Editing Optical microscope (section)

===Surpassing the resolution limit===
Multiple techniques are available for reaching resolutions higher than the transmitted light limit described above. Holographic techniques, as described by Courjon and Bulabois in 1979, are also capable of breaking this resolution limit, although resolution was restricted in their experimental analysis.<ref>{{cite journal|title=Real Time Holographic Microscopy Using a Peculiar Holographic Illuminating System and a Rotary Shearing Interferometer|author1 =Courjon, D. |author2 =Bulabois, J. |year=1979|volume=10|issue=3|pages=125 |journal=Journal of Optics|doi=10.1088/0150-536X/10/3/004|bibcode=1979JOpt...10..125C}}</ref>

Using fluorescent samples more techniques are available. Examples include [[Vertico SMI]], [[near field scanning optical microscopy]] which uses [[evanescent waves]], and [[STED microscope|stimulated emission depletion]]. In 2005, a microscope capable of detecting a single molecule was described as a teaching tool.<ref>{{cite web|title = Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope|url = http://chemeducator.org/bibs/0010004/1040269mk.htm|access-date = 25 February 2009|url-status = live|archive-url = https://web.archive.org/web/20090306183820/http://chemeducator.org/bibs/0010004/1040269mk.htm|archive-date = 6 March 2009|df = dmy-all}}</ref>

Despite significant progress in the last decade, techniques for surpassing the diffraction limit remain limited and specialized.{{cn|date=December 2024}}

While most techniques focus on increases in lateral resolution there are also some techniques which aim to allow analysis of extremely thin samples. For example, [[sarfus]] methods place the thin sample on a contrast-enhancing surface and thereby allows to directly visualize films as thin as 0.3 nanometers.{{cn|date=December 2024}}

On 8 October 2014, the [[Nobel Prize in Chemistry]] was awarded to [[Eric Betzig]], [[William Moerner]] and [[Stefan Hell]] for the development of super-resolved [[Fluorescence microscope|fluorescence microscopy]].<ref name="AP-20141008-KR">{{cite news |last1=Ritter |first1=Karl |last2=Rising |first2=Malin |title=2 Americans, 1 German win chemistry Nobel |url=http://apnews.excite.com/article/20141008/nobel-chemistry-e759dff699.html |date=8 October 2014 |work=[[AP News]] |access-date=8 October 2014 |url-status=live |archive-url=https://web.archive.org/web/20141011003419/http://apnews.excite.com/article/20141008/nobel-chemistry-e759dff699.html |archive-date=11 October 2014  }}</ref><ref name="NYT-20141008-KC">{{cite news |last=Chang |first=Kenneth |title=2 Americans and a German Are Awarded Nobel Prize in Chemistry |url=https://www.nytimes.com/2014/10/09/science/nobel-prize-chemistry.html |date=8 October 2014 |work=[[New York Times]] |access-date=8 October 2014 |url-status=live |archive-url=https://web.archive.org/web/20141009095518/http://www.nytimes.com/2014/10/09/science/nobel-prize-chemistry.html |archive-date=9 October 2014  }}</ref>

====Structured illumination SMI====
SMI (spatially modulated illumination microscopy) is a light optical process of the so-called [[point spread function]] (PSF) engineering. These are processes which modify the PSF of a [[microscope]] in a suitable manner to either increase the optical resolution, to maximize the precision of [[distance]] measurements of fluorescent objects that are small relative to the [[wavelength]] of the illuminating light, or to extract other structural parameters in the nanometer range.<ref>{{cite book|doi=10.1117/12.336833|title=Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating|year=1999|last1=Heintzmann|first1=Rainer|editor-first1=Irving J. |editor-first2=Herbert |editor-first3=Jan |editor-first4=Katarina |editor-first5=Pierre M. |editor-last1=Bigio |editor-last2=Schneckenburger |editor-last3=Slavik |editor-last4=Svanberg |editor-last5=Viallet |volume=3568|pages=185–196|series=Optical Biopsies and Microscopic Techniques III|s2cid=128763403 }}</ref><ref>Cremer, Christoph; Hausmann, Michael; Bradl, Joachim and Schneider, Bernhard "Wave field microscope with detection point spread function", {{US patent|7342717}}, priority date 10 July 1997</ref>

====Localization microscopy SPDMphymod====
[[File:3D Dual Color Super Resolution Microscopy Cremer 2010.png|thumb|600px|alt=3D Dual Color Super Resolution Microscopy Cremer from 2010|3D dual color super resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568 LIMON]]
SPDM (spectral precision distance microscopy), the basic localization microscopy technology is a light optical process of [[fluorescence microscopy]] which allows position, distance and angle measurements on "optically isolated" particles (e.g. molecules) well below the theoretical [[limit of resolution]] for light microscopy. "Optically isolated" means that at a given point in time, only a single particle/molecule within a region of a size determined by conventional optical resolution (typically approx. 200–250&nbsp;nm [[diameter]]) is being registered. This is possible when [[molecules]] within such a region all carry different spectral markers (e.g. different colors or other usable differences in the [[light emission]] of different particles).<ref>{{cite journal |doi=10.1007/s00340-008-3152-x|title=SPDM: light microscopy with single-molecule resolution at the nanoscale|year=2008|journal=Applied Physics B|volume=93|issue=1|pages=1–12|last1=Lemmer|first1=P.|last2=Gunkel|first2=M.|last3=Baddeley|first3=D.|last4=Kaufmann|first4=R.|last5=Urich|first5=A.|last6=Weiland|first6=Y.|last7=Reymann|first7=J.|last8=Müller|first8=P.|last9=Hausmann|first9=M.|last10=Cremer|first10=C.|bibcode=2008ApPhB..93....1L|s2cid=13805053 }}</ref><ref>{{cite book|doi=10.1117/12.260797|chapter=Comparative study of three-dimensional localization accuracy in conventional, confocal laser scanning and axial tomographic fluorescence light microscopy|year=1996|last1=Bradl|first1=Joachim|editor5-first=Pierre M|editor5-last=Viallet|editor4-first=Katarina|editor4-last=Svanberg|editor3-first=Herbert|editor3-last=Schneckenburger|editor2-first=Warren S|editor2-last=Grundfest|editor1-first=Irving J|editor1-last=Bigio|title=Optical Biopsies and Microscopic Techniques|volume=2926|pages=201–206|series=Optical Biopsies and Microscopic Techniques|s2cid=55468495 }}</ref><ref>{{cite journal|author1=Heintzmann, R.|author2=Münch, H.|author3=Cremer, C.|year=1997|title=High-precision measurements in epifluorescent microscopy – simulation and experiment|journal=Cell Vision|volume=4|pages=252–253|url=http://www.kip.uni-heidelberg.de/AG_Cremer/sites/default/files/Bilder/pdf_1997/CellVisionVol4No2Heintzmann.pdf|url-status=live|archive-url=https://web.archive.org/web/20160216030456/http://www.kip.uni-heidelberg.de/AG_Cremer/sites/default/files/Bilder/pdf_1997/CellVisionVol4No2Heintzmann.pdf|archive-date=16 February 2016}}</ref><ref>Cremer, Christoph; Hausmann, Michael; Bradl, Joachim and Rinke, Bernd "Method and devices for measuring distances between object structures", {{US patent|6424421}} priority date 23 December 1996</ref>

Many standard fluorescent dyes like [[Green fluorescent protein|GFP]], Alexa dyes, Atto dyes, Cy2/Cy3 and fluorescein molecules can be used for localization microscopy, provided certain photo-physical conditions are present. Using this so-called SPDMphymod (physically modifiable fluorophores) technology a single laser wavelength of suitable intensity is sufficient for nanoimaging.<ref>{{cite journal|author=Manuel Gunkel|pmid=19548231|year=2009|title=Dual color localization microscopy of cellular nanostructures|volume=4|issue=6|pages=927–38|doi=10.1002/biot.200900005|journal=Biotechnology Journal|s2cid=18162278 |display-authors=etal|url=https://hal.archives-ouvertes.fr/hal-00494027/file/PEER_stage2_10.1002%252Fbiot.200900005.pdf |archive-url=https://web.archive.org/web/20190503232308/https://hal.archives-ouvertes.fr/hal-00494027/file/PEER_stage2_10.1002%252Fbiot.200900005.pdf |archive-date=2019-05-03 |url-status=live}}</ref>

====3D super resolution microscopy====
3D super resolution microscopy with standard fluorescent dyes can be achieved by combination of localization microscopy for standard fluorescent dyes SPDMphymod and structured illumination SMI.<ref>{{cite journal|doi=10.1111/j.1365-2818.2010.03436.x|title=Analysis of Her2/neu membrane protein clusters in different types of breast cancer cells using localization microscopy|year=2011|journal=Journal of Microscopy|volume=242|pages=46–54|pmid=21118230|issue=1|display-authors=etal|last1=Kaufmann|first1=R|last2=Müller|first2=P|last3=Hildenbrand|first3=G|last4=Hausmann|first4=M|last5=Cremer|first5=C|citeseerx=10.1.1.665.3604|s2cid=2119158 }}</ref>

====STED====
[[File:MAX 052913 STED Phallloidin.png|thumb|right|300px|Stimulated emission depletion (STED) microscopy image of actin filaments within a cell]]
[[STED microscope|Stimulated emission depletion]] is a simple example of how higher resolution surpassing the diffraction limit is possible, but it has major limitations. STED is a fluorescence microscopy technique which uses a combination of light pulses to induce fluorescence in a small sub-population of fluorescent molecules in a sample. Each molecule produces a diffraction-limited spot of light in the image, and the centre of each of these spots corresponds to the location of the molecule. As the number of fluorescing molecules is low the spots of light are unlikely to overlap and therefore can be placed accurately. This process is then repeated many times to generate the image. [[Stefan Hell]] of the Max Planck Institute for Biophysical Chemistry was awarded the 10th German Future Prize in 2006 and Nobel Prize for Chemistry in 2014 for his development of the STED microscope and associated methodologies.<ref>{{cite web|url = http://www.heise.de/english/newsticker/news/81528|title = German Future Prize for crossing Abbe's Limit|access-date = 24 February 2009|url-status = live|archive-url = https://web.archive.org/web/20090307040808/http://www.heise.de/english/newsticker/news/81528|archive-date = 7 March 2009|df = dmy-all}}</ref>