Wide-field multiphoton microscopy

Last updated May 19, 2024

Imaging system depicting a wide-field multiphoton microscope Fig4w.pdf — Imaging system depicting a wide-field multiphoton microscope

Wide-field multiphoton microscopy^[2]^[3]^[4]^[5] refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 μm).^[2]^[3]^[4] The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy.^[3] Wide-field multiphoton microscopes are not yet commercially available, but working prototypes exist in several optics laboratories.

Introduction

The main characteristic of the technique is the illumination of a wide area on the sample with a pulsed laser beam. In nonlinear optics the amount of nonlinear photons (N) generated by a pulsed beam per (illuminating) area per second is proportional to^[6]^[7]

$N\varpropto {\frac {E^{2}}{\tau A}}f$ ,

where E is the energy of the beam in Joules, τ is the duration of the pulse in seconds, A is the illuminating area in square meters, and f is the repetition rate of the pulsed beam in Hertz. Increasing the illumination area thus reduces the amount of generated nonlinear photons unless the energy is increased. Optical damage depends on the energy density, i.e. peak intensity per area I_p=E/(τA). Therefore, both the area and energy can be easily increased without the risk of optical damage if the peak intensity per area is kept low, and yet a gain in the amount of generated nonlinear photons can be obtained because of the quadratic dependence. For example, increasing both the area and energy 1000 fold, leaves the peak intensity unchanged but increases the generated nonlinear photons by 1000 fold. This 1000 extra photons are indeed generated over a larger area. In imaging this means that the extra 1000 photons are spread over the image, which at first might not seem an advantage over multiphoton scanning microscopy. The advantage however becomes evident when the size of the image and the scanning time are considered.^[3] The amount of nonlinear photons per image frame per second generated by a wide-field multiphoton microscope compared to a scanning multiphoton microscope is given by^[3]

${\frac {N_{\mathrm {wide-field} }}{N_{\mathrm {scanning} }}}=n{\frac {f_{\mathrm {wide-field} }}{f_{\mathrm {scanning} }}}$ ,

when assuming that the same peak intensity is used in both systems. Here n is the number of scanning points such that ${\textstyle A_{\mathrm {wide-field} }=nA_{\mathrm {scanning} }}$ .

Limitations

The technique is not suitable for imaging deep in scattering tissue (e.g. brain), as the image quality rapidly degrades with increasing depth
The limit to which the energy can be increased depends on laser system. Optical amplifiers such as a regenerative amplifier, can typically yield energies of up to mJ with lower repetition rates compared to oscillator based systems (e.g. Ti:sapphire laser).
Possible damage of the optics if the beam is focused somehow somewhere in the optical system to a small area. Different methods exist to achieve the required illumination without risk of damaging the optics (see Methods).
Depth cross-sectioning may be missing.

Advantages

Ultrafast imaging. A single laser shot is needed to produce one image. The frame rate is thus limited to the repetition rate of the laser system or the frame rate of the CCD camera.
Lower damage in cells. In aqueous systems (such as cells), medium to low repetition rates (1 – 200 kHz) allow for thermal diffusion to occur between illuminating pulses so that the damage threshold is higher than with high repetition rates (80 MHz).^[8]^[9]
The whole object can be observed simultaneously because of the wide-field illumination.
Larger penetration depth in biological imaging compared to one-photon fluorescence due to the longer wavelengths required.
Higher resolution than wide-field one-photon fluorescence microscopy. The optical resolution can be comparable or better than multiphoton scanning microscopes [].

Methods

There is the technical difficulty of achieving a large illumination area without destroying the imaging optics. One approach is the so-called spatiotemporal focusing^[4]^[5] in which the pulsed beam is spatially dispersed by a diffraction grating forming a 'rainbow' beam that is subsequently focused by an objective lens.^[5] The effect of focusing the 'rainbow' beam while imaging the diffraction grating forces the different wavelengths to overlap at the focal plane of the objective lens. The different wavelengths then only interfere at the overlapping volume, if no further spatial or temporal dispersion is introduced, so that the intense pulsed illumination is retrieved and capable of yielding cross-sectioned images. The axial resolution is typically 2-3 μm^[4]^[5] even with structured illumination techniques.^[10]^[11] The spatial dispersion generated by the diffraction grating ensures that the energy in the laser is spread over a wider area in the objective lens, hence reducing the possibility of damaging the lens itself.

In contrast to what was initially thought, temporal focusing is remarkably robust to scattering.^[12] Its ability to penetrate through turbid media with minimal speckle was used in optogenetics, enabling photo-excitation of arbitrary light patterns through tissue.^[12] Temporal focusing was later combined with single-pixel detection to overcome the effect of scattering on fluorescent photons.^[13] This technique, called TRAFIX, enabled wide-field imaging through biological tissue at great depths with higher signal-to-background ratio and lower photobleaching than standard point-scanning two-photon microscopy.^[13]

Another simpler method consists of two beams that are loosely focused and overlapped onto an area (~100 μm) on the sample.^[2]^[3] With this method it is possible to have access to all the elements of the $\chi ^{(2)}$ tensor thanks to the capability of being able to change the polarisation of each beam independently.

Related Research Articles

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

<span class="mw-page-title-main">Nonlinear optics</span> Branch of physics

Nonlinear optics (NLO) is the branch of optics that describes the behaviour of light in nonlinear media, that is, media in which the polarization density P responds non-linearly to the electric field E of the light. The non-linearity is typically observed only at very high light intensities (when the electric field of the light is >10⁸ V/m and thus comparable to the atomic electric field of ~10¹¹ V/m) such as those provided by lasers. Above the Schwinger limit, the vacuum itself is expected to become nonlinear. In nonlinear optics, the superposition principle no longer holds.

$<span class="mw-page-title-main">Diffraction-limited system</span> Optical system with resolution performance at the instruments theoretical limit$

In optics, any optical instrument or system – a microscope, telescope, or camera – has a principal limit to its resolution due to the physics of diffraction. An optical instrument is said to be diffraction-limited if it has reached this limit of resolution performance. Other factors may affect an optical system's performance, such as lens imperfections or aberrations, but these are caused by errors in the manufacture or calculation of a lens, whereas the diffraction limit is the maximum resolution possible for a theoretically perfect, or ideal, optical system.

Ti:sapphire lasers (also known as Ti:Al₂O₃ lasers, titanium-sapphire lasers, or Ti:sapphs) are tunable lasers which emit red and near-infrared light in the range from 650 to 1100 nanometers. These lasers are mainly used in scientific research because of their tunability and their ability to generate ultrashort pulses thanks to its broad light emission spectrum. Lasers based on Ti:sapphire were first constructed and invented in June 1982 by Peter Moulton at the MIT Lincoln Laboratory.

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

In optics, an ultrashort pulse, also known as an ultrafast event, is an electromagnetic pulse whose time duration is of the order of a picosecond or less. Such pulses have a broadband optical spectrum, and can be created by mode-locked oscillators. Amplification of ultrashort pulses almost always requires the technique of chirped pulse amplification, in order to avoid damage to the gain medium of the amplifier.

In optics, various autocorrelation functions can be experimentally realized. The field autocorrelation may be used to calculate the spectrum of a source of light, while the intensity autocorrelation and the interferometric autocorrelation are commonly used to estimate the duration of ultrashort pulses produced by modelocked lasers. The laser pulse duration cannot be easily measured by optoelectronic methods, since the response time of photodiodes and oscilloscopes are at best of the order of 200 femtoseconds, yet laser pulses can be made as short as a few femtoseconds.

<span class="mw-page-title-main">Two-photon excitation microscopy</span> Fluorescence imaging technique

Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation 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 non-linearity 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 confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

Multiphoton intrapulse interference phase scan (MIIPS) is a method used in ultrashort laser technology that simultaneously measures, and compensates femtosecond laser pulses using an adaptive pulse shaper. When an ultrashort laser pulse reaches a duration of less than a few hundred femtosecond, it becomes critical to characterize its duration, its temporal intensity curve, or its electric field as a function of time. Classical photodetectors measuring the intensity of light are still too slow to allow for a direct measurement, even with the fastest photodiodes or streak cameras.

<span class="mw-page-title-main">Harmonic generation</span> Nonlinear optical process

Harmonic generation is a nonlinear optical process in which $photons with the same frequency interact with a nonlinear material, are "combined", and generate a new photon with times the energy of the initial photons.$

High-harmonic generation (HHG) is a non-linear process during which a target is illuminated by an intense laser pulse. Under such conditions, the sample will emit the high harmonics of the generation beam. Due to the coherent nature of the process, high-harmonics generation is a prerequisite of attosecond physics.

3D optical data storage is any form of optical data storage in which information can be recorded or read with three-dimensional resolution.

Second-harmonic imaging microscopy (SHIM) is based on a nonlinear optical effect known as second-harmonic generation (SHG). SHIM has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. A second-harmonic microscope obtains contrasts from variations in a specimen's ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in optical density, path length, or refractive index of the specimen. SHG requires intense laser light passing through a material with a noncentrosymmetric molecular structure, either inherent or induced externally, for example by an electric field.

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

Super-resolution photoacoustic imaging is a set of techniques used to enhance spatial resolution in photoacoustic imaging. Specifically, these techniques primarily break the optical diffraction limit of the photoacoustic imaging system. It can be achieved in a variety of mechanisms, such as blind structured illumination, multi-speckle illumination, or photo-imprint photoacoustic microscopy in Figure 1.

Pump–probe microscopy is a non-linear optical imaging modality used in femtochemistry to study chemical reactions. It generates high-contrast images from endogenous non-fluorescent targets. It has numerous applications, including materials science, medicine, and art restoration.

Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by $, which is much faster than that of two-photon microscopy by . In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three-photon microscopy to have higher resolution than conventional microscopy.$

Coherent Raman scattering (CRS) microscopy is a multi-photon microscopy technique based on Raman-active vibrational modes of molecules. The two major techniques in CRS microscopy are stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS). SRS and CARS were theoretically predicted and experimentally realized in the 1960s. In 1982 the first CARS microscope was demonstrated. In 1999, CARS microscopy using a collinear geometry and high numerical aperture objective were developed in Xiaoliang Sunney Xie's lab at Harvard University. This advancement made the technique more compatible with modern laser scanning microscopes. Since then, CRS's popularity in biomedical research started to grow. CRS is mainly used to image lipid, protein, and other bio-molecules in live or fixed cells or tissues without labeling or staining. CRS can also be used to image samples labeled with Raman tags, which can avoid interference from other molecules and normally allows for stronger CRS signals than would normally be obtained for common biomolecules. CRS also finds application in other fields, such as material science and environmental science.

Ultrafast scanning electron microscopy (UFSEM) combines two microscopic modalities, Pump-probe microscopy and Scanning electron microscope, to gather temporal and spatial resolution phenomena. The technique uses ultrashort laser pulses for pump excitation of the material and the sample response will be detected by an Everhart-Thornley detector. Acquiring data depends mainly on formation of images by raster scan mode after pumping with short laser pulse at different delay times. The characterization of the output image will be done through the temporal resolution aspect. Thus, the idea is to exploit the shorter DeBroglie wavelength in respect to the photons which has great impact to increase the resolution about 1 nm. That technique is an up-to-date approach to study the dynamic of charge on material surfaces.

In atomic physics, non-degenerate two-photon absorption or two-color two-photon excitation is a type of two-photon absorption (TPA) where two photons with different energies are (almost) simultaneously absorbed by a molecule, promoting a molecular electronic transition from a lower energy state to a higher energy state. The sum of the energies of the two photons is equal to, or larger than, the total energy of the transition.

References

↑ Macias-Romero, Carlos; Didier, Marie E. P.; Jourdain, Pascal; Marquet, Pierre; Magistretti, Pierre; Tarun, Orly B.; Zubkovs, Vitalijs; Radenovic, Aleksandra; Roke, Sylvie (2014-12-15). "High throughput second harmonic imaging for label-free biological applications" (PDF). Optics Express. 22 (25): 31102–12. doi: 10.1364/oe.22.031102 . ISSN 1094-4087. PMID 25607059.
1 2 3 Peterson, Mark D.; Hayes, Patrick L.; Martinez, Imee Su; Cass, Laura C.; Achtyl, Jennifer L.; Weiss, Emily A.; Geiger, Franz M. (2011-05-01). "Second harmonic generation imaging with a kHz amplifier [Invited]". Optical Materials Express. 1 (1): 57. doi:10.1364/ome.1.000057.
1 2 3 4 5 6 Macias-Romero, Carlos; Didier, Marie E. P.; Jourdain, Pascal; Marquet, Pierre; Magistretti, Pierre; Tarun, Orly B.; Zubkovs, Vitalijs; Radenovic, Aleksandra; Roke, Sylvie (2014-12-15). "High throughput second harmonic imaging for label-free biological applications" (PDF). Optics Express. 22 (25): 31102. doi: 10.1364/oe.22.031102 . PMID 25607059.
1 2 3 4 Cheng, Li-Chung; Chang, Chia-Yuan; Lin, Chun-Yu; Cho, Keng-Chi; Yen, Wei-Chung; Chang, Nan-Shan; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen (2012-04-09). "Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning". Optics Express. 20 (8): 8939–48. doi: 10.1364/oe.20.008939 . PMID 22513605.
1 2 3 4 Oron, Dan; Tal, Eran; Silberberg, Yaron (2005-03-07). "Scanningless depth-resolved microscopy". Optics Express. 13 (5): 1468–76. doi: 10.1364/opex.13.001468 . PMID 19495022.
↑ Shen, Y. R. (1989-02-09). "Surface properties probed by second-harmonic and sum-frequency generation". Nature. 337 (6207): 519–525. doi:10.1038/337519a0. S2CID 4233043.
↑ Dadap, J. I.; Hu, X. F.; Russell, M.; Ekerdt, J. G.; Lowell, J. K.; Downer, M. C. (1995-12-01). "Analysis of second-harmonic generation by unamplified, high-repetition-rate, ultrashort laser pulses at Si(001) interfaces". IEEE Journal of Selected Topics in Quantum Electronics. 1 (4): 1145–1155. doi:10.1109/2944.488693. ISSN 1077-260X.
↑ Macias-Romero, C.; Zubkovs, V.; Wang, S.; Roke, S. (2016-04-01). "Wide-field medium-repetition-rate multiphoton microscopy reduces photodamage of living cells". Biomedical Optics Express. 7 (4): 1458–1467. doi:10.1364/boe.7.001458. ISSN 2156-7085. PMC 4929654 . PMID 27446668.
↑ Harzic, R. Le; Riemann, I.; König, K.; Wüllner, C.; Donitzky, C. (2007-12-01). "Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345nm". Journal of Applied Physics. 102 (11): 114701. doi: 10.1063/1.2818107 . ISSN 0021-8979.
↑ Choi, Heejin; Yew, Elijah Y. S.; Hallacoglu, Bertan; Fantini, Sergio; Sheppard, Colin J. R.; So, Peter T. C. (2013-07-01). "Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination". Biomedical Optics Express. 4 (7): 995–1005. doi:10.1364/boe.4.000995. PMC 3704103 . PMID 23847726.
↑ Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C. (2011-01-01). "Wide-field two-photon microscopy with temporal focusing and HiLo background rejection". In Periasamy, Ammasi; König, Karsten; So, Peter T. C (eds.). Multiphoton Microscopy in the Biomedical Sciences XI. Vol. 7903. pp. 79031O–79031O–6. doi:10.1117/12.876068. hdl:1721.1/120979. S2CID 120466973.
1 2 Papagiakoumou, Eirini; Bègue, Aurélien; Leshem, Ben; Schwartz, Osip; Stell, Brandon M.; Bradley, Jonathan; Oron, Dan; Emiliani, Valentina (2013-02-17). "Functional patterned multiphoton excitation deep inside scattering tissue" (PDF). Nature Photonics. 7 (4): 274–278. doi:10.1038/nphoton.2013.9. ISSN 1749-4885.
1 2 Escobet-Montalbán, Adrià; Spesyvtsev, Roman; Chen, Mingzhou; Saber, Wardiya Afshar; Andrews, Melissa; Herrington, C. Simon; Mazilu, Michael; Dholakia, Kishan (2018-10-01). "Wide-field multiphoton imaging through scattering media without correction". Science Advances. 4 (10): eaau1338. arXiv: 1712.07415 . doi:10.1126/sciadv.aau1338. ISSN 2375-2548. PMC 6184782 . PMID 30333995.

This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.

[1] Macias-Romero, Carlos; Didier, Marie E. P.; Jourdain, Pascal; Marquet, Pierre; Magistretti, Pierre; Tarun, Orly B.; Zubkovs, Vitalijs; Radenovic, Aleksandra; Roke, Sylvie (2014-12-15). "High throughput second harmonic imaging for label-free biological applications" (PDF). Optics Express. 22 (25): 31102–12. doi: 10.1364/oe.22.031102 . ISSN 1094-4087. PMID 25607059.

[:1-2] 1 2 3 Peterson, Mark D.; Hayes, Patrick L.; Martinez, Imee Su; Cass, Laura C.; Achtyl, Jennifer L.; Weiss, Emily A.; Geiger, Franz M. (2011-05-01). "Second harmonic generation imaging with a kHz amplifier [Invited]". Optical Materials Express. 1 (1): 57. doi:10.1364/ome.1.000057.

[:0-3] 1 2 3 4 5 6 Macias-Romero, Carlos; Didier, Marie E. P.; Jourdain, Pascal; Marquet, Pierre; Magistretti, Pierre; Tarun, Orly B.; Zubkovs, Vitalijs; Radenovic, Aleksandra; Roke, Sylvie (2014-12-15). "High throughput second harmonic imaging for label-free biological applications" (PDF). Optics Express. 22 (25): 31102. doi: 10.1364/oe.22.031102 . PMID 25607059.

[:2-4] 1 2 3 4 Cheng, Li-Chung; Chang, Chia-Yuan; Lin, Chun-Yu; Cho, Keng-Chi; Yen, Wei-Chung; Chang, Nan-Shan; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen (2012-04-09). "Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning". Optics Express. 20 (8): 8939–48. doi: 10.1364/oe.20.008939 . PMID 22513605.

[:3-5] 1 2 3 4 Oron, Dan; Tal, Eran; Silberberg, Yaron (2005-03-07). "Scanningless depth-resolved microscopy". Optics Express. 13 (5): 1468–76. doi: 10.1364/opex.13.001468 . PMID 19495022.

[6] Shen, Y. R. (1989-02-09). "Surface properties probed by second-harmonic and sum-frequency generation". Nature. 337 (6207): 519–525. doi:10.1038/337519a0. S2CID 4233043.

[7] Dadap, J. I.; Hu, X. F.; Russell, M.; Ekerdt, J. G.; Lowell, J. K.; Downer, M. C. (1995-12-01). "Analysis of second-harmonic generation by unamplified, high-repetition-rate, ultrashort laser pulses at Si(001) interfaces". IEEE Journal of Selected Topics in Quantum Electronics. 1 (4): 1145–1155. doi:10.1109/2944.488693. ISSN 1077-260X.

[8] Macias-Romero, C.; Zubkovs, V.; Wang, S.; Roke, S. (2016-04-01). "Wide-field medium-repetition-rate multiphoton microscopy reduces photodamage of living cells". Biomedical Optics Express. 7 (4): 1458–1467. doi:10.1364/boe.7.001458. ISSN 2156-7085. PMC 4929654 . PMID 27446668.

[9] Harzic, R. Le; Riemann, I.; König, K.; Wüllner, C.; Donitzky, C. (2007-12-01). "Influence of femtosecond laser pulse irradiation on the viability of cells at 1035, 517, and 345nm". Journal of Applied Physics. 102 (11): 114701. doi: 10.1063/1.2818107 . ISSN 0021-8979.

[10] Choi, Heejin; Yew, Elijah Y. S.; Hallacoglu, Bertan; Fantini, Sergio; Sheppard, Colin J. R.; So, Peter T. C. (2013-07-01). "Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination". Biomedical Optics Express. 4 (7): 995–1005. doi:10.1364/boe.4.000995. PMC 3704103 . PMID 23847726.

[11] Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C. (2011-01-01). "Wide-field two-photon microscopy with temporal focusing and HiLo background rejection". In Periasamy, Ammasi; König, Karsten; So, Peter T. C (eds.). Multiphoton Microscopy in the Biomedical Sciences XI. Vol. 7903. pp. 79031O–79031O–6. doi:10.1117/12.876068. hdl:1721.1/120979. S2CID 120466973.

[:4-12] 1 2 Papagiakoumou, Eirini; Bègue, Aurélien; Leshem, Ben; Schwartz, Osip; Stell, Brandon M.; Bradley, Jonathan; Oron, Dan; Emiliani, Valentina (2013-02-17). "Functional patterned multiphoton excitation deep inside scattering tissue" (PDF). Nature Photonics. 7 (4): 274–278. doi:10.1038/nphoton.2013.9. ISSN 1749-4885.

[:5-13] 1 2 Escobet-Montalbán, Adrià; Spesyvtsev, Roman; Chen, Mingzhou; Saber, Wardiya Afshar; Andrews, Melissa; Herrington, C. Simon; Mazilu, Michael; Dholakia, Kishan (2018-10-01). "Wide-field multiphoton imaging through scattering media without correction". Science Advances. 4 (10): eaau1338. arXiv: 1712.07415 . doi:10.1126/sciadv.aau1338. ISSN 2375-2548. PMC 6184782 . PMID 30333995.

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]