Simplified Near-Degenerate Four-Wave-Mixing Microscopy

molecules

Article Simpliﬁed Near-Degenerate Four-Wave-Mixing Microscopy

Jianjun Wang 1,† , Xi Zhang 1,†, Junbo Deng 1, Xing Hu 1, Yun Hu 1, Jiao Mao 1, Ming Ma 1, Yuhao Gao 1, Yingchun Wei 1, Fan Li 1, Zhaohua Wang 1, Xiaoli Liu 1, Jinyou Xu 2,* and Liqing Ren 1,*

1 School of Energy Engineering, Yulin University, Yulin 719000, China; [email protected] (J.W.); [email protected] (X.Z.); [email protected] (J.D.); [email protected] (X.H.); [email protected] (Y.H.); [email protected] (J.M.); [email protected] (M.M.); [email protected] (Y.G.); [email protected] (Y.W.); [email protected] (F.L.); [email protected] (Z.W.); [email protected] (X.L.) 2 South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou 510006, China * Correspondence: [email protected] (J.X.); [email protected] (L.R.) † These authors contribute equally.

Abstract: Four-wave-mixing microscopy is widely researched in both biology and medicine. In this paper, we present a simplified near-degenerate four-wave-mixing microscopy (SNDFWM). An ultra-steep long-pass filter is utilized to produce an ultra-steep edge on the spectrum of a femtosecond pulse, and a super-sensitive four-wave-mixing (FWM) signal can be generated via an ultra-steep short-pass filter. Compared with the current state-of-the-art FWM microscopy, this SNDFWM microscopy has the advantages of simpler experimental apparatus, lower cost, and easier operation.

We demonstrate that this SNDFWM microscopy has high sensitivity and high spatial resolution in both nanowires and biological tissues. We also show that the SNDFWM microscopy can achieve an

Citation: Wang, J.; Zhang, X.; Deng, ultra-sensitive detection based on the electron-resonance effect. This method might find an important J.; Hu, X.; Hu, Y.; Mao, J.; Ma, M.; Gao, application in tracking of nano drugs in vivo. Y.; Wei, Y.; Li, F.; et al. Simplified Near-Degenerate Four-Wave-Mixing Keywords: near-degenerate; four-wave-mixing; microscopy Microscopy. Molecules 2021, 26, 5178. https://doi.org/10.3390/ molecules26175178 1. Introduction Academic Editors: Weifeng Lin and The continuous progress of optical microscopy is of great significance to the devel- Linyi Zhu opment of biomedicine. High sensitive fluorescence microscopy has been widely used in basic research at the nanoscale [1–3]. Received: 30 June 2021 FWM has been widely used in nonlinear optical microscopy for decades, such as Accepted: 23 August 2021 coherent anti-Stokes Raman scattering (CARS) [4–6] and stimulated Raman scattering Published: 26 August 2021 (SRS) [7–9]. These two kinds of label-free and non-invasive microscopy have been studied widely. CARS microscopy, developed in 1982, is a nonlinear optical FWM process, whose Publisher’s Note: MDPI stays neutral signal strength is nearly 100,000 times higher than that of spontaneous Raman signals [10]. with regard to jurisdictional claims in published maps and institutional affil- The CARS microscope was optimized by Xie’s group at Harvard University. This micro- iations. scope is composed of a laser-scanning microscope, which realizes the scanning of externally introduced laser and the collection of CARS signals through a simple modification of commercial confocal microscopy [11]. Two pulsed lasers, called pump and Stokes, which coincide in space and synchronize in time, are used as the excitation light source. The time overlap of two laser pulse sequences of different wavelengths can be achieved in a Copyright: © 2021 by the authors. variety of ways. One approach is to use two titanium: sapphire lasers to generate a pump Licensee MDPI, Basel, Switzerland. beam and a Stokes beam of the right wavelength, respectively. An external synchronizer This article is an open access article distributed under the terms and synchronizes the two pulses, and an optical delay line allows the two pulses to overlap conditions of the Creative Commons precisely. The advantage of this method is that the wavelengths of the two laser beams Attribution (CC BY) license (https:// can be tuned in a large spectral range. Its disadvantage is that the use of the external creativecommons.org/licenses/by/ synchronizer not only increases the cost, but also makes it difficult to maintain the stability 4.0/).

Molecules 2021, 26, 5178. https://doi.org/10.3390/molecules26175178 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 5178 2 of 7

of synchronization, which increases the difficulty of experiment, thus restricting the application of this method. Another method uses an optical parametric oscillator (OPO) as the key device for wavelength tuning; this has gradually become the mainstream configuration method of the CARS system over the last two decades [12]. The main reason is that this method eliminates the need for synchronous control of two independent pulse lasers and simplifies the experimental setup. However, Silberberg’s research group implemented a single-beam CARS microscope by using the pulse shaping method [13]. Later, the team further simplified the single-beam CARS microscope system by replacing the pulse shaper with a resonant photonic crystal plate or notch filter [14,15]. Recently, researchers found that the resonant photonic crystal plate or notch filter can be removed from the system by using the ultra-steep filter, proving the simplest CARS microscope to date [16]. Single-beam near-degenerate four-wave-mixing microscopy can provide two imaging contrast mechanisms, with nearly perfect phase-matching conditions, providing an effective imaging contrast mechanism [17]. In this paper, we present a simpler near-degenerate FWM microscopy by replacing the complex pulse shaper used in a previous study with a compact and low-cost long-pass filter, an ultra-steep long-pass filter (ULPF). This ULPF produces a sharp “edge” on the spectrum of the incoming femtosecond laser, achieving an infinitely close FWM signal on the spectrum to the incident laser, and thus generating a super-strong FWM signal. Simplifying the experimental apparatus and its operation, the cost of such a system is also lowered. We call this method a simplified near-degenerate four-wave-mixing (SNDFWM) microscope system.

2. Theory In the process of four-wave-mixing, when the frequency of the detection wave and the pump light are the same, that is, when the frequency of the four waves are the same, it is called degenerate four-wave mixing. In practical applications, detection light and pump light often have a certain frequency difference, and when their frequency difference is much smaller than any of the frequencies involved in mixing, it is the so-called near-degenerate four-wave-mixing process. The third-order nonlinear polarizability tensor in the process of near-degenerate four-wave-mixing is described as [18]:

N µ µ µ µ χ(3)(−ω ) = P km ml ln nk (1) 4 3 F ∑ ( − − )( − − − )( − − ) } m,l,n ωmk ω1 iγmk ωlk ω1 ω2 iγlk ωnk ω4 iγnk

where PF is the full permutation operator, µ is the transition dipole moment, ω is the energy difference between the corresponding energy levels, and γ is the uniform linewidth associ- ated with the corresponding electron transition or vibrational transition. In Equation (1), the third-order nonlinear polarizability tensor will reach the maximum value when the real part of the value inside the brackets on the denominator is the minimum (or equal to zero), especially when the incident light reaches resonance with the sample. For example, such resonance enhancement occurs when the wavelength of the incident light coincides with the absorption peak of the ﬂuorescent material. The SNDFWM spectrum measured in the experiment can be quantitatively described by continuous integral [17]:

∞ ∞ ∞ 2 Z Z Z ( ) 2 ( ) ∗ I(ω ) ∝ P 3 (ω ) ∝ dω dω dω χ 3 (−ω )E(ω )E (ω )E(ω ) (2) 4 4 1 2 3 4 1 2 3 −∞ −∞ −∞

where ω4 = ω1 − ω2 + ω3.

3. Experimental Equipment The main function of the pulse shaper is to ﬁlter out some wavelength components of the incident laser so that the spectrum of this laser can reach the near-degenerate condition. Besides being complex to adjust, the pulse shaper is also relatively expensive. MoleculesMolecules 20212021,, 2626,, 5178x FOR PEER REVIEW 33 ofof 77

Withthe progress the progress of science of science and technology, and technology, a kind of a ultra-steep kind of ultra-steep long-pass long-pass filter has come filter into has comebeing. into This being. filter Thisnot only filter has not the only function has the functionof a long-pass of a long-pass filter, but filter, can butalso can cut also off cutthe offunneeded the unneeded wavelength wavelength components components very steeply. very steeply.Therefore, Therefore, this filter this can filterreplace can the replace pulse theshaper pulse used shaper in the used near-degenerate in the near-degenerate four-wave-mixing four-wave-mixing scheme. This scheme. not only This simplifies not only simplifiesthe experimental the experimental equipment, equipment, but also realizes but also realizesthe low-cost the low-cost and easy-to-operate and easy-to-operate micro- microscopicscopic technique technique.. The SNDFWM microscope device device is is shown shown in in Figure Figure 11.. An An ultra-short ultra-short pulse pulse with with a acentral central wavelength wavelength of of800 800 nm, nm, a repetition a repetition frequency frequency of of80 80MHz, MHz, and and a pulse a pulse width width of of20 20fs was fs was used used to tocompensate compensate the the laser laser dispersi dispersionon at at the the sample sample position position through through the pulsepulse compressor, andand thenthen truncatedtruncated steeplysteeply byby thethe ultra-steepultra-steep long-passlong-pass filterfilter (ULPF).(ULPF). AfterAfter that, aa dichromaticdichromatic mirror mirror was was reflected reflected and and introduced introduced into into the microscopicthe microscopic objective objective lens tolens focus to focus the sample the sample placed placed on the on2-D the precisely-controlled2-D precisely-controlled stage. stage. The The reflected reflected part part of the of scatteredthe scattered signal signal is collected is collected by by the the same same objective objective lens lens and and then then filtered filtered by by a a band-passband-pass filter.filter. Finally, itit isis introducedintroduced intointo aa photomultiplierphotomultiplier tubetube (PMT2)(PMT2) forfor two-photontwo-photon excitedexcited fluorescencefluorescence (TPEF) (TPEF) imaging. imaging. The The transmitted transmitted pa partrt of the ofthe scattered scattered signal signal is collected is collected by a bycondenser a condenser lens and lens divided and divided into two into channels two channels by a beam by a beamsplitter splitter mirror. mirror. One way One enters way entersthe high-resolution the high-resolution spectrometer spectrometer through through an USPF an for USPF SNDFWM for SNDFWM spectrum spectrum measurement, measurement,and the other and way the enters other wayinto another enters into photom anotherultiplier photomultiplier tube (PMT1) tubeafter (PMT1)a second after USPF a secondfor SNDFWM USPF for imaging. SNDFWM The imaging. sample is The placed sample on isa two-dimensional placed on a two-dimensional movable high-preci- movable high-precisionsion platform. platform.

Figure 1. Schematic diagram of the the SNDFWM SNDFWMmicroscope. microscope. PC:PC: pulsepulse compressor;compressor; fs:fs: femtosecond; ULPF: ultra-steep long-pass ﬁlter;filter; O: objective lens;lens; S:S: Sample;Sample; C:C: condensercondenser lens;lens; DM:DM: dichromaticdichromatic mirror; BPF: band-passband-pass ﬁlter;filter; PMT:PMT: PhotomultiplierPhotomultiplier tube;tube; BS:BS: beambeam splitter.splitter.

4. Results and Discussion First, glass and air were used as samples for experimental detection ofof thethe SNDFWM microscope as shown shown in in solid solid red red and and blue blue lines lines in Figure in Figure 2, respectively.2, respectively. Equation Equation (2) was (2) wasused used for theoretical for theoretical simulation simulation calculation calculation of ofthe the SNDFWM SNDFWM spectrum spectrum of of the the glass, asas shown by the dashed greengreen lineline inin FigureFigure2 2.. The The solid solid blue blue line line in in Figure Figure2 2shows shows that that the the air does not generate a FWM signal at all. The results in Figure2 2 show show that that the the theoretical theoretical simulation resultsresults are are in in good good agreement agreement with with the the experimental experimental results. results. Therefore, Therefore, both both the theoreticalthe theoretical simulation simulation and and the experimentalthe experimental measurement measurement of the of correspondingthe corresponding samples sam- proveples prove the feasibility the feasibility of the of system. the system. Secondly, ourour experimental experimental results results show show that that the the electron-resonance electron-resonance effect effect can be can used be toused achieve to achieve ultra-sensitive ultra-sensitive spectral spectral detection. detect Thision. isThis especially is especially important important for samples for samples that havethat have only only spectral spectral absorption absorption but no but fluorescent no fluorescent signal, signal, as such assamples such samples are difficult are difficult to see withto see a with fluorescence a fluorescence microscope. microscope. According Accord to Equationing to Equation (1), when (1), thewhen wavelength the wavelength of the incidentof the incident laser is laser within is within the absorption the absorption spectrum spectrum of the sample, of the sample, one can greatlyone can enhance greatly theen- FWMhance signalthe FWM by usingsignal theby effectusing ofthe electron-resonance. effect of electron-resonance. Figure3 shows Figure the 3 shows ultra-sensitive the ultra- detection of a solution of bleach IR775 that resonates at 775 nm. The results in the figure sensitive detection of a solution of bleach IR775 that resonates at 775 nm. The results in show that when the incident light power is 1 mW and the integration time is 100 ms, the figure show that when the incident light power is 1 mW and the integration time is the detection sensitivity was similar to the previous results [17]. Therefore, the proposed 100 ms, the detection sensitivity was similar to the previous results [17]. Therefore, the SNDFWM microscope can also achieve ultra-high sensitive detection. proposed SNDFWM microscope can also achieve ultra-high sensitive detection.

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Figure 2. System test via the SNDFWM microscope. The solid red line indicates the experimental Figure 2. System test via the SNDFWM microscope. The solid red line indicates the experimental Figuremeasurement 2. System of testSNDFWM via the spectrumSNDFWM of microscope. glass. The blTheue solid linered lineindicates indicates the experimentalthe experimental meas- measurement of SNDFWM spectrum of glass. The blue solid line indicates the experimental mea- measurementurement of SNDFWM of SNDFWM spectrum spectrum of air. of glass.The green The bldottedue solid line line indicate indicatess the the theoretical experimental simulation meas- of surementurementSNDFWM of SNDFWM of glass. spectrum of air. TheThe greengreen dotteddotted lineline indicatesindicates thethe theoreticaltheoretical simulationsimulation ofof SNDFWMSNDFWM of of glass. glass.

Figure 3. Resonance-enhanced SNDFWM spectrum of IR775 solution. (a) Spectrum of the excitation FigureFigurepulse 3. 3.(red)Resonance-enhanced Resonance-enhanced and absorption spectr SNDFWM SNDFWMum (green) spectrum spectrum of IR775of of IR775IR775 solution; solution.solution. (b) SNDFWM ((aa)) SpectrumSpectrum spectra ofof thethe of excitationexcitation IR775 solu- pulse (red) and absorption spectrum (green) of IR775 solution; (b) SNDFWM spectra of IR775 solu- pulsetion (red) with and different absorption concentrations; spectrum (green) (c) Concentration of IR775 solution; dependence (b) SNDFWM of SNDFWM spectra signals. of IR775 solution tion with different concentrations; (c) Concentration dependence of SNDFWM signals. with different concentrations; (c) Concentration dependence of SNDFWM signals. To further demonstrate the high spatial resolution of the proposed system, we em- To further demonstrate the high spatial resolution of the proposed system, we em- ployedTo further an objective demonstrate lens (Olympus the high spatial×60/1.2) resolution to perform of theimaging proposed of the system, CdS nanowires we em- ployedployed(with anaan transverse objective objective lensmean lens (Olympus (Olympusdiameter ×of ×60/1.2)60/1.2) about 93to nm), perform as shown imaging in Figure of thethe CdS4.CdS CdS nanowiresnanowires nanowires (with(withwere a a transverseprepared transverse by mean mean using diameter diameter the chemical ofof aboutabout vapor 9393 nm), nm),deposition asas shownshown [19] inin method. FigureFigure4 .4.The CdS CdS growth nanowires nanowires of CdS

werewerenanowires prepared prepared was by by performed using using the the chemical inchemical a home-build vapor vapor deposition two-zonedeposition horizontal [19[19]] method.method. tube TheThe furnace. growthgrowth In ofofa typical CdSCdS nanowiresnanowiressynthesis, was wasabout performed performed 0.12 g CdS in in a apower home-build home-build (99.99%, two-zonetwo-zone Sigma-Aldrich, horizontalhorizontal St. tubetube Louis, furnace.furnace. MO, InUSA)In aa typicaltypical evapo- synthesis,synthesis,rated at about 860about °C 0.12 0.12under g CdSg CdS 300–400 power power (99.99%,mbar (99.99%, served Sigma-Aldrich, Sigma-Aldrich, as the precursor. St. Louis,St. Louis, The MO, CdS MO, USA) vapor USA) evaporated wasevapo- then atratedcarried 860 ◦atC 860to under the °C M-plane 300–400under 300–400 sapphires mbar served mbar (8 × asserved8 mm the2precursor.) aspre-coated the precursor. The with CdS Au The vapor catalysts CdS was vapor (average then was carried 50then nm) carried to the M-plane sapphires (8 ×2 8 mm2) pre-coated with Au catalysts (average 50 nm) toby the a M-planehigh-purity sapphires N2 gas(8 flow× 8 at mm a rate) pre-coated of 400 sccm. with The Au temperature catalysts (average around 50 the nm) sapphire by a by a high-purity N2 gas flow at a rate of 400 sccm. The temperature around the sapphire high-puritywas maintained N2 gas at ﬂow 560–600 at a rate °C for of 40030 min sccm. to Theenable temperature the desired around nanowire the sapphiregrowth. Figure was maintainedwas4a,b maintained are images at 560–600 atof 560–600CdS◦C nanowires for °C 30 minfor 30 toat minenabledifferen to theenablet magnifications desired the desired nanowire under nanowire growth. scanning growth. Figure electron4a,b Figure are mi- images4a,bcroscopy, are of images CdS respectively. nanowires of CdS nanowires Figure at different 4c,d at aredifferen magniﬁcations SNDFWMt magnifications imaging under scanning and under the scanningcorresponding electron microscopy,electron spectral mi- respectively.croscopy,images, respectively. respectively. Figure4c,d ItFigure are is shown SNDFWM 4c,d thatare SNDFWM this imaging method and imaging can the be corresponding andutilized the corresponding to perform spectral imaging images,spectral of a respectively.images,single nanowire.respectively. It is shown The It issingle-nanowire that shown this that method this sensitmethod can beivity utilized can of be SNDFWM utilized to perform to microscopy perform imaging imaging of means a single of that a nanowire.singlesuch annanowire. approach The single-nanowire The is a single-nanowirereliable technique sensitivity sensit for of ex SNDFWMivityamining of SNDFWM the microscopy electronic microscopy meansstructure that means of such materials that an such an approach is a reliable technique for examining the electronic structure of materials approachat the nanoscale is a reliable [20]. technique Therefore, for as examining long as the the wavelength electronic structure of the incident of materials light atis theshort at the nanoscale [20]. Therefore, as long as the wavelength of the incident light is short nanoscaleenough [(e.g.,20]. Therefore,ultraviolet aslaser) long and as the the wavelength numerical ofaperture the incident of the light microscopic is short enough objective enough (e.g., ultraviolet laser) and the numerical aperture of the microscopic objective (e.g.,lens ultraviolet is large enough, laser) and a microscopic the numerical image aperture with higher of the microscopic spatial resolution objective can lens be achieved. is large enough,lens is large a microscopic enough, a imagemicroscopic with higher image spatial with higher resolution spatial can resolution be achieved. can be achieved.

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Figure 4. SNDFWM microscopic imaging of CdS nanowires. (a) Scanning electron microscopy FigureFigure 4. 4.SNDFWM SNDFWM microscopicmicroscopic imaging imaging of of CdS CdS nanowires. nanowires. (a()a) Scanning Scanning electron electron microscopy microscopy (SEM) images of CdS nanowires with a scale of 2 µm; (b) SEM images of nanowires with a scale of (SEM)(SEM) images images of of CdS CdS nanowires nanowires with with a a scale scale of of 2 2µ m;µm; (b ()b SEM) SEM images images of of nanowires nanowires with with a a scale scale of of 10 µm; (c) SNDFWM image of CdS nanowires with a scale of 5 µm; (d) SNDFWM spectrum of CdS 1010µ µm;m; (c ()c SNDFWM) SNDFWM image image of of CdS CdS nanowires nanowires with with a a scale scale of of 5 5µ m;µm; (d ()d SNDFWM) SNDFWM spectrum spectrum of of CdS CdS nanowires; (e) Two-photon excited fluorescence (TPEF) image of CdS nanowires with 5 µm; (f) nanowires; (e) Two-photon excited fluorescence (TPEF) image of CdS nanowires withµ 5 µm; (f) nanowires;TPEF spectrum (e) Two-photon of CdS nanowires. excited fluorescence The pixel size (TPEF) of the image SNDFWM of CdS and nanowires TPEF images with 5 ism; 0.5 (f µm.) TPEF spectrumTPEF spectrum of CdS nanowires.of CdS nanowires. The pixel The size pixel of thesize SNDFWM of the SNDFWM and TPEF and images TPEF images is 0.5 µ m.is 0.5 µm. Finally, we apply the proposed system to high spatial resolution microscopic imag- Finally,Finally, we we apply apply the the proposed proposed system system to highto high spatial spatial resolution resolution microscopic microscopic imaging imaging of mouse brain tissue samples, as shown in Figure 5. The preparation methods of ofing mouse of mouse brain brain tissue tissue samples, samples, as shown as shown in Figure in 5Figure. The preparation 5. The preparation methods methods of mouse of mouse brain tissue samples were consistent with those reported in the literature [6]. Fresh brainmouse tissue brain samples tissue samples were consistent were consistent with those wi reportedth those reported in the literature in the literature [6]. Fresh [6]. mouse Fresh mouse brain tissues were commercially procured (Zyagen) and pre-mounted on charged brainmouse tissues brain were tissues commercially were commercially procured proc (Zyagen)ured (Zyagen) and pre-mounted and pre-mounted on charged on charged glass glass slides. The samples were shipped on dry ice and stored at −80 °C. They were thawed slides.glass slides. The samples The samples were shipped were shipped on dry on ice dry and ice stored and stored at −80 at◦ C.−80 They °C. They were were thawed thawed for for 10 min, and then washed twice in PBS to remove debris and residual cutting media 10for min, 10 andmin,then and washedthen washed twice intwice PBS in to PBS remove to remove debris anddebris residual and residual cutting mediacutting before media before imaging. The tissues were kept wet with PBS and sandwiched with a glass co- imaging.before imaging. The tissues The were tissues kept were wet withkept PBSwet andwith sandwiched PBS and sandwiched with a glass with coverslip a glass over coverslip over the sample and the glass slide, and then sealed with nail polish. Figure 5a is theverslip sample over and the the sample glass slide,and the and glass then slide, sealed an withd then nail sealed polish. with Figure nail5 apolish. is the SNDFWMFigure 5a is the SNDFWM image of mouse brain tissue, while Figure 5b is a two-photon excited fluo- imagethe SNDFWM of mouse image brain tissue,of mouse while brain Figure tissue,5b iswhile a two-photon Figure 5b excitedis a two-photon fluorescence excited (TPEF) fluorescence (TPEF) image obtained at the same time. Figure 5c is the result of the superposi- imagerescence obtained (TPEF) at image the same obtained time. Figureat the 5samec is the time result. Figure of the 5c superposition is the result of of the a FWM superposi- and tion of a FWM and reflected TPEF image. The spatial distribution of blood vessels and reflectedtion of a TPEF FWM image. and reflected The spatial TPEF distribution image. Th ofe blood spatial vessels distribution and white of blood matter vessels in normal and white matter in normal brain tissue can be seen clearly in this image. This is because the brainwhite tissue matter can in be normal seen clearlybrain tissue in this can image. be seen This clearly is because in this the image. TPEF This is more is because sensitive the TPEF is more sensitive to blood, while the FWM is more sensitive to the white matter. toTPEF blood, is more while sensitive the FWM to is blood, more sensitivewhile the to FWM the white is more matter. sensitive During to the the white experiment, matter. During the experiment, we only used the numerical aperture of 0.4 objective lens (New- weDuring only usedthe experiment, the numerical we only aperture used ofthe 0.4 numerical objective aperture lens (Newport of 0.4 objective×20/0.4), lens and (New- we port ×20/0.4), and we only used 1 mW incident light power and 200 ms pixel-dwell time, onlyport used ×20/0.4), 1 mW and incident we only light used power 1 mW and incident 200 ms light pixel-dwell power and time, 200 which ms pixel-dwell shows that time, the which shows that the SNDFWM microscope has high spatial resolution and high sensi- SNDFWMwhich shows microscope that the SNDFWM has high spatial microscope resolution has high and highspatial sensitivity. resolution This and ishigh of greatsensi- tivity. This is of great scientific significance for biomedical applications. scientifictivity. This significance is of great for scientific biomedical significance applications. for biomedical applications.

Figure 5. Microscopic imaging of mouse brain using SNDFWM microscope. (a) SNDFWM image; Figure 5. Microscopic imaging of mouse brain using SNDFWM microscope. (a) SNDFWM image; Figure(b) TPEF 5. Microscopic image; (c) Overlaid imaging ofimage mouse of brainSNDFWM using and SNDFWM TPEF. The microscope. white scale (a) is SNDFWM 20 µm, the image; pixel- (b) TPEF image; (c) Overlaid image of SNDFWM and TPEF. The white scale is 20 µm, the pixel- (bdwell) TPEF time image; is 200 (c) microseconds, Overlaid image the of SNDFWMpixel size is and 1 µm, TPEF. and The the white incident scale laser is 20 powerµm, the is 1 pixel-dwell mW. dwell time is 200 microseconds, the pixel size is 1 µm, and the incident laser power is 1 mW. time is 200 microseconds, the pixel size is 1 µm, and the incident laser power is 1 mW.

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Compared with the previous study [17], we replaced the pulse-shaping system with a compact ULPF, simplifying the NDFWM microscope system significantly. This simplified setup is smaller, cheaper, and easier-to-operate. In comparison with our previous work [15], less excitation energy is needed to excite the FWM signals and the imaging speed is much faster. This method might find significant application in tracking nanodrugs in vivo [21].

5. Conclusions In conclusion, we have presented a well-designed SNDFWM microscope device. We successfully observed the high-resolution multimodal imaging of brain tissue of mice, and FWM imaging of a single nanowire. The microscope system device is not only simple and low-cost, but also easy-to-operate. These advantages are the guarantee that SNDFWM microscopy can be widely used in the ﬁeld of biomedical imaging and nanomaterials.

Author Contributions: Conceptualization, J.X. and L.R.; methodology, J.W.; software, X.Z. and J.D.; validation, X.H., J.M., M.M., Y.G., Y.H., Y.W., Z.W., F.L., X.L.; formal analysis, J.W. and L.R.; investigation, J.X. and L.R.; resources, L.R.; data curation, J.X. and L.R.; writing—original draft preparation, J.W. and X.Z.; writing—review and editing, all the authors; project administration, L.R.; funding acquisition, J.X. and L.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Natural Science Foundation of China Regional Fund Project (Approval Number: 12064046), Guangdong Young Talents Project (Approval Number: 2019QN01C290), Shaanxi Province Science and Technology Development Project (Approval Number: 2014K05-11), Yulin Science and Technology Plan Project (Approval Number: CXY-2020-015-16,2019- kjj045), Special scientific research projects on campus of Yulin University (12YK25), Guangdong Basic and Applied Basic Research Foundation (Approval number: 2021A1515012235), and start-up project of the Outstanding Young Scholar at South China Normal University. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare that they have no conflict of interest.

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