{ "id": "epanz8bktx", "slug": "second-harmonic-generation-microscopy", "title": "Second-harmonic generation microscopy", "updated": "2026-10-21", "topic_path": [ { "id": "life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/topics/life" }, { "id": "life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology" }, { "id": "life.biology.cells", "label": "Cell biology", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.cells" }, { "id": "life.biology.cells.light-microscopy-techniques", "label": "Light microscopy techniques", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.cells.light-microscopy-techniques" } ], "geo": [ { "id": "geo.nongeo.t1946.life.biology.cells.light-microscopy-techniques", "label": "Non-geographic · 1946 to 2000: Light microscopy techniques", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.cells.light-microscopy-techniques", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t1946", "label": "Non-geographic · 1946 to 2000", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946" }, { "id": "geo.nongeo.t1946.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life" }, { "id": "geo.nongeo.t1946.life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology" }, { "id": "geo.nongeo.t1946.life.biology.cells", "label": "Cell biology", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.cells" }, { "id": "geo.nongeo.t1946.life.biology.cells.light-microscopy-techniques", "label": "Light microscopy techniques", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.cells.light-microscopy-techniques" } ] } ], "excerpt": "Second-harmonic generation microscopy, also called harmonic generation microscopy, is a label-free nonlinear optical imaging method that visualizes fibrillar collagen and myosin in intact tissue without stains or dyes.", "snippet": "Second-harmonic generation microscopy, also called harmonic generation microscopy, is a label-free nonlinear optical imaging method that visualizes fibrillar collagen and myosin in intact tissue without stains or dyes.", "node": "life.biology.cells.light-microscopy-techniques", "markdown": "# Second-harmonic generation microscopy\n\n[Second-harmonic generation](https://www.edgepedia.org/second-harmonic-generation) (SHG) microscopy is a label-free nonlinear optical imaging method that forms images from second-harmonic light, generated at exactly half the excitation wavelength by ordered, noncentrosymmetric structures in a sample. In biology it visualizes fibrillar collagen and myosin without stains or dyes, because the signal arises only where molecular assemblies lack a center of symmetry on the scale of the emitted wavelength.[1](https://spj.science.org/doi/10.34133/2021/3973857) • [2](https://link.springer.com/article/10.1007/s12551-022-01041-6) Because the contrast comes from an induced polarization rather than absorption, photobleaching and phototoxicity are substantially reduced relative to fluorescence.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) Laser-scanning SHG microscopy of endogenous structural proteins in intact tissue, at about 1 μm resolution, was reported by Campagnola and colleagues in 2002.[4](https://doi.org/10.1016/s0006-3495%2802%2975414-3)\n\n| Key fact | Value |\n|---|---|\n| Contrast origin | Second-order susceptibility \\( \\chi^{(2)} \\); requires noncentrosymmetric order at molecular \\( (\\beta \\neq 0) \\) and macromolecular \\( (\\chi^{(2)} \\neq 0) \\) scales[2](https://link.springer.com/article/10.1007/s12551-022-01041-6) |\n| Signal-producing structures | Fibrillar collagen (types I, II, III, V) and myosin; collagen IV, laminin, fibronectin, and elastin are invisible[1](https://spj.science.org/doi/10.34133/2021/3973857) • [2](https://link.springer.com/article/10.1007/s12551-022-01041-6) |\n| Typical excitation/emission | Tunable Ti:sapphire, 700–1000 nm; 900 nm excitation gives 450 nm SHG[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) |\n| Penetration and resolution | 100–300 μm deep with 800–1000 nm excitation[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) • [5](https://google.iopscience.iop.org/article/10.1088/2515-7647/ae37b3/meta) |\n| Emission directionality | Forward coherence length a few microns; backward only a few tens of nanometers, so backward SHG is always poorly phase matched and weaker[2](https://link.springer.com/article/10.1007/s12551-022-01041-6) |\n| Setup effort | Building and calibrating a quantitative SHG microscope from components takes an estimated 2–4 weeks[6](https://www.nature.com/articles/nprot.2012.009) |\n| Throughput option | Wide-field SHG improves imaging throughput by 2–3 orders of magnitude over scanning[2](https://link.springer.com/article/10.1007/s12551-022-01041-6) |\n\n## How it works\n\nSHG is a second-order nonlinear optical process, so it can arise only from media lacking a center of symmetry.[2](https://link.springer.com/article/10.1007/s12551-022-01041-6) Two excitation photons combine coherently to produce one photon at double the frequency; the efficiency depends on phase matching, expressed as the wave-vector mismatch\n\n\\[ \\Delta k = k_{2\\omega} - 2k_{\\omega} \\]\n\nwhere \\( k_{2\\omega} \\) is the wave vector of the SHG photon and \\( k_{\\omega} \\) that of the incident photon. Conversion is maximized at \\( \\Delta k = 0 \\) and falls for nonzero mismatch.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC2390911/) In collagenous tissue the measured dispersion is \\( \\Delta n = n(2\\omega) - n(\\omega) = 0.02 \\), which sets coherence lengths on the order of the inter-fibrillar spacing.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC2390911/)\n\nDirectionality follows directly from phase matching. Forward-emitted SHG has a coherence length of a few microns in most materials, while backward SHG has a coherence length of only a few tens of nanometers, so the backward signal is always poorly phase matched and smaller than the forward channel.[2](https://link.springer.com/article/10.1007/s12551-022-01041-6) Backward-detected images also have a mixed origin, combining backscattered forward SHG with directly generated backward SHG.[8](https://www.frontiersin.org/journals/photonics/articles/10.3389/fphot.2026.1771907/full)\n\n## How it is done\n\nThe two primary requirements are a mode-locked femtosecond laser and a laser-scanning microscope; the most common source is a tunable (700–1000 nm) titanium sapphire oscillator.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) Typical laser and SHG wavelengths are 900 and 450 nm, respectively.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325)\n\nA quantitative instrument collects both backward (epi) and forward (transmission) channels with calibrated relative detection efficiencies, a dichroic mirror, a ~10 nm full-width-at-half-maximum bandpass filter, identical photomultiplier tubes, and λ/2 and λ/4 wave plates.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) Only small modifications equip a standard two-photon laser-scanning microscope for SHG, although the best signals have historically required the transmitted-light geometry.[9](https://europepmc.org/article/MED/14595363) Conversion efficiency decreases about 3-fold across the 700–1000 nm tuning range, and collection below 380 nm is limited by glass optics.[1](https://spj.science.org/doi/10.34133/2021/3973857) At the focus, transient power densities can reach \\( 10^{18} \\,\\mathrm{W\\,m^{-2}} \\).[10](https://www.nature.com/articles/s41598-017-13752-y) Setup and calibration from component parts takes an estimated 2–4 weeks.[6](https://www.nature.com/articles/nprot.2012.009)\n\n## Origin\n\nSHG has been recognized since the earliest days of laser physics and was demonstrated through a microscope more than 25 years before it emerged as a viable imaging contrast mechanism.[9](https://europepmc.org/article/MED/14595363) SHG from collagen was reported by Shmuel Roth and Isaac Freund in 1979 in The Journal of Chemical Physics.[11](https://doi.org/10.1063/1.437677) SHG microscopy of biological samples, rat-tail tendon collagen at ~50 μm resolution, was performed by I. Freund, M. Deutsch, and A. Sprecher in 1986 in Biophysical Journal.[12](https://doi.org/10.1016/s0006-3495%2886%2983510-x) The scanning two-photon fluorescence microscope that provided the platform was reported by [Winfried Denk](https://www.edgepedia.org/winfried-denk), James H. Strickler, and [Watt W. Webb](https://www.edgepedia.org/watt-w-webb) in 1990 in Science.[13](https://doi.org/10.1126/science.2321027)\n\nModern biological SHG microscopy came in quick succession. Paul J. Campagnola and colleagues reported high-resolution SHG imaging of live cells in 1999 in Biophysical Journal[14](https://doi.org/10.1016/s0006-3495%2899%2977165-1); Campagnola and colleagues extended this to three-dimensional laser-scanning imaging of endogenous collagen, myosin, and microtubules in 2002 in Biophysical Journal[4](https://doi.org/10.1016/s0006-3495%2802%2975414-3); Campagnola and Loew named the technique second-harmonic imaging microscopy in a 2003 [Nature Biotechnology](https://www.edgepedia.org/nature-biotechnology) review.[15](https://doi.org/10.1038/nbt894) Polarization-dependent SHG imaging of rat-tail tendon was reported by Patrick Stoller and colleagues in 2002 in the Journal of Biomedical Optics[16](https://doi.org/10.1117/1.1431967), and live-tissue imaging combining native fluorescence with SHG by Warren R. Zipfel and colleagues in 2003 in PNAS.[17](https://doi.org/10.1073/pnas.0832308100)\n\n## Variants\n\n**Polarization-resolved SHG (P-SHG)** exploits the tensor character of \\( \\chi^{(2)} \\). With Kleinman symmetry and cylindrical C6v symmetry assumed, two unique susceptibility elements remain, with the ratio \\( \\rho = \\chi_{zzz}^{(2)}/\\chi_{zxx}^{(2)} \\); the anisotropy parameter is \\( \\beta = (I_{\\parallel} - I_{\\perp})/(I_{\\parallel} + 2I_{\\perp}) \\), and circular-dichroism SHG, \\( I_{\\mathrm{CD\\text{-}SHG}} = 2(I_L - I_R)/(I_L + I_R) \\), reports only chiral structures.[18](https://www.frontiersin.org/journals/physics/articles/10.3389/fphy.2021.726996/full) [Birefringence](https://www.edgepedia.org/birefringence) in the sample can distort the retrieved polarization signatures and must be accounted for.[19](https://doi.org/10.1364/oe.18.014859)\n\n**Stokes ellipsometry (NOSE)** recovers the full local-frame \\( \\chi^{(2)} \\) tensor, a 3 × 3 × 3 tensor with 27 elements of which 18 can be unique for SHG.[20](https://doi.org/10.1016/j.bpj.2016.05.055) Ab initio calculation gives a collagen ratio, so the chiral element contributes little.[20](https://doi.org/10.1016/j.bpj.2016.05.055)\n\n**Ratiometric and wide-field implementations.** Epi-generated SHG images at 920 and 860 nm discriminate collagen from myosin.[10](https://www.nature.com/articles/s41598-017-13752-y) Wide-field SHG improves throughput by 2–3 orders of magnitude, and a MHz-repetition-rate wide-field microscope for contracting muscle uses pulses as low as ~60 nJ.[2](https://link.springer.com/article/10.1007/s12551-022-01041-6)\n\n**Endoscopic and in vivo SHG.** Microendoscope systems collect up to four contrasts (SHG, THG, TPEF, and three-photon fluorescence) in real time but rely exclusively on backward-detected SHG, which is always weaker than the forward channel.[1](https://spj.science.org/doi/10.34133/2021/3973857) Polarization-resolved SHG has been demonstrated through a multimode fiber endoscope, imaging mouse tail tendon and heart tissue at depths up to 1 mm.[21](https://doi.org/10.1364/optica.430295)\n\n## Applications\n\n**Cancer.** In ovarian tissue, SHG reveals densely packed helical fibrils and acellular dense collagen in tumors, versus less tightly woven fibers with stromal cells in normal tissue.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) In breast cancer, a model-free multiscale pSHG analysis found tumor regions with liquid-crystal scalar order parameter \\( S > 0.8 \\) and substantially elevated biaxial parameter B.[22](https://link.springer.com/article/10.1007/s10439-026-04345-w)\n\n**Fibrosis and other collagenous tissue.** Polarization-modulated SHG with electro-optic modulation and lock-in detection measured collagen orientation in rat-tail tendon, mouse aorta, and fibrotic mouse liver.[23](https://www.osti.gov/servlets/purl/15013321) P-SHG has also been applied to liver fibrosis, osteoarthritis, keratoconus, corneal stroma denaturation, dysplastic skin, and colon cancer.[18](https://www.frontiersin.org/journals/physics/articles/10.3389/fphy.2021.726996/full) A standard protocol lists applications spanning cancer, fibrosis, osteogenesis imperfecta, cornea, and muscle.[6](https://www.nature.com/articles/nprot.2012.009)\n\n## Limitations and alternatives\n\n**Signal absence.** SHG is specific to ordered noncentrosymmetric assemblies: nonfibrous collagen type I, II, and VI powders show no detectable SHG, and thermal denaturation gradually destroys the signal.[24](https://discovery.ucl.ac.uk/id/eprint/10234/) Collagen IV, laminin, fibronectin, and elastin are transparent to the modality.[1](https://spj.science.org/doi/10.34133/2021/3973857)\n\n**Scattering and depth.** Polarization-resolved responses become largely scrambled within 1–2 scattering lengths (~20–50 μm) in tendon and skeletal muscle; optical clearing reduces the scattering coefficient by roughly 5- to 20-fold while retaining correct polarization signatures.[1](https://spj.science.org/doi/10.34133/2021/3973857) Penetration is limited to 100–300 μm with 800–1000 nm excitation, versus millimeters to centimeters for MRI, CT, and PET, and water absorption caps the biological transparency window near 1.3 μm.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325) Forward detection is generally limited to ex vivo thin sections because the sensor must sit behind the sample; backward detection is the in vivo route.[8](https://www.frontiersin.org/journals/photonics/articles/10.3389/fphot.2026.1771907/full)\n\n**Compared with THG and TPEF.** Third-harmonic generation microscopy, reported by Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg in 1997, requires excitation longer than about 1200 nm for signal transmission through glass optics.[25](https://doi.org/10.1063/1.118442) • [1](https://spj.science.org/doi/10.34133/2021/3973857) Two-photon fluorescence, reported by Denk, Strickler, and Webb in 1990, detects dyes or autofluorescence rather than the \\( \\chi^{(2)} \\) response.[13](https://doi.org/10.1126/science.2321027) SHG's induced-polarization contrast gives reduced photobleaching and phototoxicity relative to fluorescence.[3](https://pubs.acs.org/doi/full/10.1021/ac1032325)\n\n## References\n\n1. [Recent Advancements in Optical Harmonic Generation Microscopy: Applications and Perspectives](https://spj.science.org/doi/10.34133/2021/3973857)\n2. [Second harmonic generation microscopy: a powerful tool for bio-imaging | Biophysical Reviews](https://link.springer.com/article/10.1007/s12551-022-01041-6)\n3. [Second Harmonic Generation Imaging Microscopy: Applications to Diseases Diagnostics (Analytical Chemistry)](https://pubs.acs.org/doi/full/10.1021/ac1032325)\n4. [Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues (Biophysical Journal, 2002)](https://doi.org/10.1016/s0006-3495%2802%2975414-3)\n5. [Generalized 3D quasi-phase-matching model of image contrast in SHG microscopy of fibrillar collagen architectures (IOPscience)](https://google.iopscience.iop.org/article/10.1088/2515-7647/ae37b3/meta)\n6. [Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure (Nature Protocols)](https://www.nature.com/articles/nprot.2012.009)\n7. [Phase Matching considerations in Second Harmonic Generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology](https://pmc.ncbi.nlm.nih.gov/articles/PMC2390911/)\n8. [Correlation between forward and backward generated SHG images of biological tissue relating to collagen structure and biomechanics (Frontiers in Photonics, 2026)](https://www.frontiersin.org/journals/photonics/articles/10.3389/fphot.2026.1771907/full)\n9. [Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms (Europe PMC record)](https://europepmc.org/article/MED/14595363)\n10. [A new mode of contrast in biological second harmonic generation microscopy | Scientific Reports](https://www.nature.com/articles/s41598-017-13752-y)\n11. [Shmuel Roth, Isaac Freund (1979). Second harmonic generation in collagen. The Journal of Chemical Physics.](https://doi.org/10.1063/1.437677)\n12. [Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon (Biophysical Journal, 1986)](https://doi.org/10.1016/s0006-3495%2886%2983510-x)\n13. [Winfried Denk, James H. Strickler, Watt W. Webb (1990). Two-Photon Laser Scanning Fluorescence Microscopy. Science.](https://doi.org/10.1126/science.2321027)\n14. [High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation (Biophysical Journal, 1999)](https://doi.org/10.1016/s0006-3495%2899%2977165-1)\n15. [Paul J Campagnola, Leslie M Loew (2003). Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nature Biotechnology.](https://doi.org/10.1038/nbt894)\n16. [Patrick Stoller and colleagues (2002). Polarization-dependent optical second-harmonic imaging of a rat-tail tendon. Journal of Biomedical Optics.](https://doi.org/10.1117/1.1431967)\n17. [Warren R. Zipfel and colleagues (2003). Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proceedings of the National Academy of Sciences.](https://doi.org/10.1073/pnas.0832308100)\n18. [Polarization-Sensitive Second Harmonic Generation Microscopy for Investigations of Diseased Collagenous Tissues (Frontiers in Physics)](https://www.frontiersin.org/journals/physics/articles/10.3389/fphy.2021.726996/full)\n19. [Sophie Brasselet and colleagues (2010). Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging. Optics Express.](https://doi.org/10.1364/oe.18.014859)\n20. [Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry (Biophysical Journal, 2016)](https://doi.org/10.1016/j.bpj.2016.05.055)\n21. [Polarization-resolved second-harmonic generation imaging through a multimode fiber (aggregator record)](https://doi.org/10.1364/optica.430295)\n22. [Characterization of Collagen Fiber Organization in Breast Cancer via Model-Free Multiscale pSHG Image Analysis (Annals of Biomedical Engineering, 2026)](https://link.springer.com/article/10.1007/s10439-026-04345-w)\n23. [Polarization-modulated second harmonic generation microscopy of collagen tissues (OSTI report)](https://www.osti.gov/servlets/purl/15013321)\n24. [Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections (UCL Discovery record)](https://discovery.ucl.ac.uk/id/eprint/10234/)\n25. [Y. Barad and colleagues (1997). Nonlinear scanning laser microscopy by third harmonic generation. Applied Physics Letters.](https://doi.org/10.1063/1.118442)\n\n---\n*Topic: Encyclopedia › Life and health › Biological foundations › Cell biology › Light microscopy techniques*\n\n*Initially written Sep 29, 2026 · Edited: Oct 15, 2026; Oct 21, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/second-harmonic-generation-microscopy", "markdown_url": "https://www.edgepedia.org/second-harmonic-generation-microscopy.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. AI training is open to everyone. For other uses, organizations over USD 100M in revenue or 100M monthly users license separately.", "spdx": "LicenseRef-Edgepedia-Community-1.0" }, "credit": "\"Second-harmonic generation microscopy\", Edgepedia (EdgeChat), https://www.edgepedia.org/second-harmonic-generation-microscopy. Edgepedia Community License 1.0.", "credit_md": "\"[Second-harmonic generation microscopy](https://www.edgepedia.org/second-harmonic-generation-microscopy)\", Edgepedia (EdgeChat), [https://www.edgepedia.org/second-harmonic-generation-microscopy](https://www.edgepedia.org/second-harmonic-generation-microscopy). [Edgepedia Community License 1.0](https://www.edgepedia.org/edgepedia/license).", "credit_html": "\"Second-harmonic generation microscopy\", Edgepedia (EdgeChat), https://www.edgepedia.org/second-harmonic-generation-microscopy. Edgepedia Community License 1.0.", "speakable": "Second-harmonic generation microscopy, also called harmonic generation microscopy, is a label-free nonlinear optical imaging method that visualizes fibrillar collagen and myosin in intact tissue without stains or dyes." }