Two-Photon Polymerization: Functionalized Microstructures, Micro-Resonators, and Bio-Scaffolds

polymers

Review Two-Photon Polymerization: Functionalized Microstructures, Micro-Resonators, and Bio-Scaffolds

Adriano J. G. Otuka 1,*,†, Nathália B. Tomazio 1,2,†, Kelly T. Paula 1 and Cleber R. Mendonça 1,*

1 Photonics Group, São Carlos Institute of Physics, University of São Paulo, São Carlos 13566-590, SP, Brazil; [email protected] (N.B.T.); [email protected] (K.T.P.) 2 Device Research Laboratory, “Gleb Wataghin” Institute of Physics, University of Campinas, Campinas 13083-859, SP, Brazil * Correspondence: [email protected] (A.J.G.O.); [email protected] (C.R.M.) † Contributed equally to this work as the ﬁrst author.

Abstract: The direct laser writing technique based on two-photon polymerization (TPP) has evolved considerably over the past two decades. Its remarkable characteristics, such as 3D capability, sub- diffraction resolution, material ﬂexibility, and gentle processing conditions, have made it suitable for several applications in photonics and biosciences. In this review, we present an overview of the progress of TPP towards the fabrication of functionalized microstructures, whispering gallery mode (WGM) microresonators, and microenvironments for culturing microorganisms. We also describe the key physical-chemical fundamentals underlying the technique, the typical experimental setups, and the different materials employed for TPP.

Keywords: direct laser writing; ultrashort laser pulses; two-photon polymerization; functional microdevices; whispering gallery mode microresonators; scaffolds for biological applications

Citation: Otuka, A.J.G.; Tomazio, N.B.; Paula, K.T.; Mendonça, C.R. Two-Photon Polymerization: 1. Introduction Functionalized Microstructures, Micro-Resonators, and Bio-Scaffolds. The processing of materials by ultrashort lasers, which allows high precision-patterning Polymers 2021, 13, 1994. https:// of 2D and 3D micro/nanostructures, has become a cornerstone technology for a wide range doi.org/10.3390/polym13121994 of areas related to academic research and engineering [1]. An ultrashort laser-based fabrication technique that has been widely used is direct laser writing via two-photon polymeriza- Academic Editor: Lech Sznitko tion (TPP) [2,3]. As opposed to the most conventional micro/nanofabrication techniques, TPP is a maskless patterning tool and does not require harsh processing conditions nor a Received: 30 April 2021 cleanroom facility [4]. Furthermore, this technique is not limited to the processing materials Accepted: 3 June 2021 in the powder form, such as selective laser sintering (SLS) [5–8], another laser fabrica- Published: 18 June 2021 tion method that allows sculpting macroscopic structures with micrometric resolution. TPP leverages the nonlinear nature of two-photon absorption to fabricate 3D polymeric Publisher’s Note: MDPI stays neutral microstructures with arbitrary geometries and sub-diffraction features [9]. Moreover, a with regard to jurisdictional claims in large variety of materials can be employed as photoresist, or be incorporated into it, which published maps and institutional affil- allows customizing the microstructures’ physical, chemical or biological properties to meet iations. specific applications [10,11]. For example, improvements in the spatial resolution and the amount of dry-related shrinkage undergone by the microstructures can be accomplished by varying the photoresist components proportion [1,12–14]. The TPP technique has found applications in numerous fields, including photon- Copyright: © 2021 by the authors. ics [15–19], biosciences [20–22], and micromechanical systems [23–25]. Precise photonic Licensee MDPI, Basel, Switzerland. building blocks, such as optical microresonators [26], photonic crystals [27], and waveg- This article is an open access article uides [28], as well as photonic wire bondings [29] and free-form coupling elements [16], distributed under the terms and have been successfully fabricated by TPP. Active photonic structures have also been fab- conditions of the Creative Commons ricated [30,31]. In the field of biological sciences, TPP has mainly been used to create Attribution (CC BY) license (https:// microscaffolds suitable for culturing cells, tissues, and microorganisms [21,32,33], and creativecommons.org/licenses/by/ microneedles [22] and porous microstructures [20] for drug delivery. TPP has also been 4.0/).

Polymers 2021, 13, 1994. https://doi.org/10.3390/polym13121994 https://www.mdpi.com/journal/polymers Polymers 2021, 13, x FOR PEER REVIEW 2 of 31

croscaffolds suitable for culturing cells, tissues, and microorganisms [21,32,33], and microneedles [22] and porous microstructures [20] for drug delivery. TPP has also been a remarkable tool for fabricating optically driven micromachines that allows grabbing or Polymers 2021probing, 13, 1994 nanoscale objects [34]. Microstructures containing movable parts, such as nanot- 2 of 30 weezers [23], microgears [24], and micropump setups [35], have been demonstrated. This paper summarizes the advances of TPP towards three of its major applications: functionalized microstructures,a remarkable tool whispering for fabricating gallery optically mode driven (WGM micromachines) microresonators, that allows and grabbing microscaffolds foror culturing probing nanoscalemicroorganisms. objects [34 The]. Microstructuresfavorable properties containing of TPP, movable such parts, as such as fine-tuning of the nanotweezersstructure dimensions, [23], microgears the integration [24], and micropump of the fabricated setups [35 structures], have been into demonstrated. a range of substrates, andThis its paper material summarizes flexibility, the advanceshave pushed of TPP forward towards the three design of its majorand po- applications: tential of applicationsfunctionalized of WGM microresonators. microstructures, whispering Moreover, gallery the material mode (WGM) flexibility microresonators, of TPP, and along with its 3D microscaffoldscapability and for gentle culturing processing microorganisms. conditions, The has favorable made it propertieswell suited of for TPP, such as fine-tuning of the structure dimensions, the integration of the fabricated structures into the fabrication of bioactive microscaffolds with spatially varying properties. a range of substrates, and its material flexibility, have pushed forward the design and The further sectionspotential of of the applications present review of WGM are microresonators. organized as follows: Moreover, In theSection material 2, we flexibility of briefly describe theTPP, physical along with-chemical its 3D capability fundamentals and gentle underlying processing the conditions, TPP technique. has made We it well suited subsequently showfor the the typical fabrication experimental of bioactive setups microscaffolds and different with spatially materials varying employed properties. in TPP. Section 3 providesThe an further overview sections of the of general the present applications review are of organized TPP. Emphas as follows:is is given In Section 2, we to the fabrication brieflyof WGM describe microresonators the physical-chemical and 3D microscaffolds fundamentals underlying to study microorgan- the TPP technique. We ism’s development.subsequently Finally, Section show the 4 presents typical experimental the overall setupsconclusions and different and prospects materials employedof in TPP in the near future.TPP. Section 3 provides an overview of the general applications of TPP. Emphasis is given to the fabrication of WGM microresonators and 3D microscaffolds to study microorganism’s development. Finally, Section4 presents the overall conclusions and prospects of TPP in 2. Two-Photon Polymerization (TPP) Fundamentals the near future. 2.1. Two-Photon Absorption 2. Two-Photon Polymerization (TPP) Fundamentals The spatial confinement of TPP, which allows fabricating 3D microstructures with 2.1. Two-Photon Absorption arbitrary geometry and submicrometric features, arises from the nonlinear nature of the two-photon absorptionThe (TPA) spatial phenomenon. confinement ofThe TPP, latter which refers allows to fabricatingan electronic 3D microstructurestransition with arbitrary geometry and submicrometric features, arises from the nonlinear nature of the excited by the combined action of two photons [2,36], such that the sum of the energy of two-photon absorption (TPA) phenomenon. The latter refers to an electronic transition the individual photonsexcited must by the be combined resonant action with ofthe two transition photons [energy2,36], such [37] that. When the sum the ofpho- the energy of tons have the samethe energy, individual as photonsindicated must in be Figure resonant 1b, with the theprocess transition is called energy degenerated [37]. When the photons [38]. TPA can alsohave be observed the same energy, with photons as indicated of different in Figure1 b,energies the process since is calledthe sum degenerated of their [ 38]. TPA energies matches canthe alsoenergy be observedgap. with photons of different energies since the sum of their energies In a quantummatches mechanics the energy framework, gap. TPA is described by considering the existence of a virtual intermediateIn astate, quantum which mechanics takes part framework, in the electronic TPA is described transition by [36,39] considering. In this the existence picture, a transitionof ato virtual a real intermediate state is possible state, only which if takesthe time part interval in the electronic between transition the two- [36,39]. In this picture, a transition to a real state is possible only if the time interval between the photon is shorter than the lifetime of the virtual state [39]. Because the virtual state lifetime two-photon is shorter than the lifetime of the virtual state [39]. Because the virtual state is extremely short,lifetime in the or isder extremely of femtoseconds short, in the [37] order, TPA of femtoseconds is usually described [37], TPA as is usuallythe “sim- described as ultaneous” absorptionthe “simultaneous” of two photons. absorption of two photons.

Figure 1. Energy diagram representing the absorption of (a) one and (b) two photons with the same energy to promote the electronicFigure transition 1. Energy between diagram the states representing|ni e |n + 1 thei. Reprinted absorption with of permission (a) one and [40 (].b Licensed) two photons under awith Creative the Commons Attributionsame 4.0energy International to promote license. the electronic transition between the states |푛⟩ e |푛 + 1⟩. Reprinted with permission [40]. Licensed under a Creative Commons Attribution 4.0 International license. The TPA is a nonlinear optics phenomenon that can occur in the presence of high- The TPA is aintensity nonlinear light, optics i.e., whenphenomenon the magnitude that can of theoccur light in electricthe presence field is of comparable high- to the 11 −1 intensity light, i.e.,interatomic when the electric magnitude field ( Eofint the≈ 10lightV electric·m )[36 field]. In thisis comparable regime, the electricto the in- field induced in the material responds nonlinearly with the electric field of light, which can prompt teratomic electric field (퐸 ≈ 1011 푉 ∙ 푚−1) [36]. In this regime, the electric field induced unusual푖푛푡 effects such as light generation at frequencies other than that of the incident light and absorption of light in the frequency range of transparency of the material [36]. When

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in the material responds nonlinearly with the electric field of light, which can prompt un- Polymers 2021, 13, 1994 usual effects such as light generation at frequencies other than that of the incident 3light of 30 and absorption of light in the frequency range of transparency of the material [36]. When high-intensity light is employed, the absorption becomes dependent on the excitation in- tensityhigh-intensity according light to [36,38] is employed,: the absorption becomes dependent on the excitation intensity according to [36,38]: 훼(퐼) = 훼0 + 훽퐼, (1) α(I) = α0 + βI, (1) in which, 훼0 and 훽 are, respectively, the linear absorption and TPA coefficient, and 퐼 is in which, α0 and β are, respectively, the linear absorption and TPA coefﬁcient, and I is the light intensity. 훽 is directly associated with the TPA cross-section (휎푇푃퐴) as stated in Equationthe light intensity.(2) [38]: β is directly associated with the TPA cross-section (σTPA) as stated in Equation (2) [38]: 푁휎푇푃퐴 NσTPA (2) 훽 =β = , , (2) ℎ휈hν in which, 푁, ℎ, and 휈 are the concentration of entities that constitute the material (e.g., in which, N, h, and ν are the concentration of entities that constitute the material (e.g., atoms, molecules, etc.), the Planck constant, and the pump frequency, respectively. For atoms, molecules, etc.), the Planck constant, and the pump frequency, respectively. For most most molecules, the TPA cross-section (휎 ) is in the order of 10−50푐푚4 ∙ 푠 ∙ 푝ℎ표푡표푛−1 or molecules, the TPA cross-section (σ )푇푃퐴 is in the order of 10−50cm4·s·photon−1 or 1 GM. 1 GM. Such unit is a reference to theTPA Nobel-laureate physicist Maria Göppert–Mayer Such unit is a reference to the Nobel-laureate physicist Maria Göppert–Mayer [38,41]. [38,41]. As light propagates throughout the material, its intensity is attenuated exponen- As light propagates throughout the material, its intensity is attenuated exponentially tially [37]. For light in the frequency range of transparency of the material, for which [37]. For light in the frequency range of transparency of the material, for which 훼0 = 0, α0 = 0, the light dissipation rate along the propagation direction (z) is described by [37]: the light dissipation rate along the propagation direction (푧) is described by [37]:

dI푑퐼(z()푧) 2 ==−−β훽I퐼(2z(푧)). . ( (3)3) dz푑푧 FromFrom Equation (3),(3), wewe seesee thatthat thethedissipation dissipation rate rate for for a a TPA TPA process process depends depends quadrat- quad- raticallyically on on the the light light intensity. intensity. The The nonlinear nonlinear dependence dependence ofof TPATPA onon thethe intensity of light, light, togethertogether with with the the relatively relatively small small TPA TPA cross cross-section-section of of materials, materials, allows allows attaining attaining spatial spa- confintial conﬁnementement of the of excitation the excitation when when focused focused high-intensity high-intensity lasers are lasers used, are such used, as such Ti:sap- as phireTi:sapphire lasers [ lasers38]. An [38 illustration]. An illustration comparing comparing the light the dissipation light dissipation produced produced by one and by two one photonsand two is photons presented is presented in Figure in2. FigureFor the2 .linear For the absorption, linear absorption, the light intensity the light intensityis exponen- is tiallyexponentially attenuated attenuated as it propagates as it propagates through the through medium. the medium.In contrast, In contrast,for the TPA for case, the TPA at- tenuationcase, attenuation is confined is conﬁned to the laser to the focal laser volume. focal volume.

- -

FigureFigure 2. AttenuationAttenuation of of a a focused focused laser laser beam beam prompted prompted by by the the absorption absorption of of one one (above) (above) and and two two ((below)below) photons. The The white white color color indicates indicates attenuation. attenuation. The The horizontal horizontal axis axis is is given given in in units units of of the the confocal√ parameter (z0): the distance from the focal plane at which the beam waist increases by a factor of 2. Reprinted with permission [40]. Licensed under a Creative Commons Attribution 4.0 International license. Polymers 2021, 13, x FOR PEER REVIEW 4 of 31

confocal parameter (푧0): the distance from the focal plane at which the beam waist increases by a factor of √2. Reprinted with permission [40]. Licensed under a Creative Commons Attribution 4.0 International license.

The spatial confinement of the excitation promoted by TPA allows to restrict the

Polymers 2021, 13, 1994 polymerization reaction to the focal volume of a high-intensity laser,4 ofwhich 30 confers 3D capability and high resolution to the TPP technique [2]. Besides, the quadratic dependence of TPA on the light intensity pushes the resolution down by a factor of √2, which allows fabricatingThe spatial sub confinement-diffraction of features the excitation [42]. promoted by TPA allows to restrict the polymerization reaction to the focal volume of a high-intensity laser, which confers 3D capability2.2. Two and-Photon high resolutionPolymerization to the TPP (TPP) technique [2]. Besides, the quadratic√ dependence of TPA on the light intensity pushes the resolution down by a factor of 2, which allows fabricatingSince sub-diffraction the 1990s, TPP features technique [42]. [3] has been widely used in the development of different technological devices due to a varied range of advantages. In this technique, the 2.2.utilization Two-Photon of Polymerization high-intensity (TPP) pulse lasers allows the observation of nonlinear optical phe- nomenaSince in the the 1990s, irradiated TPP technique sample. [3 ]Besides has been that, widely the used polymerization in the development of small of volume (called different technological devices due to a varied range of advantages. In this technique, voxels) [43] occurs only near the focal spot, giving the sculpted structure finer resolution the utilization of high-intensity pulse lasers allows the observation of nonlinear optical phenomena(bellow toin the the diffractio irradiatedn sample. limit) Besides[9]. Moving that, the the polymerization laser beam ofinto small the volume sample, it is possible (calledto fabricate voxels) [three43] occurs-dimensional only near the structures focal spot, without giving the shape sculpted limitation, structure finerwith complex con- resolutiontours, including (bellow to manipulable the diffraction limit)devices [9]. Moving[9,44]. theSamples laser beam employed into the sample, in TPP it isusually are com- possibleposed toof fabricatemonomers, three-dimensional hybrid sol- structuresgels materials, without hydrogels, shape limitation, or composite with complex matrices [45,46]. contours, including manipulable devices [9,44]. Samples employed in TPP usually are composedThese compounds of monomers, are hybrid mixed sol-gels into materials, a photoinitiator, hydrogels, or compositean organic matrices molecule [45,46]. responsible for Theseabsorbing compounds the laser are mixedenergy into and a photoinitiator, starting the polymerization an organic molecule procedure. responsible The for use of different absorbingmonomers the laser or elements energy and in starting the employed the polymerization sample procedure. allows The matrices use of different with specific features monomers(Section 2.4), or elements which incan the be employed important sample for desired allows matrices applications. with specific features (Section 2.4), which can be important for desired applications. An essential optical element to TPP technique is the microscope objective, which is An essential optical element to TPP technique is the microscope objective, which is usedused to to focus focus the the laser laser beam beam into the into sample. the sample. Figure3 shows Figure a schematic 3 shows diagram a schematic for diagram for differentdifferent objectives objectives [38]. [38].

FigureFigure 3. 3.Light Light focusing focusing through through a microscope a microscope objective lens objective with different lens numericalwith different aperture numerical(NA) aperture and(푁퐴 working) and distance working(w.d. distance).(a) NA (w =. 0.17,d. ). ((ba)) NANA = = 0.42 0.17, and (b (c) )NA NA = = 0.42 0.87. and Reproduced (c) NA with= 0.87. Reproduced permissionwith permission [38]. Licensed [38]. under Licensed a Creative under Commons a Creative Attribution Commons 4.0 International Attribution license. 4.0 International license. The greater the numerical aperture, the smaller the working distance, i.e., the distance betweenThe the greater focal plane the and numerical the surface aperture, of the objective. the Thesmaller smallest the spot working size achieved distance, is i.e., the dis- giventance by between Abbe’s expression the focal [47 ]: plane and the surface of the objective. The smallest spot size 0.61λ achieved is given by Abbe’s expressionr = [47]: (4) NA in which, r is the focal spot radius, λ is the light wavelength,0.61휆 and NA is the numerical 푟 = (4) aperture of the microscope objective. The numerical aperture푁퐴 is deﬁned by Abbe as: in which, 푟 is the focal spot radius,NA = n .휆 sin is( θthe) light wavelength, and 푁퐴(5) is the numerical aperture of the microscope objective. The numerical aperture is defined by Abbe as:

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푁퐴 = 푛. sin (휃) (5) Polymers 2021, 13, 1994 5 of 30 in which, 푛 is the index of refraction in the focusing medium, and θ is the convergence angle of the beam. in which,However,n is the index most of pulsed refraction lasers in the have focusing an approximately medium, and θ isGaussian the convergence beam profile. Besides anglethat, of two the- beam.photon polymerization setups use microscope objectives with high numerical aperture,However, and most the pulsed minimum lasers havebeam an waist approximately can be expressed Gaussian beam by: proﬁle. Besides that, two-photon polymerization setups use microscope objectives with high numerical aperture, and the minimum beam waist can be expressed휆 by: 푤 = √푛2 − 푁퐴2 (6) 0 휋. 푁퐴 λ p 2 2 w0 = n − NA (6) where 푤0 is the beam waist at πthe.NA focal plane. The beam waist is defined as the distance

wherefrom wthe0 is center the beam of waist the Gaussian at the focal beam plane. Theup to beam the waist point is where deﬁned the as the amplitude distance of the electric fromfield the falls center to of1/ the푒 of Gaussian its maximum. beam up to the point where the amplitude of the electric ﬁeld fallsThe to photopolymerized 1/e of its maximum. structure resolution can also be improved when the fabrica- tionThe proc photopolymerizededure is performed structure around resolution the can polymerization also be improved whenthreshold. the fabrication When the laser beam procedure is performed around the polymerization threshold. When the laser beam using energyusing below energy the polymerization below the threshold polymerization value irradiates threshold the sample, value no polymerization irradiates the sample, no occurspolymerization (Figure4). The occurs more the (Figure peak power 4). The gets closermore tothe the peak threshold power power, gets the closer smaller to the threshold ispower, the voxel the size. smaller is the voxel size.

1.25 Energy I Energy II 1.00 Energy III

Threshold Energy

0.75

0.50 Energy (arb. units) 0.25

0.00 -1.50 -0.75 0.00 0.75 1.50 Radius (mm) FigureFigure 4. Threshold4. Threshold power power relative relative to various to Gaussianvarious proﬁles.Gaussian Polymerization profiles. Polymerization is not observed ifis not observed if peakpeak power power is below is below the threshold the threshold power. power. In the next section, we discuss the experimental features of TPP technique. In the next section, we discuss the experimental features of TPP technique. 2.3. Experimental Aspects of TPP 2.3.The Experimental optical systems Aspects used of for TPP microfabrication via TPP vary in different aspects, including the laser system itself and the optical components. A schematic showing the basic componentsThe optical of syst a TPPems system used is presentedfor microfabrication in Figure5. The via laser TPP beam vary comes in different from aspects, in- acluding mode-locked the laser Ti:sapphire system oscillator itself and isthe focused optical on components. the photoresist usingA schematic an objective showing the basic lens.components The pulse energyof a TPP can system be adjusted is presented by using a half-wavein Figure plate5. The and laser a polarizer. beam comes A set from a mode- oflocked two movable Ti:sap mirrorsphire oscillator is used to scan and the is focused laser beam on transversally the photoresist in the photoresist.using an objective A lens. The telescope is used to expand the laser beam to match the objective aperture. As previously mentioned,pulse energy the choice can ofbe the adjusted objective by lens using is essential, a half as-wave it determines plate theand focal a polarizer. volume, A set of two allowingmovable control mirrors of the is size used and thicknessto scan ofthe the laser fabricated beam structures. transversally A translational in the stagephotoresist. A telescope is used to expand the laser beam to match the objective aperture. As previously mentioned, the choice of the objective lens is essential, as it determines the focal volume, allowing control of the size and thickness of the fabricated structures. A translational stage is used for the sample positioning, and a mechanical shutter controls the laser exposure.

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Controlis used programs for the sample are positioning,used to manage and athe mechanical shutter, shuttermovable controls mirrors, the translation laser exposure. stage, ex- posureControl time, programs and laser are usedscanning to manage speed. the The shutter, combination movable of mirrors, movabl translatione mirrors with stage, the 3D translationalexposure time, stage and allows laser scanning the fabrication speed. The of combinationthe microstructures of movable over mirrors a large with area the in the order3D translational of millimeters. stage The allows TPP the writing fabrication process of the is microstructures performed in a over successive a large area layer in theapproach andorder can of be millimeters. monitored The in TPPreal- writingtime with process a CCD is performed camera a innd a a successive backlight layer illumination, approach while theand refractive can be monitored index of in the real-time polymerized with a CCD structure camera is and slightly a backlight changed illumination, during the while process. the refractive index of the polymerized structure is slightly changed during the process. After the TPP fabrication, the sample is immersed in an appropriate solvent, to remove After the TPP fabrication, the sample is immersed in an appropriate solvent, to remove the theunpolymerized unpolymerized material. material.

Figure 5. Representation of the femtosecond laser microfabrication via TPP setup, showing the basic Figure 5. Representation of the femtosecond laser microfabrication via TPP setup, showing the components of the system. Reprinted with permission [40]. Licensed under a Creative Commons basic components of the system. Reprinted with permission [40]. Licensed under a Creative Com- Attribution 4.0 International license. mons Attribution 4.0 International license. 2.4. Materials Used for TPP 2.4. MaterialsIn polymerization Used for TPP systems initiated by light absorption, there are two classes of photoresistIn polymeri materialszation that systems can be initiated employed, by andlight they absorption, are denominated there are astwo positive classes or of pho- toresistnegative materials photoresists. that can A great be employed, advantage and of TPP they technique are denominated is the maskless as positive fabrication, or negative due to the focalization of the laser beam into the sample. When exposed to light, positive photoresists. A great advantage of TPP technique is the maskless fabrication, due to the photoresists, such as S1800 [48], become soluble and, in the non-irradiated region, the focmaterialalization solidifies. of the laser In the beam case ofinto negative the sample. photoresists, When such exposed as SU-8 to [light,49], the positive process photore- is sists,reversed, such andas S1800 the region [48], that become will be soluble solidified and, is the in onethe thatnon is-irradiated exposed to region, light. the material solidifieA varietys. In the of case materials of negative can be used photoresists, in the TPP such experiments. as SU-8 The [49 matrices], the process used in is this reversed, andlaser the fabrication region that technique will be shouldsolidified be selected is the one considering that is exposed the application to light. of interest. For instance,A variety acrylate of materials monomers can are be widely used usedin the in TPP TPP experiments. [34,50]. These The compounds matrices usually used in this laserpresent fabrication high optical technique quality andshould are biocompatiblebe selected considering with different the microorganisms application of [ 51interest.,52]. For instance,Besides that,acrylate these monomers resins can be are easily widely functionalized used in withTPP several [34,50] materials,. These compounds giving specific usually optical or biological features [53–56]. The combination of two acrylate monomers, for present high optical quality and are biocompatible with different microorganisms [51,52]. example, can improve the hardness and prevent the shrinking of the final structure [50]. BesidesEpoxy-based that, these compounds, resins can such be as easily the SU-8, functionalized are also attractive with several for the TPPmaterials, experiment, giving spe- cificallowing optical the or manufacture biological features of structures [53– with56]. aThe high combination aspect ratio [57of ].two Hybrid acrylate materials monomers, also for example,are noteworthy can improve in TTP the experiments hardness [ 58and,59 ].prevent These matricesthe shrinking combine of thethe advantagesfinal structure of [50]. Epoxyorganic-based compounds compounds, (for instance, such flexibility as the SU and-8, chemical are also resistance) attractive with for those the ofTPP inorganic experiment, allowing the manufacture of structures with a high aspect ratio [57]. Hybrid materials also are noteworthy in TTP experiments [58,59]. These matrices combine the advantages of organic compounds (for instance, flexibility and chemical resistance) with those of inorganic components, improving different properties of the final device (such as mechanical stability and optical quality in several wavelengths) [60].

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components, improving different properties of the final device (such as mechanical stability and optical quality in several wavelengths) [60]. Negative-tone resins produced by Nanoscribe GmbH & Co. (Eggenstein-Leopoldshafen, Germany) also are applied in the TPP setups [61–63]. These resins, called IP Photoresins, can be used to fabricate several technological devices, such as optical elements for the biological scaffolds. Other commercial matrices, such as Ormocer® and Ormocomp®, have been used to sculpt devices for photonics, optoelectronics, and biological areas [48,64,65]. However, many groups have developed their own samples, directing it to the desired application. For instance, some researchers have expended efforts to develop photocrosslinkable gelatin hydrogels for biofabrication applications [66–70]. For tissue engineering applications, photoreactive resin based on poly(trimethylenecarbonate) (PTMC) has been modified through chemical reactions, aiming at the fabrication of highly porous and biodegradable scaffolds [71]. Still, regarding the employed samples in the TPP experiments, photoinitiator compounds are essential to the technique. Many groups have been working on the development or testing of new photoinitiators for TPP experiments [72,73]. Usually, they look for compounds with a high value of TPA cross-section (σTPA). After absorbing the laser pulse energy, radicals are generated. Then, these radicals can react with other sample components (monomers) and start the photopolymerization procedure. There are two mechanisms through which the photoinitiator can trigger the polymerization reaction in the TPP: polymerization by free radicals generation and cationic polymerization [74]. The active species in the polymerization by free radicals are neutral, and their incorporation with the monomers is not influenced by the polarity of the monomer or the solvent. However, molecular oxygen can inhibit its action, which is often advantageous for better resolutions in manufactured devices [9]. In free radical polymerization, photoinitiators can be classified into two groups, according to the mechanism of radical formation. The photoinitiators classified as Norish Type I, when absorbing one or two photons, are brought to an excited state and undergo breakdown (or chemical fission), producing radicals, in general, equally reactive. Among the Norish Type I photoinitiators, the commercial compound named Lucirin TPO-L [75] is a typical material employed in TPP fabrication. The photoinitiators classified as Norish Type II, for example, benzophenone [76], do not gener- ate radicals independently, and the use of a co-initiator is necessary. Such photoinitiators undergo a bimolecular reaction with a co-initiator to create radicals. Thus, as the bond cleaves homolytically after excitation, two radicals are generated, and at least one of them will initiate the polymerization reaction [77]. In cationic photoinitiation systems, an acid is generated after laser irradiation. Unlike photopolymerization initiated by free radicals, sometimes cationic polymerization can occur without chain growth control. However, some photoinduced methods allow better control of the cationic polymerization [78]. Also, cationic photopolymerization is not inhibited by oxygen. Different classes of monomers, such as epoxy, vinyl esters, and siloxanes can be polymerized by a cationic mechanism. The variety of materials that can be employed in the TPP experiments, added to the ease of sophisticated structure fabrication, makes this technique very attractive for developing the device. In the next section, we present some applications of the sculpted structures by means of TPP technique.

3. Devices Fabricated via TPP: General Applications 3.1. Functionalized Structures One of the most attractive advantages of the TPP technique is related to the production of functionalized devices, which can be applied in several technological ﬁelds. Usually, these devices are designed with speciﬁc properties according to the application need. There are different methods able to realize the device’s functionalization. The mixture of the functionalizing compound with the host sample (used in the TPP experiment) is the Polymers 2021, 13, 1994 8 of 30

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simplest method to achieving devices with special properties. For optics and photonics applications,applications, organicorganic dyesdyes cancan easilyeasily bebe addedadded toto thethe purepure matricesmatrices onlyonly byby mixingmixing thethe desireddesired elementselements [[53,55,79]53,55,79].. SeveralSeveral worksworks werewere performedperformed toto manufacturemanufacture microstruc-microstruc- turestures dopeddoped with fluorescent ﬂuorescent dyes, dyes, such such as as Rhodamine Rhodamine B, B,Disodium Disodium Fluorescein, Fluorescein, and and Stil- Stilbenebene 420 420 [53 [53]. Specifically,]. Speciﬁcally, the the use use of of these these three three dyes dyes allows allows the fabrication ofof RGB-likeRGB-like syntonizedsyntonized devices.devices. OtherOther opticallyoptically activeactive compoundscompounds ofof considerableconsiderable interestinterest inin photon-photon- icsics areare thethe azobenzene-basedazobenzene-based dyes,dyes, suchsuch asas DisperseDisperse OrangeOrange 11 (DO1),(DO1), DisperseDisperse RedRed 1313 (DR13),(DR13), oror SudanSudan BlackBlack BB (SBB).(SBB). ThisThis kindkind ofof dyedye cancan alsoalso bebe usedused asas a a photoinitiator photoinitiator inin TPPTPP experiments,experiments, keepingkeeping theirtheir originaloriginal opticaloptical propertiesproperties eveneven afterafter allall microfabricationmicrofabrication proceduresprocedures [[8080].]. FigureFigure6 6shows shows some some 3D 3D structures structures fabricated fabricated using using azobenzene-based azobenzene-based dyesdyes asas aa photoinitiator.photoinitiator.

FigureFigure 6.6. Optical microscopy microscopy (top (top) )and and SEM SEM images images (bottom (bottom) of) of3D 3D structures structures fabricated fabricated using using azobenzene-basedazobenzene-based dyesdyes as a photoinitiator in in a a concentration concentration of of 1.00 1.00 wt%: wt%: (a ()a Disperse) Disperse Orange Orange 3 3 (DO3), (b) Disperse Red 13 (DR13), and (c) Sudan Black B (SBB). The scale bar is 20 μm. (DO3), (b) Disperse Red 13 (DR13), and (c) Sudan Black B (SBB). The scale bar is 20 µm.

AsAs cancan bebe seenseen inin FigureFigure6 ,6, structures structures fabricated fabricated through through the the free free radical radical generation generation inin azoaromaticazoaromatic compoundscompounds present good good structural structural integrity. integrity. The The final final structure structure color color is issimilar similar to tothe the pristine pristine dye dye solved solved in inethanol ethanol (the (the solution solution used used to incorporate to incorporate the thedye dyeinto intothe pure the pure resin). resin). Moreover, Moreover, due dueto a high to a highdye concentration dye concentration used usedas a photoinitiator, as a photoinitiator, a sig- anificant significant amount amount remains remains unchanged unchanged in the in produced the produced structures, structures, and some and someoptical optical prop- properties,erties, such such as optically as optically induced induced birefringence, birefringence, can canbe monitored be monitored [80] [. 80]. PolymericPolymeric materials materials sometimes sometimes have have poor poor physical-chemical physical-chemical properties, properties, which makes which difficultmakes difficult their application their application in microdevices. in microdevices. An excellent An alternative excellent alternative to solve this to problem solve this is, forproblem example, is, for the example, development the development of polymeric of materials polymeric reinforced materials with rein carbonforced with nanotubes, carbon ananotubes, material widely a material known widely to have known interesting to have electrical, interesting thermal, electrical, optical, thermal, and mechanicaloptical, and propertiesmechanical [ 81properties–84]. Even [81 a–84] low. Even concentration a low concentration of carbon of nanotubes carbon nanotubes dispersed dispersed into the polymericinto the polymeric matrices (0.01matrices wt%) (0.01 can wt significantly%) can significantly improve the impro device’sve the properties device’s properties [85]. [85].Some groups have performed the fabrication of 2D and 3D structures using polymeric matricesSome reinforced groups have with performed single-walled the orfabrication multi-walled of 2D carbon and 3D nanotubes structures (SWCNT using poly- or MWCNT)meric matrices [85–89 reinforced]. Figure 7with shows single some-walled examples or multi of different-walled carbon structures nanotubes fabricated (SWCNT via TPP,or MWCNT) using SWCNT/polymer [85–89]. Figure 7 composites. shows some examples of different structures fabricated via TPP,Still, using regarding SWCNT/polymer microstructures composites. fabricated using SWCNT/polymer composites, researchers have demonstrated that the carbon nanotubes are uniformly distributed into the sample [85], and also, they can stay aligned inside the photopolymerized structures,

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according to the laser scanning direction [88,89]. Besides that, structures fabricated with MWCNT/polymer matrices, grown upon two pairs of Au electrodes, had different electrical conductivity responses according to the laser fabrication direction. For instance, structures fabricated with parallel scanning (in respect to the Au electrodes) were 1000 times more conductive than those fabricated with perpendicular scanning [89]. An increase in the Polymers 2021, 13, x FOR PEER REVIEWelectrical conductivity of over 11 orders of magnitude can be observed in acrylate-based 9 of 31

devices functionalized with 0.2 wt% MWCNT [89].

FigureFigure 7. 7.3D 3D micro/nano micro/nano structural structural SWCNT/polymer SWCNT/polymer composites composites are fabricated are fabricated by using by the using TPP the TPP lithography.lithography. The The structures structures in ( ain–f ()a are–f) aare 8-lm-long a 8-lm-long micro micro bull, abull, micro a micro tea pod, tea a pod, micro a lizard,micro alizard, a nanowirenanowire suspended suspended between between two two micro micro boxes, boxes, magniﬁed magnified image ofimage (d), and of ( ad perspective), and a perspective view of view of thethe nanowire, nanowire, respectively. respectively. Reproduced Reproduced with permission with permission [87]. Copyright [87]. Copyright © 2013, Elsevier. © 2013, Elsevier.

MechanicalStill, regarding properties microstructures are also improved fabricated after CNT using incorporation SWCNT/polymer in acrylate composites, resins. re- Forsearchers structures have reinforced demonstrated with 1 wt% that of the SWCNT, carbon the nanotubes change of are the uniformly modulus of distributed elasticity into the andsample viscoelasticity [85], and is also, notable they [85 can]. If stay the MWCNT aligned concentrationinside the photopolymerized increases in the acrylate structures, ac- material,cording the to shrinkage the laser of scanning the photopolymerized direction [88,89] structures. Besides decreases that, [89 structures]. fabricated with The device functionalization also can be performed after the complete laser fabrication MWCNT/polymer matrices, grown upon two pairs of Au electrodes, had different electri- procedure [65,90]. Figure8 shows 3D metallic structures fabricated in a large area by TPP,cal usingconductivity for this purposeresponses a microlenses according to array the thatlaser allows fabrication the fabrication direction. of For hundreds instance, struc- oftures devices fabricated simultaneously with parallel [90]. The scanning structures’ (in metallization respect to the was Au realized electrodes) by depositing were 1000 times thinmore ﬁlms conductive of small silver than particles those fabricated via electroless with plating. perpendicular Chemical scanning changes of[89 the]. An resin increase in andthe the electrical hydrophobic conductivity coating of of the over glass 11 substrate orders avoid of magnitude metal deposition can be and observed conﬁne thein acrylate- adhesionbased devices on the polymer functionalized [90]. with 0.2 wt% MWCNT [89]. Mechanical properties are also improved after CNT incorporation in acrylate resins. For structures reinforced with 1 wt% of SWCNT, the change of the modulus of elasticity and viscoelasticity is notable [85]. If the MWCNT concentration increases in the acrylate material, the shrinkage of the photopolymerized structures decreases [89]. The device functionalization also can be performed after the complete laser fabrication procedure [65,90]. Figure 8 shows 3D metallic structures fabricated in a large area by TPP, using for this purpose a microlenses array that allows the fabrication of hundreds of devices simultaneously [90]. The structures’ metallization was realized by depositing thin films of small silver particles via electroless plating. Chemical changes of the resin and the hydrophobic coating of the glass substrate avoid metal deposition and confine the adhesion on the polymer [90].

PolymersPolymers 20212021, ,1313,, x 1994 FOR PEER REVIEW 10 of 30 10 of 31 Polymers 2021, 13, x FOR PEER REVIEW 10 of 31

Figure 8. (a) 78 × 58 μm2 SEM image of a 3D periodic silver-coated structure fabricated on a hydrophobic coated glass Figure 8. (a) 78 × 58× μm2µ SEM2 image of a 3D periodic silver-coated structure fabricated on a hydrophobic coated glass surface.Figure 8.(b()a Tilted) 78 magnified58 m SEM view image of ofana individual 3D periodic uncoated silver-coated polymer structure structure fabricated composed on a hydrophobic of a cube (2 coated μm in glass size) holding surface. (b) Tilted magnified view of an individual uncoated polymer structure composed of a cubeµ (2 μm in size) holding upsurface. a spring (b) (height Tilted magniﬁed 2.2 μm, viewinner of diameter an individual 1 μm). uncoated (c) SEM polymer image structure of an individual composed silver of a cube coated (2 m structure in size) holding after electroless up a spring (height 2.2 μm,µ inner diameter 1 μm).µ (c) SEM image of an individual silver coated structure after electroless plating.up a spring Reproduced (height 2.2 withm, permission inner diameter [90 1]. Copyrightm). (c) SEM © image 2006, ofThe an Optical individual Society. silver coated structure after electroless plating.plating. Reproduced Reproduced with with permission permission [90 [90]. ].Copyright Copyright © © 2006, 2006, The OpticalOptical Society. Society.

TheTheThe use use useof ofTPP of TPP TPP combined combined combined with with with other other other laser laser laser fabrication fabrication fabrication techniques,techniques, techniques, such such such as as Laser- Laser as Laser-In--In- ducedInducedduced Forward Forward Forward Transfer Transfer Transfer (LIFT), (LIFT), allows allowsallows selective selectiveselective structure structure structure functionalization functionalization functionalization or or even or even even selec- selec- tiveselectivetive microorganisms microorganisms microorganisms inoculation inoculation inoculation [91]. [ [ 9191Figure]. Figure 9 shows9 9shows shows a microstructure a microstructurea microstructure fabricated fabricated via via TPPvia TPP andTPPand selectively and selectively selectively coated coated coated with with witha conducting a a conducting conducting polymer polymerpolymer (PPV). (PPV). (PPV). It Itis isItimportant importantis important to to highlight to highlight that that that the transferred polymer keeps its original features, even after the laser irradiation on the ttransferredhe transferred polymer polymer keeps keeps its original its original features, features, even even after after the thelaser laser irradiation irradiation on theon the thedonor donor material. material. Besides, Besides, a a material material cancan bebe LIFTed LIFTed several several times times upon upon the photopoly-the photopolymer- donormerized material. device Besides, [92], expanding a material its use can to be functionalize LIFTed several microstructures. times upon the photopolymer- izedized device device [92], [ 92expanding], expanding its use its useto functionalize to functionalize microstructures. microstructures.

FigureFigureFigure 9. ( 9.a 9.) SEM ((aa)) SEMSEM image imageimage of a of ofpolymeric aa polymericpolymeric microstructure microstructure microstructure fabricated fabricated fabricated by by fs by- fs-laserlaser fs-laser writing writing writing via via 2PP.via 2PP. 2PP. (b) (b) Fluorescence(bFluorescence) Fluorescence microscopy microscopymicroscopy image image image of the of of microstructurethe the microstructure microstructure shown shownshown after afterafter it was it it was wasselectively selectivelyselectively coated coated coated with with PPVwithPPV via PPV fsvia-LIFT. viafs-LIFT. fs-LIFT. This This image This image imagewas wasobtained was obtained obtained with with withexcitation excitation excitation at 450 at – 450–490450490– nm490. nm. nmReproduced.Reproduced Reproduced with with with per- per- missionpermissionmission [92]. [ 92Copyright [92].]. Copyright Copyright © 2021, © 2021,2021, Springer Springer Springer Nature. Nature. Nature.

StructuresStructuresStructures that that that can can canbe be manipulable manipulablebe manipulable areare very arevery attractivevery attractive attractive to variousto various to various ﬁelds, fields, mainly fields, mainly formainly for for microﬂuidics or biomedical research [44,93,94]. The structure’s movement can be achieved microfluidicsmicrofluidics or biomedical or biomedical research research [44,93,94] [44,93,94]. The. The structure structure’s movement’s movement can canbe achieved be achieved throughthrough the the use use of opticalof optical tweezers tweezers or magnetic or magnetic manipulation. manipulation. An example An ofexample manipulable of manipula- throughstructures the use is presented of optical in Figuretweezers 10. or magnetic manipulation. An example of manipulable structuresble structures is presented is presented in Figure in Figure 10. 10.

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Figure 10. Remote control of the micro-turbine in acetone. (a) Model of the micro-turbine. (b) and Figure 10. Remote control of the micro-turbine in acetone. (a) Model of the micro-turbine. (b) and (c) SEM images of the micro-turbine. (d) Top view scheme model for circumgyration. (e–i) Optical (cmicroscopy) SEM images images of the of micro-turbine.the microturbine (d in) Topa circumgyration view scheme cycle. model For for remote circumgyration. control and (observa-e–i) Optical microscopytion of the micro images-turbine, of themicroturbine a piece of ferromagnet in a circumgyration was placed cycle. on a vortical For remote device control around and the observation objec- oftive the lens. micro-turbine, Reproduced a piecewith permission of ferromagnet [93]. wasCopyright placed © on 2010, a vortical John Wiley device and around Sons. the objective lens. Reproduced with permission [93]. Copyright © 2010, John Wiley and Sons. In Figure 10, a photopolymerizable ferrofluid was developed, and 3D micro-turbines wereIn fabricated Figure 10 via, a photopolymerizableTPP. The micro-turbine ferrofluid size is approxi was developed,mately 35 μm and in 3D diameter micro-turbines with werea central fabricated axletree via and TPP. three The blades micro-turbine [93]. These size kinds is approximately of devices can 35be µmovedm in diameter and control- with a centrallable aided axletree by andan external three blades magnet. [93 ].Moreover, These kinds these of devices devices can can turn be moved clockwise and and controllable anti- aidedclockwise. by an Based external onmagnet. these results, Moreover, the importance these devices of manipulable can turn clockwise devices andis evident, anticlockwise. and Basedis possible on these thanks results, to the the TPP importance process. of manipulable devices is evident, and is possible thanksFunctionalized to the TPP process. devices are essential for several applications. One of the most signifi- cantFunctionalized areas is the whispering devices gallery are essential mode microresonators, for several applications. which we Onediscuss of in the the most next sig- nificantsection. areas is the whispering gallery mode microresonators, which we discuss in the next section. 3.2. Whispering Gallery Mode Microresonators 3.2. WhisperingOptical microresonators Gallery Mode Microresonators are microstructures that strongly confine light, both in the timeOptical and space microresonators domains [95]. areThey microstructures have been widely that exploited strongly for confine basic light,studies both on the in the timelight and–matter space interaction domains [96,97] [95]. They, and have for numerous been widely applications, exploited ranging for basic from studies lasers on to the light–mattersensors [98–100] interaction. In particular, [96,97], optical and for microresonators numerous applications, supporting ranging whispering from gallery lasers to sensorsmodes [(WGM)98–100] .are In noteworthy particular, due optical to their microresonators unique features, supporting such as high whispering temporal con- gallery modesfinement (WGM) of light, are high noteworthy sensitivity due to to the their environment, unique features, and their such ease as high integration temporal in confine-pho- menttonic of systems light, high[95]. sensitivityA light beam to can the travel environment, over 106 roundtrips and their easeinside integration a WGM microreso- in photonic systemsnator, which [95]. A significantly light beam enhancescan travel light over–matter 106 roundtrips interactions inside and a makes WGM it microresonator, possible to whichachieve significantly a plethora of enhances scientific light–matter discoveries and interactions technological and breakthroughs makes it possible [101 to–108] achieve. a plethoraWGMs of scientific are electromagnetic discoveries waves and technological that propagate breakthroughs along the rim of [101 circular–108]. structures, e.g.,WGMs cylinders are and electromagnetic spheres, by total waves internal that propagatereflection (TIR) along [ the109]. rim In ofa geometric circular structures, optics e.g.,framework, cylinders the and WGMs spheres, can bybe totalseen as internal light rays reflection that perform (TIR) [ 109a close]. In trajectory a geometric after optics a framework,round trip in the the WGMs circular can-shaped be seen structure, as light rays as shown that perform in Figure a close11d. The trajectory theoretical after anal- a round ysis of the WGMs in a given geometry is obtained by solving Maxwell’s equations with trip in the circular-shaped structure, as shown in Figure 11d. The theoretical analysis of the WGMs in a given geometry is obtained by solving Maxwell’s equations with the proper boundary conditions [40,110]. To provide an insight on the theoretical aspects of WGM

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For fundamental radial WGM modes (푚 ≫ 1, 푙 = 1), the resonance condition can be described by [110]: 2 휋 휆 = 푛 푎 (11) 0 푚

where 휆0 is the resonance wavelength, 푚 is the azimuthal order, and 푛 and 푎 are the refractive index and the radius of the microresonator, respectively. Note that, since the fundamental radial WGM modes are strongly confined to the microcylinder’s surface, their effective index can be approximated to the microresonator’s refractive index Polymers 2021, 13, 1994 12 of 30 (푛푒푓푓~ 푛). Equation (11) is helpful to estimate the spectral position of the WGM resonances without solving the characteristic equation. From Equation (11), the free spectral range (FSR), which is the frequency/wavelength spacing between consecutive azimuthal modes, canmicroresonators, be estimated by we [98,112] will take: dielectric microstructures with cylindrical symmetry as an 2 example. For this particular case, the WGMs휆 are0 electromagnetic ﬁelds that propagate in 퐹푆푅 = , (12) the azimuthal direction and have a zero axial2 휋 propagation 푎 푛 factor [111].

Figure 11. 퐸푧 field amplitude over a transverse plane of the cylinder for the azimuthal order m = Figure 11. Ez field amplitude over a transverse plane of the cylinder for the azimuthal order m = 120 120 and the following radial orders: (a,b) 푙 = 1 (휆 = 1668.2 nm) and (c) 푙 = 2 (휆 = 1579 and the following radial orders: (a,b) l = 1 (λ 120=,1 1668.2 nm) and (c) l = 2 (λ 120=,2 1579 nm). Item nm). Item (b) shows a zoomed-in view of the field120,1 distribution displayed in (a), along120,2 with the color(b) shows bar for a all zoomed-in the field distributions. view of the field The distributioncircumference displayed corresponds in (a to), alongthe cylinder’s with the edge. color To bar for all obtainthe field these distributions. field profiles, The the circumferencerefractive indices corresponds of both media to the and cylinder’s microcylinder’s edge. Toradius obtain were these set field toprofiles, 푛1 = 1 the.51, refractive푛2 = 1.0 and indices 푎 = of22 both.5 µm media in Equation and microcylinder’s (9). (d) Geometric radius Optics were picture set to nof1 the= 1.51 , n2 = 1.0 WGMsand a = propagating 22.5 µm in Equation along the (9). rim ( dof) a Geometric microcylinder. Optics Adapted picture ofwith the permission WGMs propagating [40]. Licensed along the rim underof a microcylinder. a Creative Commons Adapted Attribution with permission 4.0 International [40]. Licensed license. under a Creative Commons Attribution 4.0 International license.

The resonance takes place when the ﬁelds interfere constructively after a roundtrip in the structure, i.e., [40]:

→ → → → iβφ iβ(φ+2π) E(ρ, φ) = E0(ρ)e = E after a roundtrip(ρ, φ) = E0(ρ)e . (7)

∴ β = m; m = 1, 2, 3, . . . → where (ρ, φ, z) are the radial, azimuthal, and longitudinal coordinates; E0 is the electric field amplitude; and β is the propagation constant in the azimuthal direction. The condition given by Equation (7) takes into account the selectivity of the fields allowed in the microstructure. The fields for which β is an integer constitute the modes of the structure. However, there is a spectrum of modes for each polarization of the electromagnetic field, which are expressed as TM(z) modes when the magnetic field in the longitudinal (z) direction is null (Hz = 0), and TE modes when the electric field in the longitudinal Polymers 2021, 13, 1994 13 of 30

direction is null (Ez = 0)[40]. For a microcylinder, the longitudinal component of the electric ﬁeld for a TM(z) mode is given by [110]:  a J (k ρ)ei(mφ−ωt), ρ < a.  m m 1 Ez(ρ, φ, t) = (8)  (2) i(mφ−ωt)  bm Hm (k2ρ)e , ρ > a.

for which: k = n 2π , i = 1, 2. i i λ0 (2) In the Equation (8), a is the microcylinder’s radius, Jm(k1ρ) and Hm (k2ρ) are Bessel function of first kind and Hankel function of second kind, respectively, m is the azimuthal order, am and bm are the field amplitude coefficients, ni is the refractive index of each medium, and λ0 is the wavelength of light. The boundary conditions of the tangential fields at the dielectric interface are applied to obtain the characteristic equation for the TM(z) mode [110]:

(2) 1 Jm(k1a) 1 Hm (k2a) 2 π 0 = 0 ; ki = ni (i = 1, 2). (9) n J (k a) n (2) λ 1 m 1 2 Hm (k2a) 0

For each azimuthal order (m), Equation (9) gives a set of complex wave numbers that constitute the TM(z) modes of the cylinder. Their real part gives the resonant wave numbers, whereas their imaginary part accounts for radiation losses resulting from the cylinder’s curvature [40,110,111]. The total internal reﬂection imposes an additional boundary condition that constrains the allowed modes to the interval [110]:

2π k2a < m < k1a, in which ki = ni . (10) λ0 Thus, the WGMs are the roots of the characteristic equation (Equation (9)) that meet the TIR constraint. As an example, in Figure 11a–c it is shown the Ez distributions over the transverse plane of a microcylinder, obtained for the WGMs of azimuthal order m = 120 and different radial orders (l = 1, 2). The azimuthal order corresponds to the number of field amplitude maxima along the azimuthal direction, while the radial order corresponds to the number of field amplitude maxima along the radial direction. As shown in Figure 11a, the field distribution of fundamental radial modes is more confined to the microcylinder’s surface. Albeit, higher-order radial modes (Figure 11c) exhibit field distributions spread out inside the resonator, making them more susceptible to losses and rendering lower peak-intensity. The electromagnetic field confined in the microcylinder exhibits an exponentially decaying tail that extends to the surrounding medium, with a decay length of a few hundred nanometers. This characteristic allows light coupling in and out of the structure, being exploited, for instance, in the development of sensors. For fundamental radial WGM modes (m 1, l = 1), the resonance condition can be described by [110]: 2 π λ = n a (11) 0 m

where λ0 is the resonance wavelength, m is the azimuthal order, and n and a are the refractive index and the radius of the microresonator, respectively. Note that, since the fundamental radial WGM modes are strongly conﬁned to the microcylinder’s surface, their effective index can be approximated to the microresonator’s refractive index ( ne f f ∼ n). Equation (11) is helpful to estimate the spectral position of the WGM resonances without solving the characteristic equation. From Equation (11), the free spectral range (FSR), Polymers 2021, 13, 1994 14 of 30

which is the frequency/wavelength spacing between consecutive azimuthal modes, can be estimated by [98,112]: λ2 FSR = 0 , (12) 2 π a n One of the most important parameters used to measure the time conﬁnement of microresonators is the quality factor (Q), which is given by the ratio between the total energy stored and the power dissipated in the resonator in one cycle of the ﬁeld [112,113]:

stored energy ω τ ν Q = ω = 0 = 0 , (13) 0 dissipated power 2 ∆ν

where ωo is the angular resonance frequency, τ is the time for the stored energy to decay by a factor of 1/e, and ν0 and ∆ν are, respectively, the resonance frequency and its corresponding linewidth. The energy inside the microresonator dissipates through many routes, being intrinsic or external to the resonator. The overall losses can be written as [114]:

−1 −1 −1 −1 −1 −1 −1 Q = Qin + Qext = Qrad + Qmat + Qsur f + Qext , (14)

−1 −1 −1 −1 −1 in which, Qin (Qext) are the intrinsic (extrinsic/coupling) losses, and Qrad, Qmat and Qsur f are the major contributions to the intrinsic losses due to radiation, material, and surface scattering losses, respectively. Moreover, power dissipation in the microresonator can also occur due to the presence of surface contaminants (e.g., water adsorption) [115,116], and through nonlinear processes, such as multiphoton absorption [117], prompted by the high intensities achieved in WGM microresonators. The overall Q-factor is limited by the dominant source of loss. WGM microresonators with high Q are able to confine light for a relatively long time, such that high circulating intensities are achieved even from moderate excitation levels, because of the extremely small volume of WGMs in these structures [99]. WGM microresonators with Q in the range of 103–106 are called high-Q, and those surpassing 107 are named as ultra-high-Q [96]. The materials used for fabricating WGM microresonators are diverse and depend on the application and fabrication technique. In particular, polymeric microresonators have become increasingly competitive with microresonators made of other materials due to their structural flexibility, ease of processing and functionalization, and low cost [99]. Moreover, there are polymers with a wide range of refractive indices and with electro-optic properties available [118]. Various techniques have been used to fabricate WGM polymeric microresonators, including direct lithography patterning [118,119], molding from a master structure [120,121], surface-tension-based approach [122], solution-printing technique [123], and femtosecond laser writing via two-photon polymerization (TPP) [17,51]. In the past two decades, the latter technique has attracted significant interest due to its 3D fabrication capability, fine- tuning of the structure dimensions, and the integration of the fabricated structures into a range of substrates [2,9]. Polymeric microresonators with several geometries have been fabricated by TPP, including toroids [124], disks [125–127], cylinders [17,128], rings [26,51,129], and spheres [130]. Moreover, the flexibility of geometry afforded by this technique allows the fabrication of suspended parts (Figure 12a,c), varying the height of structures on the same chip (Figure 12b), and wire bonding fiber-to-chip coupling elements (Figure 12d and Ref. [18]), which opens up new degrees of freedom for microresonator designing. For example, the microresonators displayed in Figure 12b, which were incorporated with a photoresponsive deformable material, are vertically coupled to integrated waveguides [131]. Owing to the 3D integration scheme, the microresonator spectral response can be dynamically tuned not only through its refractive index variation but also through its mechanical deformation by a Polymers 2021, 13, 1994 15 of 30

remote optical stimulus. Another example is the integrated microresonator displayed in Figure 12d, which is coupled to a fiber coupler through a photonic bonding wire [129]. In addition to compactness, this architecture avoids alignment difficulties and reduces the losses related to fiber-to-chip coupling [18,29]. The integration of the microresonators into different substrates represents a great advantage of TPP over the other techniques. Figure 12c,e,f respectively shows microresonators fabricated inside a microfluidic channel, onto a silicone-based substrate, and onto the polished facet of a multicore optical fiber. The interface between microresonator systems and microfluidic channels/optical fibers gives rise to portable and easy-to-handle sensing units, which are promising tools for medical care, chemical manufacturing, and environmental monitoring [26,132]. Besides, the integration of microresonators into multicore fibers allows light coupling into and out through the different fiber cores, thus making the microresonator interface with laboratory instrumentation very convenient. The use of silicone as a substrate can enable various photonic applications by allowing reversible and precise tuning of the gap distance between pairs of microresonators through mechanical stretching [133,134]. It has been shown that this method affords a tuning precision comparable to a nano-positioning piezo stage but with the significant advantage that the microresonators are on the same substrate [133]. Another convenience of the TPP technique for the fabrication of microresonators is its material flexibility. Several polymeric formulations have been employed as photoresists, such as acrylic [51,135] and epoxy resins [130,132], and various materials have been incorporated into them to meet specific applications. Polymeric microresonators fabricated by TPP have been incorporated with organic dyes to realize optically active microdevices [125,130,134], with liquid crystal networks to confer elasticity for optical tunability [131,136], and with nanodiamonds to enable quantum photonics applications [135], among other materials [137]. WGM microresonators fabricated by TPP can achieve Q-factors, in the near-infrared, from 102 until 106)[124,125,138–140]. Since the WGMs are strongly confined to the microresonator’s edge, the Q-factor in such structures is primarily limited by light scattering at the microresonator surface. Therefore, the major challenge behind accomplishing a high-Q microresonator by TPP lies in ensuring optically smooth surfaces. As described in Section2, the laser writing process gives rise to polymerized volumes (voxels), which overlap to create the geometrical shapes that constitute the microstructure. In order to produce microstructures with smooth surfaces, the exposure and laser scanning parameters must be adjusted to ensure that the voxels are properly overlapped, i.e., the surface roughness associated with the individual characteristics of the voxels is significantly reduced. Figure 13a shows one of the highest Q-factor WGM microresonators fabricated by TPP thus far [17]. It is a 22.5-µm-radius hollow microcylinder fabricated from an acrylate polymer using a 0.25 NA objective lens, 0.1 nJ pulses from a Ti:sapphire oscillator, and 40 µm/s of laser scan speed. Such parameters resulted in a longitudinal and transversal voxel overlap of 85% and 90%, respectively, translating into an average surface roughness as small as 2 nm. A morphology chart of the microresonator sidewall surface is shown in Figure 13b. The microresonator was interrogated by evanescent coupling through a 2 µm-waist tapered optical fiber, as depicted in Figure 13c. Its broadband spectral response around 1550 nm (Figure 13d) contains resonances accounting for different polarization states and different azimuthal and radial order WGMs. The TE and TM fundamental radial order WGMs and their corresponding FSR are highlighted in Figure 13d. The microresonator loaded Q-factor was found to be 1 × 105, which is very close to the fundamental 5 limit imposed by the acrylate material losses (Qmat = 1.9 × 10 ). Such Q-factor is competitive with the current semiconductor platforms and holds great prospects for several applications in photonics. Polymers 2021, 13, x FOR PEER REVIEW 15 of 31

The integration of the microresonators into different substrates represents a great advantage of TPP over the other techniques. Figure 12c,e,f respectively shows microresonators fabricated inside a microfluidic channel, onto a silicone-based substrate, and onto the polished facet of a multicore optical fiber. The interface between microresonator systems and microfluidic channels/optical fibers gives rise to portable and easy-to-handle sensing units, which are promising tools for medical care, chemical manufacturing, and environmental monitoring [26,132]. Besides, the integration of microresonators into multicore fibers allows light coupling into and out through the different fiber cores, thus making the microresonator interface with laboratory instrumentation very convenient. The use of silicone as a substrate can enable various photonic applications by allowing reversible and precise tuning of the gap distance between pairs of microresonators through mechanical stretching [133,134]. It has been shown that this method affords a tuning precision comparable to a nano-positioning piezo stage but with the significant advantage that the microresonators are on the same substrate [133]. Another convenience of the TPP technique for the fabrication of microresonators is its material flexibility. Several polymeric formulations have been employed as photoresists, such as acrylic [51,135] and epoxy resins [130,132], and various materials have been incorporated into them to meet specific applications. Polymeric microresonators fabricated by TPP have been incorporated with organic dyes to realize optically active microdevices [125,130,134], with liquid crystal networks to confer elasticity for optical tunability Polymers 2021, 13, 1994 16 of 30 [131,136], and with nanodiamonds to enable quantum photonics applications [135], among other materials [137].

FigureFigure 12. Polymeric 12. Polymeric microresonators microresonators fabricated fabricated by by femtosecond femtosecond laser laser writing via via TPP. TPP. (a )(a microdisk) microdisk resonators resonators fabricated fabricated from afrom nanodiamond a nanodiamond photoresist photoresist to toenable enable quantum quantum photonics applications, applications, (b) ( twob) two vertically-coupled vertically-coupled microring microring resonators resonators incorporatedincorporated with liquidliquid crystal crystal networks networks and and an azo an dye azo to dye afford to light-drivenafford light tunability,-driven tunability, (c) microring (c) resonator microring inside resonator a insideglass a glass microﬂuidic microfluidic chip chip for biosensing, for biosensing, and (d and) integrated (d) integrated microring microring resonator resonator used for ultrasound used for ultrasound detection. (e detection.) Goblet- (e) Gobletshaped-shaped microresonators microresonators fabricated fabricated on a on PDMS a PDMS elastomer elastomer substrate substrate for coupling for coupling tunability, tunability, and (f) microring and (f) microring resonators reso- natorsfabricated fabricated on on the the facet facet of a of multicore a multicore optical optica ﬁber forl fiber vapor for sensing. vapor sensing. Figure 12 aFigure Reproduced 12a Reproduced with permission with [135 permission]. Licensed [135]. under a Creative Commons Attribution 4.0 International license. (b) Reproduced with permission [131]. Copyright © 2018, American Chemical Society. (c) Reproduced with permission [132]. Copyright © 21019, The Royal Society of Chemistry. (d) Reproduced with permission [129]. Copyright © 2017, The Optical Society. (e) Reproduced with permission [133]. Licensed under a Creative Commons Attribution 4.0 International license. (f) Reproduced with permission [26]. Copyright © 2019, John Wiley and Sons.

The microresonators with cylindrical geometry do not support light confinement in the vertical direction, which brings alignment difficulties to the coupling and leads to a more spread out mode volume. However, it can be challenging to accomplish more confined geometries, such as spheres, toroids, or even disks and integrated microrings, with smooth surfaces by TPP. Indeed, the Q-factor of microresonators with spherical [130] and toroid-like [124] geometries is in the order of 104 or lower, and the Q-factor of integrated microrings is in the range 102 − 103 [51,129,138,141]. Microdisks and integrated microrings suffer from additional scattering losses at their top surface, which is usually rougher than their sidewall surface due to the pointed shape of the voxel tips. One of the strategies used to reduce light scattering in a microdisk consists of designing its edge in the shape of a wedge and engineering the wedge angle to minimize the mode overlap with the top and sidewall surfaces [139]. One of the most studied applications of microresonators fabricated by TPP is lasing. The unique combination of Q-factor and tight mode confinement provided by WGM mi- Polymers 2021, 13, 1994 17 of 30

croresonators enables strong interaction between the light and the gain medium, which makes it possible to achieve low threshold and narrow linewidth microlasers [99]. More- Polymers 2021, 13, x FOR PEER REVIEW over, its small size and the possibility of on-chip integration make them promising17 of 31 as bright

sources for integrated photonics applications.

Figure 13.FigurePolymeric 13. Polymeric microcylinder microcylinder resonator fabricated resonator by fabricated TPP. (a) Scanning by TPP. electron (a) Scanning micrographs electron of the micro- microresonator. graphs of the microresonator. (b) Atomic force microscopy of the microresonator outer sidewall (b) Atomic force microscopy of the microresonator outer sidewall surface. (c) Schematics of the evanescent light coupling to surface. (c) Schematics of the evanescent light coupling to the microresonators through the use of the microresonators through the use of tapered fibers. (d) Spectral response of the microresonator. The attenuation peaks tapered fibers. (d) Spectral response of the microresonator. The attenuation peaks represent the represent the WGMs accounting for different azimuthal and radial orders. Highlighted in the figure are the TE and TM WGMs accounting for different azimuthal and radial orders. Highlighted in the figure are the TE fundamental radial WGMs, with their corresponding FSR. Adapted with permission [17]. Copyright © 2017, John Wiley and TM fundamental radial WGMs, with their corresponding FSR. Adapted with permission [17]. and Sons. Copyright © 2017, John Wiley and Sons. To realize microlasers, the microresonators are incorporated with organic dyes, such One of the asmost Rhodamine studied Bapplications (RhB) [125] and of microresonators Pyrromethene 597 fabricated [134]. The microresonatorsby TPP is lasing. displayed The unique combinationin Figure 14 ofa wereQ-factor fabricated and tight from mode an acrylic-based confinement photoresist provided incorporated by WGM with mi- RhB [31]. croresonators enablesTheir hollow strong cylindrical interaction geometry between was strategicallythe light and chosen the gain since medium, it supports which a smaller num- makes it possibleber to of achieve high-order low radial threshold WGMs and that narrow would otherwise linewidth compete microlasers with the [99 most]. More- fundamental over, its small sizeWGMs and for the the possibility pump energy. of A on 3D-chip reconstructed integration confocal make fluorescence them promising micrograph as confirmed the homogeneity of the dye distribution throughout the microresonator (Figure 14b), bright sources for integrated photonics applications. and absorbance/fluorescence analyses showed the neutrality of the polymeric matrix, i.e., To realize microlasers,it was shown the not microresonators to affect the emission are propertiesincorporated of the with dye organic significantly. dyes, such as Rhodamine B (RhB) [125] and Pyrromethene 597 [134]. The microresonators displayed in Figure 14a were fabricated from an acrylic-based photoresist incorporated with RhB [31]. Their hollow cylindrical geometry was strategically chosen since it supports a smaller number of high-order radial WGMs that would otherwise compete with the most fundamental WGMs for the pump energy. A 3D reconstructed confocal fluorescence micrograph confirmed the homogeneity of the dye distribution throughout the microresonator (Figure 14b), and absorbance/fluorescence analyses showed the neutrality of the polymeric matrix, i.e., it was shown not to affect the emission properties of the dye significantly. The setup used to pump and collect the emission of the RhB-doped microlasers is shown in Figure 14c. In this setup, 100 ps-pulses from a Q-switched and mode-locked Nd:YAG laser centered at 532 nm are loosely focused on the microresonator top surface,

Polymers 2021, 13, x FOR PEER REVIEW 18 of 31

and the microresonator emission is collected with a multimode optical fiber connected to a 250-pm-resolution spectrometer. Most of the other polymeric microlasers are also pumped with pulsed lasers with a time duration in the order of 10 − 100 ps [125,127,142]. The pulsed excitation prevents population transfer to the triplet state of the dye molecules, which would hinder its fluorescence quantum yield [143]. Overall, since the fluorescence lifetime of organic dyes is in the order of nanoseconds [144], the excitation being performed in the ps regime leads to less molecular cycles, thus extending the device lifetime. The emission spectra of the RhB-doped microlasers feature a set of well-defined peaks within a narrow spectral range around 600 nm. The lasing threshold was measured at 12 nJ of pulse energy, which is well within the energy levels afforded by conventional ps-lasers. Besides, such a low lasing threshold is comparable to that which has been Polymers 2021, 13, 1994 18 of 30 achieved for polymeric microlasers fabricated by other techniques [123,145,146], thus reinforcing the potential of TPP in fabricating active microresonators.

FigureFigure 14. 14.Rhodamine Rhodamine B-doped B-doped WGM WGM polymeric polymeric microlaser microlaser fabricatedfabricated byby TPP.TPP. ( a(a)) Scanning Scanning electron, electron, and and ( b(b)) 3D 3D recon- recon- structedstructed confocal confocal fluorescence fluorescence micrographs micrographs of of the the microlaser. microlaser. ( c()c) Setup Setup used used to to excite excite the the microlasers microlasers and and collect collect their their emission. (d) Emission spectra of a microlaser for the excitation energy levels indicated on the right hand side of the graph. emission. (d) Emission spectra of a microlaser for the excitation energy levels indicated on the right hand side of the graph. To make the visualization easier, an offset was applied to the y-axis of each curve. The first curve represents the spectrum To make the visualization easier, an offset was applied to the y-axis of each curve. The first curve represents the spectrum below lasing threshold. The inset shows a graph of the amplitude of the peak highlighted in red as a function of excitation belowenerg lasingy. The threshold.graph is plotted The inset on a shows linear a scale. graph The of thethreshold amplitude energy, of the which peak was highlighted obtained in by red fitting as a functionthe experimental of excitation data energy.with a Thebilinear graph curve is plotted (black on line), a linear is indicated scale. The in threshold the graph. energy, Adapted which with was permission obtainedby [31 fitting]. Licensed the experimental under a Creative data withCommons a bilinear Attribution curve (black 4.0 International line), is indicated license. in the graph. Adapted with permission [31]. Licensed under a Creative Commons Attribution 4.0 International license. Some strategies have been developed to improve directionality, monochromaticity, andThe tunability setup for used WGM to pump microlasers and collect fabricated the emission by TPP. ofOne the of RhB-doped the strategies microlasers that has been is shownused to in overcome Figure 14 thec. Inlack this of setup, directionality 100 ps-pulses of such from microl a Q-switchedasers consists and in mode-lockedbreaking their Nd:YAGrotational laser symmetry. centered This at 532 can nm be areaccomplished loosely focused by designing on the microresonator a spiral-shaped top microdisk, surface, andwhich the has microresonator been shown to emission exhibit directional is collected lasing with aemission multimode with opticala far-field fiber divergence connected of toabout a 250-pm-resolution 20° − 40° [142,147] spectrometer.. To ensure Most single of-mode the other operation polymeric without microlasers significantly are also de- − pumpedgrading withthe microlaser pulsed lasers Q-factor, with a coupled time duration WGM inmicrolasers the order ofhave10 been100 psemployed. [125,127 ,It142 has]. The pulsed excitation prevents population transfer to the triplet state of the dye molecules, which would hinder its fluorescence quantum yield [143]. Overall, since the fluorescence lifetime of organic dyes is in the order of nanoseconds [144], the excitation being performed in the ps regime leads to less molecular cycles, thus extending the device lifetime. The emission spectra of the RhB-doped microlasers feature a set of well-defined peaks within a narrow spectral range around 600 nm. The lasing threshold was measured at 12 nJ of pulse energy, which is well within the energy levels afforded by conventional ps-lasers. Besides, such a low lasing threshold is comparable to that which has been achieved for polymeric microlasers fabricated by other techniques [123,145,146], thus reinforcing the potential of TPP in fabricating active microresonators. Some strategies have been developed to improve directionality, monochromaticity, and tunability for WGM microlasers fabricated by TPP. One of the strategies that has been used to overcome the lack of directionality of such microlasers consists in breaking their rotational symmetry. This can be accomplished by designing a spiral-shaped microdisk, which has been shown to exhibit directional lasing emission with a far-field divergence of Polymers 2021, 13, x FOR PEER REVIEW 19 of 31 Polymers 2021, 13, 1994 19 of 30

been demonstrated that the coupling of two microdisks with different diameters sup- about 20◦ − 40◦ [142,147]. To ensure single-mode operation without signiﬁcantly degrading pressesthe microlaser some resonances Q-factor, coupledand increases WGM the microlasers effective have FSR been, which employed. allows accommodating It has been onlydemonstrated one mode thatwithin the couplingthe gain of region two microdisks [142,148] with. Laser different tunability diameters has suppresses been achieved some by usingresonances elastomer and-based increases materials the effective either FSR, as the which substrate allows accommodating[134] or integrated only into one modethe micro- laserswithin [136] the. For gain example, region [142 it has,148 ].been Laser reported tunability tha hast the been lasing achieved modes by of using a WGM elastomer- microlaser integratedbased materials with photo either-responsive as the substrate liquid [134 crystal] or integrated elastomers into the reversibly microlasers reach [136 a]. 0.51 For nm- shiftexample, for every it hasincrease been reportedin the pump that theenergy lasing by modes0.14 mJ/pulse. of a WGM microlaser integrated withAnother photo-responsive important liquidapplication crystal of elastomers WGM microresonators reversibly reach a fabricated 0.51 nm- shift by forTPP every is in sen- increase in the pump energy by 0.14 mJ/pulse. sor technology. The operational principle of WGM sensors is to monitor changes in their Another important application of WGM microresonators fabricated by TPP is in spectralsensor properties technology. prompted The operational by environmental principle of WGM changes. sensors Sensing is to monitor of refractive changes inindex their [128], temperaturespectral properties [141], volatile prompted organic by environmental compounds changes. [26], Sensingultrasound of refractive waves index [129], [128 and], the labeltemperature-free detection [141], volatile of biological organic compounds specimens [[132]26], ultrasound have been waves accomplished [129], and the with label- these structures.free detection The sensing of biological mechanism specimens usually [132] have relies been on accomplished resonance wavelength with these structures. shifts and/or resonanceThe sensing linewidth mechanism variations. usually An relies example on resonance of WGM wavelength sensor fabricated shifts and/or by resonance TPP is shown in Figurelinewidth 15. variations. The multicore An example fiber ofcontaining WGM sensor the fabricated WGM polymer by TPP is microresonator shown in Figure 15(Figure. 15a)The is multicoreplaced in ﬁbera vapor containing environment the WGM produced polymer microresonatorby an aqueous (Figure solution 15 a)of is propylene placed in gly- a vapor environment produced by an aqueous solution of propylene glycol monomethyl col monomethyl ether acetate (PGMEA) [26]. As shown in Figure 15b, by increasing the ether acetate (PGMEA) [26]. As shown in Figure 15b, by increasing the concentration of concentrationPGMEA, the of WGMs PGMEA, are shifted the WGMs towards are longer shifted wavelengths towards inlonger response wavelengths to microresonator in response to microresonatorswelling and refractive swelling index and variation refractive of itsindex surrounded variation medium. of its surrounded Besides PGMEA, medium. the Be- sidesWGM PGMEA, sensor the showed WGM in sensor Figure showed15a was usedin Figure to detect 15a was isopropanol used to anddetect alcohol isopropanol vapors, and alcoholexhibiting vapors, a sensitivity exhibiting of 21.7,a sensitivity 3.38, and 3.87of 21.7, pm/ppm 3.38, forand each 3.87 of pm/ppm these vapors for in each the lowof these vaporsconcentration in the low range concentration [26]. range [26].

FigureFigure 15. Microring 15. Microring resonators resonators fabricated fabricated by by TPP TPP on on the the facet facet of a multicoremulticore optical optical ﬁber fiber for for vapor vapor sensing. sensing. (a) Illustration (a) Illustration of theof light the lightpropagation propagation in the in thephotonic photonic setup. setup. (b) ( bSpectral) Spectral response response of of the the microresonator microresonator when when the the ﬁber fiber tip tip is placedis placed in the vaporin the environment vapor environment produced produced by an byaqueous an aqueous solution solution of propylene of propylene glycol glycol monomethyl monomethyl ether ether acetate acetate (PGMEA) (PGMEA) with the concentrationwith the concentration varying from varying 0% from to 2%, 0% in to steps 2%, in of steps 0.2%. of 0.2%.Adapted Adapted with withpermission permission [26]. [26 Copyright]. Copyright © ©2019,2019, John John Wiley and Sons.Wiley and Sons.

In Inthe the context context of of WGM WGM sensors, sensors, itit isis desirable desirable to to have have a clean a clean resonance resonance spectrum spectrum to to prevent distortions associated with the overlap of multiple resonance peaks. One proposed prevent distortions associated with the overlap of multiple resonance peaks. One pro- solution to achieve mode cleaning is by incorporating a lossy element in a low concentra- posedtion solution into the microresonator. to achieve mode It was cleaning shown is that by theincorporating incorporation a lossy of graphene element oxide in a (GO) low con- centrationto WGM into microresonators the microresonator. led to the It suppressionwas shown ofthat a number the incorporation of resonances of ingraphene the spec- oxide (GO)trum to withoutWGM microresonators signiﬁcantly reducing led to the the Q-factor suppression of the most of a prominent number resonancesof resonances [137 ].in the spectrumAnother without route to significantly mode cleaning reducing is by decreasing the Q-factor the microresonator of the most prominent diameter to resonances a few [137]. Another route to mode cleaning is by decreasing the microresonator diameter to a few microns [149]. In this case, radiation losses would be considerably raised for all the modes, but the higher-order radial WGMs would be the most affected and ultimately the ones to be filtered out. The most loss-free strategy towards mode cleaning consists of re-

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microns [149]. In this case, radiation losses would be considerably raised for all the modes, ducing the sidewallbut thickness the higher-order of hollow radial microcylinders WGMs would beor themicrorings most affected to dimensions and ultimately com- the ones to parable to light wavelengthsbe filtered out. [121,149] The most. Such loss-free thin strategy sidewall towards microresonators mode cleaning would consists only of reducing support the fundamentthe sidewallal radial thickness WGMs. of hollowHowever, microcylinders it can be challenging or microrings to toachieve, dimensions by the comparable TPP technique, microresonatorsto light wavelengths with [121 such,149 a]. small Such thinsidewall sidewall thickness microresonators that still wouldmaintain only support the fundamental radial WGMs. However, it can be challenging to achieve, by the TPP their structural integrity. technique, microresonators with such a small sidewall thickness that still maintain their structural integrity. 3.3. Three-Dimensional Scaffolds for Biological Investigations At present, a3.3. significant Three-Dimensional part of the Scaffolds TPP forsculpted Biological devices Investigations is dedicated to biomedical applications due to theAt free present, form a significantfabrication part achieved of the TPP by sculptedthis technique, devices iscombined dedicated with to biomedical ease functionalizationapplications of the duemicroenvironment. to the free form fabrication The design achieved and fabr byication this technique, of biological combined with scaffolds can be easeoptimized functionalization to obtain ofplatforms the microenvironment. with biomimetic The designshapes and and fabrication responses. of biological scaffolds can be optimized to obtain platforms with biomimetic shapes and responses. Several researchers have investigated the geometry (or porosity) influence of photopoly- Several researchers have investigated the geometry (or porosity) influence of photopolymer- merized scaffoldsized in the scaffolds microorganisms in the microorganisms’’ development development [21,150,151] [21,.150 Besides,,151]. Besides, the mechan- the mechanical ical and chemicaland resistance chemical are resistance also important are also importantfeatures that features should that be should characterized be characterized be- before fore applying theseapplying scaffolds these in scaffolds biological in biological studies. studies.Usually, Usually, to optimize to optimize the scaffold the scaffold pro- production, duction, many parametersmany parameters have been have defined, been defined, and andcomputational computational simulation simulation is is required. required. Figure 16 Figure 16 shows anshows example an example of three of types three typesof human of human cells cellsprofiles, profiles, red redblood blood cell cell—Figure—Figure 16a–d, 16a–d, smooth musclesmooth cell muscle (SMC) cell— (SMC)—FigureFigure 16e–h, 16 ande–h, ciliated and ciliated columnar columnar epithelial epithelial cell cell (CEC)– (CEC)–Figure 16hFigure–l, which 16h–l were, which produced were produced by TPP by technique, TPP technique, with witha negative a negative-tone-tone com- commercial acrylic photoresist from Nanoscribe [152]. mercial acrylic photoresist from Nanoscribe [152].

FigureFigure 16. Images 16. Images showing showing the designed the designed and fabricated and fabricated cell scaffolds cell scaffolds using additive using manufacturing additive manufactur- with optimized parameters.ing with (a–d )optimized Red blood cellparameters. (RBC) model. (a–d (e)– Redh) Smooth blood muscle cell (RBC) cell (SMC) model. model. (e–h ()i –Smoothl) Ciliated muscle columnar cell epithelial cell (CEC) model.(SMC) model. (a,e,i)—Rendered (i–l) Ciliated images columnar of the 3Depithelial designs cell for (CEC) a single model. cell. (b (,af,ej)—preview,i)—Rendered of a images single cell of generatedthe in the DeScribe3D designs software for a single after deﬁningcell. (b,f the,j)— parameterspreview of for a single the additive cell generated manufacturing in the DeScribe fabrication software process. (c,g,k)— Scanningafter electron defining micrographs the parameters of the fabricated for the additive individual manufacturing cells, acquired usingfabrication a 30◦ stage process. tilt, scale (c,g, bark)— isScan- 5 µm. (d,h,l)— 120 × 120ningµm electron2 cell arrays, micrographs scale bar isof 25theµ m.fabricated Reproduced individual with permission cells, acqui [152red]. Licensedusing a 30° under stage a Creative tilt, scale Commons 2 Attributionbar is 4.0 5 Internationalμm. (d,h,l)— license.120 × 120 μm cell arrays, scale bar is 25 μm. Reproduced with permission [152]. Licensed under a Creative Commons Attribution 4.0 International license.

The growing interest in the tissue engineering and regenerative medicine fields has motivated the development of several bio-applicable materials. For instance, the use of a highly reactive photopolymer in TPP experiments, with their fabrication parameters optimized, allows the production of scaffolds with large size (in the order of millimeters in the three dimensions), with relevant size for clinical application [71].

Polymers 2021, 13, 1994 21 of 30

The growing interest in the tissue engineering and regenerative medicine ﬁelds has motivated the development of several bio-applicable materials. For instance, the use of Polymers 2021, 13, x FOR PEER REVIEW 21 of 31 a highly reactive photopolymer in TPP experiments, with their fabrication parameters optimized, allows the production of scaffolds with large size (in the order of millimeters in the three dimensions), with relevant size for clinical application [71]. ScaffoldsScaffolds sculptedsculpted from from a biodegradablea biodegradable and and biocompatible biocompatible poly(trimethylene-carbonate) poly(trimethylene-carbonate)(PTMC)-based (PTMC) material-based material by TPP by (Figure TPP ( 17Figurea) supported 17a) supported the adhesion the adhesion and proliferation and prolifer- of ationhuman of adipose-derivedhuman adipose-derived stem cells stem (hASCs) cells (hASCs) [71]. Two [71 days]. Two post-seeding, days post-seeding, hASCs attached hASCs attachedto the scaffold to the exhibitedscaffold exhibited elongated elongated ﬁlopodia filopodia (Figure 17(Figureb). Over 17b). 28 Over days 28 of days culture, of cul- the ture,hASCs the colonized hASCs colonized the entire scaffoldthe entire and scaffold formed an ad dense formed cellular a dense layer cellular on the surfacelayer on of the the surfacescaffold of (Figure the scaffold 17c,d). (Figure 17c,d).

a. b. c. d.

f. g.

Figure 17. TPP-produced PTMC-based scaffolds support cell invasion and proliferation: (a) SEM images of the tri-layered Figure 17. TPP-produced PTMC-based scaffolds support cell invasion and proliferation: (a) SEM images of the tri-layered PTMC-based scaffold at various magnifications (×50, ×150 and ×300). SEM images of cell-seeded scaffolds day 2 (b) and PTMC-based scaffold at various magnifications (×50, ×150 and ×300). SEM images of cell-seeded scaffolds day 2 (b) and day 28 (c) post seeding. (d) Bright field and (e) live/dead illustrations of the colonized scaffold at day 28. (f) Cellular proliferationday 28 (c) post quantified seeding. by (d DNA) Bright assay field (§ indicates and (e) live/dead statistical illustrationssignificance) ofand the (g colonized) Ki67 immunostaining scaffold at day (nuclei 28. (off) Cellularpositive proliferativeproliferation cells quantified are stained by DNA in black). assay (§Adapted indicates with statistical permission significance) [71]. Licensed and (g) under Ki67 immunostaining a Creative Commons (nuclei Attribution of positive 4.0proliferative International cells license. are stained in black). Adapted with permission [71]. Licensed under a Creative Commons Attribution 4.0 International license. Figure 17e presents live/dead staining conducted after 28 days, showing a high cellular Figureviability. 17 Thee presents proliferation live/dead of the staining hASCs was conducted validated after by 28the days,significant showing increase a high of DNAcellular (Figure viability. 17f) Theand proliferationthe presence ofof theki67 hASCs-positive was cells validated (Figure by 17g) the [71] significant. increase of DNACultured (Figure neuronal 17f) and networks the presence are the of ki67-positivesubject of many cells biological (Figure 17 studiesg) [71]. because they allowCultured researchers neuronal to investigate networks neuronal are the activity subject in of a many controlled biological environment, studies because in compar- they isonallow to researchersin a live organism. to investigate These experiments neuronal activity provide, in a controlledfor instance, environment, a better understanding in compari- ofson the to primary in a live disease organism. mechanisms These experiments and investigating provide, the for response instance, to a bettermedicines understanding [153]. TPP of the primary disease mechanisms and investigating the response to medicines [153]. can be employed to produce 3D scaffolds, with high-resolution, using biocompatible and TPP can be employed to produce 3D scaffolds, with high-resolution, using biocompatible nondegradable photoreactive resins [153]. Figure 18 shows examples of 3D scaffolds fab- and nondegradable photoreactive resins [153]. Figure 18 shows examples of 3D scaffolds ricated using Dental LT Clear (DClear) resin employed in 3D neural cell culture and their fabricated using Dental LT Clear (DClear) resin employed in 3D neural cell culture and characterization. their characterization.

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a. b. c. d.

e. f. g. h.

Figure 18. Generation of a 3D neuronal cell culture using laser-fabricated scaffolds. (a) General view of the scaffold struc- Figure 18. Generation of a 3D neuronal cell culture using laser-fabricated scaffolds. (a) General view of the scaffold structure ture (″honeycomb″) used for 3D neuronal culture development. (b) Phase-contrast image showing uniformly distributed (”honeycomb”) used for 3D neuronal culture development. (b) Phase-contrast image showing uniformly distributed cells cells within the scaffold 5 days after seeding. (c) Immunofluorescence image depicting well-accommodated NSCs in the poreswithin of thethe scaffoldscaffold. 5 The days labeled after seeding. proteins (c are) Immunofluorescence Nestin and PAX6, characteristic image depicting of cortical well-accommodated stem cells. (d NSCs) Quantitative in the pores ratio of ofthe young scaffold. (DCX+) The and labeled mature proteins (NeuN+) are Nestinneurons and as PAX6,well as characteristic SOX2+ multipotent of cortical neural stem progenitor cells. (d) Quantitativecells in the scaffold ratio of at young day 25(DCX+) of culture/differentiation. and mature (NeuN+) (e– neuronsg) Expression as well of asyoung SOX2+ and multipotent mature neuronal neural markers progenitor DCX/MAP cells in2/NeuN the scaffold and SOX2, at day indi- 25 of catingculture/differentiation. a healthy formation (e– ofg) a Expression neuronal ofnetwork. young and(h) Differentiation mature neuronal of markersearly-born DCX/MAP2/NeuN CTIP2+ cortical neurons and SOX2, from indicating neural stema healthy cells. formationReproduced of awith neuronal permission network. [153 (h]. )Copyright Differentiation © 2021, of early-bornAmerican CTIP2+Chemical cortical Society. neurons from neural stem cells. Reproduced with permission [153]. Copyright © 2021, American Chemical Society. Figure 18a presents a 3D honeycomb-like scaffold fabricated via TPP, while Figure 18b showsFigure a 18phasea presents-contrast a 3D image honeycomb-like demonstrating scaffold uniformly fabricated distributed via TPP, cells while within Figure the 18b scaffoldshows a5 phase-contrast days after seeding. image Still, demonstrating in Figure 18b uniformly the scaffold distributed design cellsallows within a noninvasive the scaffold high5 days-resolution after seeding. visualization Still, in of Figure real-time 18b cell the growth scaffold and design neuronal allows network a noninvasive formation high- in aresolution 3D environment. visualization Immunofluorescence of real-time cell analysis growth andshowed neuronal that the network fabricated formation scaffold in pro- a 3D videsenvironment. a suitable Immunofluorescence 3D environment for analysis the rapid showed development that the of fabricated neural stem scaffold cells provides (NSCs), a facilitatingsuitable 3D the environment growth and for long the- rapidterm developmentviability of neuronal of neural networks stem cells ( (NSCs),Figure 18c) facilitating [153], whichthe growth may be and related long-term to the viability increased of neuronalcell density networks in the cylindrical (Figure 18 c)areas [153 of], whichthe scaffolds may be thatrelated leads to to the strengthened increased cell neurons density communications. in the cylindrical Figure areas 18d of therepresents scaffolds a quantitative that leads to ratiostrengthened of young neurons(DCX+) and communications. mature (NeuN+) Figure neurons 18d represents as well as a SOX2+ quantitative multipotent ratio of neural young progenitor(DCX+) and cells mature in the (NeuN+) scaffold neurons at day as25 wellof culture/differentiation. as SOX2+ multipotent neuralIn Figure progenitor 18, the cellsex- pressionin the scaffold of young at dayand 25mature of culture/differentiation. neuronal markers DCX/MAP2/NeuN In Figure 18, the and expression SOX2 indicat of younge a healthyand mature formation neuronal of a markersneuronal DCX/MAP2/NeuN network. Finally, on and day SOX2 25 of indicate3D culture, a healthy a large formation propor- tionof aof neuronal neurons network.already expressed Finally,on the day early 25-born of 3D cortical culture, neuronal a large marker proportion CTIP2 of neurons(Figure 18h)already [153] expressed. the early-born cortical neuronal marker CTIP2 (Figure 18h) [153]. MicroenvironmentsMicroenvironments functionalized functionalized with with biological biological comp compoundsounds can can also also be be sculpted sculpted usingusing TPP. TPP. To To evaluate thetheE. E. coli colibacteria bacteria development, development, microenvironments microenvironments were were developed devel- opedwith with selective selective functionalization functionalization using using ciprofloxacin ciprofloxacin antibiotic. antibiotic. The The inhibition inhibition of bacterial of bac- terialgrowth growth was primarilywas primarily observed observed around around the doped the doped element. element. By analyzing By analyzing images obtainedimages from the microenvironment, it was possible to determine the maximum range of inhibition obtained from the microenvironment, it was possible to determine the maximum range of (12 µm), as well as to quantitatively determine the bacterial density in the inhibition inhibition (12 μm), as well as to quantitatively determine the bacterial density in the inhi- halo [56]. bition halo [56]. The natural biopolymer named bacterial cellulose (BC) is an attractive material for The natural biopolymer named bacterial cellulose (BC) is an attractive material for biomedical experiments because it presents uniform structure and morphology, besides biomedical experiments because it presents uniform structure and morphology, besides unique features, such as high purity, good mechanical properties, chemical stability, and unique features, such as high purity, good mechanical properties, chemical stability, and others [154,155]. This polymer is completely biocompatible and can be produced in practi-

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Polymers 2021others, 13, 1994 [154,155]. This polymer is completely biocompatible and can be produced in prac-23 of 30 tically any shape due to its high moldability. Figure 19 shows 3D acrylate-based structures fabricated via TPP used to evaluate the BC formation through the development of Koma- gataeibacter xylinuscally bacteria any shape [156 due]. In to itsFigure high moldability. 19, it is possible Figure 19 to shows observe 3D acrylate-based the biofilms structures when bacteria are inoculatedfabricated in the via TPPmicrostructure used to evaluate for the 6 h BC (Figure formation 19a) through and 24 the h development (Figure 19c). of Koma-As displayed in Figuregataeibacter 19a, even xylinus afterbacteria a short [156 incubation]. In Figure 19 time,, it is possibleit is possible to observe to observe the biofilms a net- when work of nanofibersbacteria around are inoculatedthe structures. in the microstructureAfter 24 h, it for can 6 hbe (Figure seen 19a a)thin and film 24 h covering (Figure 19 c). As displayed in Figure 19a, even after a short incubation time, it is possible to observe a the microstructures.network Here, of nanofibersit is important around to the highlight structures. that After the 24 h,BC it cangrown be seen in microenviron- a thin film covering ments, such as thethe one microstructures. presented in Here, the itFigure is important 19, have to highlight the same that chemical the BC grown composition in microenviron- as BC grown in macroscale.ments, such The as theuse one of presentedBC as a flexible in the Figure substrate 19, have in theTPP same experiments chemical composition is very interesting for theas fabrication BC grown in of macroscale. versatile devices. The use ofThe BC natural as a flexible polymer substrate was in not TPP altered experiments by the laser irradiation,is very such interesting that its fororiginal the fabrication properties of versatile were kept devices. until The the natural end of polymer the photo- was not altered by the laser irradiation, such that its original properties were kept until the end of polymerization. Thethe photopolymerization. structures well adhered The structures to the wellnatural adhered substrate, to the natural even substrate, after washing even after the sample in heatedwashing ethanol the sample (a procedure in heated ethanolrequired (a procedureto remove required the unpolymerized to remove the unpolymer- resin). BC flexible substratesized resin). are potential BC flexible platforms substrates arefor potential liquid diffusion platforms formechanisms liquid diffusion in microen- mechanisms vironments, opening,in microenvironments, for example, novel opening, approaches for example, for novel drug approaches delivery forand drug tissue delivery engi- and tissue engineering. neering.

FigureFigure 19. Bacterial 19. Bacterial cellulose cellulose (BC) grown (BC) on polymeric grown on materials. polymeric (a) BC materials. ﬁlm formed (a after) BC 6 film h of bacteriaformed incubation after 6 h andof (b) SEMbacteria image incubation of the BC network and (b grown) SEM in image these structures. of the BC ( cnetwork) BC ﬁlm grown formed afterin these 24 h structures. of bacteria incubation (c) BC film and (d) SEMformedimage after of the 24 BC h network of bacteria grown incubation in these structures. and (d Reproduced) SEM image with of permission the BC network [156]. Licensed grown under in athese Creative Commonsstructures. Attribution Reproduc 4.0 Internationaled with license.permission [156]. Licensed under a Creative Commons Attribution 4.0 International license. Often, biological studies require several simultaneous analyses, and the TPP experiments (in their most straightforward setup) are not attractive to the microenvironments Often, biological studies require several simultaneous analyses, and the TPP experi- fabrication. However, the TPP technique can be used as a tool to create the main environ- ments (in their mostment, straightforward which can be replicated setup) several are timesnot attractive later. As an to example, the micr Figureoenvironments 20a,b shows SEM fabrication. However,images the of the TPP master technique nanograss can and be Figure used 20asc,d a tool nanograss to create replica the [157 main]. environment, which can be replicated several times later. As an example, Figure 20a,b shows SEM images of the master nanograss and Figure 20c,d nanograss replica [157].

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Figure 20. Replication of nanograss. (a,b) SEM images of the master nanograss. (c,d) nanograss Figure 20. Replication of nanograss. (a,b) SEM images of the master nanograss. (c,d) nanograss replica. Scale bars, 200 nm. replica. Scale bars, 200 nm. Reproduced with permission [157]. Licensed under a Creative Com- Reproduced with permission [157]. Licensed under a Creative Commons Attribution 4.0 International license. mons Attribution 4.0 International license. The mass production of nanotopographic surfaces opens new possibilities to study the The mass productionmechanism of related nanotopographic to cell migration surfaces and development. opens new Besides, possibilities several to replica study experiments the mechanism relatedprovide to morecell migration accurate results, and development. which can ease theBesides, understanding several ofreplicamicroorganisms exper- growth. iments provide more accurateBased on theresults, fewexamples which can presented ease the in thisunderstanding review, it is possible of microorgan- to see the substantial isms growth. importance of TPP technique to device manufacturing. The versatility of this fabrication Based on the methodfew examples allows severalpresented advantages in this review, for sophisticated it is possible technological to see the applications. substantial importance of 4.TPP Final technique Remarks to and device Perspectives manufacturing. The versatility of this fabrication method allows severalThis review advantages discussed for thesophisticated technological technological potential of the app laserlications. micromachining method known as Two-Photon Polymerization (TPP), which has a unique set of advantages. Several 4. Final Remarks andmaterials Perspectives have been used in TPP experiments, such as polymeric matrices, composites, This review and discussed gelatins. the The technological search for new potential materials that of the can laser be used micromachining in this technique is one of method known asthe Two major-Photon efforts Polymerization of different current (TPP), researches. which Structures has a unique fabricated set ofby thisad- technique have great applicability in optics, photonics, and biology, mainly due to the possibility of vantages. Several materials have been used in TPP experiments, such as polymeric matri- functionalization with several compounds of interest or the manipulable devices fabrica- ces, composites, andtion. gelatins. Combining The TPP search technique for new with materials other laser-processing that can be used methodologies, in this tech- such as Laser nique is one of theInduced major efforts Forward of Transfer different (LIFT), current allows researches. the selective Structures functionalization fabricated of by the sculpted this technique havestructures, great applicability creating new in op approachestics, photonics, to functionalize and biology, the devices.mainly due Even to living the microor- possibility of functionalizationganisms can be with transferred several compounds for 3D scaffolds, of interest without or any the damage. manipulable The wide de- variety of vices fabrication. Combining3D structures TPP that technique can be fabricated with other using laser TPP is-processing one of the attractivemethodologies, advantages of this such as Laser Inducedmanufacturing Forward technique.Transfer (LIFT), The growth allows of microdevicesthe selective manufacturedfunctionalization via TPP of is notable, the sculpted structures,principally creating regarding new approaches photonics devices to functionalize and bio-platforms the devices. development. Even living For instance, high-Q WGM microresonators with distinct geometries can be fabricated by TPP in few microorganisms can be transferred for 3D scaffolds, without any damage. The wide vari- steps, achieving performance comparable to resonators fabricated using other fabrication ety of 3D structuresmethods. that can Bioscaffolds be fabricated also using can be TPP sculpted is one using of the TPP attractive and replicated advantages several of times, keep- this manufacturinging technique. their original The features. growth Manyof microdevices microenvironments manufactured have been via created TPP is for no- studies and table, principally regarding photonics devices and bio-platforms development. For instance, high-Q WGM microresonators with distinct geometries can be fabricated by TPP in few steps, achieving performance comparable to resonators fabricated using other fabrication methods. Bioscaffolds also can be sculpted using TPP and replicated several times, keeping their original features. Many microenvironments have been created for

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monitoring of microorganisms development and can be ease functionalized with biological compounds of interest. Certainly, TPP is a consolidated laser fabrication technique with large applicability in the most diverse technological ﬁelds and will continue to be employed in sophisticated devices fabrication.

Author Contributions: Writing—original draft preparation, A.J.G.O., N.B.T. and K.T.P.; writing— review and editing, A.J.G.O., N.B.T., K.T.P. and C.R.M. supervision, C.R.M.; funding acquisition, A.J.G.O. and C.R.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by FAPESP—Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant #2018/11283-7, #2016/20094-8, #2020/04686-8); CAPES—Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CNPq—Conselho Nacional de Desenvolvimento Científico e Tecnológico. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The study did not report any new data. Acknowledgments: The authors acknowledge the financial support from Brazilian agencies FAPESP, CAPES, and CNPq. Conflicts of Interest: The authors declare no conflict of interest.

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