View source for Second-harmonic imaging microscopy

{{Distinguish|two-photon excitation microscopy}}

'''Second-harmonic imaging microscopy''' ('''SHIM''') is based on a [[nonlinear]] optical effect known as [[second-harmonic generation]] (SHG).  SHIM has been established as a viable [[microscope]] imaging contrast mechanism for visualization of [[Cell (biology)|cell]] and [[Tissue (biology)|tissue]] structure and function.<ref>{{cite book|author=Juan Carlos Stockert, Alfonso Blázquez-Castro|chapter=Chapter 19 Non-Linear Optics|pages=642–686|title=Fluorescence Microscopy in Life Sciences|chapter-url=https://ebooks.benthamscience.com/book/9781681085180/|access-date=24 December 2017|year=2017|publisher=Bentham Science Publishers|isbn=978-1-68108-519-7}}</ref>  A second-harmonic microscope obtains contrasts from variations in a specimen's ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in [[optical density]], path length, or [[refractive index]] of the specimen.  SHG requires intense [[laser]] light passing through a material with a [[centrosymmetric|noncentrosymmetric]] molecular structure, either inherent or induced externally, for example by an electric field.<ref name="WaterPerspectiveRoke">{{Cite journal | last1=Roesel | first1=D. | last2=Eremchev | first2=M. | last3=Schönfeldová | first3=T. | last4=Lee | first4=S. | last5=Roke | first5=S. | title=Water as a contrast agent to quantify surface chemistry and physics using second harmonic scattering and imaging: A perspective | journal=Applied Physics Letters | publisher=AIP Publishing | volume=120 | issue=16 | date=2022-04-18 | issn=0003-6951 | doi=10.1063/5.0085807 | page=160501| bibcode=2022ApPhL.120p0501R | s2cid=248252664 | doi-access=free }}</ref>

[[Second-harmonic generation|Second-harmonic light]] emerging from an SHG material is exactly half the wavelength (frequency doubled) of the light entering the material.  While [[Two-photon excitation microscopy|two-photon-excited fluorescence]] (TPEF) is also a two photon process, TPEF loses some energy during the relaxation of the excited state, while SHG is energy conserving. Typically, an inorganic crystal is used to produce SHG light such as [[lithium niobate]] (LiNbO<sub>3</sub>), [[potassium titanyl phosphate]] (KTP = KTiOPO<sub>4</sub>), or [[lithium triborate]] (LBO = LiB<sub>3</sub>O<sub>5</sub>). Though SHG requires a material to have specific molecular orientation in order for the incident light to be frequency doubled, some biological materials can be highly polarizable, and assemble into fairly ordered, large noncentrosymmetric structures. While some biological materials such as collagen, [[microtubules]], and muscle [[myosin]]<ref name="NucciottiStringari2010">{{cite journal|last1=Nucciotti|first1=V.|last2=Stringari|first2=C.|last3=Sacconi|first3=L.|last4=Vanzi|first4=F.|last5=Fusi|first5=L.|last6=Linari|first6=M.|last7=Piazzesi|first7=G.|last8=Lombardi|first8=V.|last9=Pavone|first9=F. S.|title=Probing myosin structural conformation in vivo by second-harmonic generation microscopy|journal=Proceedings of the National Academy of Sciences|volume=107|issue=17|year=2010|pages=7763–7768|issn=0027-8424|doi=10.1073/pnas.0914782107|pmid=20385845|pmc=2867856|bibcode=2010PNAS..107.7763N|doi-access=free}}</ref> can produce SHG signals, even water can become ordered and produce second-harmonic signal under certain conditions, which allows SH microscopy to image surface potentials without any labeling molecules.<ref name="WaterPerspectiveRoke" /> The SHG pattern is mainly determined by the phase matching condition.  A common setup for an SHG imaging system will have a [[Laser scanning confocal microscopy|laser scanning microscope]] with a [[Ti-sapphire laser|titanium sapphire]] [[Modelocking|mode-locked]] laser as the excitation source.  The SHG signal is propagated in the forward direction.  However, some experiments have shown that objects on the order of about a tenth of the [[wavelength]] of the SHG produced signal will produce nearly equal forward and backward signals.

[[File:SHG collagen in liver.jpg|300px|right|thumb|Second-harmonic image of collagen (shown in white) in liver]]

==Advantages==

SHIM offers several advantages for live cell and tissue imaging. [[Second-harmonic generation|SHG]] does not involve the excitation of molecules like other techniques such as [[fluorescence microscopy]] therefore, the molecules shouldn't suffer the effects of [[phototoxicity]] or [[photobleaching]].  Also, since many biological structures produce strong SHG signals, the labeling of molecules with [[exogenous]] probes is not required which can also alter the way a biological system functions.  By using [[near infrared]] wavelengths for the incident light, SHIM has the ability to construct [[3D computer graphics|three-dimensional]] images of specimens by imaging deeper into thick tissues.

===Difference and complementarity with two-photon fluorescence (2PEF)===

Two-photons [[autofluorescence|fluorescence]] ([[Two-photon excitation microscopy|2PEF]]) is a very different process from [[Second-harmonic generation|SHG]]: it involves excitation of electrons to higher energy levels, and subsequent de-excitation by photon emission (unlike SHG, although it is also a 2-photon process). Thus, [[Two-photon excitation microscopy|2PEF]] is a non [[Coherence (physics)|coherent]] process, spatially (emitted isotropically) and temporally (broad, sample-dependent spectrum). It is also not specific to certain structure, unlike SHG.<ref name="chap2PEFSHGbook">{{Cite book|chapter= SHG Microscopy and Its Comparison with THG, CARS, and Multiphoton Excited Fluorescence Imaging |title= Second Harmonic Generation Imaging, 2nd edition|last1= Chen |first1= Xiyi |last2= Campagnola |first2= P.J.| year=2016|isbn=978-1-4398-4914-9|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=EiTOBQAAQBAJ&q=Second+Harmonic+Generation+Imaging+Pavone+Francesco}}</ref>

It can therefore be coupled to SHG in multiphoton imaging to reveal some molecules that do produce [[autofluorescence]], like [[elastin]] in tissues (while SHG reveals [[collagen]] or [[myosin]] for instance).<ref name="chap2PEFSHGbook"/>

==History==

Before SHG was used for imaging, the first demonstration of SHG was performed in 1961 by P. A. Franken, [[Gabriel Weinreich|G. Weinreich]], C. W. Peters, and A. E. Hill at the University of Michigan, Ann Arbor using a quartz sample.<ref>{{cite journal|last=Franken|first=Peter|author2=Weinreich, G |author3=Peters, CW |author4= Hill, AE |title=Generation of Optical Harmonics|journal=Physical Review Letters|year=1961|volume=7|issue=4|pages=118–119|doi=10.1103/PhysRevLett.7.118|bibcode = 1961PhRvL...7..118F |doi-access=free}}</ref>   In 1968, SHG from interfaces was discovered by Bloembergen <ref>{{cite journal|last=Bloembergen|first=N.|author2= Chang, R. K. |author3= Jha, S. S. |author4= Lee, C. H. |title=Optical Second-Harmonic Generation in Reflection from Media with Inversion Symmetry|journal=Physical Review|year=1968|volume=174|issue=813|pages=813–822|doi=10.1103/PhysRev.174.813|bibcode = 1968PhRv..174..813B}}</ref> and has since been used as a tool for characterizing surfaces and probing interface dynamics. In 1971, Fine and Hansen reported the first observation of SHG from biological tissue samples.<ref>{{cite journal|author=Fine, S.|author2=Hansen, W. P.|year=1971|title=Optical second harmonic generation in biological systems|journal=Applied Optics|volume=10|issue=10|pages=2350–2353|doi=10.1364/AO.10.002350|pmid=20111328|bibcode=1971ApOpt..10.2350F}}</ref>  In 1974, Hellwarth and Christensen first reported the integration of SHG and microscopy by imaging SHG signals from polycrystalline [[ZnSe]].<ref>{{cite journal|author=Hellwarth, Robert|author2=Christensen, Paul|title=Nonlinear optical microscopic examination of structure in polycrystalline ZnSe|journal=Optics Communications|volume=12|issue=3|year=1974|pages=318–322|doi=10.1016/0030-4018(74)90024-8|bibcode=1974OptCo..12..318H}}</ref>  In 1977, [[Colin Sheppard]] imaged various SHG crystals with a scanning optical microscope.  The first biological imaging experiments were done by Freund and Deutsch in 1986 to study the orientation of [[collagen]] fibers in [[rat]] tail [[tendon]].<ref name="freund">{{cite journal|author=Freund, I.|author2=Deutsch, M.|year=1986|title=Second-harmonic microscopy of biological tissue|journal=Optics Letters|volume=11|issue=2|pages=94–96|doi=10.1364/OL.11.000094|pmid=19730544|bibcode=1986OptL...11...94F}}</ref>  In 1993, Lewis examined the second-harmonic response of styryl [[dye]]s in [[electric field]]s. He also showed work on imaging live cells. In 2006, [[Goro Mizutani]] group developed a non-scanning SHG microscope that significantly shortens the time required for observation of large samples, even if the two-photons wide-field microscope was published in 1996 <ref>{{Cite journal|last1=Brakenhoff|first1=G.J.|last2=Sonoda|first2=Y.|last3=Squier|first3=J.|last4=Norris|first4=T.|last5=Bliton|first5=A.C.|last6=Wade|first6=M.H.|last7=Athey|first7=B.|date=1996|title=Real-time two-photon confocal microscopy using afemtosecond, amplified Tisapphire system|journal=Journal of Microscopy|volume=181|issue=3|pages=253–259|doi=10.1046/j.1365-2818.1996.97379.x|pmid=8642584|hdl=2027.42/71623|s2cid=12174100|hdl-access=free}}</ref> and could have been used to detect SHG. The non-scanning SHG microscope was used for observation of plant [[starch]],<ref>{{Cite journal|last1=Mizutani|first1=G.|last2=Sonoda|first2=Y.|last3=Sano|first3=H.|last4=Sakamoto|first4=M.|last5=Takahashi|first5=T.|last6=Ushioda|first6=S.|date=2000|title=Detection of starch granules in a living plant by optical second harmonic microscopy|journal=Journal of Luminescence|volume=87|pages=824–826|doi=10.1016/S0022-2313(99)00428-7|bibcode=2000JLum...87..824M}}</ref><ref>{{Cite journal|last1=Zhao|first1=Yue|last2=Takahashi|first2=Shogo|last3=Li|first3=Yanrong|last4=Hien|first4=K. T. T.|last5=Matsubara|first5=Akira|last6=Mizutani|first6=Goro|last7=Nakamura|first7=Yasunori|date=2018|title=Ungerminated Rice Grains Observed by Femtosecond Pulse Laser Second-Harmonic Generation Microscopy|url=https://pubs.acs.org/doi/10.1021/acs.jpcb.8b04610|journal=J. Phys. Chem. B|volume=122|issue=32|pages=7855–7861|arxiv=1808.05449|doi=10.7566/JPSJ.86.124401|pmid=30040415|s2cid=51687400}}</ref> megamolecule,<ref name="megamol">{{Cite journal|last1=Zhao|first1=Yue|last2=Hien|first2=Khuat Thi Thu|last3=Mizutani|first3=Goro|last4=Rutt|first4=Harvey N.|last5=Amornwachirabodee|first5=Kittima|last6=Okajima|first6=Maiko|last7=Kaneko|first7=Tatsuo|date=2017|title=Optical second-harmonic images of sacran megamolecule aggregates|journal=Journal of the Optical Society of America A|volume=34|issue=2|pages=146–152|arxiv=1702.07165|doi=10.1364/JOSAA.34.000146|pmid=28157840|bibcode=2017JOSAA..34..146Z|s2cid=4533122}}</ref> [[spider silk]]<ref name="spider">{{Cite journal|last1=Zhao|first1=Yue|last2=Hien|first2=Khuat Thi Thu|last3=Mizutani|first3=Goro|last4=Rutt|first4=Harvey N.|date=June 2017|title=Second-order nonlinear optical microscopy of spider silk|journal=Applied Physics B|volume=123|issue=6|pages=188|arxiv=1706.03186|doi=10.1007/s00340-017-6766-z|bibcode=2017ApPhB.123..188Z|s2cid=51684427}}</ref><ref>{{Cite journal|last1=Zhao|first1=Yue|last2=Li|first2=Yanrong|last3=Hien|first3=K. T. T.|last4=Mizutani|first4=Goro|last5=Rutt|first5=Harvey N.|year=2019|title=Observation of Spider Silk by Femtosecond Pulse Laser Second Harmonic Generation Microscopy|journal=Surf. Interface Anal.|volume=51|issue=1|pages=50–56|arxiv=1812.10390|doi=10.1002/sia.6545|s2cid=104921418}}</ref>  and so on. In 2010 SHG was extended to whole-animal [[in vivo]] imaging.<ref>{{Cite journal| last1 = Cohen | first1 = B. E.| title = Biological imaging: Beyond fluorescence| journal = Nature| volume = 467| issue = 7314| pages = 407–8| year = 2010| pmid = 20864989| doi = 10.1038/467407a|bibcode = 2010Natur.467..407C | s2cid = 205058963| doi-access = free}}</ref><ref>{{Cite journal| last1 = Pantazis | first1 = P.| last2 = Maloney | first2 = J.| last3 = Wu | first3 = D.| last4 = Fraser | first4 = S.| title = Second harmonic generating (SHG) nanoprobes for in vivo imaging| journal = Proceedings of the National Academy of Sciences of the United States of America| volume = 107| issue = 33| pages = 14535–14540| year = 2010| pmid = 20668245| pmc = 2930484| doi = 10.1073/pnas.1004748107|bibcode = 2010PNAS..10714535P | doi-access = free}}</ref> In 2019, SHG applications widened when it was applied to the use of selectively imaging agrochemicals directly on leaf surfaces to provide a way to evaluate the effectiveness of pesticides.<ref name="grubbs">{{Cite journal|last1=Grubbs|first1=Benjamin|last2=Etter|first2=Nicholas|last3=Slaughter|first3=Wesley|last4=Pittsford|first4=Alexander|last5=Smith|first5=Connor|last6=Schmitt|first6=Paul|date=August 2019|title=A Low-Cost Beam-Scanning Second Harmonic Generation Microscope with Application for Agrochemical Development and Testing|journal=Analytical Chemistry|volume = 91|issue = 18|pages = 11723–11730|doi = 10.1021/acs.analchem.9b02304|pmid=31424922|s2cid=201099822 }}</ref>

==Quantitative measurements==

===Orientational anisotropy===

SHG [[Polarization (waves)|polarization]] [[anisotropy]] can be used to determine the orientation and degree of organization of proteins in tissues since SHG signals have well-defined polarizations.  By using the anisotropy equation:<ref name="CampagnolaLoew2003">{{cite journal|last1=Campagnola|first1=Paul J|last2=Loew|first2=Leslie M|title=Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms|journal=Nature Biotechnology|volume=21|issue=11|year=2003|pages=1356–1360|issn=1087-0156|doi=10.1038/nbt894|pmid=14595363|s2cid=18701570}}</ref>

<math>\frac{I_{par}-I_{perp}}{I_{par}+2I_{perp}}=r</math>

and acquiring the intensities of the polarizations in the parallel and perpendicular directions.  A high <math>r</math> value indicates an anisotropic orientation whereas a low <math>r</math> value indicates an isotropic structure.  In work done by Campagnola and Loew,<ref name="CampagnolaLoew2003" /> it was found that collagen fibers formed well-aligned structures with an <math>r=0.7</math> value.

===Forward over backward SHG===
{{See also|Second-harmonic generation#Radiation pattern}}

[[Second-harmonic generation|SHG]] being a [[Coherence (physics)|coherent]] process ([[Coherence (physics)#Spatial coherence|spatially]] and [[Coherence (physics)#Temporal coherence|temporally]]), it keeps information on the direction of the excitation and is not emitted isotropically. It is mainly emitted in forward direction (same as excitation), but can also be emitted in backward direction depending on the [[Second-harmonic generation#Critical phase-matching|phase-matching condition]]. Indeed, the coherence length beyond which the conversion of the signal decreases is:

<math>l_c = 2/\Delta k </math>

with <math>\Delta k \propto 1/(n_{2\omega}-n_{\omega}) </math> for forward, but <math>\Delta k_{bwd} \propto 1/(n_{2\omega}+n_{\omega}) </math> for backward such that <math>l_c</math> >> <math>l_{c,bwd}</math>. Therefore, thicker structures will appear preferentially in forward, and thinner ones in backward: since the SHG conversion depends at first approximation on the square of the number of nonlinear converters, the signal will be higher if emitted by thick structures, thus the signal in forward direction will be higher than in backward. However, the tissue can scatter the generated light, and a part of the SHG in forward can be retro-reflected in the backward direction.<ref name="ChenNadiarynkh2012">{{cite journal|last1=Chen|first1=Xiyi|last2=Nadiarynkh|first2=Oleg|last3=Plotnikov|first3=Sergey|last4=Campagnola|first4=Paul J|title=Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure|journal=Nature Protocols|volume=7|issue=4|year=2012|pages=654–669|issn=1754-2189|doi=10.1038/nprot.2012.009|pmid=22402635|pmc=4337962}}</ref>
Then, the forward-over-backward ratio F/B can be calculated,<ref name="ChenNadiarynkh2012" /> and is a metric of the global size and arrangement of the SHG converters (usually collagen fibrils). It can also be shown that the higher the out-of-plane angle of the scatterer, the higher its F/B ratio (see fig. 2.14 of <ref name="chapSHGbook">{{Cite book|title= "How to Build an SHG Apparatus" in Second Harmonic Generation Imaging, 2nd edition|last1= Cicchi|first1= Riccardo |last2= Sacconi|first2= Leonardo |last3= Vanzi|first3= Francesco|last4= Pavone|first4= Francesco S. | year=2016|isbn=978-1-4398-4914-9|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=EiTOBQAAQBAJ&q=Second+Harmonic+Generation+Imaging+Pavone+Francesco}}</ref>).

===Polarization-resolved SHG===

The advantages of [[polarimetry]] were coupled to SHG in 2002 by Stoller et al.<ref>{{Cite journal | last1 = 	Stoller| first1 = P. | last2 = Reiser | first2 = K. | last3 = Celliers | first3 = P. | last4 = Rubenchik | first4 = A. | year = 2002 | title = Polarization-modulated second harmonic generation in collagen | journal = Biophys. J.| volume = 82 | issue = 6 | pages = 3330–3342   | doi = 10.1016/S0006-3495(02)75673-7 | pmid = 12023255 | pmc = 1302120 | bibcode = 2002BpJ....82.3330S }}</ref> Polarimetry can measure the orientation and order at molecular level, and coupled to SHG it can do so with the specificity to certain structures like collagen: polarization-resolved SHG microscopy (p-SHG) is thus an expansion of SHG microscopy.<ref name="DuboissetAït-Belkacem2012">{{cite journal|last1=Duboisset|first1=Julien|last2=Aït-Belkacem|first2=Dora|last3=Roche|first3=Muriel|last4=Rigneault|first4=Hervé|last5=Brasselet|first5=Sophie|author5-link=Sophie Brasselet|title=Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation|journal=Physical Review A|volume=85|issue=4|year=2012|pages=043829|issn=1050-2947|doi=10.1103/PhysRevA.85.043829|bibcode=2012PhRvA..85d3829D|s2cid=85559569 |url=https://hal.archives-ouvertes.fr/hal-00690339/file/Duboisset_PRA.pdf}}</ref>
p-SHG defines another anisotropy parameter, as:<ref name="TeulonGusachenko2015">{{cite journal|last1=Teulon|first1=Claire|last2=Gusachenko|first2=Ivan|last3=Latour|first3=Gaël|last4=Schanne-Klein|first4=Marie-Claire|authorlink4=Marie-Claire Schanne-Klein|title=Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy|journal=Optics Express|volume=23|issue=7|year=2015|pages=9313–28|issn=1094-4087|doi=10.1364/OE.23.009313|pmid=25968762|bibcode=2015OExpr..23.9313T|url=https://hal-polytechnique.archives-ouvertes.fr/hal-01173949/file/oe-23-7-9313.pdf|doi-access=free}}</ref>

<math>\rho = \sqrt{\frac{I_{par}}{I_{perp}}} </math>

which is, like ''r'', a measure of the principal orientation and disorder of the structure being imaged. Since it is often performed in long cylindrical filaments (like collagen), this anisotropy is often equal to <math>\rho = \frac{\chi^{(2)}_{XXX}}{\chi^{(2)}_{XYY}} </math>
,<ref name="GusachenkoTran2012">{{cite journal|last1=Gusachenko|first1=Ivan|last2=Tran|first2=Viet|last3=Houssen|first3=Yannick Goulam|last4=Allain|first4=Jean-Marc|last5=Schanne-Klein|first5=Marie-Claire|title=Polarization-Resolved Second-Harmonic Generation in Tendon upon Mechanical Stretching|journal=Biophysical Journal|volume=102|issue=9|year=2012|pages=2220–2229|issn=0006-3495|doi=10.1016/j.bpj.2012.03.068|pmid=22824287|pmc=3341536|bibcode=2012BpJ...102.2220G}}</ref> where <math>\chi^{(2)}</math> is the [[Electric susceptibility#Nonlinear susceptibility|nonlinear susceptibility tensor]] and X the direction of the filament (or main direction of the structure), Y orthogonal to X and Z the propagation of the excitation light.
The orientation ''ϕ'' of the filaments in the plane XY of the image can also be extracted from p-SHG by [[Fast Fourier transform|FFT analysis]], and put in a map.<ref name="GusachenkoTran2012" /><ref name="MazumderDeka2017">{{cite journal|last1=Mazumder|first1=Nirmal|last2=Deka|first2=Gitanjal|last3=Wu|first3=Wei-Wen|last4=Gogoi|first4=Ankur|last5=Zhuo|first5=Guan-Yu|last6=Kao|first6=Fu-Jen|title=Polarization resolved second harmonic microscopy|journal=Methods|volume=128|year=2017|pages=105–118|issn=1046-2023|doi=10.1016/j.ymeth.2017.06.012|pmid=28624539}}</ref>

===Fibrosis quantization===

[[Collagen]] (particular case, but widely studied in SHG microscopy), can exist in various forms : 28 different types, of which 5 are fibrillar. One of the challenge is to determine and quantify the amount of fibrillar collagen in a tissue, to be able to see its evolution and relationship with other non-collagenous materials.<ref name="chapSHGbookMCSK">{{Cite book|title= "SHG Imaging of Collagen and Application to Fibrosis Quantization" in Second Harmonic Generation Imaging, 2nd edition|author1= Marie-Claire Schanne-Klein |year=2016|isbn=978-1-4398-4914-9|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=EiTOBQAAQBAJ&q=Second+Harmonic+Generation+Imaging+Pavone+Francesco}}</ref>

To that end, a SHG microscopy image has to be corrected to remove the small amount of residual fluorescence or noise that exist at the SHG wavelength. After that, a [[Mask (computing)|mask]] can be applied to quantify the collagen inside the image.<ref name="chapSHGbookMCSK" /> Among other quantization techniques, it is probably the one with the highest specificity, reproductibility and applicability despite being quite complex.<ref name="chapSHGbookMCSK" />

===Others===

It has also been used to prove that backpropagating action potentials invade dendritic spines without voltage attenuation, establishing a sound basis for future work on [[Long-term potentiation]]. Its use here was that it provided a way to accurately measure the voltage in the tiny dendritic spines with an accuracy unattainable with standard two-photon microscopy.<ref>
{{Cite journal | last1 = Nuriya | first1 = Mutsuo | last2 = Jiang | first2 = Jiang | last3 = Nemet | first3 = Boaz | last4 = Eisenthal | first4 = Kenneth B. | last5= Yuste | first5 = Rafael | year = 2006 | title = Imaging membrane potential in dendritic spines | journal = [[Proceedings of the National Academy of Sciences|PNAS]] | volume = 103 | issue = 3 | pages = 786–790 | doi =10.1073/pnas.0510092103 | pmid = 16407122 | bibcode = 2006PNAS..103..786N | pmc =1334676 | doi-access = free }}
</ref> Meanwhile, SHG can efficiently convert near-infrared light to visible light to enable imaging-guided photodynamic therapy, overcoming the penetration depth limitations.<ref>{{cite journal |last1=Gu |first1=Bobo |last2=Pliss |first2=Artem |last3=Kuzmin |first3=Andrey N. |title=In-situ second harmonic generation by cancer cell targeting ZnO nanocrystals to effect photodynamic action in subcellular space |journal=Biomaterials |volume=104 |pages=78–86 |doi=10.1016/j.biomaterials.2016.07.012 |pmid=27442221 |year=2016 |doi-access=free }}</ref>

==Materials that can be imaged==

[[File:Cornea muscle-ad tendon mice cart-horse-surf.png|thumb|Biological tissues imaged by second-harmonic generation (SHG) microscopy. (a) Transverse cut of a human cornea. (b) Skeletal muscle from zebrafish (myosin). (c) Adult mice-tail tendon. (d) Surface cartilage from a knee of a mature horse.]]
SHG microscopy and its expansions can be used to study various tissues: some example images are reported in the figure below: collagen inside the extracellular matrix remains the main application. It can be found in tendon, skin, bone, cornea, aorta, fascia, cartilage, meniscus, intervertebral disks...

Myosin can also be imaged in skeletal muscle or cardiac muscle.

{| class="wikitable"
|+Table 1: Materials visible by or that efficiently generate SHG.
|-
! Type !! Material !! Found in !! SHG signal !! Specificity
|-
| valign="middle" rowspan=3| Carbohydrate || [[Cellulose]] || [[Wood]], [[green plant]], [[algae]]. || Quite weak in normal cellulose,<ref name="grubbs"/> but substantial in crystalline or [[Nanocellulose|nanocrystalline]] cellulose. || -
|-
| [[Starch]] ||  [[Staple foods]], [[green plant]] || Quite intense signal <ref name="PsilodimitrakopoulosAmat-Roldan2010">{{cite journal|last1=Psilodimitrakopoulos|first1=Sotiris|last2=Amat-Roldan|first2=Ivan|last3=Loza-Alvarez|first3=Pablo|last4=Artigas|first4=David|title=Estimating the helical pitch angle of amylopectin in starch using polarization second harmonic generation microscopy|journal=Journal of Optics|volume=12|issue=8|year=2010|pages=084007|issn=2040-8978|doi=10.1088/2040-8978/12/8/084007|bibcode=2010JOpt...12h4007P|hdl=2117/10342|s2cid=120603827 |hdl-access=free}}</ref> || chirality is at micro and macro level, and the SHG is different under right or left-handed [[Circular polarization#Handedness conventions|circular polarization]]
|-
| Megamolecular [[polysaccharide]] sacran || [[Cyanobactery]] || From sacran cotton-like lump, fibers, and cast films || signal from films is weaker <ref name="megamol" />
|-
|valign="middle" rowspan=4| Protein || [[Fibroin]] and [[sericin]] || Spider silk || Quite weak || <ref name="spider"/>
|-
| [[Collagen]]<ref name="freund"/> || [[tendon]], [[skin]], [[bone]], [[cornea]], [[aorta]], [[fascia]], [[cartilage]], [[meniscus (anatomy)|meniscus]], [[intervertebral disks]] ; connective tissues || Quite strong, depends on the type of the collagen (does it form fibrils, fibers ?) || [[Electric susceptibility#Nonlinear susceptibility|nonlinear susceptibility]] tensor components are <math>d_{33}</math>, <math>d_{31}</math>, <math>d_{15}</math>, with <math>d_{31}</math> ~ <math>d_{15}</math> and <math>d_{33}</math>/<math>d_{15}</math> ~ 1.4 in most cases
|-
| [[Myosin]] || Skeletal or cardiac [[muscle]]<ref name="NucciottiStringari2010"/> || Quite strong || [[Electric susceptibility#Nonlinear susceptibility|nonlinear susceptibility]] tensor components are <math>d_{33}</math>, <math>d_{31}</math>, <math>d_{15}</math> with <math>d_{31}</math> ~ <math>d_{15}</math> but <math>d_{33}</math>/<math>d_{15}</math> ~ 0.6 < 1 contrary to collagen
|-
|  [[Tubulin]] || [[Microtubules]] in [[mitosis]] or [[meiosis]],<ref name="SHGmicbook">{{Cite book|title= Second Harmonic Generation Imaging, 2nd edition|last1= Pavone|first1= Francesco S. |last2= Campagnola|first2= P.J. | year=2016|isbn=978-1-4398-4914-9|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=EiTOBQAAQBAJ&q=Second+Harmonic+Generation+Imaging+Pavone+Francesco}}</ref> or in [[neurite]]s (mainly axons)<ref name="Van SteenbergenBoesmans2019">{{cite journal|last1=Van Steenbergen|first1=V.|last2=Boesmans|first2=W.|last3=Li|first3=Z.|last4=de Coene|first4=Y.|last5=Vints|first5=K.|last6=Baatsen|first6=P.|last7=Dewachter|first7=I.|last8=Ameloot|first8=M.|last9=Clays|first9=K.|last10=Vanden Berghe|first10=P.|title=Molecular understanding of label-free second harmonic imaging of microtubules|journal=Nature Communications|volume=10|issue=1|year=2019|pages=3530|issn=2041-1723|doi=10.1038/s41467-019-11463-8|pmid=31387998|pmc=6684603|bibcode=2019NatCo..10.3530V}}</ref> || Quite weak || The microtubules have to be aligned to efficiently generate
|-
| Minerals || [[Piezoelectric]] crystals || Also called nonlinear [[crystals]] || Strong if [[Nonlinear optics#Phase matching|phase-matched]] || Different [[Second-harmonic generation#Types in crystals|types]] of phase-matching, critical of non-critical
|-
| Polar liquids || [[Water]] || Most living organisms || Barely detectable (requires wide-field geometry and ultra-short laser pulses <ref name="RoeselEremchevSchönfeldová2022">{{cite journal | last1 = Roesel | first1 = D. | last2 = Eremchev | first2 = M. | last3 = Schönfeldová | first3 = T. | last4 = Lee | first4 = S. | last5 = Roke | first5 = S. | title = Water as a contrast agent to quantify surface chemistry and physics using second harmonic scattering and imaging: A perspective | journal = Applied Physics Letters | date = 18 April 2022 | volume = 120 | issue = 16 | page = 160501 | issn = 0003-6951 | eissn = 1077-3118 | doi = 10.1063/5.0085807 | pmid = | bibcode = 2022ApPhL.120p0501R | s2cid = 248252664 | url = | doi-access = free }}</ref>) || Directly probing electrostatic fields, since oriented [[water]] molecules satisfy [[Nonlinear optics#Phase matching|phase-matching]] condition <ref name="RoeselEremchevPoojari2022">{{cite journal | last1 = Roesel | first1 = David | last2 = Eremchev | first2 = Maksim | last3 = Poojari | first3 = Chetan S. | last4 = Hub | first4 = Jochen S. | last5 = Roke | first5 = Sylvie | title = Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes | journal = Journal of the American Chemical Society | date = 15 December 2022 | volume = 144 | issue = 51 | pages = 23352–23357 | issn = 0002-7863 | eissn = 1520-5126 | doi = 10.1021/jacs.2c08543 | pmid = 36521841 | pmc = 9801421 | url = }}</ref>
|}

==Coupling with THG microscopy==

[[Third-harmonic generation|Third-Harmonic Generation]] (THG) microscopy can be complementary to SHG microscopy, as it is sensitive to the transverse interfaces, and to the 3rd order nonlinear susceptibility <math>\chi^{(3)}</math> <ref name="BaradEisenberg1997">{{cite journal|last1=Barad|first1=Y.|last2=Eisenberg|first2=H.|last3=Horowitz|first3=M.|last4=Silberberg|first4=Y.|title=Nonlinear scanning laser microscopy by third harmonic generation|journal=Applied Physics Letters|volume=70|issue=8|year=1997|pages=922–924|issn=0003-6951|doi=10.1063/1.118442|bibcode=1997ApPhL..70..922B}}</ref><ref name="OlivierLuengo-Oroz2010">{{cite journal|last1=Olivier|first1=N.|last2=Luengo-Oroz|first2=M. A.|last3=Duloquin|first3=L.|last4=Faure|first4=E.|last5=Savy|first5=T.|last6=Veilleux|first6=I.|last7=Solinas|first7=X.|last8=Debarre|first8=D.|last9=Bourgine|first9=P.|last10=Santos|first10=A.|last11=Peyrieras|first11=N.|last12=Beaurepaire|first12=E.|title=Cell Lineage Reconstruction of Early Zebrafish Embryos Using Label-Free Nonlinear Microscopy|journal=Science|volume=329|issue=5994|year=2010|pages=967–971|issn=0036-8075|doi=10.1126/science.1189428|pmid=20724640|bibcode=2010Sci...329..967O|s2cid=6971291|url=https://hal.archives-ouvertes.fr/hal-00519834/file/Olivier2010.pdf }}</ref>

==Applications==

===Cancer progression, tumor characterization===

The [[mammographic]] density is correlated with the [[collagen]] density, thus SHG can be used for identifying [[breast cancer]].<ref name="AlowamiTroup2003">{{cite journal|last1=Alowami|first1=Salem|last2=Troup|first2=Sandra|last3=Al-Haddad|first3=Sahar|last4=Kirkpatrick|first4=Iain|last5=Watson|first5=Peter H|title=Mammographic density is related to stroma and stromal proteoglycan expression|journal=Breast Cancer Research|volume=5|issue=5|year=2003|pages=R129-35|issn=1465-542X|doi=10.1186/bcr622|pmid=12927043|pmc=314426 |doi-access=free }}</ref> SHG is usually coupled to other nonlinear techniques such as [[Coherent anti-Stokes Raman spectroscopy|Coherent anti-Stokes Raman Scattering]] or [[Two-photon excitation microscopy]], as part of a routine called multiphoton microscopy (or tomography) that provides a non-invasive and rapid in vivo [[histology]] of [[biopsies]] that may be cancerous.<ref name="MPTbook">{{Cite book|title= "Multiphoton Tomography (MPT)" Chap.13 in Multiphoton Microscopy and Fluorescence Lifetime Imaging - Applications in Biology and Medicine|last1= König|first1= Karsten | year=2018|isbn=978-3-11-042998-5|publisher= De Gruyter|url=https://www.degruyter.com/view/product/458320}}</ref>

====Breast cancer====

The comparison of forward and backward SHG images gives insight about the microstructure of collagen, itself related to the grade and stage of a [[tumor]], and its progression in [[breast]].<ref name="KeikhosraviBredfeldt2014">{{cite book|last1=Keikhosravi|first1=Adib|last2=Bredfeldt|first2=Jeremy S.|last3=Sagar|first3=Abdul Kader|last4=Eliceiri|first4=Kevin W.|chapter=Second-harmonic generation imaging of cancer |title=Quantitative Imaging in Cell Biology|series=Methods in Cell Biology|volume=123|year=2014|pages=531–546|issn=0091-679X|doi=10.1016/B978-0-12-420138-5.00028-8|pmid=24974046|isbn=978-0-12-420138-5 |chapter-url=https://www.sciencedirect.com/science/article/pii/B9780124201385000288}}</ref> Comparison of SHG and [[Two-photon excitation microscopy|2PEF]] can also show the change of [[collagen]] orientation in [[tumors]].<ref name="bmc">{{cite journal|last1=Provenzano|first1=Paolo P|last2=Eliceiri|first2=Kevin W|last3=Campbell|first3=Jay M|last4=Inman|first4=David R|last5=White|first5=John G|last6=Keely|first6=Patricia J|title=Collagen reorganization at the tumor-stromal interface facilitates local invasion |journal=BMC Medicine|volume=4|issue=38|year=2006|pages=38|doi=10.1186/1741-7015-4-38|pmid=17190588|pmc=1781458 |doi-access=free }}</ref>
Even if SHG microscopy has contributed a lot to breast cancer research, it is not yet established as a reliable technique in [[hospitals]], or for diagnostic of this [[pathology]] in general.<ref name="KeikhosraviBredfeldt2014" />

====Ovarian cancer====

Healthy ovaries present in SHG a uniform [[epithelial]] layer and well-organized collagen in their [[stroma of ovary|stroma]], whereas abnormal ones  show an epithelium with large cells and a changed collagen structure.<ref name="KeikhosraviBredfeldt2014" /> The r ratio {{Crossreference|selfref=no|(see [[#Orientational anisotropy]])}} is also used <ref name="NadiarnykhLaComb2010">{{cite journal|last1=Nadiarnykh|first1=Oleg|last2=LaComb|first2=Ronald B|last3=Brewer|first3=Molly A|last4=Campagnola|first4=Paul J|title=Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy|journal=BMC Cancer|volume=10|issue=1|year=2010|pages=94|issn=1471-2407|doi=10.1186/1471-2407-10-94|pmid=20222963|pmc=2841668 |doi-access=free }}</ref> to show that the alignment of fibrils is slightly higher for cancerous than for normal tissues.

====Skin cancer====

SHG is, again, combined to [[Two-photon excitation microscopy|2PEF]] is used to calculate the ratio:

<math> MFSI=(\text{shg}-\text{tpef})/(\text{shg}+\text{tpef}) </math>

where shg (resp. tpef) is the number of thresholded pixels in the SHG (resp. 2PEF) image,<ref name="LinJee2006">{{cite journal|last1=Lin|first1=Sung-Jan|last2=Jee|first2=Shiou-Hwa|last3=Kuo|first3=Chien-Jui|last4=Wu|first4=Ruei-Jr|last5=Lin|first5=Wei-Chou|last6=Chen|first6=Jau-Shiuh|last7=Liao|first7=Yi-Hua|last8=Hsu|first8=Chih-Jung|last9=Tsai|first9=Tsen-Fang|last10=Chen|first10=Yang-Fang|last11=Dong|first11=Chen-Yuan|title=Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging|journal=Optics Letters|volume=31|issue=18|year=2006|pages=2756–8|issn=0146-9592|doi=10.1364/OL.31.002756|pmid=16936882|bibcode=2006OptL...31.2756L|url=http://ntur.lib.ntu.edu.tw//handle/246246/132819 }}</ref> a high MFSI meaning a pure SHG image (with no fluorescence). The highest MFSI is found in cancerous tissues,<ref name="KeikhosraviBredfeldt2014" /> which provides a contrast mode to differentiate from normal tissues.

SHG was also combined to [[third-harmonic generation|Third-Harmonic Generation]] (THG) to show that backward {{Crossreference|selfref=no|(see [[#Forward over backward SHG]])}} THG is higher in tumors.<ref name="Chen">{{cite journal|last1=Chen|first1=Szu-Yu |last2= Chen|first2=Shee-Uan| last3=Wu| first3=Hai-Yin |last4=Lee|first4=Wen-Jeng |last5=Liao |first5=Yi-Hua |last6=Sun |first6=Chi-Kuang|title= In Vivo Virtual Biopsy of Human Skin by Using Noninvasive Higher Harmonic Generation Microscopy |journal= IEEE Journal of Selected Topics in Quantum Electronics|volume=16|issue=3|year=2009|pages=478–492 |doi=10.1109/JSTQE.2009.2031987|s2cid=21644641 |url=http://ntur.lib.ntu.edu.tw/bitstream/246246/237659/-1/09.pdf }}</ref>

====Pancreatic cancer====

Changes in collagen ultrastructure in [[pancreatic]] cancer can be investigated by multiphoton fluorescence and polarization-resolved SHIM.<ref name="TokarzCisek2019">{{cite journal|last1=Tokarz|first1=Danielle|last2=Cisek|first2=Richard|last3=Joseph|first3=Ariana|last4=Golaraei|first4=Ahmad|last5=Mirsanaye|first5=Kamdin|last6=Krouglov|first6=Serguei|last7=Asa|first7=Sylvia L.|last8=Wilson|first8=Brian C.|last9=Barzda|first9=Virginijus|title=Characterization of Pancreatic Cancer Tissue Using Multiphoton Excitation Fluorescence and Polarization-Sensitive Harmonic Generation Microscopy|journal=Frontiers in Oncology|volume=9|year=2019|pages=272|issn=2234-943X|doi=10.3389/fonc.2019.00272|pmid=31058080|pmc=6478795|doi-access=free}}</ref>

====Other cancers====

SHG microscopy was reported for the study of [[lung]], [[colon (organ)|colon]]ic, esophageal [[stroma (tissue)|stroma]] and [[cervix|cervical]] cancers.<ref name="KeikhosraviBredfeldt2014" />

===Pathologies detection===

Alterations in the organization or polarity of the [[collagen]] fibrils can be signs of pathology,.<ref name="konig">{{cite book|title= Multiphoton Microscopy and Fluorescence Lifetime Imaging - Applications in Biology and Medicine| last1= König|first1= Karsten | year=2018|isbn=978-3-11-042998-5|publisher= De Gruyter|url=https://www.degruyter.com/view/product/458320}}</ref><ref name="Cicchi2014">{{cite journal|last1=Cicchi|first1=Riccardo|title=The New Digital Pathology: Just Say NLO|journal=Digestive Diseases and Sciences|volume=59|issue=7|year=2014|pages=1347–1348|issn=0163-2116|doi=10.1007/s10620-014-3165-8|pmid=24817337|doi-access=free}}</ref>

In particular, the anisotropic alignment of [[collagen]] fibers allowed the discrimination of healthy [[dermis]] from pathological scars in [[skin]].<ref name="CicchiVogler2013">{{cite journal|last1=Cicchi|first1=Riccardo|last2=Vogler|first2=Nadine|last3=Kapsokalyvas|first3=Dimitrios|last4=Dietzek|first4=Benjamin|last5=Popp|first5=Jürgen|last6=Pavone|first6=Francesco Saverio|title=From molecular structure to tissue architecture: collagen organization probed by SHG microscopy|journal=Journal of Biophotonics|volume=6|issue=2|year=2013|pages=129–142|issn=1864-063X|doi=10.1002/jbio.201200092|pmid=22791562|doi-access=free}}{{open access}}</ref> Also, pathologies in [[cartilage]] such as [[osteoarthritis]] can be probed by polarization-resolved SHG microscopy,.<ref name="MansfieldWinlove2008">{{cite journal|last1=Mansfield|first1=Jessica C.|last2=Winlove|first2=C. Peter|last3=Moger|first3=Julian|last4=Matcher|first4=Steve J.|title=Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy|journal=Journal of Biomedical Optics|volume=13|issue=4|year=2008|pages=044020|issn=1083-3668|doi=10.1117/1.2950318|pmid=19021348|bibcode=2008JBO....13d4020M|hdl=10036/4485|s2cid=25096045 |hdl-access=free}}{{open access}}</ref><ref name="YehHammer-Wilson2005">{{cite journal|last1=Yeh|first1=Alvin T.|last2=Hammer-Wilson|first2=Marie J.|last3=Van Sickle|first3=David C.|last4=Benton|first4=Hilary P.|last5=Zoumi|first5=Aikaterini|last6=Tromberg|first6=Bruce J.|last7=Peavy|first7=George M.|title=Nonlinear optical microscopy of articular cartilage|journal=Osteoarthritis and Cartilage|volume=13|issue=4|year=2005|pages=345–352|issn=1063-4584|doi=10.1016/j.joca.2004.12.007|pmid=15780648|s2cid=20052077 |url=http://www.escholarship.org/uc/item/69w5n96p|doi-access=free}}{{open access}}</ref> SHIM was later extended to fibro-cartilage ([[Meniscus (anatomy)|meniscus]]).<ref name="HanHeo2016">{{cite journal|last1=Han|first1=Woojin M.|last2=Heo|first2=Su-Jin|last3=Driscoll|first3=Tristan P.|last4=Delucca|first4=John F.|last5=McLeod|first5=Claire M.|last6=Smith|first6=Lachlan J.|last7=Duncan|first7=Randall L.|last8=Mauck|first8=Robert L.|last9=Elliott|first9=Dawn M.|author9-link=Dawn Elliott|title=Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage|journal=Nature Materials|volume=15|issue=4|year=2016|pages=477–484|issn=1476-1122|doi=10.1038/nmat4520|pmid=26726994|pmc=4805445|bibcode=2016NatMa..15..477H}}</ref>

===Tissue engineering===

The ability of [[second-harmonic generation|SHG]] to image specific molecules can reveal the structure of a certain tissue one material at a time, and at various scales (from macro to micro) using microscopy. For instance, the [[collagen]] (type I) is specifically imaged from the [[extracellular matrix]] (ECM) of cells, or when it serves as a scaffold or conjonctive material in tissues.<ref name="chapEnginSHGbook">{{Cite book|chapter= SHG Imaging for Tissue Engineering Applications| title = Second Harmonic Generation Imaging, 2nd edition|last1= Chen|first1= W.L. |last2= Lee |first2= H.S. | year=2016|isbn=978-1-4398-4914-9|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=EiTOBQAAQBAJ&q=Second+Harmonic+Generation+Imaging+Pavone+Francesco}}</ref> SHG also reveals fibroin in [[silk]], [[myosin]] in [[muscles]] and biosynthetized [[cellulose]].
All of this imaging capability can be used to design artificials tissues, by targeting specific points of the tissue : SHG can indeed quantitatively measure some orientations, and material quantity and arrangement.<ref name="chapEnginSHGbook"/> Also, SHG coupled to other multiphoton techniques can serve to monitor the development of engineered tissues, when the sample is relatively thin however.<ref name="chapEnginbook">{{Cite book|chapter= Use of Multiphoton Microscopy for Tissue Engineering Applications| title = Imaging in Cellular and Tissue Engineering, 1st edition|last1= Enejder|first1= A. |last2= Brackmann |first2= C. | year=2020|isbn=9780367445867|publisher= CRC Taylor&Francis|chapter-url=https://books.google.com/books?id=eHTRBQAAQBAJ}}</ref> Of course, they can finally be used as a quality control of the fabricated tissues.<ref name="chapEnginbook"/>

===Structure of the eye===

[[Cornea]], at the surface of the [[eye]], is considered to be made of plywood-like structure of [[collagen]], due to the self-organization properties of sufficiently dense [[collagen]].<ref name="cornea">{{Cite book|title = Cornea, Fundamentals, Diagnosis and Management. 2nd edition |last1= Krachmer |first1= J.H. |last2= Mannis |first2= M.J.|last3= Holland |first3= E.J. | year=2005|isbn= 0323023150|publisher= Elsevier Mosby|url=http://scielo.isciii.es/scielo.php?script=sci_arttext&pid=S0365-66912005000600014}}</ref> Yet, the collagenous orientation in lamellae is still under debate in this [[Tissue (biology)|tissue]].<ref name="BuenoÁvila2019">{{cite journal|last1=Bueno|first1=Juan M.|last2=Ávila|first2=Francisco J.|last3=Martínez-García|first3=M. Carmen|title=Quantitative Analysis of the Corneal Collagen Distribution after In Vivo Cross-Linking with Second Harmonic Microscopy|journal=BioMed Research International|volume=2019|year=2019|pages=3860498|issn=2314-6133|doi=10.1155/2019/3860498|pmid=30756083|pmc=6348900|doi-access=free}}</ref>
[[Keratoconus]] cornea can also be imaged by SHG to reveal morphological alterations of the [[collagen]].<ref name="MorishigeShin-gyou-uchi2014">{{cite journal|last1=Morishige|first1=N.|last2=Shin-gyou-uchi|first2=R.|last3=Azumi|first3=H.|last4=Ohta|first4=H.|last5=Morita|first5=Y.|last6=Yamada|first6=N.|last7=Kimura|first7=K.|last8=Takahara|first8=A.|last9=Sonoda|first9=K.-H.|title=Quantitative Analysis of Collagen Lamellae in the Normal and Keratoconic Human Cornea by Second Harmonic Generation Imaging Microscopy|journal=Investigative Ophthalmology & Visual Science|volume=55|issue=12|year=2014|pages=8377–8385|issn=0146-0404|doi=10.1167/iovs.14-15348|pmid=25425311|doi-access=}}</ref>
[[Third-harmonic generation|Third-Harmonic Generation]] (THG) microscopy is moreover used to image the [[cornea]], which is complementary to SHG signal as THG and SHG maxima in this tissue are often at different places.<ref name="chapTHGSHGbook">{{Cite book|chapter= THG Microscopy of Cells and Tissues: Contrast Mechanisms and Applications | title = Second Harmonic Generation Imaging, 2nd edition|last1= Olivier |first1= N. |last2= Débarre |first2= D.|last3= Beaurepaire |first3= E. | year=2016|isbn=978-1-4398-4914-9|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=EiTOBQAAQBAJ}}</ref>

==See also==
* [[Nonlinear optics]]
* [[Second-harmonic generation]]
* [[Two-photon excitation microscopy]]

==Sources==
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*{{Cite journal | last1 = Campagnola | first1 = Paul J. | last2 = Loew | first2 = Leslie M | year = 2003 | title = Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms | journal = Nature Biotechnology | volume = 21 | issue = 11 | pages = 1356–1360 | doi = 10.1038/nbt894 | url = http://www.chemphys.lu.se/courses/Microscopy/second%20harmonic%20imaging%202003.pdf | pmid = 14595363 | s2cid = 18701570 | archive-url = https://web.archive.org/web/20160304031100/http://www.chemphys.lu.se/courses/Microscopy/second%20harmonic%20imaging%202003.pdf | archive-date = 2016-03-04 | url-status = dead }}
*{{cite journal | last1 = Stoller | first1 = P. | last2 = Reiser | first2 = K.M. | last3 = Celliers | first3 = P.M. | last4 = Rubenchik | first4 = A.M. | year = 2002 | title = Polarization-modulated second harmonic generation in collagen | journal = Biophys. J. | volume = 82 | issue = 6| pages = 3330–3342 | doi=10.1016/s0006-3495(02)75673-7| pmid = 12023255 | pmc = 1302120 | bibcode = 2002BpJ....82.3330S }}
*{{cite journal | last1 = Han | first1 = M. | last2 = Giese | first2 = G. | last3 = Bille | first3 = J. F. | year = 2005 | title = Second harmonic generation imaging of collagen fibrils in cornea and sclera | journal = Opt. Express | volume = 13 | issue = 15| pages = 5791–5797 | doi=10.1364/opex.13.005791 | pmid=19498583| bibcode = 2005OExpr..13.5791H | doi-access = free }}
*{{Cite book|title= Multiphoton Microscopy and Fluorescence Lifetime Imaging - Applications in Biology and Medicine|last1= König|first1= Karsten | year=2018|isbn=978-3-11-042998-5|publisher= De Gruyter|url=https://www.degruyter.com/view/product/458320}}{{open access}}
*{{cite book|last1=Keikhosravi|first1=Adib|last2=Bredfeldt|first2=Jeremy S.|last3=Sagar|first3=Abdul Kader|last4=Eliceiri|first4=Kevin W.|chapter=Second-harmonic generation imaging of cancer |title=Quantitative Imaging in Cell Biology|series=Methods in Cell Biology|volume=123|year=2014|pages=531–546|issn=0091-679X|doi=10.1016/B978-0-12-420138-5.00028-8|pmid=24974046|isbn=978-0-12-420138-5 |chapter-url=https://www.sciencedirect.com/science/article/pii/B9780124201385000288}}
*{{Cite book| title = Imaging in Cellular and Tissue Engineering, 1st edition|author1= Hanry Yu | author2=Nur Aida Abdul Rahim | year=2013|isbn=9780367445867|publisher= CRC Taylor&Francis|url=https://books.google.com/books?id=eHTRBQAAQBAJ}}
*{{cite journal|last1=Cicchi|first1=Riccardo|last2=Vogler|first2=Nadine|last3=Kapsokalyvas|first3=Dimitrios|last4=Dietzek|first4=Benjamin|last5=Popp|first5=Jürgen|last6=Pavone|first6=Francesco Saverio|title=From molecular structure to tissue architecture: collagen organization probed by SHG microscopy|journal=Journal of Biophotonics|volume=6|issue=2|year=2013|pages=129–142|doi=10.1002/jbio.201200092|pmid=22791562|doi-access=free}}{{open access}}
*{{Cite journal | last1=Roesel | first1=D. | last2=Eremchev | first2=M. | last3=Schönfeldová | first3=T. | last4=Lee | first4=S. | last5=Roke | first5=S. | title=Water as a contrast agent to quantify surface chemistry and physics using second harmonic scattering and imaging: A perspective | journal=Applied Physics Letters | publisher=AIP Publishing | volume=120 | issue=16 | date=2022-04-18 | issn=0003-6951 | doi=10.1063/5.0085807 | page=160501| bibcode=2022ApPhL.120p0501R | s2cid=248252664 | doi-access=free }} {{open access}}

==References==
<references/>

{{Optical microscopy}}
{{Lasers}}

[[Category:Microscopy]]
[[Category:Cell imaging]]
[[Category:Laboratory equipment]]
[[Category:Optical microscopy]]