232:. 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 visible spectrum). 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
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224:. 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.
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than the wide field. The 2PEF distribution is larger due to the fact that a wavelength twice as long as in the case of a wide or confocal field is responsible for the intensity distribution. These intensity distributions are also known as point spread functions. Optical conditions: the excitation wavelengths are 488 nm and 900 nm respectively for 1PEF and 2PEF; the emission wavelength is 520 nm; the
150:
Optical response from a point source. From left to right: calculated intensity distributions xy (top) and rz (bottom), with logarithmic scale, for a point source imaged by means of a wide field (a), 2PEF (b) and confocal microscopy (c). The 2PEF and confocal forms have a better signal-to-noise ratio
1304:
Kovács, Dénes
Szepesi; Kontra, Bence; Chiovini, Balázs; Müller, Dalma; Tóth, Estilla Zsófia; Ábrányi-Balogh, Péter; Wittner, Lucia; Várady, György; Turczel, Gábor; Farkas, Ödön; Owen, Michael C.; Katona, Gergely; Győrffy, Balázs; Keserű, György Miklós; Mucsi, Zoltán; Rózsa, Balázs J.; Kovács, Ervin
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tissue has added benefits. 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
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Two-photon microscopy has been involved in numerous fields including: physiology, neurobiology, embryology and tissue engineering. Even thin, nearly transparent tissues (such as skin cells) have been visualized with clear detail due to this technique. Two-photon microscopy's high speed imaging
215:
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.
336:
for two-photon fluorescence and second harmonic generation, which are otherwise thought to occur from the same transition dipole moment. Non-degenerative two-photon excitation, or using 2 photons of unequal wavelengths, was shown to increase the fluorescence of all tested small molecules and
1344:
Kovács, Dénes
Szepesi; Chiovini, Balázs; Müller, Dalma; Tóth, Estilla Zsófia; Fülöp, Anna; Ábrányi-Balogh, Péter; Wittner, Lucia; Várady, György; Farkas, Ödön; Turczel, Gábor; Katona, Gergely; Győrffy, Balázs; Keserű, György Miklós; Mucsi, Zoltán; Rózsa, Balázs J.; Kovács, Ervin (Jun 2023).
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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
1596:
Máthé, Domokos; Szalay, Gergely; Cseri, Levente; Kis, Zoltán; Pályi, Bernadett; Földes, Gábor; Kovács, Noémi; Fülöp, Anna; Szepesi, Áron; Hajdrik, Polett; Csomos, Attila; Zsembery, Ákos; Kádár, Kristóf; Katona, Gergely; Mucsi, Zoltán; Rózsa, Balázs József; Kovács, Ervin (Jul 2024).
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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
141:
Schematic representation of the energy levels (Jabłoński diagrams) of the fluorescence process, example of a fluorescent dye that emits light at 460 nm. One (purple, 1PEF), two (light red, 2PEF) or three (dark red, 3PEF) photons are absorbed to emit a photon of fluorescence
462:
Simultaneous absorption of three or more photons is also possible, allowing for higher-order multiphoton excitation microscopy. So-called "three-photon excitation fluorecence microscopy" (3PEF) is the most used technique after 2PEF, to which it is complementary. Localized
2230:
Sortino, Rosalba; Cunquero, Marina; Castro-Olvera, Gustavo; Gelabert, Ricard; Moreno, Miquel; Riefolo, Fabio; Matera, Carlo; Fernàndez-Castillo, Noèlia; Agnetta, Luca; Decker, Michael; Lluch, José Maria; Hernando, Jordi; Loza-Alvarez, Pablo; Gorostiza, Pau (2023-10-12).
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Przhonska, Olga V.; Webster, Scott; Padilha, Lazaro A.; Hu, Honghua; Kachkovski, Alexey D.; Hagan, David J.; Van
Stryland, Eric W. (2010). "Two-Photon Absorption in Near-IR Conjugated Molecules: Design Strategy and Structure–Property Relations".
101:. Using infrared light minimizes scattering in the tissue because infrared light is scattered less in typical biological tissues. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased
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normally used has a pulse width of approximately 100 femtoseconds (fs) and a repetition rate of about 80 MHz, allowing the high photon density and flux required for two-photon absorption, and is tunable across a wide range of wavelengths.
1211:
Sadegh, Sanaz; Yang, Mu-Han; Ferri, Christopher G. L.; Thunemann, Martin; Saisan, Payam A.; Wei, Zhe; Rodriguez, Erik A.; Adams, Stephen R.; Kiliç, Kivilcim; Boas, David A.; Sakadžić, Sava; Devor, Anna; Fainman, Yeshaiahu (18 September 2019).
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Pittolo, Silvia; Lee, Hyojung; Lladó, Anna; Tosi, Sébastien; Bosch, Miquel; Bardia, Lídia; Gómez-Santacana, Xavier; Llebaria, Amadeu; Soriano, Eduardo; Colombelli, Julien; Poskanzer, Kira E.; Perea, Gertrudis; Gorostiza, Pau (2019-07-02).
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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
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requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the
485:(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.
297:
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.
227:
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
454:. 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.
305:
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.
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Liu, Lingzhi; Shao, Mei; Dong, Xiaohu; Yu, Xuefeng; Liu, Zhihong; He, Zhike; Wang, Ququan (15 October 2008). "Homogeneous
Immunoassay Based on Two-Photon Excitation Fluorescence Resonance Energy Transfer".
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Izquierdo-Serra, Mercè; Gascón-Moya, Marta; Hirtz, Jan J.; Pittolo, Silvia; Poskanzer, Kira E.; Ferrer, Èric; Alibés, Ramon; Busqué, Félix; Yuste, Rafael; Hernando, Jordi; Gorostiza, Pau (2014-06-18).
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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
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Several green, red and NIR emitting dyes (probes and reactive labels) with extremely high 2-photon absorption cross-sections have been reported. Due to the donor-acceptor-donor type structure,
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capabilities may also be utilized in noninvasive optical biopsy. Two-photon microscopy has been aptly used for producing localized chemical reactions, and effect that has been used also for
373:
2PEF has also been used in visualization of difficult-to-access cell types, especially in regards to kidney cells. It has been used in better understanding fluid dynamics and filtration.
1394:
Tanaka, Koji; Toiyama, Yuji; Okugawa, Yoshinaga; Okigami, Masato; Inoue, Yasuhiro; Uchida, Keiichi; Araki, Toshimitsu; Mohri, Yasuhiko; Mizoguchi, Akira; Kusunoki, Masato (15 May 2014).
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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
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of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with
392:
Diagram of in vivo brain function imaging. Shows the general schematic for imaging, along with neuronal and vascular images. Imaging was performed using various fluorescent dyes.
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in vitro. It had also been shown to reveal tumor cell arrest, tumor cell-platelet interaction, tumor cell-leukocyte interaction and metastatic colonization processes.
1269:
Paoli, John; Smedh, Maria; Ericson, Marica B. (September 2009). "Multiphoton Laser
Scanning Microscopy—A Novel Diagnostic Method for Superficial Skin Cancers".
2010:
Demas, Jeffrey; Manley, Jason; Tejera, Frank; Barber, Kevin; Kim, Hyewon; Traub, Francisca Martínez; Chen, Brandon; Vaziri, Alipasha (September 2021).
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516:, 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.
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128:. The excitement is at 840 nm, and the red and blue colors represent other channels of multiphoton techniques which have been superimposed.
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are used to characterize intact neural tissues in the brain of living and even behaving animals. In particular, the method is advantageous for
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Keikhosravi, Adib; Bredfeldt, Jeremy S.; Sagar, Abdul Kader; Eliceiri, Kevin W. (2014). "Second-harmonic generation imaging of cancer".
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of photoswitchable drugs, and for the imaging of other genetically encoded sensors that report the concentration of neurotransmitters.
1307:"Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging"
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with the use of a laser scanner. In two-photon excitation microscopy an infrared laser beam is focused through an objective lens. The
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in the eventual image; the focal point is scanned throughout a desired region of the sample to form all the pixels of the image.
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Currently, two-photon microscopy is widely used to image the live firing of neurons in model organisms including fruit flies (
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90:, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.
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1347:"Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging"
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Denk, Winifried; Strickler, James H.; Webb, Watt W. (6 April 1990). "Two-Photon Laser
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Schmitt, Michael; Mayerhöfer, Thomas; Popp, Jürgen; Kleppe, Ingo; Weisshart, Klaus (2013). "Light-Matter
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Denk W.; Delaney K. (1994). "Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy".
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Two-photon excitation fluorescence microscopy has similarities to other confocal laser microscopy techniques such as
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2012:"High-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy"
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Huang, Cheng; Maxey, Jessica R.; Sinha, Supriyo; Savall, Joan; Gong, Yiyang; Schnitzer, Mark J. (December 2018).
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Podgorski, Kaspar; Terpetschnig, Ewald; Klochko, Oleksii P.; Obukhova, Olena M.; Haas, Kurt (14 December 2012).
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1599:"Monitoring correlates of SARS-CoV-2 infection in cell culture using a two-photon-active calcium-sensitive dye"
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202:. These techniques use focused laser beams scanned in a raster pattern to generate images, and both have an
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2069:"A Miniature Head-Mounted Two-Photon Microscope: High-Resolution Brain Imaging in Freely Moving Animals"
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2297:"Ultra-Bright and -Stable Red and Near-Infrared Squaraine Fluorophores for In Vivo Two-Photon Imaging"
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Bewersdorf, Jörg; Pick, Rainer; Hell, Stefan W. (1 May 1998). "Multifocal multiphoton microscopy".
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2173:"Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy"
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1437:"Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability"
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Multiphoton
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Squirrell, Jayne M.; Wokosin, David L.; White, John G.; Bavister, Barry D. (August 1999).
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1001:"Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin"
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of photoswitchable drugs in vivo using three-photon excitation has also been reported.
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exhibit very high 2-photon absorption (2PA) efficiencies in comparison to other dyes,
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Zong W, Obenhaus HA, Skytøen ER, Eneqvist H, de Jong NL, Vale R; et al. (2022).
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1691:"Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience"
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171:(1906–1972) in her doctoral dissertation in 1931, and first observed in 1961 in a CaF
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47:
1740:"Two-Photon Neuronal and Astrocytic Stimulation with Azobenzene-Based Photoswitches"
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1494:"Two-photon fluorescence excitation and related techniques in biological microscopy"
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Abella, I. D. (December 1962). "Optical Double-Photon
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Ashworth, S. L.; Sandoval, R. M.; Tanner, G. A.; Molitoris, B. A. (2007-08-02).
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of excitation photons is typically required, usually generated by femtosecond
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Stockert, Juan Carlos; Blazquez-Castro, Alfonso (2017). "Non-linear Optics".
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2PEF was also proven to be valuable tool for monitoring correlates of viral (
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due to its deeper tissue penetration, efficient light detection, and reduced
1906:"Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues"
1822:
1214:"Efficient non-degenerate two-photon excitation for fluorescence microscopy"
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for this technique. Two-photon excitation can be a superior alternative to
77:, where the excitation wavelength is shorter than the emission wavelength,
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Reeve JE, Corbett AD, Boczarow I, Wilson T, Bayley H, Anderson HL (2012).
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Xu, C; Zipfel, W; Shear, J B; Williams, R M; Webb, W W (1 October 1996).
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2233:"Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo"
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Two-photon Fluorescence Light Microscopy, ENCYCLOPEDIA OF LIFE SCIENCES
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Kaiser, W.; Garrett, C. (September 1961). "Two-Photon Excitation in CaF
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imaging technique that is particularly well-suited to image scattering
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Acquisition of Multiple Real-Time Images for Laser Scanning Microscopy
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Helmchen, Fritjof; Fee, Michale; Tank, David; Denk, Winfried (2001).
1865:"Neuromodulator Dynamics In Vivo with Genetically Encoded Indicators"
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Diaspro, Alberto; Chirico, Giuseppe; Collini, Maddalena (May 2005).
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Khadria A, Coene Y, Gawel P, Roche C, Clays K, Anderson HL (2017).
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when it comes to long-term live-cell imaging of mammalian embryos.
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Fundamentals and Applications in Multiphoton Excitation Microscopy
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Webinar: Setting Up a Simple and Cost-Efficient 2Photon Microscope
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Helmchen F.; Denk W. (2005). "Deep tissue two-photon microscopy".
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Dyes and fluorescent proteins for two-photon excitation microscopy
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2PEF has shown to be advantageous over other techniques, such as
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including localized uncaging of components such as glutamate or
385:) infection in cell culture using a 2P-active Ca sensitive dye.
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of up to about one millimeter in thickness. Unlike traditional
2110:"Large-scale two-photon calcium imaging in freely moving mice"
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vapor that two-photon excitation of single atoms is possible.
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245:
176:
35:
626:. Methods in Cell Biology. Vol. 123. pp. 531–546.
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2PEF was also proven to be very valuable for characterizing
120:
Two-photon fluorescence image (green) of a cross section of
1955:"Long-term optical brain imaging in live adult fruit flies"
1393:
1109:"Push–pull pyropheophorbides for nonlinear optical imaging"
998:
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217:
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Advanced Fluorescence Reporters in Chemistry and Biology I
1303:
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1549:"Two-photon microscopy: Visualization of kidney dynamics"
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Total internal reflection fluorescence microscopy (TIRF)
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2009:
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Benninger, Richard K.P.; Piston, David W. (June 2013).
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Photo-activated localization microscopy (PALM/STORM)
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The use of infrared light to excite fluorophores in
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Multiple-photon excitation fluorescence microscopy.
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1648:Grienberger, Christine; Konnerth, Arthur (2012).
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401:2PEF as well as the extension of this method to
244:tube. This observed light intensity becomes one
93:Two-photon excitation microscopy typically uses
1903:
1802:Proceedings of the National Academy of Sciences
1641:
2508:"Two-photon absorption (2PA) spectra database"
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2643:Interference reflection microscopy (IRM/RICM)
2534:
2453:Build Your Own Video-Rate 2-photon Microscope
2353:
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778:"Über Elementarakte mit zwei Quantensprüngen"
328:–based microscopy, it was shown that organic
97:(NIR) excitation light which can also excite
2156:: CS1 maint: multiple names: authors list (
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1689:Svoboda, Karel; Yasuda, Ryohei (June 2006).
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409:of a neuron or populations of neurons, for
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1400:American Journal of Translational Research
1271:Seminars in Cutaneous Medicine and Surgery
659:Imaging in Cellular and Tissue Engineering
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30:Two-photon excitation microscopy of mouse
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657:Yu, Hanry; Rahim, Nur Aida Abdul (2013).
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745:Fluorescence Microscopy in Life Sciences
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264:Two-photon microscopy was pioneered and
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3071:Multiple-prism grating laser oscillator
2237:Angewandte Chemie International Edition
2164:
656:
3127:
2608:Quantitative phase-contrast microscopy
2548:
2442:introduction to multiphoton microscopy
1863:Sabatini, Bernardo; Tian, Lin (2020).
855:
607:. Walter de Gruyter GmbH & Co KG.
2791:
2522:
2464:"Multiphoton Fluorescence Microscopy"
602:
551:Two-photon photoelectron spectroscopy
356:
260:A diagram of a two-photon microscope.
2770:
2735:Stimulated emission depletion (STED)
1311:Organic & Biomolecular Chemistry
624:Quantitative Imaging in Cell Biology
324:. Using two-photon fluorescence and
900: "Two-photon laser microscopy."
377:Viral infection level determination
332:-type molecules can have different
280:in 1990. They combined the idea of
13:
2500:"Two-photon action cross sections"
1113:Organic and Biomolecular Chemistry
632:10.1016/B978-0-12-420138-5.00028-8
536:Second-harmonic imaging microscopy
368:
340:
272:and James Strickler in the lab of
196:laser scanning confocal microscopy
21:second-harmonic imaging microscopy
14:
3166:
2707:Lightsheet microscopy (LSFM/SPIM)
2427:Simplifying two-photon microscopy
2420:
1910:Current Protocols in Cell Biology
556:Wide-field multiphoton microscopy
236:is the key advantage compared to
3109:
3108:
2769:
2758:
2757:
2656:
55:Two-photon excitation microscopy
2223:
2060:
2003:
1946:
1897:
1856:
1788:
1731:
1589:
1540:
1498:Quarterly Reviews of Biophysics
1485:
1428:
1337:
1297:
1204:
396:
309:
167:, a concept first described by
2980:Amplified spontaneous emission
2712:Lattice light-sheet microscopy
2623:Second harmonic imaging (SHIM)
2449:(Sanderson microscopy article)
884:
849:
810:
753:10.2174/9781681085180117010023
736:
578:10.1002/9783527643981.bphot003
481:In general, all commonly used
251:
163:Two-photon excitation employs
16:Fluorescence imaging technique
1:
2086:10.1016/S0896-6273(01)00421-4
1025:10.1016/s0006-3495(97)78886-6
676:
46:. Obtained at 780 nm using a
2322:10.1371/journal.pone.0051980
1922:10.1002/0471143030.cb0411s59
1882:10.1016/j.neuron.2020.09.036
1708:10.1016/j.neuron.2006.05.019
1667:10.1016/j.neuron.2012.02.011
1650:"Imaging calcium in neurons"
970:10.1016/0165-0270(94)90189-9
322:two-photon-based lithography
7:
3036:Chirped pulse amplification
2401:10.1007/978-3-642-04702-2_4
519:
10:
3171:
2840:List of laser applications
2817:
2470:. Florida State University
2126:10.1016/j.cell.2022.02.017
2028:10.1038/s41592-021-01239-8
1979:10.1038/s41467-018-02873-1
1616:10.1186/s11658-024-00619-0
1283:10.1016/j.sder.2009.06.007
776:Goeppert-Mayer M. (1931).
561:
512:, a new type of squaraine-
469:
326:second-harmonic generation
234:localization of excitation
132:
18:
3104:
3018:
2965:
2853:
2825:
2753:
2720:
2665:
2654:
2578:
2556:
1510:10.1017/S0033583505004129
1180:10.1016/j.bpj.2012.08.003
878:10.1103/PhysRevLett.9.453
843:10.1103/PhysRevLett.7.229
349:, in addition monitoring
334:transition dipole moments
2486:University of Wisconsin.
2437:Two-photon suitable dyes
2198:10.1073/pnas.93.20.10763
1363:10.1021/acsomega.3c01796
803:10.1002/andp.19314010303
570:Handbook of Biophotonics
19:Not to be confused with
3145:Fluorescence techniques
2673:Fluorescence microscopy
2633:Structured illumination
2588:Bright-field microscopy
1823:10.1073/pnas.1900430116
858:Physical Review Letters
823:Physical Review Letters
714:10.1126/science.2321027
603:König, Karsten (2018).
541:Three-photon microscopy
526:3D optical data storage
472:Three-photon microscopy
458:Higher-order excitation
423:Drosophila melanogaster
314:
216:Therefore, a high peak
157:oil immersion objective
75:fluorescence microscopy
2830:List of laser articles
2745:Near-field (NSOM/SNOM)
2683:Multiphoton microscopy
2250:10.1002/anie.202311181
393:
337:fluorescent proteins.
261:
160:
143:
129:
51:
2598:Dark-field microscopy
1959:Nature Communications
1916:(1): Unit 4.11.1–24.
1566:10.1038/sj.ki.5002315
546:Two-photon absorption
391:
282:two-photon absorption
259:
209:point spread function
165:two-photon absorption
149:
140:
119:
79:two-photon excitation
29:
3150:Laboratory equipment
3005:Population inversion
2666:Fluorescence methods
2357:Analytical Chemistry
2120:(7): 1240–1256.e30.
1553:Kidney International
1441:Nature Biotechnology
1238:10.1364/OE.27.028022
1078:10.1364/ol.23.000655
747:. pp. 642–686.
483:fluorescent proteins
169:Maria Goeppert Mayer
3056:Laser beam profiler
2975:Active laser medium
2915:Free-electron laser
2835:List of laser types
2697:Image deconvolution
2678:Confocal microscopy
2618:Dispersion staining
2593:Köhler illumination
2495:Nikon MicroscopyU .
2313:2012PLoSO...751980P
2189:1996PNAS...9310763X
2183:(20): 10763–10768.
1971:2018NatCo...9..872H
1814:2019PNAS..11613680P
1808:(27): 13680–13689.
1357:(25): 22836–22843.
1230:2019OExpr..2728022S
1224:(20): 28022–28035.
1172:2012BpJ...103..907R
1160:Biophysical Journal
1070:1998OptL...23..655B
1017:1997BpJ....72.2405M
1005:Biophysical Journal
870:1962PhRvL...9..453A
835:1961PhRvL...7..229K
794:1931AnP...401..273G
706:1990Sci...248...73D
363:confocal microscopy
107:confocal microscopy
88:confocal microscopy
3155:Optical microscopy
2569:Optical microscopy
2550:Optical microscopy
2243:(51): e202311181.
1323:10.1039/D3OB01471A
1125:10.1039/C6OB02319C
958:J Neurosci Methods
394:
357:Embryonic research
303:optical resolution
278:Cornell University
262:
230:Ti-sapphire lasers
204:optical sectioning
187:showed in 1962 in
175::Eu crystal using
161:
153:numerical aperture
144:
130:
126:lily of the valley
52:
3122:
3121:
3076:Optical amplifier
2925:Solid-state laser
2785:
2784:
2730:Diffraction limit
2468:Microscopy Primer
2410:978-3-642-04700-8
2369:10.1021/ac801106w
2363:(20): 7735–7741.
1756:10.1021/ja5026326
1750:(24): 8693–8701.
1317:(44): 8829–8836.
782:Annals of Physics
762:978-1-68108-518-0
668:978-1-4398-4804-3
641:978-0-12-420138-5
614:978-3-11-042998-5
587:978-3-527-64398-1
411:photopharmacology
286:Ti-sapphire laser
103:penetration depth
48:Ti-sapphire laser
42:. Blue: mucus of
3162:
3112:
3111:
3086:Optical isolator
3051:Injection seeder
3031:Beam homogenizer
3010:Ultrashort pulse
3000:Lasing threshold
2812:
2805:
2798:
2789:
2788:
2773:
2772:
2761:
2760:
2723:limit techniques
2660:
2581:contrast methods
2579:Illumination and
2543:
2536:
2529:
2520:
2519:
2515:
2503:
2478:
2476:
2475:
2415:
2414:
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2168:
2162:
2161:
2155:
2147:
2137:
2105:
2099:
2098:
2088:
2064:
2058:
2057:
2039:
2022:(9): 1103–1111.
2007:
2001:
2000:
1990:
1950:
1944:
1943:
1933:
1901:
1895:
1894:
1884:
1860:
1854:
1853:
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1586:
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1391:
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1384:
1374:
1341:
1335:
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1266:
1260:
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1191:
1151:
1145:
1144:
1104:
1098:
1097:
1053:
1047:
1046:
1036:
1011:(6): 2405–2412.
996:
990:
989:
953:
944:
943:
924:10.1038/nmeth818
907:
901:
899:
898:
894:
888:
882:
881:
853:
847:
846:
814:
808:
807:
805:
773:
767:
766:
740:
734:
733:
689:
672:
653:
618:
599:
531:Nonlinear optics
294:light-scattering
200:Raman microscopy
99:fluorescent dyes
3170:
3169:
3165:
3164:
3163:
3161:
3160:
3159:
3125:
3124:
3123:
3118:
3100:
3014:
2995:Laser linewidth
2985:Continuous wave
2961:
2854:Types of lasers
2849:
2821:
2816:
2786:
2781:
2749:
2722:
2721:Sub-diffraction
2716:
2661:
2652:
2580:
2574:
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2547:
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2008:
2004:
1951:
1947:
1902:
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1861:
1857:
1793:
1789:
1736:
1732:
1687:
1683:
1646:
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1267:
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1105:
1101:
1054:
1050:
997:
993:
954:
947:
908:
904:
896:
890:
889:
885:
864:(11): 453–455.
854:
850:
820:
815:
811:
774:
770:
763:
741:
737:
700:(4951): 73–76.
690:
683:
679:
669:
642:
615:
588:
564:
522:
479:
474:
460:
407:calcium imaging
399:
379:
371:
369:Kidney research
359:
343:
341:Cancer research
317:
312:
254:
242:photomultiplier
181:Wolfgang Kaiser
174:
155:is 1.3 with an
135:
24:
17:
12:
11:
5:
3168:
3158:
3157:
3152:
3147:
3142:
3137:
3120:
3119:
3117:
3116:
3105:
3102:
3101:
3099:
3098:
3093:
3091:Output coupler
3088:
3083:
3081:Optical cavity
3078:
3073:
3068:
3063:
3058:
3053:
3048:
3043:
3041:Gain-switching
3038:
3033:
3028:
3022:
3020:
3016:
3015:
3013:
3012:
3007:
3002:
2997:
2992:
2990:Laser ablation
2987:
2982:
2977:
2971:
2969:
2963:
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2954:
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2911:
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2900:
2895:
2890:
2888:Carbon dioxide
2880:
2879:
2878:
2876:Liquid-crystal
2873:
2863:
2861:Chemical laser
2857:
2855:
2851:
2850:
2848:
2847:
2845:Laser acronyms
2842:
2837:
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2800:
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2655:
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2650:
2645:
2640:
2635:
2630:
2628:4Pi microscope
2625:
2620:
2615:
2610:
2605:
2603:Phase contrast
2600:
2595:
2590:
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2576:
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2421:External links
2419:
2417:
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2409:
2382:
2346:
2307:(12): e51980.
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2222:
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2100:
2079:(6): 903–912.
2059:
2016:Nature Methods
2002:
1945:
1896:
1855:
1787:
1730:
1701:(6): 823–839.
1681:
1660:(5): 862–885.
1640:
1588:
1559:(4): 416–421.
1539:
1484:
1447:(8): 763–767.
1427:
1406:(3): 179–187.
1386:
1336:
1296:
1277:(3): 190–195.
1261:
1218:Optics Express
1203:
1166:(5): 907–917.
1146:
1119:(4): 947–956.
1099:
1064:(9): 655–657.
1058:Optics Letters
1048:
991:
945:
918:(12): 932–40.
902:
883:
848:
829:(6): 229–231.
818:
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490:squaraine dyes
478:
475:
470:Main article:
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2967:Laser physics
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238:single-photon
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123:
118:
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108:
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85:
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80:
76:
72:
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68:
64:
60:
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45:
41:
37:
33:
28:
22:
3140:Cell imaging
3066:Mode locking
3019:Laser optics
2774:
2762:
2691:Three-photon
2686:
2682:
2567:
2560:
2511:
2489:
2482:
2472:. Retrieved
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2076:
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2019:
2015:
2005:
1962:
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1948:
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397:Neuroscience
380:
372:
360:
344:
318:
310:Applications
299:
291:
274:Watt W. Webb
263:
233:
226:
222:pulsed laser
193:
185:Isaac Abella
162:
142:(turquoise).
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3096:Q-switching
2957:X-ray laser
2950:Ti-sapphire
2920:Laser diode
2898:Helium–neon
2260:2445/203764
912:Nat Methods
347:skin cancer
252:Development
213:fluorophore
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3135:Microscopy
3129:Categories
2687:Two-photon
2562:Microscope
2474:2018-03-03
1965:(1): 872.
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383:SARS-CoV-2
3061:M squared
2883:Gas laser
2866:Dye laser
2269:1433-7851
2054:237366015
1832:0027-8424
1764:0002-7863
1575:0085-2538
1518:1469-8994
1461:1546-1696
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452:zebrafish
433:songbirds
330:porphyrin
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1997:29491443
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596:93908151
520:See also
514:rotaxane
502:Seta-660
498:Seta-700
494:Seta-670
492:such as
443:, mice (
437:primates
266:patented
2893:Excimer
2776:Commons
2332:3522634
2309:Bibcode
2217:8855254
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2135:8970296
2037:8958902
1988:5830414
1967:Bibcode
1931:4004770
1841:6613107
1810:Bibcode
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978:7869748
940:3339971
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