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Article Optical Design of an LED Source for

Tai-Chih Kuo 1, Ting-Jou Ding 2, Jui-Hui Lin 3 and Shih-Hsin Ma 3,*

1 Department of Biochemistry and Molecular , Taipei Medical University, Taipei City 11031, Taiwan; [email protected] 2 Department of Materials and Energy Engineering, Ming Dao University, No.369, Wen-Hua Rd., Pitou, Chang Hua 52345, Taiwan; [email protected] 3 Department of Photonics, Feng Chia University, No. 100, Wenhwa Rd., Seatwen, Taichung City 40724, Taiwan; [email protected] * Correspondence: [email protected]

 Received: 1 October 2019; Accepted: 22 October 2019; Published: 28 October 2019 

Abstract: In this study, we reveal an LED source model applied in fluorescence microscopes. This optical model is composed of a confocal total internal reflection array system (CTLAS) with a nine-LED array. The CTLAS optical system that we designed consists of a total internal reflection (TIR) lens array and a confocal system. The electrical power of the nine-LED array is 7.9 watts, which is lower than traditional light sources, such as the original 120-watt lamps used in fluorescence microscopes (Zeiss, Axio Imager 2). We have successfully applied the CTLAS system to an Axio Imager 2 fluorescence to observe the vascular bundle organization, modified with Cy3 fluorescence , and have found that in the process of system assembly, the fabrication errors of optical could have a critical effect on the CTLAS system. The results of our experiment show that, in order to achieve the same illuminance as that of the , the displacement error tolerances of the lateral x-axis and the longitudinal z-axis must be controlled within 1.3 mm and 1.7 mm, respectively.

Keywords: LED light source; optical model; uniformity; optical design

1. Introduction For the past decade, scientists have been interested in the field of [1]. In this field, the application of imaging is a particularly fascinating research topic. Bio-imaging technology provides an excellent for acquiring high quality images. Usually, when scientists observe germs, cells, tissues and other objects by microscopes, high resolution and sufficient contrast are the primary factors which determine the microscopic image quality [2,3]. However, in the study of biology, many analytes exceed the diffraction limit of an in the visible light band. In order to see those small analytes, scientists modify the fluorescence modules and excite them to emit light of specific . Accordingly, we can use conventional optical microscopes to observe nanoscale objects. As a fluorescence microscope can excite fluorescence modules and detect the emitted light, the light source of the fluorescence microscope that is used to generate fluorescent light is an essential component that must be sufficiently powerful, and which must have the correct light . Halogen lamps, or xenon arc lamps with broad band light used in commercial fluorescence microscopes [4–10], are usually employed as light sources [11]. The broad band light is used for both conventional visual observation and fluorescence excitement. Considering the energy efficiency of light excitation, we understand that only a part of all emitted light, in the specified spectrum that

Appl. Sci. 2019, 9, 4574; doi:10.3390/app9214574 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 4574 2 of 14 can be absorbed by the (fluorescence modules), is used in the excitation process. An amount of light from the light source is blocked by different types of optical filters which cannot ideally block all undesired light, due to fabrication defects. The leaked light will generate noise and reduce image quality. Additionally, optical filters would waste most of the lamp’s energy because most light energy is outside the region of the absorption spectrum. This means that we need more powerful light sources when operating microscopes in fluorescence mode. Without such light sources, the image in the view field will become darker and blurry. If the emitted light spectrum can fit into the energy absorption dye range, we can have a brighter view field and a stronger signal. In this way, selecting a light source that has excellent luminous efficiency to achieve the goal is very important in . For the past ten years, light emitting diode (LED) techniques have been developing rapidly with a major breakthrough when the luminous efficacy exceeded the 100 Lm/W mark. The high luminous efficiency has enabled LEDs to replace the traditional lamp in many applications. In addition, LEDs have other advantages, such as excellent , longer lifespan, etc. Moreover, using monochromatic LEDs is a good solution to the excitation of fluorescent molecules. To work on various types of fluorescent molecules, we can choose LEDs with a corresponding spectrum to install into the microscopes. How to design an excellent LED light source has been an important issue among researchers. Herman proposed a -domain fluorescence microscope using LEDs as a light source in 2001 [12]. Hohman incorporated this new LED technique into the Colibri illumination system in 2007 [13]. Bormuth applied different color LEDs in fluorescence microscopes that effectively suppress the peripheral light of video-enhanced DIC in 2007 [14]. Hanscheid took low-cost and long-life span LEDs to replace more expensive lamps in fluorescence microscopes to detect tuberculosis in resource-poor areas in 2008 [15]. Albeanu introduced the use of a simple light pipe to couple the light emitted from an LED light source to a fluorescence microscope in 2008 [16]. Martin et al. and Gerhardt et al. used LED light sources to detect single molecules in 2009 and 2011, respectively [17,18]. Compact optical microscopes have been developed based on the idea that researchers built mobile phone-mounted light microscopes to detect diseases in developing regions where health treatment is unaffordable, due to expensive equipment and training costs, but which are well served with phone networks [19]. The small-size characteristic of LEDs is the key that has contributed to the designs of many compact, portable optical or fluorescence microscopes [20,21]. The functionality of LED light sources in fluorescence microscopes is multipurpose. For example, they provide a multi- spectrum, and combine with a system, and they are suitable for the built-in function of commercial devices [22–24]. Moreover, LEDs in high-power applications are designed in array form to enhance the illumination of the images [25–31]. However, both single LED and LED array designs suffer from the critical problem that the light from LEDs has a large divergent angle and a wide emitting surface area, making the étendue of the light source large, so that the light energy is not easily sufficiently concentrated. Hence, a LED lighting system with excellent light energy concentrating skill is necessary for a fluorescence microscope, and can provide brighter illumination for the specimen in lower electrical power. Given that the optical design of LEDs is important, especially for the light energy absorption of fluorescent molecules, we propose a novel LED light source system composed of a 3 3 LED array × to replace the original light source system of the Zeiss fluorescence microscope (Axio Imager 2). The original light source of the Axio Imager fluorescence microscope is a light halogen lamp with 120-watt electrical power and a circle project pattern with a 27 mm diameter, while the distance between the light source and the specimen of the microscope is 550 mm. The central illuminance value of its halogen lamp projected on the specimen is approximately 10,578 lux. In order to use LEDs to replace the halogen lamp, we have designed a confocal total internal reflection lens array system (CTLAS), which is composed of a total internal reflection (TIR) lens array [32–39] and a confocal LED array system [40–42]. This system can efficiently collect light energy and project it onto the specimen. The assembly tolerance of the LED light sources will be discussed in the latter part of this paper. Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 14

Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 14 Appl.2. Optical Sci. 2019 Design, 9, 4574 of the LED Light Source System 3 of 14 2. Optical Design of the LED Light Source System 2.2.1. Optical The Optical Design Model of theBuilding LED of Light a Single Source LED System 2.1. TheIn Optical the design Model setup, Building we ofuse a SingleCy3 fluorescence LED molecules as a reference material for fluorescence 2.1. The Optical Model Building of a Single LED absorption.In the design To achieve setup, wethe uselargest Cy3 light fluorescence flux passin moglecules through as thea reference excitation material filters offor the , absorption.the spectrumIn the To design achieveof the setup, selected the welargest LED use light Cy3 must fluorescenceflux fit thepassin absorptiong moleculesthrough spectrum the as excitation a reference of the filtersCy3 material fluorescent of the for microscope, fluorescence molecules theabsorption.as spectrum closely as of Topossible. the achieve selected A the Lumile LED largest dsmust LED light fit (LXML-PX02-0000) fluxthe absorption passing through spectrum made the excitationofby thePhilips Cy3 filters companyfluorescent of the was molecules microscope, chosen to asthebuild closely spectrum up as our possible. oflight the source selectedA Lumile system. LEDds LED must The (LXML-PX02-0000) fitabsorption the absorption and emission spectrummade by Philipsspectrum of the Cy3company of fluorescent the Cy3was chosenfluorescent molecules to buildasmolecules closely up our asand light possible. the source spectrum A system. Lumileds of the The selected LED absorption (LXML-PX02-0000) LED are and shown emission in made Figure spectrum by 1. Philips of the company Cy3 fluorescent was chosen moleculesto build upand our the lightspectrum source of system.the selected The LED absorption are shown and in emission Figure 1. spectrum of the Cy3 fluorescent molecules and the spectrum of the selected LED are shown in Figure1.

Figure 1. The absorption and of Cy3 fluorescent molecules, and the emission Figure 1. The absorption and emission spectrum of Cy3 fluorescent molecules, and the emission spectrum of the selected LED. Figurespectrum 1. The of theabsorption selected and LED. emission spectrum of Cy3 fluorescent molecules, and the emission spectrum of the selected LED. The luminous fluxflux of the LED, which is measured by a 10-inch integrating sphere, is 143 lumens at 2.75The Vluminous (operating flux voltage) of the LED, and 0.32which A (operatingis measured current). by a 10-inch This luminousintegrating value sphere, will is be 143 used lumens in the atsimulation 2.75 V (operating to calculate voltage) the actualand 0.32 number A (operating of LEDs current). required. This Based luminous on the limitations value will of be the used fabrication fabrication in the simulationand the measuredmeasured to calculate luminousluminous the actual flux, flux, number we we take take of 9LEDs 9 LEDs LEDs required. in in a 3a 3×33 Basedarray array on to constructthe to limitationsconstruct the lightingthe of thelighting fabrication system. system. The × anddistanceThe the distance measured between between luminous each each LED flux,LED in the wein 3 the take3 3×3 array 9 LEDs is array 38.5 in isa mm. 38.53×3The mm. array layout The to layout construct is shown is shown inthe Figure lighting in Figure2. system. 2. × The distance between each LED in the 3×3 array is 38.5 mm. The layout is shown in Figure 2.

Figure 2. The layout of the 3 3 LEDs array. Figure 2. The layout of the 3 × 3 LEDs array. The Lumileds LED is a quasi-LambertianFigure 2. The layout LED. of the The 3 × emitting 3 LEDs array. area of the LED is 1.06 mm 1.06 mm, The Lumileds LED is a quasi-Lambertian LED. The emitting area of the LED is 1.06 ×mm × 1.06 and the shape of the packaging lens is dome-like, with a radius of about 2.8 mm. Jacobson proposed a mm, and the shape of the packaging lens is dome-like, with a radius of about 2.8 mm. Jacobson simpleThe LEDLumileds light sourceLED is model a quasi-Lambertian construction in LED. 2001 [43The]. Accordingemitting area to previous of the LED research is 1.06 by mm × [44 1.06–47 ], proposed a simple LED light source model construction in 2001 [43]. According to previous research mm,precision and the simulation shape of resultsthe packaging require constructing lens is dome-like, the structure with a of radius the LEDs of about to get 2.8 the opticalmm. Jacobson model of proposed a simple LED light source model construction in 2001 [43]. According to previous research Appl. Sci. 2019, 9, x FOR PEER REVIEW 4 of 14 Appl.Appl. Sci. Sci. 20192019, 9, ,9 x, 4574FOR PEER REVIEW 4 4of of 14 14 by Sun [44–47], precision simulation results require constructing the structure of the LEDs to get the by Sun [44–47], precision simulation results require constructing the structure of the LEDs to get the theoptical single model LED. of The the optical single model LED. ofThe the optical LED is model shown of in the Figure LED3a. is Further,shown in the Figure rays emitted 3a. Further, from thethe optical model of the single LED. The optical model of the LED is shown in Figure 3a. Further, the LEDrays areemitted traced, from and the the LED corresponding are traced, normalizedand the corresponding intensity distribution normalized is calculatedintensity distribution by the ASAP is rays emitted from the LED are traced, and the corresponding normalized intensity distribution is opticalcalculated analysis by the software ASAP (Breaultoptical analysis Research software Organization, (Breault Inc. Research Tucson, Organization, AZ 85706 USA). Inc. The Tucson, precision AZ calculated by the ASAP optical analysis software (Breault Research Organization, Inc. Tucson, AZ of8570 the6 modelUSA). The is verified precision by comparingof the model the is LED verified intensity by comparing distribution the from LED the intensity experimental distribution result from with 85706 USA). The precision of the model is verified by comparing the LED intensity distribution from thatthe experimental from the simulation result with result. that from the simulation result. the experimental result with that from the simulation result. The normalizednormalized intensityintensity distribution distribution comparison comparison from from the the experimental experimental result result and and that that from from the The normalized intensity distribution comparison from the experimental result and that from simulationthe simulation result result is shown is shown in Figure in Figure3b, in 3b, which in which the blackthe black and redand dotsred dots respectively respectively represent represent the the simulation result is shown in Figure 3b, in which the black and red dots respectively represent experimentalthe experimental and and simulation simulation values. values. The The accuracy accuracy of of the the optical optical model model can can also also be be verifiedverified byby thethe the experimental and simulation values. The accuracy of the optical model can also be verified by the normalized correlationcorrelation coe coefficienfficient (NCC)t (NCC) value value that represents that represents the similarity the betweensimilarity the between experimental the normalized correlation coefficient (NCC) value that represents the similarity between the resultexperimental and the result simulation and the result. simulation It is obtained result. asIt is follows, obtained as follows, experimental result and the simulation result. It is obtained as follows, P [(AABBθθ )-][( )-] [[(AAABB(θθθn) )-][(nnA][B(θ )-]n) B] = n n nn− − , NCCNCC== n , ,(1) (1) NCC r [(AABBθθ )-][(22 )-] (1) P[(AABBθθ )-][(nn222 )-] 2 n[A(θnnn) A] [B(θn) B] nn − −

where A(θn) and B(θn) are the experimental and simulation results, respectively, while A and B n n wherewhere A(A(θθn)) andand B(B(θn)) areare the experimentalexperimental andand simulation simulation results, results, respectively, respectively, while whileA andA Bandare B the are the mean values. θn is the divergent angle of the LED. After calculation, the NCC value is 99.5%, are the mean values. θn is the divergent angle of the LED. After calculation, the NCC value is 99.5%, meanwhich values. means thatθn is this the optical divergent model angle is accurate of the LED. enough After to calculation, be used in thesubsequent NCC value optical is 99.5%, designs. which whichmeans means that this that optical this optical model model is accurate is accurate enough enough to be usedto be inused subsequent in subsequent optical optical designs. designs.

(a) (b) (a) (b) Figure 3. (a) The optical model of the LED, (b) the normalized intensity distribution comparison of FigureFigure 3. 3. (a(a) )The The optical optical model model of thethe LED,LED, ((bb)) thethe normalized normalized intensity intensity distribution distribution comparison comparison of theof the experimental result and the simulation result. theexperimental experimental result result and and the the simulation simulation result. result.

2.2. Optical Design of the CTLAS System 2.2. Optical Design of the CTLAS System The light emitted from the quasi-Lambertian LEDs always diverges outward and requires TheThe light light emitted emitted from from the the qu quasi-Lambertianasi-Lambertian LEDs LEDs always always di divergesverges outward outward and and requires requires convergence on the specimen to enhance the values. The CTLAS system we designed is convergenceconvergence on on the the specimen specimen to to enhance enhance the the irra irradiationdiation values. values. The The CTLAS CTLAS system system we we designed designed is is composed of a TIR-lens array and a confocal system to efficiently collect the light energy of the LEDs composedcomposed of of a a TIR-lens TIR-lens array array and and a aconfocal confocal system system to to efficiently efficiently collect collect the the light light energy energy of of the the LEDs LEDs array, as shown in Figure 4. In the CTLAS system, lens 1 is built up by attaching the TIR-lens array array,array, as as shown shown in in Figure 44.. InIn thethe CTLASCTLAS system,system, lens lens 1 1 is is built built up up by by attaching attaching the the TIR-lens TIR-lens array array to theto the front front lens lens of theof the confocal confocal system system as a as one-piece a one-piece lens. lens. The The left sideleft side of lens of lens 1 is a1 3is a3 3×3TIR-lens TIR-lens array to the front lens of the confocal system as a one-piece lens. The left side of lens 1 is ×a 3×3 TIR-lens array used to assemble the 9 LEDs. arrayused used to assemble to assemble the 9 the LEDs. 9 LEDs.

Figure 4. Optical setup of the confocal total internal reflection lens array system (CTLAS) system. Figure 4. Optical setup of the confocal total internal reflection lens array system (CTLAS) system. Figure 4. Optical setup of the confocal total internal reflection lens array system (CTLAS) system. Appl.Appl. Sci. Sci.2019 2019, 9, ,9 4574, x FOR PEER REVIEW 55 ofof 14 14 Appl. Sci. 2019, 9, x FOR PEER REVIEW 5 of 14 The TIR lens is designed to collimate the divergent light of a single LED with a total internal reflectionTheThe TIR TIR effect, lens lens is as is designed designedshown into to Figurecollimate collimate 5a. the theThe diverg divergentheightent and light light diameter of of a asingle single of LEDthe LED designed with with a atotal totalTIR internal internallens are, reflectionreflectionrespectively, effect, effect, 17.5 as as mmshown shown and in 35.5 in Figure Figure mm, and55a.a. Thethe The materialhe heightight and andis PMMA diameter diameter with of ofa the1.49 the designedrefractive designed TIRindex. TIR lens lens are, are, respectively,respectively, 17.5 17.5 mm mm and and 35.5 35.5 mm, mm, and and the the material material is is PMMA PMMA with with a a 1.49 1.49 refractive refractive index. index.

(a) (b) (a) (b) Figure 5. (a) The structure of the designed total internal reflection (TIR) lens (the values are in mm), FigureFigure(b) the 5. 5. ray(a()a )Thetracing The structure structure the result of of the theof designedthe designed TIR lens. total total internal internal re reflectionflection (TIR) (TIR) lens lens (the (the values values are are in in mm), mm), (b(b) )the the ray tracing the the result result of of the the TIR TIR lens. lens. By ASAP, the ray tracing the result of the TIR lens is shown in Figure 5b. We can see that, after passingByBy ASAP, ASAP, through the the ray raythe tracing tracingTIR lens,the the result resultthe divergentof of the the TIR TIR lenslight lens is isis shown shownwell incollimated. in Figure Figure 5b.5b. InWe We light can can see seeof that, that,the aftertesting after passingpassingperformance, through we thethe further TIR TIR lens, acquiredlens, the the divergent the divergent result light that islight the well TIR-lensis collimated. well collimated.array In can light collimate ofIn the light testing the of divergent performance,the testing light performance,wefrom further the 3 acquired × we 3 LED further array the acquired result and thatturn the theit resultinto TIR-lens quasi-collimating that arraythe TIR-lens can collimate light array with can the acollimate divergent larger beam the light divergentsize, from as theshown light 3 3in × fromLEDFigure the array 36a. × andThe3 LED turnside array itlength into and quasi-collimatingof turn the TIR-lensit into quasi-collimating array light is with106.5 a mm, larger light and beamwith the anormalized size, larger as shownbeam intensity size, in Figure as distributionshown6a. The in Figuresideof the length 6a. TIR-lens The of side the array TIR-lenslength is shownof array the TIR-lens in is 106.5Figure array mm, 6b. andAccord is 106.5 theing normalizedmm, to andthe simulationthe intensity normalized distributionresults, intensity the half-width-half- of distribution the TIR-lens ofarray magnitudethe TIR-lens is shown (HWHM) array in Figure is shownvalue6b. of in According the Figure norm 6b.alized to Accord the intensity simulationing to distributionthe results,simulation theof theresults, half-width-half-magnitude TIR-lens the half-width-half-array is ±2.8°. The magnitude(HWHM)optical efficiency, (HWHM) value of defined the value normalized of as the the norm ratio intensityalized of the intensityluminous distribution distribution flux of of the the TIR-lens TIR-lens of the TIR-lens array array is to array the2.8 ◦total is. The±2.8°. luminous optical The ± opticalefluxfficiency, of efficiency, the defined LED, defined is as 91.7%. the ratioas the of ratio the luminousof the luminous flux of flux the of TIR-lens the TIR-lens array toarray the to total the luminous total luminous flux of fluxthe of LED, the isLED, 91.7%. is 91.7%.

(a) (b) (a) (b) FigureFigure 6. 6.The The designed designed 3 3 × 33 TIR-lensTIR-lens array,array, ((aa)) thethe rayray tracingtracing simulationsimulation result, result, ( b(b)) the the corresponding corresponding × Figurenormalizednormalized 6. The intensitydesigned intensity distribution.3 distribution. × 3 TIR-lens array, (a) the ray tracing simulation result, (b) the corresponding normalized intensity distribution. InIn order order to to enhance enhance the the illuminance, illuminance, the the second second surfaces surfaces of of lens lens 1 1 and and lens lens 2 2 form form a a confocal confocal system.system.In order TheThe quasi-collimatingto quasi-collimating enhance the illuminance, lightlight ofof a a larger largerthe second beam beam size surfacessize passing passing of lens through through 1 and the the lens second second 2 form surface surface a confocal of of lens lens system.11 is is focused focused The quasi-collimating on on its its focal focal point, point, light which which of a is islarger also also the thebeam front front size focal focal passing point point through of of lens lens 2. the2. After After second that, that, surface the the focal focal of lens light light 1will iswill focused re-emerge re-emerge on its from from focal the the point, focal focal pointwhich point and isand also form form the a a small-sizefront small-size focal quasi-collimating pointquasi-collimating of lens 2. After light light that, beam. beam. the As Asfocal shown shown light in in willFigureFigure re-emerge7 ,7, lens lens 2from 2 is is a a plano-convexthe plano-convex focal point lens. andlens. form a small-size quasi-collimating light beam. As shown in Figure 7, lens 2 is a plano-convex lens. Appl. Sci. 9 Appl. Sci.2019 2019,, 9,, 4574 x FOR PEER REVIEW 6 ofof 1414

Figure 7. The setup of the CTLAS system. It is composed of a two-lens confocal system. Figure 7. The setup of the CTLAS system. It is composed of a two-lens confocal system. The back surfaces of lenses 1 and 2 are both aspherical in order to eliminate the spherical aberration.The back Those surfaces surfaces of are lenses also calculated1 and 2 are by theboth sag aspherical function [48in ]order which to can eliminate represent the the spherical shape of theaberration. aspherical Those surface surfaces and which are also is obtainedcalculated by by the sag function [48] which can represent the shape of the aspherical surface and which is obtained by 2 cr 4 z(r) = p cr 2 + Ar , (2) =+1 + 1 (1 + k)c2r2 4 zr() − Ar , (2) 11-(1)++kcr22 where z(r), k, and A are the sag, conic constant, and the four-order aspherical coefficient of the aspherical surface,where z(r) respectively., k, and Ac areis the the curvature sag, conic of theconstant, aspherical and surfacethe four-order at its vertex, aspherical and r iscoefficient the axial heightof the onaspherical the aspherical surface, surface. respectively. Thus, the c is second the curvature surfaces of lensesthe aspherical 1 and 2 aresurface both at designed its vertex, and and optimized r is the byaxial the height Zemax on software. the aspherical The optimization surface. Thus, values, the secondc, k, and surfacesA, of the of secondlenses 1 surfaces and 2 are of lensesboth designed 1 and 2 areand shown optimized in Table by the1. In Zemax the confocal software. system, The optimization the distance values, between c, k the, and second A, of surfacethe second of lens surfaces 1 and of thelenses bottom 1 and plane 2 are of shown lens 2 in is Table 114 mm; 1. In moreover, the confoc aal mutual system, focal the distance point is between located 24the mm second away surface from theof lens bottom 1 and plane the bottom of lens 2.plane The of diameters lens 2 is 114 of lens mm; 1 andmoreover, lens 2 area mutual 150 mm focal and point 28 mm, is located respectively. 24 mm Theaway size from of lens the 2bottom is set to plane satisfy of thelens size 2. The of the diamet observationers of lens area 1 onand the lens specimen 2 are 150 of mm the fluorescence and 28 mm, microscoperespectively. because The size the of diameter lens 2 is ofset the to satisfy observation the size area of sizethe observation must be 27 mm. area on the specimen of the fluorescence microscope because the diameter of the observation area size must be 27 mm. Table 1. The c, k, and A values of the 2nd surfaces of lens 1 and lens 2.

Table 1. The c, k, and Ac values of the 2nd surfaces k of lens 1 and Alens 2. Lens1 46.65 c 2.3k 0 A − − Lens1Lens2 15.3−46.65 0.654−2.3 3.928 10 6 0 − − − × − Lens2 −15.3 −0.654 −3.928 × 10−6 3. Results and Analyses 3. Results and Analyses The ray tracing result of our designed CTLAS system by the ASAP software, shown in Figure8. The ray tracing result of our designed CTLAS system by the ASAP software, shown in Figure 8. Appl. Sci. 2019, 9, x FOR PEER REVIEW 7 of 14 Appl. Sci. 9 Appl. Sci. 20192019, 9,, x, FOR 4574 PEER REVIEW 7 of7 of14 14

FigureFigure 8. The8. The ray ray tracing tracing result result of of the the CTLAS CTLAS system. system. Figure 8. The ray tracing result of the CTLAS system. TheThe optical optical effi efficiency,ciency, defined defined as as the the ratio ratio of of the the luminous luminous flux flux of of the the LEDs LEDs and and the the output output luminousluminousThe optical flux flux emergingefficiency, emerging from definedfrom lens lens 2,as is2, the obtainedis obtainedratio of by theby luminous flux of the LEDs and the output luminous flux emerging from lens 2, is obtained by FO FO OpticalOptical E f Efficiencyf iciency = = ,, (3)(3) FFOL F Optical Efficiency = , L (3) F F FL wherewhereL FandL andO FOare, are, respectively, respectively, the the luminous luminous flux flux of of the the LEDs LEDs and and the the output output luminous luminous flux flux emerging from lens 2. In Figure9a, the light pattern on the specimen is a 27 mm diameter circular area whereemerging FL and fromFO are, lens respectively, 2. In Figure the9a, theluminous light patt fluxern of on the the LEDs specimen and the is aoutput 27 mm luminous diameter fluxcircular emergingthatarea is 550that from mm is 550lens away mm 2. fromIn away Figure the from bottom 9a, the the of bottom light the LEDs. patt of ernthe Figure onLE Ds.the9b showsFigurespecimen the9b shows uniformityis a 27 themm uniformity ofdiameter the light circularof patterns the light areacalculatedpatterns that is 550 bycalculated themm illuminance away by fromthe illuminance valuesthe bottom of 17 values of tested the ofLE points. 17Ds. tested Figure points. 9b shows the uniformity of the light patterns calculated by the illuminance values of 17 tested points.

(a) (b) (a) (b) FigureFigure 9. (9.a) The(a) The circular circular light patternlight pattern on the specimen,on the specimen, (b) the uniformity (b) the uniformity of the light of pattern the light calculated pattern Figurebycalculated the 9. illuminance (a) The by thecircular values illuminance light of the pattern 17values tested ofon redthe the points.17 specimen, tested red (points.b) the uniformity of the light pattern calculated by the illuminance values of the 17 tested red points. TheThe red red points points show show the the testing testing positions. positions. The The uniformity uniformity of of the the light light pattern pattern is obtainedis obtained by by The red points show the testing positions. The uniformity of the light pattern is obtained by EaveE Uni f ormity = ,ave (4) Uniformity E= , (4) EminaveE Uniformity = ,min (4) where Eave and Emin are the average and the minimumE irradiationmin values of the light pattern on the where Eave and Emin are the average and the minimum irradiation values of the light pattern on the specimen. After the calculation, the optical efficiency of this system is 72.7%. The uniformity of the wherespecimen. Eave and After Emin are the the calculat averageion, andthe opticalthe minimum efficiency irradiat of thision sy valuesstem is of 72.7%. the light The pattern uniformity on the of the light pattern is 1.14, and the central irradiation value of the light pattern is 62,950 lux. The result specimen.light pattern After theis 1.14,calculat andion, the the central optical irradiation efficiency value of this of sy thestem light is 72.7%.pattern The is 62,950 uniformity lux. The of the result surpassed the requirement of the illumination value of our initial setting. lightsurpassed pattern is the 1.14, requirement and the central of the illuminationirradiation value value of of the our light initial pattern setting. is 62,950 lux. The result In a commercial fluorescence microscope system, most LED light sources are just used to illuminate surpassedIn the a commercialrequirement fluorescenceof the illumination microscope value ofsystem our initial, most setting. LED light sources are just used to the specimen without any additional components to collect light. Some LED light sources are attached illuminateIn a commercial the specimen fluorescence without microscopeany additional system components, most LED to collect light light.sources Some are LED just lightused sources to with a converging lens to enhance the illuminance. Therefore, we take three examples, a nine-LED illuminateare attached the specimen with a converging without any le additionalns to enhance components the illuminance. to collect Therefore, light. Some we LED take light three sources examples, are attached with a converging lens to enhance the illuminance. Therefore, we take three examples, Appl. Sci. 2019, 9, 4574 8 of 14 Appl.Appl. Sci. Sci. 2019 2019, 9,, 9x, FORx FOR PEER PEER REVIEW REVIEW 8 8of of 14 14 a aarray,nine-LED nine-LED a nine-LED array, array, a array anine-LED nine-LED with aar convergingarrayray with with a lens,aconverging converging and the lens, CTLASlens, and and system, the the CTLAS CTLAS to compare system, system, the to to illuminance,compare compare the the as illuminance,illuminance,shown in Figure as as shown shown 10. in in Figure Figure 10. 10.

(a()a ) (b()b ) (c()c )

FigureFigure 10. 10. The The three three types types of of LED LED light light sources, sources, (a ( )a the)) the nine-LED nine-LED array array array only, only, only, (b ( )b the) the nine-LED nine-LED array array withwith the the converging converging lens, lens, and and (c ()c the)) thethe nine-LED nine-LEDnine-LED array arrayarray with withwith the thethe CTLAS CTLASCTLAS system. system.system.

InIn Figure Figure 10b, 10b,10b, a a 150 150 mm mm diameter diameter and and 0.7 0.7 NA NA value value converging converging lens lens are are assembled assembled at at the the center center ofof the the nine-LED nine-LED array; array; the the distance distance between between the the two two is isthe the focal length of of the thethe converging convergingconverging lens. lens.lens. The The correspondingcorresponding central central illuminance illuminance and and and the the the uniformity uniformity uniformity of of ofthese these these three three three examples examples examples are are are shown shown shown in in inTable Table Table 2. 22. . TheThe result result shows shows that that the the CTLAS CTLAS system system obviously obviously has has the the best best uniformity uniformity and and the the greatest greatest center center illuminationillumination value, value, which which is is 46 46 times times brighter brighter than than the the nine-LED nine-LED array array and and 40 40 times times brighter brighter than than the the nine-LEDnine-LED array array with with the the converging converging lens. lens.

Table 2. The simulation results of the central illuminance values and the uniformity of the nine-LED TableTable 2. 2. The The simulation simulation results results of of the the central central illuminanc illuminance evalues values and and the the uniformity uniformity of of the the nine-LED nine-LED array, the nine-LED array with the converging lens and the CTLAS system. array,array, the the nine-LED nine-LED array array with with the the co convergingnverging lens lens and and the the CTLAS CTLAS system. system. 9-LED Array with 9-LED Array with the 9-LED9-LED9-LED Array 9-LED9-LED Array Array with with 9-LED9-LED Array Array with with the the Converging Lens CTLAS System ArrayArray ConvergingConverging Lens Lens CTLASCTLAS System System Central illuminance CentralCentral illuminance illuminance 1377 1590 62,950 value (Lux) 13771377 15901590 62,950 62,950 valuevalue (Lux) (Lux) Uniformity 2 1.81 1.14 UniformityUniformity 2 2 1.811.81 1.14 1.14

WeWe successfully successfullysuccessfully assembled assembledassembled the the the LED LED LED array array array and and th the the e CTLAS CTLAS system.system. system. The The systemsystem system picturepicture picture isis shownis shown shown in ininFigure Figure Figure 11 11. .11.

FigureFigure 11. 11. The The assembled assembled CTLAS CTLAS system. system.

AllAll the the lenses lenseslenses in inin this thisthis system system system are are are made made by by mold mold injection.injection. injection. WeWe We used used used the the the CTLAS CTLAS CTLAS system system system to toobserve to observe observe the thethefluorescent fluorescent fluorescent images images images of the of of vascularthe the vascular vascular bundle bundle bundle organization organization organization modified modified modified with the with Cy3with the fluorescencethe Cy3 Cy3 fluorescence fluorescence molecules. molecules.molecules. The The light light pattern pattern and and fluorescent fluorescent images images of of the the vascular vascular bundle bundle organization organization modified modified Appl. Sci. 2019, 9, 4574 9 of 14 Appl. Sci. 2019, 9, x FOR PEER REVIEW 9 of 14 Appl. Sci. 2019, 9, x FOR PEER REVIEW 9 of 14 with the Cy3 fluorescent molecules are shown in Figure 12. As shown, this CTLAS light source system The lightAppl. pattern Sci. 2019,and 9, x FOR fluorescent PEER REVIEW images of the vascular bundle organization modified with9 theof 14 Cy3 with canthe Cy3provide fluorescent sufficient molecules light for are fluorescence shown in Figu microscopes.re 12. As shown, this CTLAS light source system fluorescent molecules are shown in Figure 12. As shown, this CTLAS light source system can provide can providewith the sufficient Cy3 fluorescent light for molecules fluorescence are shown microscopes. in Figure 12. As shown, this CTLAS light source system sufficient light for fluorescence microscopes. can provide sufficient light for fluorescence microscopes.

(a) (b) (a) (a) ((bb) ) Figure 12. (a) The light pattern of the CTLAS system, (b) fluorescent images of the vascular bundle Figure 12. (a) The light pattern of the CTLAS system, (b) fluorescent images of the vascular bundle Figureorganization 12.12. (a) TheThe lightmodifiedlight patternpattern with ofof th thethee Cy3 CTLASCTLAS fluorescent system,system, molecules. ((b)) fluorescentfluorescent imagesimages ofof thethe vascularvascular bundlebundle organization modified with the Cy3 fluorescent molecules. organization modifiedmodified withwith thethe Cy3Cy3 fluorescentfluorescent molecules.molecules. In the illumination test of the CTLAS light source system, the measurement data are lower than In the illuminationIn the illumination test oftest the of CTLASthe CTLAS light light source source system, system, thethe measurementmeasurement data data are are lower lower than than thoseIn the ofillumination the simulation. test ofTherefore, the CTLAS we light checked source the system, lens used the in measurement the system and data found are lower that there than were those ofthose the of simulation. the simulation. Therefore, Therefore, we wechecked checked the the le lensns used used in the in system the system and found and foundthat there that were there thoseminor ofminor the deformations simulation. deformations Therefore, and errors errors wein in the checkedthe two two lenses. lenses.the leThns eseTh used esedeformations deformationsin the system and errors and errorsfoundcome from comethat the there from mold were the mold were minor deformations and errors in the two lenses. These deformations and errors come from minorinjection deformationsinjection fabrication fabrication and errorsprocess. in The theThe shapetwo shape lenses. errors errors ofTh theseofe thsecond deformationse second surfaces surfaces andof lenses errors of lenses 1 andcome 21 are andfrom shown 2 theare mold inshown in the mold injection fabrication process. The shape errors of the second surfaces of lenses 1 and 2 are injectionFigureFigure fabrication 13. 13. The The blackblackprocess. and The redred linesshape lines in inerrors Figure Figure of13 th13represente representsecond the surfaces measuredthe measured of andlenses simulated and 1 and simulated 2 lens are shapes,shown lens shapes, in shown in Figure 13. The black and red lines in Figure 13 represent the measured and simulated lens Figurerespectively. respectively.13. The black and red lines in Figure 13 represent the measured and simulated lens shapes, shapes,respectively. respectively.

(a) (b) (a) (b) Figure 13. The shape errors between fini shed lens and simulation, (a) lens 1, (b) lens 2. (a) (b) Figure 13. The shape errors between finished lens and simulation, (a) lens 1, (b) lens 2. Figure 13.14 showsThe shape the shapeerrors betweenerror of the finishedfini shedfirst surf lensace and of simulation, lens 1, which (a)) lenslensis composed 1,1, ((b)) lenslens of 2.2. TIR-lens arraysFigure and 14the shows normalized the shape light intensityerror of distributhe firsttions surf fromace ofthe lens simulation 1, which and is the composed experimental of TIR-lens results. The measured light intensity in Figure 14b shows that the full width at half maximum Figurearrays 14and14 shows shows the normalized the the shape shape error lighterror of intensity theof the first first surface distribu surf ofacetions lens of lens 1,from which 1, the which is simulation composed is composed ofand TIR-lens the of experimentalTIR-lens arrays (FWHM) of the assembled CTLAS light source system is wider than that of the simulation, which andarrays theresults. and normalized the The normalized measured light intensity lightlight intensity distributionsintensity distribuin Figure fromtions the 14b simulationfrom shows the thatsimulation and the experimentalfull and width the atexperimental results. half maximum The indicates that the energy of the projecting light beam from the system is not sufficiently concentrated. measuredresults.(FWHM) The light measured of intensity the assembled light in Figure intensity CTLAS 14b in shows light Figure source that 14b the systemshows full width isthat wider atthe half thanfull maximum widththat of atthe (FWHM)half simulation, maximum of the which assembled(FWHM)indicates of CTLAS the that assembled the light energy source CTLAS ofsystem the projectinglight is source wider light thansystem beam that is from of wider the the simulation, than system that is of whichnot the sufficiently simulation, indicates concentrated. that which the energyindicates of that the projectingthe energy light of the beam projecting from the light system beam is from not sutheffi systemciently is concentrated. not sufficiently concentrated.

(a) (b)

Figure 14. The comparison results of (a) the shapes of the experimental and the simulation TIR lenses, (b) the corresponding normalized light intensity distributions. (a) (b) (a) (b) FigureFigure 14. The 14. comparison The comparison results results of (a) of the (a shapes) the shapes of the of experimental the experimental and the and simulation the simulation TIR lenses, TIR lenses, (Figureb) the(b corresponding14.) the The corresponding comparison normalized resultsnormalized lightof (a) intensity lightthe shapes intensity distributions. of the distributions. experimental and the simulation TIR lenses, (b) the corresponding normalized light intensity distributions. Appl. Sci. 2019, 9, 4574 10 of 14 Appl. Sci. 2019, 9, x FOR PEER REVIEW 10 of 14

TheThe errors errors of of the the finished finished TIR-lens TIR-lens array array cause cause the thedivergence divergence of the of light the light beam; beam; moreover, moreover, the energythe energy of the of light the lightpattern pattern is not is sufficiently not sufficiently dense dense,, due dueto the to larger the larger divergent divergent angle. angle. Hence, Hence, the measuredthe measured illumination illumination at the at thecenter center of the of theligh lightt beam beam dropped dropped to to 980 980 lux. lux. Taking Taking the the wrong wrong parametersparameters in in the the simulation, simulation, we we acquired acquired the the central central illumination illumination of of the the light light pattern pattern of of 1307 1307 lux. lux. TheThe measured measured and and simulated simulated values values are are approximatel approximatelyy in in the the same same order order of of magnitude. magnitude. This This result result demonstratesdemonstrates that that the the design design method method of of the the CTLAS CTLAS sy systemstem is is feasible feasible in in the the applications applications of of LEDs LEDs appliedapplied in in fluorescence fluorescence microscopes. microscopes.

4.4. Tolerance Tolerance Analyses Analyses of of the the Light Light Source Source Assembly Assembly InIn order order to to achieve achieve the the highest highest yield yield rate rate of of the the production production in in the the future, future, the the alignment alignment precision precision ofof the the LEDs LEDs and and TIR TIR lens lens array array is is also also a a key key point point of of the the central central illumination illumination values. values. In In the the system system assemblyassembly processes, processes, owing owing to to the the symmetric symmetric rotati rotationalonal shape shape of of the the TIR TIR lens, lens, the the effects effects of of LEDs LEDs movingmoving along along the the x-axis x-axis and and y-axis y-axis are are the the same. same. Hence, Hence, LEDs LEDs can canmove move in two in twodirections, directions, along along the x-axisthe x-axis and andthe z-axis, the z-axis, as shown as shown in Figure in Figure 15. 15 We. We evaluated evaluated and and analyzed analyzed the the impact impact on on precision precision affectedaffected by by the the misalignment. misalignment.

FigureFigure 15. 15. TheThe movements movements of of the the 9-LED 9-LED array array along along the the x-axis x-axis and and the the z-axis. z-axis.

TheThe corresponding uniformityuniformity and and the the central central illumination illumination values values of the of light the patternlight pattern will change will changealong withalong the with increasing the increasing or decreasing or decreasing distance dist betweenance between the LEDs the LEDs and and the TIR-lensthe TIR-lens array. array. In theIn theexample example of of 9 LEDs9 LEDs moving moving along along the the x-direction, x-direction, Figure Figure 16 16a,ca,c showshow thethe simulationsimulation results results of of the the lightlight patterns patterns and and the the central central illuminance illuminance values, values, respectively, respectively, and and Figure Figure 16b,d 16b,d show show the the simulation simulation resultsresults for for moving moving along along the the z-direction. z-direction. In In both both of of these these examples, examples, although although moving moving in in different different directions,directions, their their central central illumination illumination values values gradua graduallylly reduced reduced with with increasing increasing displacement displacement from from 0 mm0 mm to toapproximately approximately 2 mm. 2 mm. After After 2 mm 2 mm of ofdisplacement, displacement, the the values values tend tend to tobe bestable. stable. Appl. Sci. 2019, 9, 4574 11 of 14 Appl. Sci. 2019, 9, x FOR PEER REVIEW 11 of 14

(a) (b)

(c) (d)

FigureFigure 16. 16. TheThe light patternspatterns ofof the the 9-LED 9-LED array array move move along along (a) the(a) x-axis,the x-axis, and (andb) the (b z-axis.) the z-axis. The central The centralillumination illumination values values and uniformity and uniformity of different of different displacements displacements along (alongc) the x-axis,(c) the x-axis, and (d )and the ( z-axis.d) the z-axis. The uniformity of the light patterns shows some oscillations with increasing displacement, and the uniformityThe uniformity still of exhibits the light a gradually patterns shows rising some trend. oscillations This means with that increasing the LED displacement, misalignment and will thecause uniformity light patterns still exhibits to be a darker gradually and rising non-uniform. trend. This Moreover, means that regarding the LED bothmisalignment uniformity will and cause the lightcenter patterns illuminance, to be thedarker increasing and non-uniform. displacement Moreover, of the x-axis regarding causes bigger both uniformity values to change and the compared center illuminance,with the situation the increasing of the z-axis. displacement On the one of the hand, x-axis in thecauses x-axis, bigger the values non-uniformity to change andcompared the reduced with thebrightness situation come of the from z-axis. both theOn skewnessthe one hand, of the in projecting the x-axis, light the and non-uniformity the hot spot laterally and the leaving reduced the brightnesscentral point. come On from the otherboth hand,the skewness these variations of the projecting are affected light by and the defocusingthe hot spot e fflaterallyect of the leaving LED array the centralin the z-axis.point. On The the defocused other hand, light these from variations the adjacent are TIR affected lenses by is ditheff used,defocusing and there effect is interferenceof the LED arraybetween in the light z-axis. from diThefferent defocused lenses, whichlight from reduces the the adjacent uniformity. TIR lenses is diffused, and there is interferenceTo meet between the initial light specifications from different that lenses, the which center reduces illuminance the uniformity. of a fluorescence microscope is 10,578To lux,meet the the tolerances initial specifications of the LED that displacement the center along illuminance the x-axis of a and fluorescence along the z-axismicroscope must beis 10,578controlled lux, withinthe tolerances 1.3 mm andof the 1.7 LED mm, displacement respectively, and along the the corresponding x-axis and uniformityalong the z-axis is 1.39 must and1.26. be controlled within 1.3 mm and 1.7 mm, respectively, and the corresponding uniformity is 1.39 and 5. Conclusions 1.26. Research on the light sources of commercial microscopes or for other specific purposes plays a 5.significant Conclusions role in biomedical scientists’ study. In this paper, we reveal a design method for LED light sourcesResearch applied on inthe fluorescence light sources microscopes. of commercial In micr our case,oscopes the or target for other fluorescent specific molecules purposes areplays Cy3; a significanthowever, thisroledesign in biomedical method scientists’ is not limited study. to Cy3In this molecules. paper, we In reveal light ofa design the design method concept, for LED all oflight the sourcescommercial applied fluorescent in fluorescence molecules microscopes. can be applied. In ou Wer case, have the demonstrated target fluorescent and assembled molecules the are CTLAS Cy3; however,light source this system design successfully.method is not Our limited CTLAS to Cy3 light molecules. source system In light is of composed the design of concept, a nine-LED all of array, the commercialthe corresponding fluorescent TIR molecules lens array can and be the applied. confocal We system have thatdemonstrated can collimate and divergentassembled light the CTLAS emitted lightfrom source the LED system array successfully. and reduce the Our beam CTLAS size light to provide source a system circular is light composed pattern of on a the nine-LED specimen array, with thehigher corresponding illuminance. TIR lens array and the confocal system that can collimate divergent light emitted from theAll LED recipes array and and optical reduce component the beam models size to are provide simulated a circular in ASAP light optical pattern software. on the specimen We have takenwith higherboth common illuminance. cases, a nine-LED array only and a nine-LED array with a converging lens with our Appl. Sci. 2019, 9, 4574 12 of 14 designed CTLAS light source system to compare the illuminance. The simulation result shows that this CTLAS system has the best uniformity and the greatest center illumination which is 46 times brighter than only using the nine-LED array mode and 40 times brighter than using the nine-LED array with a converging lens. In the evaluation of the misalignment for the CTLAS system assembly, the skewness of the projecting light and the hot spot laterally leaving the central point on the x-axis have a greater impact on the uniformity and illuminance than the defocusing effect on the z-axis. The tolerances of the LED displacement on the x-axis and z-axis must be controlled within 1.3 mm and 1.7 mm, respectively, so that the illuminance can reach the initial settings; the corresponding uniformity is 1.39 and 1.26. In consideration of energy saving, the total electric power of the LED array is 7.9 watts, which is approximately 6.6% of the original white halogen lamp used in an Axio Imager 2 fluorescence microscope. Moreover, according to the simulation results, the central illuminance value of the light pattern projected by the microscope that we designed is 62,950 lux, which is about 5.95 times that of a microscope with the original white halogen lamp (10,578 lux). The results of our study provide an optical design that demonstrates the use of LEDs to replace the traditional energy-consuming light source.

Author Contributions: T.-C.K. provided the research resources, the fluorescence microscopy and the Cy3 test sample. T.-J.D. was responsible for the completion of the experimental and simulation work. J.-H.L. assisted in the writing of this article. S.-H.M. proposed the whole ideas and controlled the progress of this research. Funding: This research is funded by the Ministry of Science and Technology of the Republic of China. Acknowledgments: This work was supported by the Ministry of Science and Technology of the Republic of China, project MOST 107-2221-E-035-056. Conflicts of Interest: The authors declare no conflict of interest.

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