Multiphoton microscopy combined with genetically encoded ﬂuorescent indicators is a central tool in biology. Three- photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor ﬂuorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green ﬂuorescent protein (GFP) and red ﬂuorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a ﬁxed mouse brain, chick embryo spinal cord, and live adult zebraﬁsh brain, with an improved signal-to-background ratio compared to multicolor two- photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues. Introduction out-of-focus ﬂuorescence, which degrades the signal-to- background ratio and effectively limits the imaging Multiphoton microscopy is now established as the 3,4 reference method for both deep and live ﬂuorescence depth . One recently demonstrated effective strategy for imaging of biological tissues. Indeed, such an approach deeper multiphoton imaging is to use three-photon (3P) delivers a sub-cellular resolution at depths of hundreds of excitation while shifting the excitation to the short- micrometers inside intact tissues. Together with the rapid wavelength infrared (SWIR) range to approximately 1300 5 4 progress in genetically engineered probes, two-photon nm (ref. ) or 1700 nm (ref. ). This strategy has two key microscopy is a key enabling technology in ﬁelds such as advantages: (i) when the laser is focused at a depth neuroscience, developmental biology, immunology, and equivalent to several times the scattering mean free path others. However, tissue penetration for two-photon inside the tissue, 3P excitation shows a greatly improved 2,3 3,4 microscopy is limited by scattering . When laser power rejection of the out-of-focus ﬂuorescence background ; is not a limiting parameter, compensating for the expo- (ii) the wavelength windows at approximately 1300 and nential decrease in unscattered light with depth results in 1700 nm offer a better combination of tissue scattering and absorption properties compared to the 700–1100 nm wavelength range commonly used in two-photon-excited Correspondence: Emmanuel Beaurepaire (emmanuel. ﬂuorescence (2PEF) microscopy , enabling superior firstname.lastname@example.org) or Frédéric. Druon (frederic. penetration. In addition, 1300 nm was found to be a email@example.com) nearly optimal wavelength for 3P excitation of green Laboratory Charles Fabry, Institut d’Optique Graduate School, CNRS, Université Paris-Saclay, 91128 Palaiseau, France ﬂuorescent protein (GFP) and derived calcium indica- Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS, INSERM, tors , and 1700 nm is appropriate for 3P excitation of 91128 Palaiseau, France widely used genetically encoded red probes, such as red Full list of author information is available at the end of the article These authors contributed equally: Khmaies Guesmi, Lamiae Abdeladim. © The Author(s) 2018 Open Access This article is licensed under a Creative Comemons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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The ﬁrst 7,8 2 weakness of 3P absorption cross-sections, however , the crystal was 3 mm long with a 10 × 1 mm section, opti- pulsed excitation regime typically used for 2P microscopy mized to obtain a gain of 130 for a pump peak irradiance (80 MHz, 100 fs, up to 2 nJ pulses at the sample surface) is of 50 GW/cm and an energy of 3.2 µJ. The second crystal not appropriate for 3P microscopy. Instead, pulse trains in was a 3-mm-long MgO:PPLN with a 12 × 3 mm section the MHz, sub-100 fs, and few hundreds nJ range are and an aperture scaled up to sustain 33 µJ pump pulses necessary to realize rapid deep-tissue 3P imaging while while avoiding the onset of beam distortions. The pump 9,10 2 minimizing tissue heating . and signal beam diameters at 1/e in the ﬁrst OPCPA Optimized laser sources are required to develop such an crystal were 190 × 180 and 270 × 260 µm , respectively. 6,11–13 imaging approach to its full potential . In particular, The evolution of signal pulse energy with respect to pump as many potential applications require study of interac- energy is shown in Supplementary Figure S1. We tions between cells/tissue components labeled with dif- observed a ring-shaped distortion of the output beam ferent ﬂuorophores, one important challenge is to develop spatial proﬁle at pump energies exceeding 3.2 µJ, corre- laser sources that enable efﬁcient two-color three-photon sponding to pump powers >4 W. The threshold for the imaging. Given the wavelength separation between the appearance of this degradation depends on the average SWIR bands relevant for 3P microscopy (1.3 and 1.7 µm), and peak power and may be attributed to thermal effects a single excitation band is insufﬁcient for simultaneous and nonlinear absorption as reported in ref. . Alter- imaging of, for example, GFP-labeled and RFP-labeled natively, linear absorption, the photorefractive effect and structures. pyroelectric effects could account for this beam distortion In this article, we report on the ﬁrst multiband SWIR at high pump power . The optimum pump energy source that is able to simultaneously emit ultrashort eventually used in the ﬁrst stage was 3.2 µJ, corresponding pulses at 1.3 and 1.7 µm with characteristics appropriate to a pump irradiance of 50 GW/cm and resulting in a for 3P microscopy (sub-70 fs duration, 1.25 MHz repeti- gain of 130. tion rate, and µJ-range pulse energy). This design, which is In the second nonlinear crystal, beam sizes were scaled based on optical parametric chirped-pulse ampliﬁcation up to accommodate the available pump energy of 33 µJ. (OPCPA), outperforms current alternative sources and The pump and input signal beams were collimated with has intrinsically stable characteristics. The combined diameters of 2 × 1.7 and 1.8 × 1.6 mm , respectively, output enables simultaneous 3P ﬂuorescence imaging of enabling operation at full energy without the onset of various combinations of GFPs and RFPs along with label- ring-like beam distortions. 14–16 free third-harmonic generation (THG) imaging of To reach the wavelength of interest of 1.3 µm, second- the tissue structure. We demonstrate in-depth dual-color harmonic generation (SHG) for the idler pulses was car- three-photon imaging in ﬁxed mouse brains and in live ried out in a third MgO:PPLN crystal. The idler was developing spinal cord explants and compare these data focused by a 100-mm CaF lens in a crystal with a poling with those obtained by two-photon microscopy. period of 35.8 µm and a working temperature of 30 °C. Since the 2.6 µm idler beam can be perturbed by air Materials and methods humidity, we minimized the propagation distance OPCPA implementation between the OPA and frequency-doubling stage. The For the simultaneous generation of 1.3 and 1.7 µm overall efﬁciency of the SHG process was 32%. This efﬁ- pulses, we started with a pump beam at a wavelength of ciency was limited by the parasitic SHG of the 1.3 µm 1.03 µm provided by an Ytterbium-doped ﬁber ampliﬁer beam because the poling period was close to the third- (YDFA) (Satsuma HP3, Amplitude Systemes) and imple- order quasi-phase matching for this interaction. The mented a two-stage OPCPA. We generated a signal beam average power generated at 650 nm under standard at 1.7 µm and therefore a simultaneous idler beam at ≈2.6 operating conditions was 500 mW compared with 890 µm, which we then frequency-doubled to obtain 1.3 pul- mW at 1.3 µm. Finally, the 1.3 and 1.7 µm beams were ses. The input signal to the OPCPA was obtained by recombined using a dichroic mirror (HR1300/HT1700, supercontinuum (SC) generation driven by the pump Altechna, Vilnius, Lithuania). pulses at 1.03 µm, resulting in a spectrum extending up to 2 µm (Supplementary Figure S1). We optimized SC gen- Multimodal multiphoton imaging eration by moving the YAG crystal along the focal plane Imaging was carried out using a lab-built laser scanning and measuring the output power after a 1400-nm-long microscope. The excitation source was either the dual- pass ﬁlter. We estimated the seed energy contained in the band OCPCA described in this article or a Ti:Sapphire + 1.6–1.8 spectral band to be 1 nJ. optical parametric oscillator (OPO) chain optimized for The optical parametric ampliﬁcation (OPA) stages were two-photon microscopy (Chameleon Ultra2 and MPX, composed of two MgO:PPLN crystals (30.05 µm poling Coherent, CA, USA). Source selection was achieved using Guesmi et al. Light: Science & Applications (2018) 7:12 Page 3 of 9 a movable mirror. Power in the four beams was inde- expressing GFP and one of the red FPs were mixed at a 1:1 pendently controlled using motorized wave plates and ratio, deposited onto a 13 mm glass coverslip coated with polarizers. The beams sizes at the microscope input were collagen (50 µg/mL, Sigma), and cultured for an addi- controlled with adjustable two-lens telescopes. Scanning tional 24 h. The cells were ﬁxed with 4% paraformalde- was carried out using galvanometric scanners (series 6215, hyde (PFA, Antigenﬁx, Diapath) and mounted in Cambridge Technology, Bedford, MA, USA). Excitation Vectashield medium (Vector laboratories). beams were focused into the sample using a water immersion objective optimized for IR transmission (×25, Mouse brain samples 1.05NA, XLPLN25XWMP2, Olympus, Tokyo, Japan). Animal procedures were carried out according to the The objective transmission was higher than 65% at all of institutional guidelines. Mice were housed in a 14 h light/ the wavelengths used. Scanning and acquisition were 10 h dark cycle with free access to food. synchronized using lab-written LabVIEW software and a To compare the performance of 3PEF vs. 2PEF for RFP multichannel I/O board (PCI-6115, National Instruments, imaging, we used a transgenic mouse line expressing a Austin, TX, USA). Signals were detected in the backward Cytbow transgene under the broadly active CAG pro- (epi) direction by GaAsP modules (H7422P-40, Hama- moter. Cre recombination, induced by intercrossing with IRES-Cre 21 matsu, Japan) using lab-designed electronics enabling Emx1 mice , yielded expression of tdTomato mixed counting/analog detection. Fluorescence and har- throughout the cerebral cortex of the resulting offspring. monic light was collected using a dichroic mirror (BLP01- An 1-month-old animal was deeply anesthetized with 830R, Semrock, Rochester, NY, USA) and directed toward sodium pentobarbital (150 mg/kg) and perfused trans- three independent detectors using dichroic mirrors cardially with 4% PFA. After dissection, the brain was (Semrock 495 and 561 nm). Bandpass ﬁlters were used in postﬁxed in PFA and mounted in 3% agarose. The block front of the detectors to collect blue (Semrock 433/24), of tissue was immersed in phosphate-buffered saline green (Semrock 520/15), and red (Semrock 607/70) light. (PBS), and the agarose covering the cortex was sliced off Excitation power was increased with the imaging depth prior to imaging of the cortical surface. (see Supplementary movies, legends). We note that the To generate brain samples co-expressing RFPs and dispersion introduced by the microscope components was GFPs, we used in utero electroporation as previously not fully compensated during the imaging experiments. described . Brieﬂy, timed-pregnant females (E13) were We optimized the duration of the 1700 nm pulse at the anesthetized with ketamine/xylazine and a midline focus of the objective by incorporating silicon slabs of laparotomy was carried out to expose uterine horns. One various thickness into the beam path while monitoring microliter of DNA transfection mix was injected with a the THG signal obtained from a glass–water interface. We glass capillary pipette into the lateral ventricle of the measured the pulse duration of the 1300 nm pulse at the embryos. To maximize the number of labeled cells, we focus using a microscopy autocorrelator (CARPE, APE, used DNA transposable elements based on the Tol2 and Germany). Overall, we estimated that the pulse durations PiggyBac transposition systems, respectively, encoding a at the focus were approximately 80–100 and 130–150 fs nuclear-addressed cytoplasmic GFP (Tol2-CAG-H2B- for the 1700 and 1300 nm pulses, respectively. The pixel GFP, 1 µg) and a cytoplasmic RFP (PiggyBac-CAG- dwell time was typically 5 µs, i.e., the acquisition times mRFP, 1 µg) along with plasmids encoding the corre- were typically 1 to 4 s for a 500 × 500 pixel image. sponding transposases (CAG-Tol2ase and CAG-PBase, 0.2 µg). DNA was combined 9:1 with Fast Green dye Image analysis and processing (Sigma). An anode with a diameter of 3 mm, Tweezer- Data were analyzed using FIJI. Flat-ﬁeld correction was trodes (Sonidel Limited), was placed above the dorsal applied to the three-channel (red/green/THG) time-lapse telencephalon before the application of three 35 V pulses dataset to correct for illumination inhomogeneity. For with a 50 ms duration, followed by three additional pulses each channel, the illumination proﬁle was computed by in the reverse orientation. The incision site was then averaging all of the intensity images for the t-stack fol- closed with sutures (4-0, Ethicon) and mice were allowed lowed by Gaussian blurring. Each image was then divided to recover in a clean cage. A brain from a P15 pup was by the corresponding normalized illumination proﬁle. prepared and mounted in agarose as described above. The block was sectioned into two parts in the coronal orien- Cell experiments tation at mid-brain level, and imaging of the cerebral HEK 293 cells were grown for 24 h in 6-well plates (5 × cortex carried out on the resulting surface. 10 cells per well) and transfected with 1 µg of DNA plasmids encoding either EGFP, mCherry, or tdTomato Chick embryo spinal cord samples under the strong CAG or CMV promoter using Lipo- Electroporation in the chick neural tube was performed fectamine 2000 (Invitrogen). After 24 h, transfected cells at embryonic day 2 following the procedure described in a Guesmi et al. Light: Science & Applications (2018) 7:12 Page 4 of 9 3 µJ Yb:fiber laser system YAG Si 1030 nm 40 µJ Supercontinuum Stretcher 1.25 MHz 310 fs DM 3.2 µJ PPLN 3 mm 33 µJ Delay lines OPA1 0.71 µJ 69 fs DM DM @ 1300 nm 2600 nm PPLN 1 mm Si PPLN 3 mm SHG Compressor OPA2 DM SiO 1700 nm 3.1 µJ 65 fs Compressor 3PEF excitation @ 1700 nm Ti:S+OPO laser system 940 nm or 1100 nm 1.3 µm 1.7 µm XY 80 MHz 150 fs 2PEF excitation 69 fs 65 fs DM ×1.44 ×1.44 Obj THG GFP RFP –250 0 250 500 750 Sample Delay (fs) 1.3 µm 1.7 µm Scattering + absorption Absorption Scattering 800 1200 1600 2000 2400 2800 Wavelength (nm) Fig. 1 Dual-band SWIR laser source optimized for three-photon microscopy. a Experimental setup showing the source design. A Yb:ﬁber laser providing 1030 nm pulses at 1.25 MHz is used for supercontinuum generation in a YAG crystal and for ampliﬁcation in a two-stage OPCPA arrangement. Signal and idler beams are produced at 1.7 and 2.6 µm and the idler is frequency-doubled, resulting in simultaneous emission at 1.7 and 1.3 µm with pulse energies in the µJ range. The beams are injected into a scanning microscope for three-photon microscopy. Alternatively, an 80 MHz pulse train at 920 or 1100 nm is used for comparison with two-photon excitation. DM dichroic mirrors, OPA optical parametric ampliﬁcation stages, XY beam scanning, Obj microscope objective. b Measured temporal proﬁles for the two SWIR beams. c Parameters limiting deep-tissue imaging in multiphoton microscopy. The solid black curve combining tissue scattering and water absorption indicates the interest in use of the 1.3 and 1.7 µm wavelength ranges for in-depth imaging. The red and brown graphs reproduce the measured spectra for our source outputs, targeting the spectral regions of interest –1 Absorption and scattering (cm ) Intensity (norm) Guesmi et al. Light: Science & Applications (2018) 7:12 Page 5 of 9 previous study . Brieﬂy, DNA was injected with a glass a signal beam at λ = 1700 nm and therefore a simulta- 1 1 capillary in the neural tube and ﬁve 50 ms pulses of 25 V neous idler beam at λ ¼ λ λ 2600 nm, 0 1 with 100 ms interval were applied using a pair of 5 mm which we then frequency-doubled to obtain 1.3 µm pul- gold-plated electrodes (BTX Genetrode model 512) ses. Therefore, the main challenge was to reach µJ-range separated by 4 mm and a square-wave electroporator pulse energies in both beams at a MHz repetition rate. In (Nepa Gene, CUY21SC). particular, the idler ampliﬁcation and subsequent For ﬁxed spinal cord samples, plasmids encoding EGFP frequency-doubling stages were required to produce a 1.3 (CAG-EGFP) and mRFP (CAG-mRFP) were co- µm pulse train with sufﬁcient energy for efﬁcient 3P electroporated at an equal concentration (1 µg/µL). imaging. After ﬁxation in 4% PFA, E9 spinal cords were dissected Our experimental layout is schematically illustrated in and pinned on a silicone-coated dish ﬁlled with PBS, with Fig. 1a and involved an YDFA pump at 1030 nm and two the dorsal horn on top, which was positioned under the successive OPCPA stages (see also Materials and methods microscope objective for imaging. section). The pump was a commercially available YDFA For live imaging experiments, a plasmid encoding the delivering 50 W of average power in the form of a 1.25 proneural gene Neurog2 (CAG-Neurog2, 1 µg/µL) was MHz, 310 fs pulse train. The signal beam (1700 nm) was used to induce neural differentiation; this plasmid was co- obtained from an SC produced by focusing one portion of transfected with CAG-EGFP (1 µg/µL) and CAG-H2B- the 1030 nm pump into a YAG crystal. Stretching and mRFP (0.5 µg/µL) to visualize the cell cytoplasm and compression of the signal and idler pulses was achieved chromatin, respectively. Embryos were harvested one day using Si and SiO plates. Therefore, the system relied only after electroporation, transferred to F12 medium, and slit on compact and stable bulk elements. along their midline from the hindbrain to the caudal end. One key issue in the SC generation step was to reach The electroporated side of the neural tube was peeled off long wavelengths, such as 1.7 µm, before the occurrence and mounted in a glass-bottom culture dish (MatTek, of multi-ﬁlamentation, which has been reported to be P35G-0-14-C) using a thin layer of 1% low-melting dependent on the initial pulse duration and focusing 30,31 agarose dissolved in F12 medium. After polymerization conditions . We obtained a good-quality SC by of the agarose, the dishes were ﬁlled with 3 mL of culture focusing 3 µJ, 310 fs pulses using a 150-mm lens into a 10- medium (F12/penicillin streptomycin/sodium pyruvate) mm-long YAG crystal. The SC spectrum extended up to and transferred to a heating plate (37 °C) positioned under 2 µm, thereby providing a seed between 1.6 and 1.7 µm for the microscope objective. the parametric ampliﬁcation stages. Telencephalon live imaging in adult zebraﬁsh Parametric ampliﬁcation is limited by beam distortions Three-month-old adult zebraﬁsh (Danio rerio) were appearing at high peak and/or average pump power in a used. The transgenic line was created by crossing Tg(gfap: single-stage setup. We therefore implemented two stages 23 24 dTomato) with Tg(deltaA:GFP) in a casper double of ampliﬁcation to achieve a sufﬁcient gain. This conﬁg- mutant background (roy−/−;nacre−/−) and referred to uration provides additional degrees of freedom, with two as gfap:dTomato; deltaA:GFP. This line, therefore, exhi- MgO: PPLN crystals designed to optimize the gain and bits dTomato labeling of neural stem cells, which are conversion efﬁciency. We stretched the signal pulse using radial glia cells, and GFP labeling of neural progenitors a 3-mm-long Si plate before sending it to the ﬁrst cells, in particular in the telencephalon. Anesthesia and ampliﬁcation stage. The ﬁrst MgO:PPLN crystal was mounting for imaging were conducted as in previous designed to obtain a high gain (>100 typically) with a 26,27 studies . Brieﬂy, anesthesia was induced with 0.02% pump pulse energy in the 1–5 µJ range. The evolution of MS222 (Sigma) for approximately 90 s and maintained the signal pulse energy with respect to the pump energy is during imaging with 0.01% MS222. Anesthetized ﬁsh were shown in Supplementary Figure S1. We observed a ring- mounted in a home-made plastic dish between pieces of shaped distortion of the output beam proﬁle at pump sponge. All experiments and husbandry were carried out energies exceeding 3.2 µJ, corresponding to average pump in accordance with the institutional guidelines. powers >4 W. The threshold for the appearance of this degradation depends both on the average and peak power Results and discussion and may be attributed to thermal effects and nonlinear 17,18 Multiband SWIR source with MHz repetition rate absorption . Alternatively, linear absorption, the pho- OPCPA is an attractive approach for developing a MHz torefractive effect, and pyroelectric effects can also lead to source for 3P microscopy since it is, in principle, com- such a beam distortion at a high pump power. Taking this patible with energetic short pulses and can enable access distortion into account, the nominal pump energy used in 28,29 to long wavelengths . Our original strategy for simul- the ﬁrst stage was 3.2 µJ, corresponding to a pump irra- taneously generating 1.3 and 1.7 µm pulses was as follows: diance of 50 GW/cm and resulting in a gain of 130. The starting from a pump beam at λ = 1030 nm, we generated central signal wavelength was adjusted to be between 0 Guesmi et al. Light: Science & Applications (2018) 7:12 Page 6 of 9 1700 and 1710 nm using a delay line to produce an The size and divergence for the 1.3 and 1.7 µm beams optimal signal-idler combination for our purpose, that is, were adjusted using independent telescopes, and the corresponding to an idler center wavelength between beams were recombined using a dichroic mirror before 2590 and 2610 nm. The signal beam bandwidth at the entering the microscope. We estimated the axial resolu- output of the ﬁrst stage reached 107 nm full-width at half- tions provided by both beams by recording THG image maximum (FWHM) (Figure 1 of Supplement 1). The stacks from a glass–water interface. The FWHM of the z- scans were measured to be 2.8 ± 0.1 and 3.8 ± 0.1 µm for crystal for the second stage of ampliﬁcation was chosen to have a larger aperture to sustain 33 µJ pump pulses and to 1.3 and 1.7 µm excitation, respectively, with an axial provide an additional gain of 30 while avoiding beam mismatch smaller than 0.5 µm between the two foci distortion. This conﬁguration provided a good trade-off (Supplementary Figure S2). Under these conditions, up to between the ampliﬁcation bandwidth and conversion 110 and 100 mW of excitation power was available after efﬁciency. The achieved power conversion efﬁciencies the objective for the 1.3 and 1.7 µm beams, respectively. from the pump to signal and idler were 11 and 7%, We ﬁrst veriﬁed that our source can provide efﬁcient 3,4 respectively. The signal was then recompressed using a deep-tissue three-photon imaging. Previous studies 50-mm-long slab of fused silica from 220 fs down to 65 fs noted that the depth advantage of three-photon over two- FWHM (Fig. 1b). The output energy at 1700 nm was 3.1 photon excitation is maximized in densely labeled, scat- µJ, which corresponds to a peak power of approximately tering samples. We recorded two-photon and three- 4.8 GW. The stability of the output signal was typically photon images from the cerebral cortex of ﬁxed trans- 1.7% RMS over a few hours (Supplementary Figure S1). genic mice that were densely labeled with tdTomato at The beam quality of the signal (1700 nm) was well pre- depths ranging from 200 to 800 µm from the tissue sur- served, even at full pump energy. The beam proﬁle was face. Movie 1 and Fig. 2a conﬁrm that the signal-to- circular and without rings at its periphery, as seen in background ratio for the 2PEF images progressively (Supplementary Figure S1). The quality factor M for the degraded with depth, whereas three-photon excitation signal beam at full energy (3.1 µJ) was measured in the provided an appropriate contrast to visualize cell bodies at horizontal and vertical directions to be 1.1 and 1.15, all of the depths investigated. Figure 2a illustrates the respectively, indicating almost no degradation of the obvious beneﬁt provided by 3P excitation over 2P exci- beam. tation at a depth of 600 µm in this sample, despite the fact Finally, the idler was focused into a third PPLN crystal that 2P imaging was carried out here using an excitation optimized for SHG (see the Materials and methods sec- wavelength of 1100 nm, that is, under favorable condi- tion). We obtained up to 890 mW of average power at tions for deep imaging. Of note, the imaging depth in a 1300 nm in the form of 69 fs, 712 nJ pulses. The quality given sample is related to the transparency of that sample, factor M for the 1.3 µm beam at full energy (0.71 µJ) was so that such a direct comparison is important to conﬁrm measured in the horizontal and vertical directions to be the beneﬁt of changing the excitation mode. In particular, 1.7 and 1.6, respectively, indicating a small degradation of ﬁxed brain tissue is generally more scattering than live the SHG beam that can be attributed to the large con- tissue . version efﬁciency and parasitic conversion to 650 nm. We then explored the performance of our SWIR source However, even at maximum idler energy, a well-behaved for dual-color 3P imaging. We found that combined 1.3/ beam shape (i.e., circular, uniform and without satellites) 1.7 µm excitation enables dual-color three-photon ima- for the 1300 nm output beam was preserved (see Supple- ging of HEK cells and mouse brain tissue labeled with mentary protocol). several often-used GFP/RFP combinations: EGFP/ Overall, the simultaneous production of two sub-70 fs mCherry, EGFP/tdTomato, and EGFP/mRFP (Fig. 2b, c). pulses with a 1.25 MHz rate, one at 1.7 µm and another We next imaged a ﬁxed avian embryonic spinal cord co- one at 1.3 µm, makes this laser design uniquely suited for labeled with EGFP and mRFP. We recorded two-color dual-color three-photon imaging and takes advantage of image stacks over a depth of 600 µm using two-photon the two main tissue transparency windows in the SWIR and three-photon excitation sequentially (Fig. 2d, e, range. Movie 2–4). Two-photon imaging of EGFP and mRFP was done using 80 MHz pulse trains at 920 and 1100 nm, Dual-color 3P microscopy of nervous tissues respectively; 3P imaging of EGFP and mRFP was done The dual SWIR source was coupled to a custom-built using 1.2 MHz pulse trains at 1300 and 1700 nm, upright multiphoton microscope (Fig. 1c) that was also respectively. In addition, the THG signal from the 1300 equipped with a commercial Ti:Sapphire and OPO source nm beam provided a label-free image highlighting tissue for comparison with two-photon microscopy (80 MHz, morphology. Figure 2d shows XZ projections of the THG, 130 fs, 920 or 1100 nm, see Materials and methods 3P-EGFP, and 2P-EGFP image stacks. As also visible in section). Movies 3 and 4, the two-photon contrast is rapidly Guesmi et al. Light: Science & Applications (2018) 7:12 Page 7 of 9 a d THG 3PEF EGFP 3PEF tdTomato 2PEF EGFP exc 1700 nm Z = 600 µm 2PEF tdTomato exc 1100 nm Z = 500 µm GFP/mCherry GFP/tdTomato 3PEF EGFP 2PEF EGFP 3PEF mRFP 2PEF mRFP exc 1300 nm exc 950 nm exc 1700 nm exc 1100 nm 1 1 –2PEF –2PEF EGFP/mRFP –3PEF –3PEF 0 10 20 30 40 50 µm 010 20 30 40 50 µm Fig. 2 Dual-color and in-depth 3P imaging of nervous tissues. a Comparison of 3P and 2P excitation for imaging mouse brain tissue. 3PEF and 2PEF imaging of a tdTomato-labeled ﬁxed mouse brain cortex at depths of 200 and 600 µm. See also Movie 1. b, c Dual-color 3PEF imaging for several combinations of green-red ﬂuorescent proteins in b HEK cells and c mouse brain tissue at a depth of 500 µm. d, f Correlative 2PEF, 3PEF, and THG imaging of an intact chick embryo spinal cord (stage E9) co-labeled with EGFP and mRFP. Fluorescence images in each XY plane were normalized after acquisition for contrast comparison. See also Movies 2–4, and related information. d XZ projections of the THG, 3PEF EGFP, and 2PEF EGFP image stacks show the general morphology of the sample and the loss of 2PEF contrast with depth. e 3P and 2P mRFP and EGFP images recorded at a depth of 500 µm. Intensity proﬁles measured along the dashed lines illustrate the superior contrast provided in both channels by 3PEF excitation. Scale bars and arrows, 100 µm degraded with depth in the central regions of the neural labeling. The tissue developed normally, revealing the tube while the contrast in the three-photon and third- dynamics of cell migration and process formation during harmonic images remains sufﬁcient to visualize cells and the two-to-four hour duration of the experiments (Fig. 3b, labeled processes throughout the entire stack (Movie 2). Movies 5 and 6). We then recorded multimodal three- Figure 2e shows 2P and 3P-EGFP and mRFP images photon and THG images from the brain of a live adult recorded at a depth of 500 µm and intensity proﬁles zebraﬁsh that contained dual-color labeling for neural recorded across labeled cells. Both green and red channels stem cells and neural progenitor cells (see the Materials exhibit superior contrast when using 3P excitation. and methods section) from the dorsal telencephalon Finally, we explored the possibility of using our source (pallium), which lay below the surface, underneath the for live three-photon imaging of dual-labeled live tissues skull. As shown in Fig. 3a and Movies 7 and 8, the relative (Fig. 3). We ﬁrst recorded simultaneous GFP-RFP-THG distribution of the two ﬂuorescence-labeled cell popula- time-lapse movies of chick embryonic spinal cord tions could be visualized at a single-cell level resolution, explants in samples that contained dual-compartment directly in their native environment through the skin and RFP signal GFP signal Guesmi et al. Light: Science & Applications (2018) 7:12 Page 8 of 9 a b XZ 3PEF/THG Chick embryo HH18 (day 3) THG Z =57 µm THG Skin GFP dTomato THG Skull GFP RFP Skin Neural Y tube NSCs Adult zebrafish Heating plate T 0 3PEF/THG Z =207 µm deltaA/gfap/THG 3PEF T 0+2 h Z =237 µm Fig. 3 Live dual-color 3PEF and THG neural tissue imaging. a In vivo imaging through the skull in adult zebraﬁsh telencephalon. The ﬁgures show representative XY, XZ, and 3D views for a volume encompassing two labeled cell populations: red dTomato-labeled radial glia and green GFP-labeled neural stem cells expressing the deltaA neurogenic gene (see Materials and methods section). Simultaneously acquired THG signals provide additional label-free contextual information (skin and skull morphology, blood vessels, lipid accumulations). See also Movies 7 and 8 and related information. b Simultaneous dual-color 3PEF and THG imaging of developing chick embryo spinal cord tissue (stage E3) expressing cytoplasmic GFP labeling and nuclear RFP labeling. The images are extracted from a 2-h-long experiment and illustrate developmental processes such as cell migration and process formation. See also Movies 5 and 6 and related information. Scale bars, 100 µm skull. In addition, the label-free THG signals provided a tdTomato), with superior contrast at large depths com- complete morphological context, highlighting skin cells, pared to multicolor two-photon microscopy. We note red blood cells in vessels, lipid accumulations, and skull that three-photon microscopy should be generally viewed boundaries in the intact ﬁsh head. as complementary to two-photon microscopy: although 3PEF microscopy can provide deeper imaging compared Conclusions to 2PEF microscopy, it relies on a lower excitation repe- Recent studies have revealed novel perspectives offered tition rate and, as a consequence, produces a smaller by SWIR three-photon excitation for deep-tissue imaging. ﬂuorescence ﬂux, resulting in slower imaging or a lower The development of biomedical applications relies on the signal. In addition, the use of higher-energy pulses may availability of turnkey sources that are able to provide promote photoperturbation routes through higher-order efﬁcient three-photon excitation of biologically relevant absorption processes . Systematic studies of perturbation chromophores. Our robust OPCPA design provides thresholds with MHz pulse trains in live tissues are cur- simultaneous excitation in the two most promising rently needed to deﬁne safe excitation windows in this spectral windows for deep-tissue 3P microscopy at regime. However, multiple applications of dual-color approximately 1.3 and 1.7 µm, with near-optimal pulse three-photon imaging can be envisioned in systems biol- characteristics. In turn, we have shown that our source ogy. Indeed, this approach provides a unique method to can be used to readily provide simultaneous two-color 3P study interactions between molecular or cellular compo- (and THG) images of nervous tissues that are dual-labeled nents in intact tissues at depths complementing those with commonly used RFPs/GFPs (EGFP, mRFP, mCherry, reached using multicolor two-photon imaging. Dissection plane Guesmi et al. Light: Science & Applications (2018) 7:12 Page 9 of 9 Acknowledgements 11. Xu, C. & Wise, F. W. Recent advances in ﬁbre lasers for nonlinear microscopy. We thank Xavier Solinas, Jean-Marc Sintes, Mickaël Le, Jason Durand, Júlia Nat. Photonics 7,875–882 (2013). Ferrer Ortas, Sébastien Bédu, Laure Bally-Cuif, and Chiara Stringari for technical 12. Rowlands, C. J. et al. Wide-ﬁeld three-photon excitation in biological samples. help and discussions. We also thank members of the microscopy group at LOB Light Sci. Appl. 6, e16255 (2017). for scientiﬁc discussions. 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