second-harmonic imaging microscopy

SHIM offers several advantages for live cell and tissue imaging. 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 three-dimensional images of specimens by imaging deeper into thick tissues.

Two-photons fluorescence (2PEF) is a very different process from 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, 2PEF is a non coherent process, spatially (emitted isotropically) and temporally (broad, sample-dependent spectrum). It is also not specific to certain structure, unlike SHG.{{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}}

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).

Before SHG was used for imaging, the first demonstration of SHG was performed in 1961 by P. A. Franken, G. Weinreich, C. W. Peters, and A. E. Hill at the University of Michigan, Ann Arbor using a quartz sample.{{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}} In 1968, SHG from interfaces was discovered by Bloembergen {{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}} 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.{{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}} In 1974, Hellwarth and Christensen first reported the integration of SHG and microscopy by imaging SHG signals from polycrystalline ZnSe.{{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}} 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.{{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}} In 1993, Lewis examined the second-harmonic response of styryl dyes in electric fields. 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 {{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}} and could have been used to detect SHG. The non-scanning SHG microscope was used for observation of plant starch,{{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}}{{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}} megamolecule,{{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}} spider silk{{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}}{{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}} and so on. In 2010 SHG was extended to whole-animal in vivo imaging.{{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}}{{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}} 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.{{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 }}

SHG 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:{{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}}

$\backslash frac\{I\_\{par\}-I\_\{perp\}\}\{I\_\{par\}+2I\_\{perp\}\}=r$

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

{{See also|Second-harmonic generation#Radiation pattern}}

SHG being a coherent process (spatially and 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 phase-matching condition. Indeed, the coherence length beyond which the conversion of the signal decreases is:

$l\_c\; =\; 2/\backslash Delta\; k$

with $\backslash Delta\; k\; \backslash propto\; 1/(n\_\{2\backslash omega\}-n\_\{\backslash omega\})$ for forward, but $\backslash Delta\; k\_\{bwd\}\; \backslash propto\; 1/(n\_\{2\backslash omega\}+n\_\{\backslash omega\})$ for backward such that $l\_c$ >> $l\_\{c,bwd\}$. 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.{{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}}

Then, the forward-over-backward ratio F/B can be calculated, 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 {{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}}).

The advantages of polarimetry were coupled to SHG in 2002 by Stoller et al.{{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 }} 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.{{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}}

p-SHG defines another anisotropy parameter, as:{{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}}

$\backslash rho\; =\; \backslash sqrt\{\backslash frac\{I\_\{par\}\}\{I\_\{perp\}\}\}$

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 $\backslash rho\; =\; \backslash frac\{\backslash chi^\{(2)\}\_\{XXX\}\}\{\backslash chi^\{(2)\}\_\{XYY\}\}$

,{{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}} where $\backslash chi^\{(2)\}$ is the 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 FFT analysis, and put in a map.{{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}}

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.{{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}}

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 can be applied to quantify the collagen inside the image. Among other quantization techniques, it is probably the one with the highest specificity, reproductibility and applicability despite being quite complex.

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.

{{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 = PNAS | volume = 103 | issue = 3 | pages = 786–790 | doi =10.1073/pnas.0510092103 | pmid = 16407122 | bibcode = 2006PNAS..103..786N | pmc =1334676 | doi-access = free }}

Meanwhile, SHG can efficiently convert near-infrared light to visible light to enable imaging-guided photodynamic therapy, overcoming the penetration depth limitations.{{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 }}

File:Cornea muscle-ad tendon mice cart-horse-surf.png

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.

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 $\backslash chi^\{(3)\}$ {{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}}{{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 }}

The mammographic density is correlated with the collagen density, thus SHG can be used for identifying breast cancer.{{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 }} SHG is usually coupled to other nonlinear techniques such as 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.{{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}}

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.{{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}} Comparison of SHG and 2PEF can also show the change of collagen orientation in tumors.{{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 }}

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.

Healthy ovaries present in SHG a uniform epithelial layer and well-organized collagen in their stroma, whereas abnormal ones show an epithelium with large cells and a changed collagen structure. The r ratio {{Crossreference|selfref=no|(see #Orientational anisotropy)}} is also used {{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 }} to show that the alignment of fibrils is slightly higher for cancerous than for normal tissues.

SHG is, again, combined to 2PEF is used to calculate the ratio:

$MFSI=(\backslash text\{shg\}-\backslash text\{tpef\})/(\backslash text\{shg\}+\backslash text\{tpef\})$

where shg (resp. tpef) is the number of thresholded pixels in the SHG (resp. 2PEF) image,{{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 }} a high MFSI meaning a pure SHG image (with no fluorescence). The highest MFSI is found in cancerous tissues, which provides a contrast mode to differentiate from normal tissues.

SHG was also combined to Third-Harmonic Generation (THG) to show that backward {{Crossreference|selfref=no|(see #Forward over backward SHG)}} THG is higher in tumors.{{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 }}

Changes in collagen ultrastructure in pancreatic cancer can be investigated by multiphoton fluorescence and polarization-resolved SHIM.{{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}}

SHG microscopy was reported for the study of lung, colonic, esophageal stroma and cervical cancers.

Alterations in the organization or polarity of the collagen fibrils can be signs of pathology,.{{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}}{{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}}

In particular, the anisotropic alignment of collagen fibers allowed the discrimination of healthy dermis from pathological scars in skin.{{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}} Also, pathologies in cartilage such as osteoarthritis can be probed by polarization-resolved SHG microscopy,.{{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}}{{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}} SHIM was later extended to fibro-cartilage (meniscus).{{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}}

The ability of 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.{{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}} 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. Also, SHG coupled to other multiphoton techniques can serve to monitor the development of engineered tissues, when the sample is relatively thin however.{{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}} Of course, they can finally be used as a quality control of the fabricated tissues.

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.{{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}} Yet, the collagenous orientation in lamellae is still under debate in this tissue.{{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}}

Keratoconus cornea can also be imaged by SHG to reveal morphological alterations of the collagen.{{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=}}

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.{{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}}

• Nonlinear optics
• Second-harmonic generation
• Two-photon excitation microscopy

• {{Cite book |title= Handbook of Biophotonics, Chap.3 Light–Matter Interaction|last1= Schmitt|first1= Michael |last2= Mayerhöfer|first2= Thomas |last3= Popp|first3=Jürgen |last4= Kleppe|first4=Ingo|last5= Weisshartannée|first5=Klaus| year=2013|isbn=9783527643981

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• {{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}}

{{Optical microscopy}}

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Category:Microscopy

Category:Cell imaging

Category:Optical microscopy