Color_temperature" title="Color temperature">color temperature</a> of the lamp). This adjustment is always coupled to an alteration of the numerical aperture of the system, as stated above, and so adjustment of the illumination source intensity by other means is still necessary. </p><p>By adjustment of the field diaphragm, the image of the field diaphragm aperture in the sample plane is set to a size slightly larger than the imaged region of the sample (which corresponds in turn to the portion of the sample image thrown into the eyepiece <a href="/wiki/Eyepiece#Field_of_view" title="Eyepiece">field stop</a>). As the field diaphragm, sample, and eyepiece field stop all lie on <a href="/wiki/Conjugate_image_plane" class="mw-redirect" title="Conjugate image plane">conjugate image planes</a>, this adjustment allows the illuminating rays to completely fill the eyepiece field of view, while minimizing the amount of extraneous light which must be blocked by the eyepiece field stop. Such extraneous light scatters inside the system and degrades contrast. </p> <div class="mw-heading mw-heading2"><h2 id="Testing_for_and_setting_up_Köhler_illumination"><span id="Testing_for_and_setting_up_K.C3.B6hler_illumination"></span>Testing for and setting up Köhler illumination</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=K%C3%B6hler_illumination&action=edit&section=4" title="Edit section: Testing for and setting up Köhler illumination"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <p>Microscopes using Köhler illumination must be routinely checked for correct alignment. The realignment procedure tests whether the correct optical components are in focus at the two sets of conjugate image planes; the light source image planes and the specimen image planes. </p><p>Alignment of optical components on the specimen image plane is typically performed by first loading a test specimen and bringing it into focus by moving the objective or the specimen. The field diaphragm is then partially closed; the edges of the diaphragm should be in the same conjugate image planes as the specimen, therefore should appear in focus. The focus can be adjusted by raising or lowering the condenser lenses and diaphragm. Finally, the field diaphragm is reopened to just beyond the field of view. </p><p>In order to test the alignment of components on the light source image plane, the eyepiece must be removed to allow observation of the intermediate image plane (the position of the eyepiece diaphragm) either directly or by using a <a href="/wiki/Phase_telescope" title="Phase telescope">phase telescope</a>/<a href="/wiki/Bertrand_lens" class="mw-redirect" title="Bertrand lens">Bertrand lens</a>. The light source (e.g. the bulb filament) and the edges of the condenser diaphragm should appear in focus. Any optical components at the back focal plane of the objective (e.g. the phase ring for phase contrast microscopy) and at the condenser diaphragm (e.g. the annulus for phase contrast microscopy) should also appear in focus. </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=K%C3%B6hler_illumination&action=edit&section=5" title="Edit section: See also"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <ul><li><a href="/wiki/Nonimaging_optics#Köhler_integration" title="Nonimaging optics">Köhler integration</a></li> <li><a href="/wiki/Spatial_filter" title="Spatial filter">Spatial filtering</a>, the optical principle implemented in the Köhler illuminator's field diaphragm</li> <li><a href="/wiki/Polarized_light_microscopy" title="Polarized light microscopy">Polarized light microscopy</a></li> <li><a href="/wiki/Hoffman_modulation_contrast_microscopy" title="Hoffman modulation contrast microscopy">Hoffman modulation contrast microscopy</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=K%C3%B6hler_illumination&action=edit&section=6" title="Edit section: References"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <div class="mw-references-wrap"><ol class="references"> <li id="cite_note-wissenschaft_1893-1"><span class="mw-cite-backlink"><b><a href="#cite_ref-wissenschaft_1893_1-0">^</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="CITEREFKöhler1893" class="citation journal cs1">Köhler, August (1893). <a rel="nofollow" class="external text" href="https://archive.org/stream/zeitschriftfrw10stut">"Ein neues Beleuchtungsverfahren für mikrophotographische Zwecke"</a>. <i>Zeitschrift für wissenschaftliche Mikroskopie und für Mikroskopische Technik</i>. <b>10</b> (4): <span class="nowrap">433–</span>440.</cite><span title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.jtitle=Zeitschrift+f%C3%BCr+wissenschaftliche+Mikroskopie+und+f%C3%BCr+Mikroskopische+Technik&rft.atitle=Ein+neues+Beleuchtungsverfahren+f%C3%BCr+mikrophotographische+Zwecke&rft.volume=10&rft.issue=4&rft.pages=%3Cspan+class%3D%22nowrap%22%3E433-%3C%2Fspan%3E440&rft.date=1893&rft.aulast=K%C3%B6hler&rft.aufirst=August&rft_id=https%3A%2F%2Farchive.org%2Fstream%2Fzeitschriftfrw10stut&rfr_id=info%3Asid%2Fen.wikipedia.org%3AK%C3%B6hler+illumination" class="Z3988"></span></span> </li> <li id="cite_note-Royal_Society_1894-2"><span class="mw-cite-backlink"><b><a href="#cite_ref-Royal_Society_1894_2-0">^</a></b></span> <span class="reference-text"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1238218222"><cite id="CITEREFKoehler1894" class="citation journal cs1">Koehler, August (1894). <a rel="nofollow" class="external text" href="https://books.google.com/books?id=0O4BAAAAYAAJ&dq=journal%20of%20the%20royal%20microscopical%20society%201894&pg=PA261">"New Method of Illumination for Photomicrographical Purposes"</a>. <i>Journal of the Royal Microscopical Society</i>. <b>14</b>: <span class="nowrap">261–</span>262.</cite><span title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.jtitle=Journal+of+the+Royal+Microscopical+Society&rft.atitle=New+Method+of+Illumination+for+Photomicrographical+Purposes&rft.volume=14&rft.pages=%3Cspan+class%3D%22nowrap%22%3E261-%3C%2Fspan%3E262&rft.date=1894&rft.aulast=Koehler&rft.aufirst=August&rft_id=https%3A%2F%2Fbooks.google.com%2Fbooks%3Fid%3D0O4BAAAAYAAJ%26dq%3Djournal%2520of%2520the%2520royal%2520microscopical%2520society%25201894%26pg%3DPA261&rfr_id=info%3Asid%2Fen.wikipedia.org%3AK%C3%B6hler+illumination" class="Z3988"></span></span> </li> <li id="cite_note-IntroNio2e-3"><span class="mw-cite-backlink"><b><a href="#cite_ref-IntroNio2e_3-0">^</a></b></span> <span class="reference-text"><link rel="mw-deduplicated-inline-style" href="mw-data:TemplateStyles:r1238218222"><cite id="CITEREFChaves2015" class="citation book cs1">Chaves, Julio (2015). <a rel="nofollow" class="external text" href="https://books.google.com/books?id=e11ECgAAQBAJ"><i>Introduction to Nonimaging Optics, Second Edition</i></a>. <a href="/wiki/CRC_Press" title="CRC Press">CRC Press</a>. <a href="/wiki/ISBN_(identifier)" class="mw-redirect" title="ISBN (identifier)">ISBN</a> <a href="/wiki/Special:BookSources/978-1482206739" title="Special:BookSources/978-1482206739"><bdi>978-1482206739</bdi></a>.</cite><span title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Abook&rft.genre=book&rft.btitle=Introduction+to+Nonimaging+Optics%2C+Second+Edition&rft.pub=CRC+Press&rft.date=2015&rft.isbn=978-1482206739&rft.aulast=Chaves&rft.aufirst=Julio&rft_id=https%3A%2F%2Fbooks.google.com%2Fbooks%3Fid%3De11ECgAAQBAJ&rfr_id=info%3Asid%2Fen.wikipedia.org%3AK%C3%B6hler+illumination" class="Z3988"></span></span> </li> </ol></div> <div class="mw-heading mw-heading2"><h2 id="External_links">External links</h2><span class="mw-editsection"><span class="mw-editsection-bracket">[</span><a href="/w/index.php?title=K%C3%B6hler_illumination&action=edit&section=7" title="Edit section: External links"><span>edit</span></a><span class="mw-editsection-bracket">]</span></span></div> <ul><li><a rel="nofollow" class="external text" href="https://www.scientifica.uk.com/neurowire/koehler-illumination-what-why-and-how">A 6 step guide to Koehler Illumination by Scientifica</a></li> <li><a rel="nofollow" class="external text" href="http://microscopy.berkeley.edu/courses/TLM/condenser/kohler.html">Köhler illumination setup instructions</a></li></ul> <div class="navbox-styles"><style data-mw-deduplicate="TemplateStyles:r1129693374">.mw-parser-output .hlist dl,.mw-parser-output .hlist ol,.mw-parser-output .hlist ul{margin:0;padding:0}.mw-parser-output .hlist dd,.mw-parser-output .hlist dt,.mw-parser-output .hlist li{margin:0;display:inline}.mw-parser-output .hlist.inline,.mw-parser-output .hlist.inline dl,.mw-parser-output .hlist.inline ol,.mw-parser-output .hlist.inline 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style="padding:0 0.25em"> <ul><li><a href="/wiki/Bright-field_microscopy" title="Bright-field microscopy">Bright-field microscopy</a></li> <li><a class="mw-selflink selflink">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 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