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3D imaging and characterization of microlenses and microlens arrays using nonlinear

microscopy

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2013 J. Phys. D: Appl. Phys. 46 195101

(http://iopscience.iop.org/0022-3727/46/19/195101)

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IOP PUBLISHING JOURNAL OF PHYSICS D: APPLIED PHYSICS

J. Phys. D: Appl. Phys. 46 (2013) 195101 (9pp) doi:10.1088/0022-3727/46/19/195101

3D imaging and characterization ofmicrolenses and microlens arraysusing nonlinear microscopyAleksandar J Krmpot1,2, George J Tserevelakis2, Branka D Muric1,George Filippidis2 and Dejan V Pantelic1

1 Institute of Physics, University of Belgrade, Pregrevica 118, 11080 Belgrade, Serbia2 Institute of Electronic Structure and Lasers, Foundation for Research and Technology Hellas(IESL-FORTH), N. Plastira 100,71110, Heraklion, Greece

E-mail: krmpot@ipb.ac.rs

Received 14 December 2012, in final form 25 March 2013Published 19 April 2013Online at stacks.iop.org/JPhysD/46/195101

AbstractIn this work, nonlinear laser scanning microscopy was employed for the characterization andthree-dimensional (3D) imaging of microlenses and microlens arrays. Third-harmonicgeneration and two-photon excitation fluorescence (TPEF) signals were recorded and theobtained data were further processed in order to generate 3D reconstructions of the examinedsamples. Femtosecond laser pulses (1028 nm) were utilized for excitation. Microlenses weremanufactured on Tot’hema and eosin sensitized gelatin layers using a green (532 nm)continuous wave laser beam using the direct laser writing method. The profiles of themicrolens surface were obtained from the radial cross-sections, using a triple-Gaussian fit. Theanalytical shapes of the profiles were also used for ray tracing. Furthermore, the volumes ofthe microlenses were determined with high precision. The TPEF signal arising from thevolume of the material was recorded and the respective 3D spatial fluorescence distribution ofthe samples was mapped. Nonlinear microscopy modalities have been shown to be a powerfuldiagnostic tool for microlens characterization as they enable in-depth investigations of thestructural properties of the samples, in a nondestructive manner.

(Some figures may appear in colour only in the online journal)

1. Introduction

Microlenses and microlens arrays are extensively used invarious applications: medical devices, wavefront sensors,confocal microscopy, imaging sensors and quantum computingsystems [1–3]. Different materials are used in the productionof microlenses, such as photosensitive glass, composites andpolymers, to mention just a few.

Knowing the properties of microlenses, such as an exactsurface profile, diameter or index of refraction, is crucial in anyapplication. Also, understanding the process of fabricationof microlenses, by measuring (photo)chemical and physicalchanges in the material of the microlens, is also important.Different methods are used for microlens characterizationsuch as thermal analysis [4], stylus profilometry [5], variouselectron microscopy techniques [6], confocal microscopy[7–9], interferometric methods [10–16], optical coherence

tomography (OCT) [7] or digital holographic microscopy[17–19]. There is not a single ideal method for microlenscharacterization. All of these methods have both advantagesand drawbacks and provide different and complementaryinformation about microlenses. Stylus profilometry gives thelens profile but only along a single cross-section. Atomicforce microscopy (AFM) improves the technique by two-dimensional stylus scanning, but it tends to be slow and witha limited scanning range (along all three axes). Scanningelectron microscopy (SEM) has excellent imaging capabilitiesand resolution but one is able to obtain a three-dimensional(3D) profile of microlens only by using unreliable stereoscopictechniques. In addition, subsurface structures cannot berecorded. Optical methods (interferometric, OCT, confocaland holographic microscopy) are used to determine somerather specific characteristic of microlenses such as the focallength or morphology of either single microlens or an array

0022-3727/13/195101+09$33.00 1 © 2013 IOP Publishing Ltd Printed in the UK & the USA

J. Phys. D: Appl. Phys. 46 (2013) 195101 A J Krmpot et al

(sometimes using quite complicated algorithms). Only a fewmethods deal with changes in bulk material during the processof fabrication of microlenses [4]. It is possible to obtainsubsurface (volume) changes after microlens formation byindirect methods, such as those used in [7, 13].

In this paper, we present results for 3D imagingof microlenses and their surface and subsurface (volume)properties obtained by different modalities of nonlinearmicroscopy (NLM), simultaneously. Due to the stepwisechange in refractive index values at the microlens surface third-harmonic generation (THG) of incident light is very efficient.Thus, we preformed 3D imaging by THG microscopy usingultrashort (femtosecond, fs) laser pulses. Imaging the surfaceof microlenses by THG microscopy is a straightforward, rathersimple, process without using any complicated algorithms forreconstruction of the surface shape from the signal unlike theinterferometric methods [10–14]. Most of the interferometricmethods are indirect, giving the changes in bulk materialmeasuring the wave front deformation and sometimes usingrather complicated numerical algorithms. Our method couldbe used for sectioning of a micrometre-sized object slice byslice. In addition, the THG method is not sensitive to materialvariation, and the method allows 3D imaging regardless of thematerial type. Unlike THG signals, which allow morphologyto be determined, (photo)chemical changes, created during theprocess of microlens manufacturing, give rise to two-photonexcitation fluorescence (TPEF) signals. After imaging themicrolenses by the two modalities of NLM we used the data forobtaining other properties, such as profile at an arbitrary cross-section, diameter, volume, focal length, astigmatism, etc. Themethod and experimental set-up used in this work are universal,versatile and widely used, not only for microlens inspection butin a broad range of biophysical and material science problems.

The microlenses used in this work are made by directlaser writing in Tot’hema and eosin sensitized gelatin (TESG)layers [20]. Tot’hema is the trade name of a drinkable solutionused in medicine for treatment of anaemia, while eosin is anorganic dye, used in medicine too. The resulting material ischeap, easy to use and biocompatible.

2. Experimental set-up

2.1. NLM set-up

The experimental set-up used to measure the properties ofmicrolenses using nonlinear effects, THG and TPEF, is shownin figure 1. The set-up was based on the one used in ourprevious work [21–23]. It consists of an Amplitude Systemsfs laser operating at 1028 nm, with an average power of 1 W,delivering 200 fs pulses at a repetition rate of 50 MHz.

The laser beam passed through a couple of adjustableneutral density (ND) filters (New Focus) to precisely controlthe power at the sample plane, and a set of galvanometricmirrors (Cambridge Tech.), which were placed on a modifiedupright optical microscope (Nikon Eclipse). The focal planewas equipped with a motorized translation stage (Standa, 1 µmminimum step). A telescope system, which expands the laserbeam, was used to fill the back aperture (entrance pupil) of the

Figure 1. Experimental set-up for the realization of the nonlinear(TPEF, THG) imaging measurements. ND—neutral density filter,L1 and L2 lenses, DM—dichroic mirror, F1 and F2—interferencefilters, O—objective lens and C—condenser lens.

objective lens. The beam was tightly focused on the sampleplane using a high numerical aperture (NA) objective lens(Carl Zeiss, Plan Apochromat 20×, NA 0.8, air immersion orCarl Zeiss, Plan Apochromat 10×, NA 0.45, air immersion).A LabVIEW-programmed interface controlled both scanningand data acquisition procedures. Samples were placed on thin(∼170 µm) square glass slides. A CCD camera (PixeLINK)was used for observation of the specimens.

THG and TPEF were simultaneously generated at thefocal plane, which were detected via different channels, TPEFin reflection and THG in transmission. TPEF signals wererecorded using a photomultiplier tube (PMT, Hamamatsu)attached at the position of the microscope eye-piece andconnected to a computer. A short pass filter (SPF 700 nm, CVI)was placed at the PMT input to cut off the reflected laser light.A condenser lens (Carl Zeiss, Plan Apochromat, 100×, NA1.4, oil immersion) was employed for the collection of THGsignals in transmission mode. After passing through a colouredglass filter (U 340-Hoya), the THG signals reached a secondPMT tube (Hamamatsu) and were recorded simultaneously onthe same computer.

A 300 × 300 pixel THG or TPEF scan was recordedin less than 400 ms. In order to improve the signal-to-noiseratio, each image was the average of 30 scans. Thus, thetime required to obtain one image was approximately 12 s.This makes our method quite fast in comparison with most ofother techniques used for microlens characterization. ImageJ (Java-based program developed by Wayne Rasband at NIHhttp://rsb.info.nih.gov/ij/) was used for viewing and processingthe recorded data. To create a 3D reconstruction of oursamples, a series of two-dimensional (2D) optical sectionsseparated by 1 µm were acquired. The whole procedure of dataacquisition, image processing and rendering needed for the 3Dreconstruction of nonlinear images from specimens lasted from5 to 15 min (30 to 80 slices depending on microlens depth). Theaverage laser power on the sample during the experiments was30 mW (0.6 nJ per pulse, 3 kW peak power).

2.2. Microlens manufacturing

Microlenses were fabricated on a gelatine layer doped withTot’hema and eosin. Tot’hema forces the layer to retain water

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(a) (b)

Figure 2. (a) An image of a regular square grid observed through the microlens array. (b) An optical system for generation of laser-inducedmicrolens arrays.

and reduces its melting temperature, while eosin is added inorder to sensitize the material to wavelengths in the green partof the spectrum. The layer was prepared on a well-cleanedsquare (22×22 mm) cover glass with 0.13–0.16 mm thickness.Details of the layer preparation procedure can be found in[4, 5]. A trace amount of NaCl is added in order to preventcrystallization of the layer. If irradiated, the material developsa concave dip, which is used as a microlens. During irradiationthe laser beam locally heats the material, which melts and flowsunder the influence of thermo-capillary forces. The resultinglens is transparent as a consequence of photobleaching of eosin,and the surface is smooth, capable of producing high-qualityimage at its centre (see figure 2(a)).

We were able to manufacture single microlenses andarrays using a device schematically shown in figure 2(b). Asample with Tot’hema and eosin sensitized gelatine was placedon the coordinate table and irradiated using an Nd-YAG laserat 532 nm, which was used as a writing beam. A beam froman He–Ne laser was used as a pilot beam in order to observethe lens production in real time.

3. Results

3.1. Characteristics of NLM measurements and 3D imagingof microlenses

We will briefly review the theory of effects relevant to NLM. Itis well known [24] that the incident electromagnetic radiationinduces macroscopic polarization of a medium. In the electricdipole approximation, the ith component of the inducedpolarization vector may be written as a Taylor expansion onthe strength of the applied electric field E:

Pi = ε0(χ(1)ij Ej + χ

(2)ijk EjEk + χ

(3)ijkl EjEkEl + · · ·) (1)

where the summation over the repeated indices is assumed,ε0 is the vacuum dielectric constant, χ(1) is the linearsusceptibility (second-rank tensor describing optical linearprocesses such as linear absorption or refraction), χ(2)

is the second-order nonlinear susceptibility (third-rank

tensor describing three-wave interactions such as second-harmonic generation—SHG) and χ(3) is the third-ordernonlinear susceptibility (fourth-rank tensor describing four-wave interactions such as THG, coherent anti-Stokes Ramanscattering—CARS or two-photon absorption—TPA). THG isa nonresonant, coherent process where the laser light is up-converted to shorter wavelengths by a factor of 3 with respectto the incident wavelength. The most important property of theTHG process, when it is used as a contrast mechanism, is thatTHG efficiency is the highest in regions where there is a largechange in refractive index. Since the THG intensity (ITHG) hasa third-order dependence on the incident intensity (Ii)

ITHG ∼ [χ(3)]2I 3i (2)

it is crucial to use short light pulses (preferably fs) in order tomaximize the signal.

The THG process is highly efficient at the opticalinterfaces, i.e. where a sudden change in the refractive indexexists [24]. As a consequence, the signal at the PMT ismaximized whenever the focus (waist) of the laser beam ispositioned at the interface of the microlens material duringthe scanning process. Otherwise (if the focal point is insideor outside of the material), the THG process is very weakand there is no signal from the PMT. Thus, one is able toreconstruct the 3D profile of the sample surface. We used NLMto analyse a number of microlenses obtained under differentmanufacturing conditions: different chemical compositionsof the TESG material (concentration of Tot’hema), variouslaser powers (40–120 mW) and exposure times (1–10 s). Theobtained results were used to make 3D reconstruction of thelenses and to analyse their surface profile and calculate opticalproperties. In figure 3, three-dimensional reconstruction of themicrolens recorded in the TESG material is shown. The lenswas made by the set-up shown in figure 2 during 5 s exposuretime and with 40 mW laser beam power. The thickness ofthe TSG layer was 60 µm and the concentration of Tot’hemawas 10% v/v. The microscope recording was made using a20× objective lens. The shape of the microlens exhibits an

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J. Phys. D: Appl. Phys. 46 (2013) 195101 A J Krmpot et al

Figure 3. 3D rendered reconstruction of a microlens obtained withTHG microscopy. All dimensions are in micrometres.

obvious toroidal bulge at the rim of the microlens that was alsofound in former measurements by other techniques [4, 5]. THGmodality could be used regardless of the material type since itsintensity depends only on the difference in refractive indicesbetween air and the material, and any material for microlensfabrication has much higher refractive index than air.

Information about the microlens is contained in a setof slices (figures 4(a)–(c)) obtained by scanning through thevolume of the material. Slices are further processed in orderto produce a 2D matrix containing the profile of the surface,which can be used for visualization and rendering. A stack ofslices is computer processed to determine the exact position ofthe surface cross-section.

At this point, it is convenient to emphasize the simplicityand versatility of the proposed method. As could be understoodfrom figure 4, the method enables slicing of the sample andthe images are obtained in a quite straightforward way: duringthe scanning process the signal in each point of the plane isrecorded and converted to the image. Thus, the method doesnot need any complicated algorithms for obtaining the sliceimage from the recorded signal.

All lenses (including those presented in figure 3) wererecorded in 300×300 dots with a lateral resolution of 0.8 µm.The scan area was 235 × 235 µm2. The axial resolution wasestimated to be around 1.5 µm from slice separation (1 µm)and the refractive index of the TESG layer was n ≈ 1.537.

From the 3D reconstruction of the lens the profiles ofthe surface in yz (vertical) cross-sections are extracted andshown in figure 5. The distance between two adjacent yzcross-sections is 25.5 µm. Since the shape of the cross-sections is symmetrical, we conclude that the microlens surfaceis also undeformed and symmetrical. It seems that thelaser beam penetrates uniformly through the material duringmicrolens formation.

3.2. Measurements of the microlens profile

The profiles of four radial cross-sections of the lens fromfigure 3 are shown in figure 6(a). The angle between planesof two adjacent cross-sections is 45◦ (see the inset). The curve

denoted with AVG is the average of the four cross-sections.Comparing profiles obtained with THG microscopy with theprofile presented in [5] obtained with stylus profilometryit could be seen that they are in good agreement and thatcharacteristic features like two symmetrically placed bulgesare present. The advantage of THG microscopy is that oneis able to obtain a number of cross-sections/profiles in anydirection from a single measurement. The profiles obtained insuch a way are used to verify the cylindrical symmetry of themicrolenses, which is important for image quality.

The residuals, i.e. the differences between the averagedcurve and each cross-section, are given in figure 6(b).Considering the given residuals it is clear that maximaldeviation is around 1–2 µm which is in the range of theaxial scanning resolution of the experiment. Any systematicdeviation of a certain cross-section from the average curve,which would indicate lens asymmetry, was not noticed withinthe accuracy of the experiment for any lens.

Certain amount of noise is present, as can be seen infigure 6(a), and it defines a confidence interval σ calculatedduring the averaging procedure. It is a consequence ofthe detection procedure and precludes the measurement ofsurface roughness. However, the overall lens profile is easilycalculated.

The central part of the lens profile follows the Gaussiancurve since the writing beam used for microlens fabricationhas the same profile. Several physical processes, occurringduring microlens formation, affect the shape of the edgesof the microlens profile. It was verified in the previouspaper [20] that the laser beam induces a Gaussian temperaturedistribution. As a consequence, the gelatin layer is liquefiedand thermocapillary forces push the liquid to the edges.

If the analytical shape of a lens profile is known then itcan be used in lens design programs for a detailed study ofthe refraction of light at the surface of the lens. Thus, weperformed a triple-Gaussian fit of the averaged lens profile(tick magenta curve from figure 6(a)) and the results of the fitare shown in figure 7. Each of the three Gaussian peaks and thecumulative fit curve are shown in figure 7(a). The goodness ofthe fit obtained from the numerical procedure is R2 = 0.9989.

In order to test the imaging quality of the microlens, a ray-tracing analysis was performed (figure 7(b)). A number of lensparameters were also calculated: the focal length, the positionof principal and nodal planes, aberration diagrams, resolution,diffraction limit, influence of a toroidal rim around the lens onthe image quality. All these calculations are a useful guide inconstructing lenses with better imaging characteristics. Fromthe production point of view all these are used to ensure thereproducibility of microlens shape.

3.3. Volume properties of microlens

From the data of 3D picture we were able to determine volumesV2 of the outer toroidal ring, which is above the unaffectedsurface, and V1 of the lens (see the sketch in figure 8). Onewould expect that the material from volume V1 is transferred tovolume V2, and therefore, they should be equal. However, theV2/V1 ratio is in the range 30–40% regardless of the microlens

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Figure 4. Three THG slice images through the microlens taken at different depths (denoted at the bottom left corner of each picture) insidethe TESG layer. The inset in (a) is the microlens observed through an ordinary optical microscope.

(a) (b)

Figure 5. Cross-sectional view of the microlens from figure 3. (a) 3D and (b) individual axial cross-sections extracted from the 3Dreconstruction. The distance between two adjacent cross-sections is 25.5 µm.

formation conditions (writing laser power and exposure time).This indicates that evaporation of water and/or compression(due to crosslinking) of the material occurs during microlensformation since volume V2 is always smaller than V1.

In addition, volume V1 of a lens increases in proportionto the laser power or/and exposure time (figure 8(b)). But,surprisingly volume V2 of the outer ring drops as exposure timegoes up, for the same laser power (figure 8(c)). This could bean indication of the nature of the complex processes occurringduring laser-induced material melting. We think that this is anindication of material crosslinking and consequent shrinkage.Another possible mechanism—water evaporation—is not veryprobable, since our thermographic measurements show that themaximum temperature at the centre of the beam is about 50◦.

The nonlinear-microscopy techniques (TPEF and THG)are powerful enough to reveal defects in microlenses. Wewere able to observe high-aspect-ratio imperfections in thesamples of the TESG layer with Tot’hema concentration 20%v/v and thickness 120 µm. At the 100 mW laser power and5 s exposure time, a deep hole (with the approximate aspectratio of 1 : 5) was drilled in the material. 3D reconstruction of

such lens with a hole at its centre is shown in figure 9. Weshow the 3D reconstruction obtained from both THG signal(figures 9(a) and (b)) and TPEF signal (figures 9(c) and (d)).The recording was performed by using a 10× objective lens,with 500×500 pixel resolution, and with a lateral resolution of1.6 µm. The scan area was 470×470 µm. The axial resolutionwas estimated to be around 3 µm as apparent depth from sliceseparation (2 µm) and the refractive index of the TESG layerwas n ≈ 1.537. As stated before, THG signal is producedefficiently at the interface and is vanishingly small (due todestructive interference) inside the volume of the material.Consequently, the signal is obtained from the microlens surfaceand from the glass surface observable through the hole atthe bottom of the microlens (small green dot in figure 9(b)).Since the walls of the hole are parallel to the incident beampropagation, i.e. the optical axis, the THG signal originatedfrom the wall’s surface cannot be collected due to the Gouyphase shift [25]. As in [26], the deflection of the THGradiation maximum from the optical axis cause that little signalis detected from an interface parallel to the optical axis. Thus,for an interface parallel to the optical axis the THG radiation

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J. Phys. D: Appl. Phys. 46 (2013) 195101 A J Krmpot et al

(a)

(b)

Figure 6. (a) Radial cross-sections of the microlens from figure 3extracted from the 3D reconstruction. The angle betweencross-sections is 45◦ (see the inset). The tick magenta curve is theaverage of all four cross-sections. (b) Residuals, the differenceamong the average curve and each of the cross-sections. Thehorizontal red lines denote the axial measurement uncertainty(σ ≈ 1.5 µm).

propagates off the optical axis, since the Gouy phase shiftdeflects the phase matching direction [25].

Since eosin in the TESG layer exhibits strong absorptionin the region 470–570 nm [4], due to the high peak power ofthe ultrashort pulses, the probability for two-photon absorptionof 1028 nm photons is high enough, and consequentlyfluorescence occurs. The intensity distribution of the TPEFsignal obtained from the volume of the material reveals thepotential photochemical changes in the material. The TPEFsignal arising from the volume of the material enables oneto visualize the walls of the hole in TPEF 3D reconstruction.Depending on the material type and excitation laser wavelengththe TPEF modality could be used for detection of possible(photo)chemical changes and impurities.

For the lens from figure 9, the corresponding central cross-section profiles are given in figure 10. The central cross-sectionshown in figure 10(a) is the profile extracted from the TPEFsignal only and it is shown in pseudo-colours. The profilein figure 10(b) is extracted from the THG signal (white) anda combination of THG (white) and TPEF (pseudo-colours)

(a)

(b)

Figure 7. (a) Triple-Gaussian fit of the average curve of the fourcross-sections from the previous figure. The goodness of the fitmeasured after coefficient of determination R2 = 0.9989.(b) Ray-tracing analysis performed with obtained analytical shapeof microlens profile. The distances are given in arbitrary units.

is shown in figure 10(c). Also, the distribution of the TPEFsignal, which is visible from the cross-section presented inpseudo-colours, indicates the appearance of the mechanical orphotochemical changes during microlens formation, inside thevolume of the material.

As shown in figures 9 and 10, there is no limitation for thepenetration depth of the proposed method, except for technicalones (e.g. working distance of microscopic objective). Thus,one is able to make a 3D image of a microbject of an arbitrarydepth or relief. The reason lies in the tight localization ofnonlinear effects within the focal volume of the laser beam.It should be stated that the accuracy and precision of themeasurement results depend on the number of experimentalfactors: focal spot diameter, i.e. NA of the objective lens,sampling interval, the mechanical movement of the laser beamin the lateral (XY) and the sample in the axial (Z) dimension,wavelength of the incident laser light and refractive index of the

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(a)

(b) (c)

Figure 8. Volumes V1 of the microlens and V2 of the external ring depending on the laser power and exposure time during microlensmanufacturing.

sample. In our case, for an objective lens with NA = 0.8, thelateral focal spot diameter was estimated to be 2w0 = 0.8 µmusing the well-known relation

w0 � λ

π · NA(3)

where λ = 1028 nm is the wavelength of the incident laserlight. Since the probability of producing a nonlinear effectdepends on the mth power of the illumination intensity (m isthe order of the nonlinear process) [24] the effective focal spotdiameter is estimated to be 2w0/

√2 = 0.57 µm for second-

order nonlinarities (TPEF in our case) and to be 2w0/√

3 =0.46 µm for third-order nonlinarities (THG in our case). Thelatter diameters would be the lower limit for lateral resolutionif the spatial sampling rate is such that the Nyquist criterion isfulfilled. Using a microscopic objective lens with higher NAone can improve the lateral resolution but usually the field ofview is reduced. For volume measurements, accuracy of therefractive index determines the overall measurement accuracy.Along the vertical axis, precision also depends on the depthof the focus of the laser beam and the THG or the TPEFgeneration volume. With 20× objective (NA = 0.8) and ourdetection system we were able to detect changes in the THGsignal for 1 µm vertical displacement of the interface (apparentdistance). With a given index of refraction for the TESG layerthis apparent distance refers to ≈1.5 µm real distance. With

10× objective (NA = 0.45), changes in the THG signal weredetected for 2 µm vertical movement of the sample, whichrefers to ≈3 µm real distance.

The TPEF signal can be used to get information about thehomogeneity of the microlens volume (see figures 10(a) and(c)). As can be seen the material volume becomes particularlyinhomogeneous around the defect (a hole in the material).

3.4. 3D imaging of the microlens arrays

For microlenses with a smaller diameter we were able to recordthe whole microlens array and to make 3D reconstruction.This was done by detecting either the THG or TPEF signal.There is no significant difference between these two modalitiesconcerning the morphology of the array, thus we show only thereconstruction made from the THG signal (figure 11). The scanarea was 235 × 235 µm2 for both, figures 11(a) and (b). Theresolution was 300 × 300 pixels with a pixel size of 0.8 µmin the lateral direction, and 1.5 µm resolution in the axialdirection, as in all previous cases. The microlens matrix wasmade in the direct laser writer set-up enabling tight focusing ofthe writing laser beam and precise movement (of the order of2 µm) of the sample. The writing laser operates at 473 nm andthus gelatin was sensitized with an aqueous solution of yellowfood dye (tartrazine E102) instead of eosin.

Figure 11 shows that tartrazine could be used insteadof eosin for microlens fabrication and that the obtained

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Figure 9. (a), (b) THG (green) and (c), (d) TPEF (yellow) images of the microlens with the defect (a hole) extending from the bottom of themicrolens through the material to the glass surface. The small green dot in (a) and (b) is due to the THG signal from the air–glass interface.

Figure 10. Radial profiles of the lens from the previous figure. (a) TPEF signal shown in pseudo-colours where the blue is less and the redis more intense signal, (b) THG signal only, and (c) combined THG (white) and TPEF (pseudo-colours) signals.

Figure 11. 3D reconstruction of (a) 2 × 2 and (b) 4 × 4 microlens array, performed using THG signals. The scanning area is the same as infigure 3 (235 × 235 µm2).

microlenses are of good quality within the possibilities ofdetection and resolution of our method. Furthermore, thelenses in arrays are reproducible and regularly positioned on asquare grid.

4. Conclusions

We performed 3D imaging and characterization of microlensesproduced in biocompatible, organic and elastic Tot’hema and

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eosin sensitized gelatin (TESG) layers. Measurements weremade using a nonlinear microscopic set-up with two pathwaysof signal detection: THG and TPEF. The proposed method andexperimental set-up are versatile, already used in many otherapplications and they are not specially designed for microlenscharacterization. The method is reliable, suitable for surfaceand volume measurements, and compatible with other methodsoffering additional information about the samples (subsurfacefeatures and volumetric information). After the 3D image isobtained one is able to extract other information useful formicrolens analysis (volume, aspect ratio, homogeneity). Themorphology of the microlenses was determined and then thetriple-Gaussian fit of the surface profile was performed. Raytracing was used to calculate the imaging properties of themicrolens.

The most important advantage of NLM used for microlenscharacterization is that it enables investigations of the volumeproperties of the sample regardless of its thickness/depth. Themethod is straightforward and it does not use any complicatednumerical algorithms for obtaining slice images from recordedsignals. We were able to determine surface shape at any depthof microlenses and to calculate the volumes of the microlenses.For microlenses with defects (like very narrow holes at thecentre, see figure 9) the depths of microlenses and the holeare determined and it is verified that the writing laser beamdrilled the hole to the glass substrate surface. Even more, byemploying the TPEF modality, the signal from the volume ofthe material can be detected, which is not possible, to the bestof our knowledge, with other methods.

Acknowledgments

The authors thank the FP7 projects ‘LASERLAB-EUROPE’(228334) and the ‘HERACLITUS II-University of Crete’funded by the European Social Fund and national resources.The authors also acknowledge the help from the grants III45016 and OI 171038 of the Ministry of Education, Scienceand Technological Development of the Republic of Serbia.

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