Two-photon excitation microscopy

<h2 id="concept">Concept</h2>

Two-photon excitation employs <a href="/facts/Two-photon_absorption/BaEbrAKj">two-photon absorption</a>, a concept first described by <a href="/facts/Maria_Goeppert_Mayer/Ung0ch6E">Maria Goeppert Mayer</a> (1906–1972) in her doctoral dissertation in 1931,<a class="footnote-ref" id="fnref:3" href="#fn:3">3</a> and first observed in 1961 in a CaF2:Eu2+ crystal using <a href="/facts/Laser/jk6PTvvT">laser</a> excitation by <a href="/facts/Wolfgang_Kaiser_(physicist)/Jzt4129Q">Wolfgang Kaiser</a>.<a class="footnote-ref" id="fnref:4" href="#fn:4">4</a> <a href="/facts/Isaac_Abella/mfUJdIo9">Isaac Abella</a> showed in 1962 in <a href="/facts/Caesium/dXKN1S5k">caesium</a> vapor that two-photon excitation of single atoms is possible.<a class="footnote-ref" id="fnref:5" href="#fn:5">5</a>
Two-photon excitation fluorescence microscopy has similarities to other confocal laser microscopy techniques such as <a href="/facts/Laser_scanning_confocal_microscopy/Gh7pY9Cl">laser scanning confocal microscopy</a> and <a href="/facts/Raman_microscope/7Tmmj2Uz">Raman microscopy</a>. These techniques use focused laser beams scanned in a raster pattern to generate images, and both have an <a href="/facts/Optical_sectioning/2cXJUF4m">optical sectioning</a> effect. Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures that give confocal microscopes their optical sectioning quality. The optical sectioning produced by multiphoton microscopes is a result of the <a href="/facts/Point_spread_function/9cUtf9P0">point spread function</a> of the excitation.
The concept of two-photon excitation is based on the idea that two photons, of comparably lower photon energy than needed for one-photon excitation, can also excite a <a href="/facts/Fluorophore/wDWBmYhn">fluorophore</a> in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. The emitted photon is at a higher energy (shorter wavelength) than either of the two exciting photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore, a high peak <a href="/facts/Flux/Aw3P5Bvb">flux</a> of excitation photons is typically required, usually generated by femtosecond <a href="/facts/Pulsed_laser/PIcW85dx">pulsed laser</a>. For example, the same average laser power but without pulsing results in no detectable fluorescence compared to fluorescence generated by the pulsed laser via the two-photon effect.
The longer wavelength, lower energy (typically infrared) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than the short-wavelength lasers typically used for single-photon excitation, so living tissues may be observed for longer periods with fewer toxic effects.
The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the two-photon fluorescence lies in the ~700–1100 nm (infrared) range produced by <a href="/facts/Ti-sapphire_laser/pUWECwSc">Ti-sapphire lasers</a>. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into the excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used (typically in the <a href="/facts/Visible_spectrum/frPBh8o5">visible spectrum</a>). Because two photons are absorbed during the excitation of the fluorophore, the probability of fluorescent emission from the fluorophores increases quadratically with the excitation intensity. Therefore, much more two-photon fluorescence is generated where the laser beam is tightly focused than where it is more diffuse. Effectively, excitation is restricted to the tiny focal volume (~1 femtoliter), resulting in a high degree of rejection of out-of-focus objects. This localization of excitation is the key advantage compared to <a href="/facts/Confocal_microscopy/Gh7pY9Cl">single-photon</a> excitation microscopes, which need to employ elements such as pinholes to reject out-of-focus fluorescence. The fluorescence from the sample is then collected by a high-sensitivity detector, such as a <a href="/facts/Photomultiplier/lVDdAH5d">photomultiplier</a> tube. This observed light intensity becomes one <a href="/facts/Pixel/UzwBvXBV">pixel</a> in the eventual image; the focal point is scanned throughout a desired region of the sample to form all the pixels of the image.

<h2 id="development">Development</h2>

Two-photon microscopy was pioneered and <a href="/facts/Patent/LjT2bG3A">patented</a> by <a href="/facts/Winfried_Denk/gEJcxgj0">Winfried Denk</a> and James Strickler in the lab of <a href="/facts/Watt_W._Webb/vSMQjNpM">Watt W. Webb</a> at <a href="/facts/Cornell_University/FGyXuXeI">Cornell University</a> in 1990. They combined the idea of <a href="/facts/Two-photon_absorption/BaEbrAKj">two-photon absorption</a> with the use of a laser scanner.<a class="footnote-ref" id="fnref:6" href="#fn:6">6</a><a class="footnote-ref" id="fnref:7" href="#fn:7">7</a> In two-photon excitation microscopy an infrared laser beam is focused through an objective lens. The <a href="/facts/Ti-sapphire_laser/pUWECwSc">Ti-sapphire laser</a> normally used has a pulse width of approximately 100 femtoseconds (fs) and a repetition rate of about 80 <a href="/facts/Hertz/6Wshj5qm">MHz</a>, allowing the high photon density and flux required for two-photon absorption, and is tunable across a wide range of wavelengths.
The use of infrared light to excite fluorophores in <a href="/facts/Light_scattering_by_particles/pyNaTyrj">light-scattering</a> tissue has added benefits.<a class="footnote-ref" id="fnref:8" href="#fn:8">8</a> Longer wavelengths are scattered to a lesser degree than shorter ones, which is a benefit to high-resolution imaging. In addition, these lower-energy photons are less likely to cause damage outside the focal volume. Compared to a confocal microscope, photon detection is much more effective since even scattered photons contribute to the usable signal. These benefits for imaging in scattering tissues were only recognized several years after the invention of two-photon excitation microscopy.<a class="footnote-ref" id="fnref:9" href="#fn:9">9</a>
There are several caveats to using two-photon microscopy: The pulsed lasers needed for two-photon excitation are much more expensive than the continuous wave (CW) lasers used in confocal microscopy. The two-photon absorption spectrum of a molecule may vary significantly from its one-photon counterpart. Higher-order photodamage becomes a problem and bleaching scales with the square of the laser power, whereas it is linear for single-photon (confocal). For very thin objects such as isolated cells, single-photon (confocal) microscopes can produce images with higher <a href="/facts/Optical_resolution/TcLkPYFw">optical resolution</a> due to their shorter excitation wavelengths. In scattering tissue, on the other hand, the superior optical sectioning and light detection capabilities of the two-photon microscope result in better performance.

<h2 id="applications">Applications</h2>
<h3>Main</h3>
Two-photon microscopy has been involved in numerous fields including: physiology, neurobiology, embryology and <a href="/facts/Tissue_engineering/Tx77KG1S">tissue engineering</a>. Even thin, nearly transparent tissues (such as skin cells) have been visualized with clear detail due to this technique.<a class="footnote-ref" id="fnref:10" href="#fn:10">10</a> Two-photon microscopy's high speed imaging capabilities may also be utilized in noninvasive optical biopsy.<a class="footnote-ref" id="fnref:11" href="#fn:11">11</a> Two-photon microscopy has been aptly used for producing localized chemical reactions,<a class="footnote-ref" id="fnref:12" href="#fn:12">12</a> an effect that has been used also for <a href="/facts/Multiphoton_lithography/QScbSQrA">two-photon-based lithography</a>. Using two-photon fluorescence and <a href="/facts/Second-harmonic_generation/NzWSrT3J">second-harmonic generation</a>–based microscopy, it was shown that organic <a href="/facts/Porphyrin/lKNtdXp1">porphyrin</a>-type molecules can have different <a href="/facts/Transition_dipole_moment/SN3ztKJ6">transition dipole moments</a> for two-photon fluorescence and second harmonic generation,<a class="footnote-ref" id="fnref:13" href="#fn:13">13</a> which are otherwise thought to occur from the same transition dipole moment.<a class="footnote-ref" id="fnref:14" href="#fn:14">14</a> Non-degenerative two-photon excitation, or using 2 photons of unequal wavelengths, was shown to increase the fluorescence of all tested small molecules and fluorescent proteins.<a class="footnote-ref" id="fnref:15" href="#fn:15">15</a>

<h3>Cancer research</h3>
2PEF was also proven to be very valuable for characterizing <a href="/facts/Skin_cancer/r9BWEX7W">skin cancer</a>,<a class="footnote-ref" id="fnref:16" href="#fn:16">16</a> It had also been shown to reveal tumor cell arrest, tumor cell-platelet interaction, tumor cell-leukocyte interaction and metastatic colonization processes.<a class="footnote-ref" id="fnref:17" href="#fn:17">17</a>

<h3>Embryonic research</h3>
2PEF has shown to be advantageous over other techniques, such as <a href="/facts/Confocal_microscopy/Gh7pY9Cl">confocal microscopy</a> when it comes to long-term live-cell imaging of mammalian embryos.<a class="footnote-ref" id="fnref:18" href="#fn:18">18</a>

<h3>Kidney research</h3>
2PEF has also been used in visualization of difficult-to-access cell types, especially in regards to kidney cells.<a class="footnote-ref" id="fnref:19" href="#fn:19">19</a> It has been used in better understanding fluid dynamics and filtration.<a class="footnote-ref" id="fnref:20" href="#fn:20">20</a>

<h3>Viral infection level determination</h3>
2PEF was also proven to be valuable tool for monitoring correlates of viral (<a href="/facts/SARS-CoV-2/DxF8rDc1">SARS-CoV-2</a>) infection in cell culture using a 2P-active Ca2+ sensitive dye.<a class="footnote-ref" id="fnref:21" href="#fn:21">21</a>

<h3>Neuroscience</h3>
2PEF as well as the extension of this method to <a href="/facts/Three-photon_microscopy/uwEyDp3l">3PEF</a> are used to characterize intact neural tissues in the brain of living and even behaving animals. In particular, the method is advantageous for <a href="/facts/Calcium_imaging/3Y85smYK">calcium imaging</a> of a neuron or populations of neurons,<a class="footnote-ref" id="fnref:22" href="#fn:22">22</a> for <a href="/facts/Photopharmacology/2Mk7Hurw">photopharmacology</a> including localized uncaging of components such as glutamate,<a class="footnote-ref" id="fnref:23" href="#fn:23">23</a> GABA<a class="footnote-ref" id="fnref:24" href="#fn:24">24</a> or <a href="/facts/Isomerization/sxv23uKl">isomerization</a> of photoswitchable drugs,<a class="footnote-ref" id="fnref:25" href="#fn:25">25</a><a class="footnote-ref" id="fnref:26" href="#fn:26">26</a> and for the imaging of other genetically encoded sensors that report the concentration of neurotransmitters.<a class="footnote-ref" id="fnref:27" href="#fn:27">27</a>
Currently, two-photon microscopy is widely used to image the live firing of neurons in model organisms including fruit flies (<a href="/facts/Drosophila_melanogaster/KtnHVCcB">Drosophila melanogaster</a>), <a href="/facts/Rat/J2daVSTB">rats</a>, <a href="/facts/Zebra_finch/XWL7nVKe">songbirds</a>, <a href="/facts/Primates/Hl8RNE2X">primates</a>, <a href="/facts/Ferret/rqrBoAu8">ferrets</a>, mice (<a href="/facts/Mus_musculus_domesticus/Unyh5MAs">Mus musculus</a>), <a href="/facts/Zebrafish/Nxg1F1mG">zebrafish</a>.<a class="footnote-ref" id="fnref:28" href="#fn:28">28</a><a class="footnote-ref" id="fnref:29" href="#fn:29">29</a><a class="footnote-ref" id="fnref:30" href="#fn:30">30</a> The animals are typically head-fixed due to the size of the microscope and scan devices, but also miniatured microscopes are being developed that enable imaging of neurons in the moving and freely behaving animals.<a class="footnote-ref" id="fnref:31" href="#fn:31">31</a><a class="footnote-ref" id="fnref:32" href="#fn:32">32</a>

<h2 id="higher-order-excitation">Higher-order excitation</h2>
Simultaneous absorption of three or more photons is also possible, allowing for higher-order multiphoton excitation microscopy.<a class="footnote-ref" id="fnref:33" href="#fn:33">33</a> So-called "three-photon excitation fluorescence microscopy" (3PEF) is the most used technique after 2PEF, to which it is complementary. Localized <a href="/facts/Isomerization/sxv23uKl">isomerization</a> of photoswitchable drugs in vivo using three-photon excitation has also been reported.<a class="footnote-ref" id="fnref:34" href="#fn:34">34</a>

Main article: <a href="/facts/Three-photon_microscopy/uwEyDp3l">Three-photon microscopy</a>
<h2 id="dyes-and-fluorescent-proteins-for-two-photon-excitation-microscopy">Dyes and fluorescent proteins for two-photon excitation microscopy</h2>
In general, all commonly used <a href="/facts/Fluorescent_protein/WQv6UQ7T">fluorescent proteins</a> (CFP, GFP, YFP, RFP) and dyes can be excited in two-photon mode. Two-photon excitation spectra are often considerably broader, making it more difficult to excite fluorophores selectively by switching excitation wavelengths.
Several green, red and NIR emitting dyes (probes and reactive labels) with extremely high 2-photon absorption cross-sections have been reported.<a class="footnote-ref" id="fnref:35" href="#fn:35">35</a> Due to the donor-acceptor-donor type structure, <a href="/facts/Squaraine_dye/xYvXcaGD">squaraine dyes</a> such as Seta-670, Seta-700 and Seta-660 exhibit very high 2-photon absorption (2PA) efficiencies in comparison to other dyes,<a class="footnote-ref" id="fnref:36" href="#fn:36">36</a><a class="footnote-ref" id="fnref:37" href="#fn:37">37</a><a class="footnote-ref" id="fnref:38" href="#fn:38">38</a> SeTau-647 and SeTau-665, a new type of squaraine-<a href="/facts/Rotaxane/k2gYRpgV">rotaxane</a>, exhibit extremely high two-photon action cross-sections of up to 10,000 GM in the near IR region, unsurpassed by any other class of organic dyes.<a class="footnote-ref" id="fnref:39" href="#fn:39">39</a>

<h2 id="see-also">See also</h2>
<ul><li><a href="/facts/3D_optical_data_storage/T9rq6tqC">3D optical data storage</a></li>
<li><a href="/facts/Nonlinear_optics/maerngXd">Nonlinear optics</a></li>
<li><a href="/facts/Second-harmonic_imaging_microscopy/yNYLbeeI">Second-harmonic imaging microscopy</a></li>
<li><a href="/facts/Three-photon_microscopy/uwEyDp3l">Three-photon microscopy</a></li>
<li><a href="/facts/Two-photon_absorption/BaEbrAKj">Two-photon absorption</a></li>
<li><a href="/facts/Two-photon_photoelectron_spectroscopy/vX20xf8H">Two-photon photoelectron spectroscopy</a></li>
<li><a href="/facts/Wide-field_multiphoton_microscopy/n8p41Cwv">Wide-field multiphoton microscopy</a></li></ul>
<h2 id="sources">Sources</h2>
<ul><li>Schmitt, Michael; Mayerhöfer, Thomas; Popp, Jürgen; Kleppe, Ingo; Weisshart, Klaus (2013). "Light-Matter Interaction". Handbook of Biophotonics. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1002%2F9783527643981.bphot003">10.1002/9783527643981.bphot003</a>. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 978-3-527-64398-1. <a href="/facts/S2CID_(identifier)/ldJsHa2Y">S2CID</a> <a href="https://api.semanticscholar.org/CorpusID:93908151">93908151</a>.</li>
<li>König, Karsten (2018). Multiphoton Microscopy and Fluorescence Lifetime Imaging: Applications in Biology and Medicine. Walter de Gruyter GmbH & Co KG. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 978-3-11-042998-5.</li>
<li>Keikhosravi, Adib; Bredfeldt, Jeremy S.; Sagar, Abdul Kader; Eliceiri, Kevin W. (2014). "Second-harmonic generation imaging of cancer". Quantitative Imaging in Cell Biology. Methods in Cell Biology. Vol. 123. pp. 531–546. <a href="/facts/Doi_(identifier)/muM9Etpq">doi</a>:<a href="https://doi.org/10.1016%2FB978-0-12-420138-5.00028-8">10.1016/B978-0-12-420138-5.00028-8</a>. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 978-0-12-420138-5. <a href="/facts/PMID_(identifier)/JlHAvMHt">PMID</a> <a href="https://pubmed.ncbi.nlm.nih.gov/24974046">24974046</a>.</li>
<li>Yu, Hanry; Rahim, Nur Aida Abdul (2013). Imaging in Cellular and Tissue Engineering. CRC Press. <a href="/facts/ISBN_(identifier)/15AdSPa9">ISBN</a> 978-1-4398-4804-3.</li></ul>

<h2 id="external-links">External links</h2>
<ul><li><a href="https://analyticalscience.wiley.com/do/10.1002/was.00050105">Simplifying two-photon microscopy</a></li>
<li><a href="https://www.photonics.com/Webinars/Setting_Up_a_Simple_and_Cost-Efficient_Two-Photon/w273">Webinar: Setting Up a Simple and Cost-Efficient 2Photon Microscope</a></li>
<li><a href="http://www.twophotondyes.com">Two-photon suitable dyes</a></li>
<li><a href="http://microscopy.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonhome.html">introduction to multiphoton microscopy</a></li>
<li><a href="http://parkerlab.bio.uci.edu/attachments/build2photon/Sanderson_microscopy_article%5B1%5D.pdf">Acquisition of Multiple Real-Time Images for Laser Scanning Microscopy</a> (Sanderson microscopy article)</li>
<li><a href="http://parkerlab.bio.uci.edu/microscopy_construction/build_your_own_twophoton_microscope.htm">Build Your Own Video-Rate 2-photon Microscope</a></li>
<li><a href="http://web.mit.edu/solab/Documents/Assets/So-2PF%20light%20microscopy.pdf">Two-photon Fluorescence Light Microscopy, ENCYCLOPEDIA OF LIFE SCIENCES</a></li>
<li><a href="http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonhome.html">"Multiphoton Fluorescence Microscopy"</a>. Microscopy Primer. Florida State University. Retrieved 2018-03-03.</li>
<li><a href="http://www.loci.wisc.edu/multiphoton/mp.html">Multiple-photon excitation fluorescence microscopy.</a> University of Wisconsin.</li>
<li><a href="http://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html">Fundamentals and Applications in Multiphoton Excitation Microscopy</a>. Nikon MicroscopyU .</li>
<li><a href="https://zipfellab.bme.cornell.edu/cross_sections.html">"Two-photon action cross sections"</a>.</li>
<li><a href="https://kbfi.ee/chemical-physics/research/nonlinear-spectroscopy/multiphoton-spectra/">"Two-photon absorption (2PA) spectra database"</a>. KBFI.</li></ul>

<h2 id="references">References</h2>

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</ol>

Two-photon excitation microscopy open-in-new

Two-photon excitation microscopy