Compact UV LED Lamp with Low Heat Emissions for Biological Research Applications

electronics

Article Compact UV LED Lamp with Low Heat Emissions for Biological Research Applications

Matija Pirc 1,* , Simon Caserman 2, Polonca Ferk 3 and Marko Topiˇc 1

1 Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, SI-1000 Ljubljana, Slovenia; [email protected] 2 National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana; Slovenia; [email protected] 3 Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia; [email protected] * Correspondence: [email protected]; Tel.: +386-14768407

Received: 27 February 2019; Accepted: 19 March 2019; Published: 21 March 2019

Abstract: Much biomedical research focuses on the effects of UV light on human cells. UV light sources are a prerequisite for such research. This paper presents the design and achieved performance of a UVA (Ultraviolet A: 320–400 nm) and a UVB (Ultraviolet B: 290–320 nm) LED-based lamp suitable for use in bioassays, as well as inside an incubator. Numerical simulations were used to optimise the number, layout and output power of LEDs to achieve good irradiance homogeneity while maintaining low costs. Design was optimised for the efﬁcient transfer of generated heat away from the irradiated samples through the heatsink at the back of the lamps. The average irradiance of the target surface by the UVA lamp was 70.1 W/m2 with a maximum deviation of 4.9%, and the average irradiance by the UVB lamp was 3.1 W/m2 with a maximum deviation of 4.8%. With the UVA and UVB lamps, the temperature of samples undergoing irradiation in the incubator rises from 37 to 42 ◦C within 40 and 67 min, respectively. This by far exceeds the required UV irradiation time in most cases. Tests on Jurkat and HEK-293 cell cultures conﬁrmed the suitability of our lamps for biomedical research.

Keywords: UV lamp; LED; UVA; UVB; incubator; bioassay

1. Introduction Focusing on the field of medicine and human health, ultraviolet light (UV) is the most prominent and universal environmental human carcinogen. Research shows that as many as 90% of skin cancers are due to UV radiation [1]. Other unwanted UV effects include sunburn, immunosuppression, eye damage and skin ageing [2]. On the other hand, there are many known positive effects and uses of UV light, including its therapeutic effects (e.g., in treating infants suffering from jaundice [3]; in treating skin, sleep, mood and seasonal affective disorders [4]; in treating vitamin D deficiency [5]; wound healing [6]) and further applications in medicine, such as its use in air ventilation systems in hospitals, in air/water purification, in disinfection in surgery, and much more [7,8]. Since UV light has such a vast variety of effects on the human body, a significant amount of biomedical research focuses on it. Exposing in vitro cell cultures of selected tissue origin to UV radiation is a very convenient option for researching human body responses to UV irradiation. The responses triggered by UV irradiation are wavelength and dose dependent. The UVA (320 nm to 400 nm) and UVB (290 nm to 320 nm) ranges of the UV spectrum are of particular interest because the intensities they reach on the earth’s surface are often harmful. On the other hand, the UVC (100 nm to 290 nm) part of the solar spectrum is, for all intents and purposes, completely absorbed by

Electronics 2019, 8, 343; doi:10.3390/electronics8030343 www.mdpi.com/journal/electronics Electronics 2019, 8, 343 2 of 18 the earth’s atmosphere [9]. Therefore, reliable light sources emitting light in the UVA and UVB ranges adapted to the cell culture environment are indispensable research tools. In the past, researchers have used a wide variety of solutions for UV illumination, ranging from home solarium lamps to state-of-the-art solar simulators. By far the most common source of UV light is a lamp with one or more fluorescent tubes [10–14], ranging from simple hand-held lamps [14] or home solariums [10] to UV-crosslinkers [12] and therapeutic illuminators designed for medical use [13]. The second most common type of UV light sources for medical research are solar simulators [15–17], which usually employ a high-power Xenon arc lamp with an optical output spectrum very similar to that of natural sunlight. However, their large size and high heat output make them less suitable for bioassays. Even fluorescent tubes, which are still among the more efficient light sources, emit approximately 90% of their energy in the form of heat [18]. Researchers combat the problem of excessive heat dissipation with ventilation [10], ice blocks [11], cooling blocks connected to a thermostated bath [16], etc. In the last few decades, remarkable progress has been achieved in the field of light emitting diodes (LEDs) [19], which have already proven to be very suitable for biomedical research applications [20–23]. In the last decade, UV LEDs have progressed significantly as well. These now include UVA, UVB and UVC LEDs with emission peaks reaching all the way down to 210 nm [24]. LEDs have already surpassed all other illumination technologies in terms of conversion efficiencies [25], making them potentially an ideal replacement for current solutions. Unfortunately, UV LEDs have not yet reached the same level of maturity as white LEDs, but they are nevertheless becoming an attractive option because of their ability to operate at low temperatures, their long lifespan, almost instant on/off times, and because they do not require high voltages to operate. Up until now, the best available option for bioassays has been a purpose-built petri dish or microtiter plate UV illuminator like Vilber Bio-sun [26], with highly uniform illumination and a built-in cooling system, which is usually based on fluorescent tubes. However, such light sources still require that the samples are taken out of the incubator for the duration of illumination, risking other environmental influences like atmosphere, temperature and humidity changes that may influence the effects of the UV illumination experiment. These influences may be especially prominent when simulating long-term UV exposure. An alternative would be a handheld UV lamp, which is about the right shape and size to be placed inside an incubator. However, we couldn’t find one which would be uniform enough and designed to operate in extreme humidity environments such as an incubator. In this work, we demonstrate the development and use of UVA and UVB LED-based light sources designed for cell culture research inside an incubator. A preliminary report on the UVA lamp has already been presented at the 53rd MIDEM conference [27]. We show bottom-up illumination, applicable for microtiter plates, cell culture flasks and Petri dishes. This approach enables homogenous irradiation of the surface with flexible and precise settings of irradiance and time. Low heat emission reduces the risk of unwanted thermal effects on cell cultures. The low operating voltage and compact design allow its use in a 100% relative humidity environment inside a cell culture incubator. These achievements, along with the foreseeable further development of UV LEDs, will likely lead to a close to ideal UV irradiation source for cell culture research with the potential for a variety of other applications.

2. UV Lamp Design

2.1. UV Lamp Requirements and Optical Design A lamp intended to operate in an incubator needs to be small, it has to have a very limited heat output, and it has to be able to operate in a 100% relative humidity environment. The desired illumination area was 155 mm × 87 mm, which would accommodate the illumination of samples in various containers with sizes up to the size of the T75 cell culture ﬂasks. The desired illumination Electronics 2019, 8, 343 3 of 18 inhomogeneity was less than ±5%, which would be a slight improvement over the stated ±7% inhomogeneity of Vilber’s Bio-sun [26]. An ideal UV light source for biomedical research would have a sufﬁciently large illumination area,Electronics which 2019 would, 8, x FOR emit PEER light REVIEW uniformly. LEDs are just the opposite—light sources concentrated3 into of 18 a tiny dot. To achieve homogenous illumination, many LEDs have to be arranged in a two-dimensional array.dimensional Although array. the Although prices of white the prices and blue of white LEDs and have blue by nowLEDs become have by very now low, become and even very the low, prices and ofeven UVA the LEDs prices are of quite UVA reasonable, LEDs are quite the prices reasonable, of UVB th LEDse prices were, of UVB at the LEDs time ofwere, this at writing, the time still of very this high,writing, ranging still very from high, USD ranging 30 up from to USD USD 160 30 forup ato single USD 160 LED. for To a single keep theLED. price To keep of the the lamp price within of the reasonablelamp within limits, reasonable the number limits, of the LEDs number had toof beLEDs as low had as topossible. be as low as possible. The minimum number of LEDs was determineddetermined by numericalnumerical simulations of irradiance of the desired target area at different distances from the LEDs and with different different sizes sizes of of the the LED LED array. array. The density of the LED array and the power output of individual LEDs within the array were were varied. varied. Normalised irradiance at each point of the targettarget area was calculatedcalculated by summing up the contributions ofof allall thethe LEDsLEDs andand normalizingnormalizing thethe resultsresults withwith thethe maximummaximum calculatedcalculated value:value:

1 − =⋅1 θθ ⋅2 EPRRPrj i () ij,,,cos () ij−2 ij , (1) Ej =  PiRRP θi,j cos θi,j ·ri,j , (1) E ∑i Emax i

where Emax is the maximum irradiance in any of the target points Tj, P is the output power of an LED where Emax is the maximum irradiance in any of the target points Tj, P is the output power of an LED normalised toto its its maximum maximum possible possible output output power, power,RRP isRRP the is relative the relative radiation radiation proﬁle inprofile the direction in the fromdirection the LEDfrom to the the LED target to point,the targetr is thepoint, distance r is the between distance the between LED and the the LED target and point, the target and θ point,is the LEDand θ light is the beam’s LED light outbound beam’s polar outbound angle. polar The geometry angle. The of geometry the simulated of the problem simulated is shownproblem in is Figure shown1. in Figure 1.

Figure 1. Geometry of the simulated problem. Vectors N1 and N2 are normals to the LED plane and Figure 1. Geometry of the simulated problem. Vectors N1 and N2 are normals to the LED plane and target surface, respectively. The light beam’s outbound angle at LEDi and incident angle at the point Tj target surface, respectively. The light beam’s outbound angle at LEDi and incident angle at the point are the same, because the LED plane and the target surface are parallel. Tj are the same, because the LED plane and the target surface are parallel. Simulations were performed using the RRP (Figure2) of the selected UVA LEDs Simulations were performed using the RRP (Figure 2) of the selected UVA LEDs LTPL-C034UCH365 [28] by Lite-on Technology Corp. (Taipei, Taiwan, China). The RRP of the selected LTPL-C034UCH365 [28] by Lite-on Technology Corp. (Taipei, Taiwan, China). The RRP of the UVB LEDs, UF1HA-0F001 by Dowa Electronics Materials Co., Ltd. (Tokyo, Japan), was not available selected UVB LEDs, UF1HA-0F001 by Dowa Electronics Materials Co., Ltd. (Tokyo, Japan), was not at the design stage; therefore, the simulations were only performed for the UVA lamp, and the results available at the design stage; therefore, the simulations were only performed for the UVA lamp, and were applied to both lamps. The typical maximum optical output power of the UVA LED is 690 mW the results were applied to both lamps. The typical maximum optical output power of the UVA LED at an injected current of 500 mA and the typical maximum optical output power of the UVB LED is is 690 mW at an injected current of 500 mA and the typical maximum optical output power of the 13 mW at 100 mA. UVB LED is 13 mW at 100 mA. The optimum conﬁguration determined by the simulations consists of an array of 3 by 6 LEDs, at a distance of 30 mm from the target area. The distance between the 3 rows of LEDs is 34 mm with 31 mm between the 6 columns. The distance between rows is slightly larger than the distance between columns in order to achieve a slightly wider illumination area without having to add another row of LEDs. The relative output power of the individual LEDs for achieving homogenous illumination is given in Table1 and varies from 55% to 100%.

Figure 2. Relative radiation profile of the UVA LED (LTPL-C034UCH365).

Electronics 2019, 8, x FOR PEER REVIEW 3 of 18 dimensional array. Although the prices of white and blue LEDs have by now become very low, and even the prices of UVA LEDs are quite reasonable, the prices of UVB LEDs were, at the time of this writing, still very high, ranging from USD 30 up to USD 160 for a single LED. To keep the price of the lamp within reasonable limits, the number of LEDs had to be as low as possible. The minimum number of LEDs was determined by numerical simulations of irradiance of the desired target area at different distances from the LEDs and with different sizes of the LED array. The density of the LED array and the power output of individual LEDs within the array were varied. Normalised irradiance at each point of the target area was calculated by summing up the contributions of all the LEDs and normalizing the results with the maximum calculated value:

1 − =⋅()θθ () ⋅2 EPRRPrj i ij,,,cos ij ij , (1) Emax i where Emax is the maximum irradiance in any of the target points Tj, P is the output power of an LED normalised to its maximum possible output power, RRP is the relative radiation profile in the direction from the LED to the target point, r is the distance between the LED and the target point, and θ is the LED light beam’s outbound polar angle. The geometry of the simulated problem is shown in Figure 1.

Figure 1. Geometry of the simulated problem. Vectors N1 and N2 are normals to the LED plane and

target surface, respectively. The light beam’s outbound angle at LEDi and incident angle at the point

Tj are the same, because the LED plane and the target surface are parallel.

Simulations were performed using the RRP (Figure 2) of the selected UVA LEDs LTPL-C034UCH365 [28] by Lite-on Technology Corp. (Taipei, Taiwan, China). The RRP of the selected UVB LEDs, UF1HA-0F001 by Dowa Electronics Materials Co., Ltd. (Tokyo, Japan), was not available at the design stage; therefore, the simulations were only performed for the UVA lamp, and the results were applied to both lamps. The typical maximum optical output power of the UVA LED Electronics 2019, 8, 343 4 of 18 is 690 mW at an injected current of 500 mA and the typical maximum optical output power of the UVB LED is 13 mW at 100 mA.

FigureFigure 2. 2. RelativeRelative radiation radiation profile proﬁle of the UVA LED (LTPL-C034UCH365). Table 1. Theoretical relative output power of individual LEDs in the array for achieving the most homogenous illumination, expressed in percent.

Column 1 2 3 4 5 6 Row 1 100 75 77 77 75 100 2 75 55 56 56 55 75 3 100 75 77 77 75 100

2.2. Electrical Design Given the optical simulation results summarised in Table1, it would be theoretically possible to achieve satisfactorily homogenous illumination with five or even only four different LED driving currents. In reality, though, every LED has a different initial efficiency, which means that every LED has to have its own adjustable current source. This challenge was overcome by two TLC59401 (Texas Instruments Inc., Dallas, TX., USA) integrated circuits (IC), which have 16 individually controlled outputs each, whereby each output has 64-step linear current control and an additional 4096 steps using pulse-width modulation (PWM). These ICs were originally designed for driving large-panel LED displays, which suffer from the same problem of varying LED efficiencies. Since the lamp needs to be controlled from outside of the incubator, the control module was designed as a separate unit connected to the lamp head by a flat cable, which can be easily inserted through a crack in the closed door of an incubator. A block diagram of the lamp is shown in Figure3, and the complete schematics, along with the source code, are available in the Supplementary Materials. The lamp head contains a microcontroller ATmega324 (Microchip Technology Inc., Chandler, AZ., USA), which controls the LED driving ICs, monitors temperature, holds calibration data and logs usage time. The TLC59401 LED drivers are connected to the microcontroller via a serial interface which is compatible with the Serial Peripheral Interface (SPI). The TLC59401 ICs employ constant current sink LED drivers with 64-step linear current control and an additional 4096-step PWM brightness control. The PWM function was not used in this case in order to exclude any potential uncertainties in the biomedical research conducted with regard to pulsed UV illumination. The maximum output current per channel is set by an external resistor and is set to 120 mA for the UVA lamp and to 100 mA for the UVB lamp. The TLC59401 ICs also detect open LED events (LED disconnected), or over-temperature events, which are then signalled to the microcontroller. The on-board microcontroller measures temperature in two places using negative temperature coefficient resistors (NTC): on one of the aluminium LED carrying substrates as close to the LED as possible (TLED in Figure3), to get an estimation of the LED temperature; and on the heatsink Electronics 2019, 8, x FOR PEER REVIEW 4 of 18

The optimum configuration determined by the simulations consists of an array of 3 by 6 LEDs, at a distance of 30 mm from the target area. The distance between the 3 rows of LEDs is 34 mm with 31 mm between the 6 columns. The distance between rows is slightly larger than the distance between columns in order to achieve a slightly wider illumination area without having to add another row of LEDs. The relative output power of the individual LEDs for achieving homogenous illumination is given in Table 1 and varies from 55% to 100%.

Table 1. Theoretical relative output power of individual LEDs in the array for achieving the most homogenous illumination, expressed in percent.

Row Column 1 2 3 4 5 6 1 100 75 77 77 75 100 2 75 55 56 56 55 75 3 100 75 77 77 75 100

ElectronicsLED displays,2019, 8, 343 which suffer from the same problem of varying LED efficiencies. 5 of 18 Since the lamp needs to be controlled from outside of the incubator, the control module was designed as a separate unit connected to the lamp head by a flat cable, which can be easily inserted (THSthroughin Figure a crack3) accessed in the closed through door a of hole an inincubator. the printed A block circuit diagram board of (PCB), the lamp to evaluate is shown the in Figure average temperature3, and the ofcomplete the UV lampschematics, head. along with the source code, are available in the Supplementary Materials.

FigureFigure 3. 3.Block Block diagram diagram of of the the UV UV lamp. lamp. TheThe lamplamp head and and the the lamp lamp controller controller are are connected connected by bya a ﬂatflat cable. cable.

TheThe control lamp modulehead contains provides a microcontroller the required power ATme toga324 the lamp(Microchip head, allowsTechnology the lamp Inc., toChandler, be turned onAZ., and USA), off, shows which the controls current the status LED ofdriving the lamp, ICs, includingmonitors temperature, potential errors, holds shows calibration the illumination data and timelogs and usage radiation time. The dose TLC59401 weighted LED with drivers the are reference connec actionted to the spectrum microcontroller for erythema via a serial in human interface skin CIE1987which (Commissionis compatible Internationalewith the Serial de Peripheral l’Éclairage Interface 1987) [ 29(SPI).]. It alsoThe TLC59401 includes a ICs safety employ switch, constant which is intendedcurrent tosink be mountedLED drivers onto with the incubator64-step linear door, current to prevent control accidental and an exposureadditional to 4096-step UV radiation. PWM The heart of the control module is an ATmega324 microcontroller which communicates with the lamp head via the universal asynchronous receiver-transmitter (UART). Its function is to receive commands from the keyboard or the Bluetooth module and relay them to the lamp head and to display the status of the lamp to the user. All other functions, like measuring illumination time, calculating the illumination dose, measuring usage time, etc., are performed by the lamp head. This makes the control modules of different lamps interchangeable. UVA and UVB LEDs require different voltages to operate. It is important that the voltage drop on the TLC59401 ICs is as low as possible, because it translates directly into excess heat. To keep this voltage drop as low as possible, the voltage provided for the LEDs by the lamp controller has to be tailored for each of the two lamps. This is achieved by changing the output voltage feedback divider ratio of the switching power supply for the LEDs. The feedback divider ratio is controlled by the UVA/UVB ID line which is either closed to ground (UVB) or left ﬂoating (UVA) within the lamp head. A short circuit was selected for the UVB lamp so that in case of bad connection or similar error, the lower voltage, which cannot damage either lamp head, will be generated. Before the LED voltage is provided to the lamp head, it is fed through the safety switch, which has to be mounted onto the incubator door, to prevent accidental exposure to UV illumination. Since the LED power supply voltage is tailored for each of the two types of UV LEDs, the power supply voltage for all other components is provided by a separate 5 V switching power supply. The whole system is powered from an external 12 V power supply. The control module on its own only allows the lamp to be turned on and off and displays basic status information. All other functionality, like setting the container transparency factor, time countdown, dose countdown, temperatures check, and usage statistics, requires a Bluetooth connection. The Bluetooth connection is facilitated by a Bluetooth module with a Serial Port Proﬁle (SPP) connected to the microcontroller via a UART. An android application for control of the lamps over a Bluetooth connection is provided online in the Supplementary Materials. Electronics 2019, 8, 343 6 of 18

2.3. Mechanical Design One of the important considerations in biological research is the temperature of the sample and its immediate environment. Although LEDs are efficient power to light converters, they still emit heat. However, in contrast to other light sources, LEDs are more efficient at lower temperatures and can be effectively cooled through the structure they are mounted on. To maximise cooling efficiency, each LED was mounted on an aluminium substrate, which was in turn mounted onto a large aluminium heatsink which doubles as the body of the enclosure. The LED driver circuit with a microcontroller was manufactured on a standard fibre glass (FR-4) PCB substrate and mounted onto the same aluminium heatsink. The driver circuit PCB has cut-outs for the LED carrying aluminium substrates. Electrical connection between each of the aluminium substrate Electronics 2019, 8, x FOR PEER REVIEW 6 of 18 PCBs and the main PCB was achieved with 0 Ω resistors. The cover of the lamp is made from a PLUS UV-transparentfrom a UV-transparent acrylic sheetacrylic ACRISUN sheet ACRISUNmadePLUS made by Plastidite by Plastidite S.p.A. S.p.A. (Trieste, (Trieste, Italy), Italy), otherwise otherwise used inused solariums in solariums as protective as protective covering covering for UV lights. for UV A cross-section lights. A cross-section of the UV lamp of the and UV a photograph lamp and ofa thephotograph lamp are of shown the lamp in Figure are shown4. in Figure 4.

Acrylic cover LED Aluminum substrate FR-4 substrate

Aluminum heatsink

(a) (b)

FigureFigure 4. (a) A A cross-section cross-section drawing drawing and and (b (b) )a aphotograph photograph of of the the portable portable UV UV lamp lamp head head with with a 3 aby 3 by6 LED 6 LED array array without without the the acrylic acrylic glass glass cover. cover. On On the the right right side side of of the the middle middle row, row, one LED with itsits aluminiumaluminium substratesubstrate isis missingmissing toto betterbetter illustrateillustrate thethe design.design.

3.3. Materials and Methods 3.1. Irradiance and Homogeneity 3.1. Irradiance and Homogeneity Spatial irradiance at a speciﬁed distance from the UV lamps was measured using a calibrated Spatial irradiance at a specified distance from the UV lamps was measured using a calibrated S1133-01 photodiode (Hamamatsu Photonics K.K., Hamamatsu, Japan) with a photosensitive area S1133-01 photodiode (Hamamatsu Photonics K.K., Hamamatsu, Japan) with a photosensitive area of of 2.8 mm × 2.4 mm and a Keithley 238 Source-Measure unit (Keithley Instruments Inc., Solon, OH., 2.8 mm × 2.4 mm and a Keithley 238 Source-Measure unit (Keithley Instruments Inc., Solon, OH., USA). Homogeneity was determined by scanning the entire area of both UV lamps in 2 mm intervals USA). Homogeneity was determined by scanning the entire area of both UV lamps in 2 mm intervals at a constant distance of 18 mm above the cover of the lamp, which corresponds to a distance of 30 mm at a constant distance of 18 mm above the cover of the lamp, which corresponds to a distance of 30 above the LEDs. All the measurements were performed inside a black enclosure to prevent any stray mm above the LEDs. All the measurements were performed inside a black enclosure to prevent any light from interfering with the measurements. stray light from interfering with the measurements. 3.2. Spectrum and Transparency 3.2. Spectrum and Transparency The illumination spectrum of the UV lamps was measured using an HR2000+ spectrometer The illumination spectrum of the UV lamps was measured using an HR2000+ spectrometer (Ocean Optics Inc., Tampa, FL., USA) and CC-3-UV-S cosine corrector (Ocean Optics Inc., Tampa, FL., (Ocean Optics Inc., Tampa, FL., USA) and CC-3-UV-S cosine corrector (Ocean Optics Inc., Tampa, USA). Spectrometer calibration was performed with an LS-1-CAL-220 calibration light source (Ocean FL., USA). Spectrometer calibration was performed with an LS-1-CAL-220 calibration light source Optics Inc., Tampa, FL., USA). However, the output of the calibration light source at wavelengths (Ocean Optics Inc., Tampa, FL., USA). However, the output of the calibration light source at below 400 nm is too low to provide acceptable calibration accuracy. Therefore, only relative spectrum wavelengths below 400 nm is too low to provide acceptable calibration accuracy. Therefore, only measurements were performed, and the results were normalised to the maximum averaged value relative spectrum measurements were performed, and the results were normalised to the maximum for each lamp. Spectrum measurements were carried out just above the transparent cover of the UV averaged value for each lamp. Spectrum measurements were carried out just above the transparent lamps, which is 12 mm above the LEDs. The transparency of the microtiter plates and acrylic glass cover of the UV lamps, which is 12 mm above the LEDs. The transparency of the microtiter plates cover was measured with a Lambda 950 spectrophotometer (Perkin Elmer Inc., Waltham, MA., USA). and acrylic glass cover was measured with a Lambda 950 spectrophotometer (Perkin Elmer Inc., Waltham, MA., USA).

Electronics 2019, 8, 343 7 of 18

3.3. Heat Dissipation The effects of excess heat dissipation were measured in a WTC binder Type 36210180003100 incubator (BINDER GmbH, Tuttlingen, Germany) with 6 DS18B20 digital temperature sensors (Maxim Integrated Products Inc., San Jose, CA., USA), which were placed inside the wells of a 96-well microtiter plate. All the wells were ﬁlled with 100 µL of a basic cell culture medium. Four out of six sensors were dyed white in order to reduce any possible temperature effects due to light absorption. Two sensors remained black for comparison. One black and one white sensor were placed in neighbouring wells in the middle of the microtiter plate, one white sensor was placed on the edge, and two white sensors were placed into opposite corners of the microtiter plate. The remaining black sensor was positioned approximately 5 cm away from the lamp to measure the ambient temperature. The microtiter plate was placed on top of the lamp at the appropriate height above the lamp. The incubator was set to maintain a temperature of 37 ◦C. The measurements showed only small discrepancies between different sensor positions, and conﬁrmed that the wells in the centre of the microtiter plate, which are the most insulated from the ambient temperature, are the most critical with regard to unwanted temperature increases. Therefore, only the measurements of the centre well are reported. The difference between the black sensors and the ones that were dyed white was also negligible. Further investigation of heat dissipation was performed with a FLIR i7 infrared (IR) camera (FLIR Systems Inc., Wilsonville, OR., USA). A lamp was covered with a black painted cardboard box with a hole on top for the camera. Pictures were taken after 60 min of operation. Pictures from angles other than from the top were taken just after the lamp had been turned off to avoid exposure to the UV radiation. The emissivity factor was set to 0.95, which is a reasonable value for most matte surfaces. Further calibration was not performed, therefore the IR images cannot be used for accurate temperature reading, while they do provide a good understanding of temperature changes.

3.4. Cell Culture Tests Jurkat cells (E6.1) were obtained from the European Collection of Authenticated Cell Cultures (ECACC). The cells were cultured as a suspension in cell culture flasks in a RPMI 1640 medium supplemented with 10% (v:v) of heat-inactivated foetal bovine serum (HIFBS) at 37 ◦C and a 5% CO2 atmosphere in a high humidity incubator. The medium was replaced every second or third day. Cells were sub cultured through dilution in a fresh medium. HEK-293 cells (CRL-1593) were obtained from American Type Culture Collection (ATCC). Cells were cultured adherent in cell culture flasks in a DMEM medium, supplemented with 10% (v:v) of HIFBS and 1% (v:v) of L-glutamine to a final 2 mM concentration. The medium was replaced every second or third day. Cells were sub cultured when reaching about 90% confluency using a trypsin-EDTA solution. For viability testing, the cells were seeded on 96-well microtiter plates in 200 µL of growth medium a day before exposure to the UV irradiation to enable cell adaptation and adhesion in adherent HEK-293 cells. The Jurkat and HEK-293 cells were seeded at a density of 150,000 cells/mL and 100,000 cells/mL, respectively, in all wells. In microtiter plates, border wells are often omitted from experiments due to border related effects. To minimise water evaporation from edge wells and related effects on cell fitness, the microtiter plates were kept on wet paper in vented plastic containers within the high humidity incubator. To ensure equal positioning of the microtiter plates in subsequent experiments, the plate holder was fixed on top of the UV light illumination panel. Plates were irradiated with UVA (365 nm) or UVB (308 nm). The overall exposure time to UVA was 966 s, which, taking into consideration the 73.84% transparency of the microtiter well at a wavelength of 365 nm, resulted in a dose of 50000 J/m2, while for UVB, the exposure time was 666 s, which in conjunction with the 48.46% transparency at a wavelength of 308 nm, resulted in a dose of 1000 J/m2. The irradiation was performed in a dust free cabinet in the bottom-up direction. By doing so, the optical effect of medium surface was excluded. As the cells were adherent or sedimented to the bottom during irradiation, the possible medium-related Electronics 2019, 8, 343 8 of 18 absorption was neglected. The time out of the incubator during irradiation was equal for all tested and control plates. After irradiation, the cells were incubated for 21 h to develop apoptotic response and then a PrestoBlue® Cell Viability Reagent (Thermo Fisher Scientific Inc., Waltham, MA., USA) was added to them. A fluorescent signal proportional to the number of viable cells was measured after 3 h with a plate reader. Means and standard deviations of identically positioned wells from three independent experiments were calculated.

4. Results and Discussion All presented measurement results and simulations are available in an online dataset [30]. Electronics 2019, 8, x FOR PEER REVIEW 8 of 18 4.1. Simulation Results 4.1. Simulation Results Simulated normalised irradiance of the optimised conﬁguration for the UVA lamp is shown in Simulated normalised irradiance of the optimised configuration for the UVA lamp is shown in Figure5. This conﬁguration yields a usable illumination area of 148 mm × 66 mm (marked with the Figure 5. This configuration yields a usable illumination area of 148 mm × 66 mm (marked with the black line), which is slightly less than the initially desired area. However, the smaller illumination area black line), which is slightly less than the initially desired area. However, the smaller illumination is acceptable, given the cost saving. The inhomogeneity within the usable area is ±3%—slightly better area is acceptable, given the cost saving. The inhomogeneity within the usable area is ±3%—slightly than the initial requirement, which gives a bit of leeway for the real world implementation. better than the initial requirement, which gives a bit of leeway for the real world implementation.

Figure 5. Simulated normalised irradiance of the optimised configuration of the 3 by 6 UVA LED array Figure 5. Simulated normalised irradiance of the optimised configuration of the 3 by 6 UVA LED at a distance of 30 mm. The black line marks the bounds of the usable area. The vertical scale of the array at a distance of 30 mm. The black line marks the bounds of the usable area. The vertical scale of graph is limited to a range between 90 and 100% to show the inhomogeneity in greater detail. the graph is limited to a range between 90 and 100% to show the inhomogeneity in greater detail. 4.2. Irradiance Homogeneity 4.2. Irradiance Homogeneity The irradiance measurement graphs are normalised to the maximum measured value in order to facilitateThe irradiance comparison measurement of inhomogeneity. graphs are The normalis black lineed to in the graphsmaximum indicates measured the areavalue where in order the averageto facilitate optical comparison power density of inhomogeneity. and maximum The deviations black line from in the the graphs average indicates value were the area calculated. where the averageFigure optical6 shows power the density measured and irradiance maximum achieved deviations by the from UVA the lamp, average which value matches were the calculated. simulation veryFigure well. The6 shows difference the measured between the irradiance simulated ac andhieved measured by the dataUVA is lamp, below which±2%. Thematches average the irradiancesimulation producedvery well. by The the difference UVA lamp between was measured the simulated to be 70.1and W/mmeasured2. The data maximum is below irradiance ±2%. The wasaverage 73.2 irradiance W/m2 and produced the minimum by the was UVA 66.7 lamp W/m was2. These measured numbers to translatebe 70.1 W/m to an2. inhomogeneityThe maximum inirradiance the range was between 73.2 −W/m4.9%2 and +4.4%,the minimum which is slightlywas 66.7 more W/m than2. These the simulation numberspredicted translate values,to an yetinhomogeneity less than the in initial the range requirement, between albeit −4.9% on and a slightly +4.4%, reduced which is area slightly than more initially than specified. the simulation Table2 containspredicted the values, individual yet less LED than output the initial power requirement, settings required albeit on to a achieveslightly thisreduced result. area The than maximum initially discrepancyspecified. Table between 2 contains the valuesthe individual determined LEDby output the simulation power settings and therequired values to determinedachieve this byresult. the calibrationThe maximum is approximately discrepancy 10%,between which the confirms values thatdetermined the ability by to the set currentssimulation for eachand the individual values LEDdetermined is a necessary by the feature.calibration is approximately 10%, which confirms that the ability to set currents for each individual LED is a necessary feature.

Electronics 2019, 8, 343 9 of 18 Electronics 2019, 8, x FOR PEER REVIEW 9 of 18 Electronics 2019, 8, x FOR PEER REVIEW 9 of 18

Figure 6. Measured normalised irradiance of the UVA lamp at a distance of 30 mm above the Figure 6. Measured normalised irradiance of the UVA lamp at a distance of 30 mm above the LEDs. LEDs. The boxplot on the right represents the measured values within the bounds of the black The boxplot on the right represents the measured values within the bounds of the black line. The line.Figure The 6. whiskersMeasured represent normalised the extremesirradiance of of the the data UVA set. lamp at a distance of 30 mm above the LEDs. whiskersThe boxplot represent on the the right extremes represents of the the data measured set. values within the bounds of the black line. The Tablewhiskers 2. Actualrepresent relative the extremes output of power the data of individual set. LEDs of the UVA lamp after the calibration, Tableexpressed 2. Actual in percent. relative output power of individual LEDs of the UVA lamp after the calibration, expressedTable 2. Actual in percent. relative output power of individual LEDs of the UVA lamp after the calibration, Column expressed in percent. 1 2 3 4 5 6 RowRow Column 1 2 3 4 5 6

Row 11 Column 100.01 100.0 76.22 76.274.63 74.677.84 77.874.65 74.6 100.0 6100.0 221 100.0 77.8 77.8 55.676.2 55.658.774.6 58.755.677.8 55.6 60.374.6 60.3 100.0 79.479.4 3 98.4 77.8 74.6 76.2 73.0 93.7 32 98.4 77.8 77.8 55.6 74.658.7 76.255.6 73.060.3 93.7 79.4 3 98.4 77.8 74.6 76.2 73.0 93.7 The measured irradiance produced by the UVB lamp, shown in Figure 7, did not match the The measured irradiance produced by the UVB lamp, shown in Figure7, did not match the initial simulation shown in Figure 5, which was performed using the RRP of the UVA LED. A revised initialThe simulation measured shown irradiance in Figure produced5, which by was the performed UVB lamp, using shown the RRPin Figure of the UVA7, did LED. not Amatch revised the version of the UVB LED datasheet with a RRP had, in the meantime, been published. The published versioninitial simulation of the UVB shown LED datasheetin Figure 5, with which a RRP was had, performed in the meantime, using the RRP been of published. the UVA LED. Thepublished A revised RRPversion differed of the considerably UVB LED datasheet from the with RRP a RRPof the had, UVA in theLED. meantime, Using the been newly published. published The RRP published in the RRP differed considerably from the RRP of the UVA LED. Using the newly published RRP in the simulationRRP differed still considerably failed to provide from thea match RRP betwof theeen UVA the LED.measurement Using the and newly the simulation.published RRP However, in the simulation still failed to provide a match between the measurement and the simulation. However, Kheyrandishsimulation still et al.failed [31] to reported provide that a match most betwLEDseen with the a measurementheart-shaped RRP,and thelike simulation. the one provided However, for Kheyrandish et al. [31] reported that most LEDs with a heart-shaped RRP, like the one provided for theKheyrandish UVB LED, et have al. [31] an reportedazimuth thatdependant most LEDs RRP. with Therefore, a heart-shaped a 3D scan RRP, of thelike UVB the one LED’s provided RRP was for the UVB LED, have an azimuth dependant RRP. Therefore, a 3D scan of the UVB LED’s RRP was performedthe UVB LED, (Figure have 8a). an Theazimuth new simulationdependant usingRRP. Therefore,the 3D scan a 3Das an scan input of theparameter UVB LED’s was RRPfound was to performed (Figure8a). The new simulation using the 3D scan as an input parameter was found to matchperformed the measured (Figure 8a). irradiance The new of simulation the UVB lamp using very the well 3D scan(Figure as an8b). input parameter was found to match the measured irradiance of the UVB lamp very well (Figure8b). match the measured irradiance of the UVB lamp very well (Figure 8b).

Figure 7. Measured normalised irradiance of the UVB lamp at a distance of 30 mm above the LEDs. Figure 7. Measured normalised irradiance of the UVB lamp at a distance of 30 mm above the LEDs. Figure 7. Measured normalised irradiance of the UVB lamp at a distance of 30 mm above the LEDs.

Electronics 2019, 8, 343 10 of 18 Electronics 2019, 8, x FOR PEER REVIEW 10 of 18 Electronics 2019, 8, x FOR PEER REVIEW 10 of 18

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

FigureFigure 8. 8.((a ()a 3D 3D) 3D scanned scanned scanned RRP RRP RRP of ofof the thethe UVB UVB LED LED UF1HA-0F001UF1HA-0F001 UF1HA-0F001 and and and ( b( (b) simulated)) simulated simulated normalised normalised normalised irradiance irradiance of of the the UVB UVB lamp lamp using using the the newly newly scannedscannedscanned RRPRRP atat aa distancedistance of ofof 30 3030 mm mmmm above aboveabove the thethe LEDs. LEDs.LEDs.

TheThe measuredmeasured measured irradiance irradiance irradiance distribution distributiondistribution of of the the UVB UVBUVB lamp lamplamp was was was too too inhomogeneoustoo inhomogeneous inhomogeneous to meetto tomeet themeet the initial the requirementinitialinitial requirement requirement of ±5%. of Toof ±5%. improve±5%. ToTo improve theimprove homogeneity, the homohomo angeneity,geneity, optical an diffusoran opticaloptical was diffusor diffusor created was bywas engravingcreated created by anby engravingPLUS an ACRISUNPLUS acrylic sheet (Plastidite S.p.A., Trieste, Italy) with a laser. Although the ACRISUNengraving an ACRISUNacrylic sheetPLUS acrylic (Plastidite sheet S.p.A., (Plastidite Trieste, S.p.A., Italy) Trieste, with a Italy) laser. with Although a laser. the Although addition the of addition of the diffusor reduced the optical output of the lamp by approximately 40%, the theaddition diffusor of reducedthe diffusor the optical reduce outputd the ofoptical the lamp output by approximatelyof the lamp 40%,by approximately the homogeneity 40%, of the homogeneity of the lamp was significantly improved. Figure 9 shows the irradiance achieved by the lamphomogeneity was significantly of the lamp improved. was signif Figureicantly9 improved. shows the Figure irradiance 9 shows achieved the irradiance by the UVB achieved lamp by with the UVB lamp with the diffusor. The area where the lamp satisfies the initial requirements is only 120 theUVB diffusor. lamp with The the area diffusor. where The the lamparea where satisfies the the lamp initial satisfies requirements the initial is requirements only 120 mm is× only60 mm; 120 mm × 60 mm; however, it was decided that the benefits of a lamp redesign would not outweigh the however,mm × 60 mm; it was however, decided it that was the decided benefits that of athe lamp bene redesignfits of a would lamp redesign not outweigh would the not additional outweigh costs. the additional costs. The average irradiance produced by the UVB lamp with added diffusor is 3.10 W/m2, The average irradiance produced by the UVB lamp with added diffusor is 3.10 W/m2, with a maximum2 additionalwith a maximum costs. The of average 3.20 W/m irradiance2 and a producedminimum by of the2.95 UVB W/m lamp2, which with correspondsadded diffusor to maximumis 3.10 W/m , 2 2 2 2 ofwithdeviations 3.20 a W/mmaximum ofand +3.2% aof minimum and3.20 − 4.8%W/m of and 2.95the averagea W/m minimum ,optical which of power corresponds2.95 W/mdensity,, which to respectively. maximum corresponds The deviations different to maximum of RRP +3.2% − anddeviationsof the4.8% UVB of of LEDs +3.2% the average and and the − 4.8%opticaladdition of powerthe of theaverage density,diffusor optical respectively.result power in a different dens Theity, different distribution respectively. RRP of of the theThe output UVB different LEDs power RRP and theof ofthe addition individual UVB LEDs of LEDs. the and diffusor Thethe additionvalues result determined inof athe different diffusor by cali distribution resubrationlt in aare different of summarised the output distribution in power Table of of 3. the individualThe output difference power LEDs. Theof inindividual valuesthe efficiency determined LEDs. of individualThe by values calibration LEDs determined is even are summarised greate by calir inbration the incase Tableare of summarised the3. TheUVB difference LEDs in compared Table in the3. The to efficiency the difference UVA of individualin LEDs.the efficiency LEDs of is individual even greater LEDs in the is even case greate of ther UVB in the LEDs case comparedof the UVB to LEDs the UVA compared LEDs. to the UVA LEDs.

Figure 9. Measured normalised irradiance of the UVB lamp with added diffusor at a distance of 30 mm above the LEDs. The boxplot on the right represents the measured values within the bounds of Figurethe black 9. Measured line. The whiskers normalised represen irradiance t the extremes of the UVB of the lamp data withset. added diffusor at a distance of 30 mm aboveabove thethe LEDs.LEDs. TheThe boxplot boxplot on on the the right right represents represents the the measured measured values values within within the the bounds bounds of the of blacktheTable black line. 3. line. TheActual The whiskers relativewhiskers represent output represen power thet extremesthe of extremes individual of the of dataLEDsthe data set. of set.the UVB lamp after the calibration, expressed in percent. Table 3. Actual relative output power of individual LEDs of the UVB lamp after the calibration, Row Column 1 2 3 4 5 6 expressed in percent. 1 90.5 84.1 82.5 79.4 79.4 98.4 Row 2Column 79.41 65.12 71.43 74.64 63.55 77.86 13 100.0 90.5 79.484.1 100.082.5 81.079.4 82.579.4 96.898.4 2 79.4 65.1 71.4 74.6 63.5 77.8 3 100.0 79.4 100.0 81.0 82.5 96.8

Electronics 2019, 8, 343 11 of 18

Table 3. Actual relative output power of individual LEDs of the UVB lamp after the calibration, expressed in percent.

Column 1 2 3 4 5 6 Row

Electronics 2019, 8, x FOR PEER REVIEW1 90.5 84.1 82.5 79.4 79.4 98.4 11 of 18 2 79.4 65.1 71.4 74.6 63.5 77.8 3 100.0 79.4 100.0 81.0 82.5 96.8 There is a very large difference in the irradiance both lamps produce, even though the electrical input power is almost the same in both cases. The main reason is that the efficiency of UVA LEDs is moreThere than 10 is atimes very largehigher difference than that inof the UVB irradiance LEDs, which both lampsshows produce,how much even room though for improvement the electrical inputstill remains power in is almostthe area the of samedeep UV in both LED cases. development. The main In reason this case, is that however, the efﬁciency the lower of UVA efficiency LEDs of is morethe UVB than LEDs 10 times is nothigher a problem, than because that of UVBbiological LEDs, tissue which is showsmuch more how sensitive much room to UV for light improvement of shorter stillwavelengths. remains in A the good area reference of deep UVis the LED sensitivity development. of human In this skin, case, presented however, in thethe lowerCIE1987 efﬁciency reference of theaction UVB spectrum LEDs is notfor erythema a problem, in because human biological skin (Commission tissue is much Internationale more sensitive de l’Éclairage to UV light 1987) of shorter [29]. wavelengths.Taking into account A good the reference CIE1987 isreference the sensitivity action sp ofectrum, human the skin, CIE presented weighted in irradiance the CIE1987 that referencethe UVA actionlamp (peak spectrum at 365 for nm) erythema produces in is human only 0.0254 skin (CommissionW/m2CIE, while Internationale the CIE weighted de l’ irradianceÉclairage 1987)which [ 29the]. TakingUVB lamp into produces account the (peak CIE1987 at 308 reference nm) is 0.516 action W/m spectrum,2CIE. This the means CIE weightedthat the impact irradiance of the that UVB the lamp UVA 2 lampon biological (peak at samples 365 nm) should produces be ismuch only larger 0.0254 than W/m thatCIE ,of while the UVA the CIE lamp, weighted despite irradiance its much whichlower 2 theradiometric UVB lamp irradiance. produces (peak at 308 nm) is 0.516 W/m CIE. This means that the impact of the UVB lamp on biological samples should be much larger than that of the UVA lamp, despite its much lower radiometric4.3. Spectrum irradiance.

4.3. SpectrumThe spectrum of the UVA lamp is expected to be limited to the 320 nm–400 nm range and the spectrum of the UVB lamp to the 290 nm–320 nm range. The normalised measured spectrum of both lampsThe is shown spectrum in Figure of the 10. UVA The lamp UVA is lamp expected emits to most be limited of its light to the in 320the wavelength nm–400 nm range range between and the spectrum360 nm and of the390 UVBnm with lamp a topeak the 290at 368 nm–320 nm and nm a range.full width The normalisedhalf magnitude measured (FWHM) spectrum of 9 nm. of bothThe lampslight outside is shown of the in Figure UVA range 10. The is below UVA lamp the noise emits fl mostoor of of the its instrument. light in the wavelengthThe UVB lamp range spectrum between is 360slightly nm andbroader 390 nm than with that a peakof the at UVA 368 nm lamp. and Most a full of width the halflight magnitude of the UVB (FWHM) lamp is ofemitted 9 nm. Thebetween light outside295 nm ofand the 320 UVA nm, range and only is below 17% the of noiselight is ﬂoor emitted of the in instrument. the UVA range The UVBbetween lamp 320 spectrum nm and is 340 slightly nm. broaderThe peak than is at that 308 ofnm the and UVA FWHM lamp. is Most 14 nm. of the light of the UVB lamp is emitted between 295 nm and 320 nm, and only 17% of light is emitted in the UVA range between 320 nm and 340 nm. The peak is at 308 nm and FWHM is 14 nm.

Figure 10. Normalised spectrum of the UVA (blue lines) and the UVB (magenta lines) lamp. The thin Figure 10. Normalised spectrum of the UVA (blue lines) and the UVB (magenta lines) lamp. The thin lines are measurements of individual LEDs and the thick line is the average of all the measurements. lines are measurements of individual LEDs and the thick line is the average of all the measurements. 4.4. Heat Dissipation 4.4. Heat Dissipation Most of the input electrical power of any lamp is transformed into waste heat instead of light. This heatMost raises of the the input temperature electrical ofpower biological of any samples lamp is under transformed test beyond into their waste optimal heat instead temperature of light. of 37This◦C. heat Purpose-built raises the temperature microtiter plate of biological UV illuminators samples haveunder a built-intest beyond cooling their solution. optimal Ontemperature the other of 37 °C. Purpose-built microtiter plate UV illuminators have a built-in cooling solution. On the other hand, a light source designed for use inside an incubator cannot transfer excess heat outside of the incubator, and therefore has to produce as little excess heat as possible. Fans are also not desirable, because excessive air movement can increase the risk of sample trans-contamination. Since heat dissipation comparison between a light source which cannot be placed inside an incubator and one that can does not make much sense, the heat dissipation of UV LED lamps is compared to the heat dissipation of the Vilber VL-6LC [32] handheld UV lamp based on fluorescent tubes with a similar power consumption, which can also be placed inside an incubator, provided that the humidity is set low enough.

Electronics 2019, 8, 343 12 of 18 hand, a light source designed for use inside an incubator cannot transfer excess heat outside of the incubator, and therefore has to produce as little excess heat as possible. Fans are also not desirable, because excessive air movement can increase the risk of sample trans-contamination. Since heat dissipation comparison between a light source which cannot be placed inside an incubator and one that can does not make much sense, the heat dissipation of UV LED lamps is compared to the heat dissipationElectronics 2019 of, 8 the, x FOR Vilber PEER VL-6LC REVIEW [32] handheld UV lamp based on ﬂuorescent tubes with a similar12 of 18 power consumption, which can also be placed inside an incubator, provided that the humidity is set low enough.Figure 11 shows temperature traces of the centre microtiter well (a) and ambient temperature insideFigure an incubator 11 shows (b) temperature for the UVA traces (blue) of the and centre UVB microtiter(magenta) wellLED (a) lamps and ambientand the VL-6LC temperature (red) insidelamp. an The incubator optimal (b) temperature for the UVA for (blue) most and of UVBthe biological (magenta) samples LED lamps is about and the 37 VL-6LC°C. In this (red) test, lamp. we Theassume optimal a maximum temperature allowed for mosttemperature of the biological of 42 °C, whic samplesh most is aboutcells can 37 survive,◦C. In this to establish test, we assumea metric afor maximum comparison allowed of all temperaturethree lamps. ofThe 42 UVA◦C, whichlamp took most approximately cells can survive, 40 mi ton, establish the UVB alamp metric 67 min for comparisonand the VL-6LC of all threeonly 20 lamps. min Theto reach UVA a lamp 5 °C tookincrease approximately above the optimal 40 min, thetemperature UVB lamp in 67 the min central and thewell VL-6LC of the onlymicrotiter 20 min plate. to reach a 5 ◦C increase above the optimal temperature in the central well of the microtiter plate.

(a) (b)

FigureFigure 11. 11.The The temperaturetemperature of the centre centre microtiter microtiter well well (a ()a )and and ambient ambient temperature temperature (b) ( bduring) during a 120 a 120min min illumination illumination period period inside inside an an incubator incubator with with ea eachch of of the the three lamps. TheThe lampslamps werewere switched switched onon at at the the 0 0 min min mark. mark.

AlthoughAlthough the the input input electrical electrical powerpower ofof allall threethree lampslampsis isvery very similar,similar, with with12.3 12.3W Wfor forthe theUVA UVA LEDLED lamp, lamp, 13.313.3 WW for for the the UVB UVB LED LED lamp lamp and and 12.3 12.3 W W for for the the VL-6LC VL-6LC lamp lamp (VILBER (VILBER LOURMAT LOURMAT DeutschlandDeutschland GmbH, GmbH, Eberhardzell, Eberhardzell, Germany), Germany), the the temperature temperature increase increase rates rates of of the the centre centre microtiter microtiter wellwell are are veryvery different.different. This behaviour behaviour can can be be explained explained with with IR IR images images shown shown in inFigures Figures 12, 1213– and14, which14, which were were taken taken after after 60 min 60 ofmin operation. of operation. Figure Fi 12gurea shows 12a shows the UVA the lampUVA withoutlamp without the acrylic the acrylic glass cover.glass cover. Here, allHere, the all individual the individual light sources light sources can be can distinguished, be distinguished, while inwhile Figure in Figure12b the 12b acrylic the acrylic glass coverglass obscurescover obscures all the all details. the details. This suggests This suggests that the that acrylic the acrylic glass blocksglass blocks direct direct IR radiation IR radiation and thus and actsthus as acts a good as a thermalgood thermal insulator. insulator. The details The details in the in left the image left image show thatshow the that hottest the hottest parts areparts the are LED the ◦ drivingLED driving ICs, which ICs, which have a have temperature a temperature of about of 40abouCt above 40 °C roomabove temperature. room temperature. The temperature The temperature of the ◦ LEDsof the has LEDs increased has increased by only about by 14onlyC about since activation.14 °C since Each activation. LED driving Each sub-circuit LED driving consumes sub-circuit much lessconsumes power thanmuch the less LED power itself, than but becausethe LED eachitself, IC but drives because nine LEDs,each IC the drives total dissipatednine LEDs, power the total of eachdissipated IC is approximately power of each 2 toIC 3 is times approximately higher than 2 that to 3 of times each individualhigher than LED, that whichof each partially individual accounts LED, forwhich the higherpartially temperature. accounts for The the other higher reason temperat the LEDsure. are The considerably other reason cooler the isLEDs that theare LEDsconsiderably have a muchcooler better is that thermal the LEDs contact have toa much the heatsink. better thermal The same contact cooling to the solution heatsink. would The besame unpractical cooling solution for the drivingwould ICsbe unpractical because of thefor the large driving number ICs of because electrical of connections.the large number of electrical connections.

Electronics 2019, 8, x FOR PEER REVIEW 13 of 18

Electronics 2019, 8, 343 13 of 18 Electronics 2019, 8, x FOR PEER REVIEW 13 of 18

(a) (b) (c)

Figure 12. Infrared images of the UVA LED lamp after 60 min of operation: (a) top view without the acrylic glass cover; (b) top view with cover; (c) side view with cover.

Figure 12c shows a side view of the UVA LED lamp. The bottom part of the lamp, which acts as a heatsink, is considerably hotter than the acrylic glass cover. This means that a large part of the generated heat is dissipated to the ambient, away from the samples under test. (a) (b) (c) The UVB lamp has a much smaller temperature effect on samples under test than the UVA lamp becauseFigure of the12. Infrareddiffusor, images which of is the basically UVA LED a second lamp after sheet 60 ofmin acrylic of operation: glass, providing( a) top view a without second the layer of thermalacrylic insulation. glass cover; IR (imagesb) top view of th withe UVB cover; lamp (c) sideare viewshown with in cover.Figure 13.

Figure 12c shows a side view of the UVA LED lamp. The bottom part of the lamp, which acts as a heatsink, is considerably hotter than the acrylic glass cover. This means that a large part of the generated heat is dissipated to the ambient, away from the samples under test. The UVB lamp has a much smaller temperature effect on samples under test than the UVA lamp because of the diffusor, which is basically a second sheet of acrylic glass, providing a second layer of thermal insulation. IR images of the UVB lamp are shown in Figure 13.

(a) (b)

Figure 13. Infrared images of the UVB LED lamp after 60 min of operation: ( a) top view with cover; Electronics((b)) sideside2019 view,view 8, x FOR withwith PEER cover.cover. REVIEW 14 of 18

Figure 14 shows IR images of the VL-6LC lamp after 60 min of operation. This lamp is noticeably hotter than the LED lamps. Please note, the images in Figure 14 were taken later in the year when ambient temperatures were higher, which is why their colour scale is offset by 5 °C. Figure 14a shows (a) (b) the lamp from the top and Figure 14b from the side. The left side of the glass window is noticeably hotterFigure than 13.the Infrared right side images of the of windowthe UVB LEDand lampthe rest after of 60the min enclosure. of operation: The (hottesta) top view spot with was cover; measured to be( b53) side °C. viewThe sideswith cover. of this lamp are no hotter than the top of the lamp. In fact, the window seems to be the hottest part of the lamp. The reason is that a fluorescent tube radiates light in all directions equally,Figure and 14 there shows is usuallyIR images a reflec of thetor VL-6LC behind lamp the tube after to 60 reflect min of the operation. light that This is radiated lamp is awaynoticeably from hotterthe target, than backthe LED towards lamps. the Please target. note, Unfortunately, the images insuch Figure reflectors 14 were reflect taken all later the inheat the towards year when the ambientwindow temperaturesand the target were as well. higher, Since(a which) the metal is why parts their of colourthe lamp scale( conductb) is offset heat by quite5 °C. well,Figure all 14a the shows metal theparts lamp of the from lamp the seem top and to have Figure a considerably 14b from the un side.iform The temperature. left side of Thethe glassglass windowwindow, is on noticeably the other Figure 14. InfraredInfrared images images of of the the fluorescent ﬂuorescent tube-b tube-basedased lamp lamp after after 60 60 min min of of operation: operation: ( (aa)) top top view; view; hotterhand, doesthan thenot rightconduct side heat of the very window well, which and the is whresty of the the IR enclosure. images can The reveal hottest which spot of was the measuredtwo tubes (b) side view. tois currentlybe 53 °C. Thein operation. sides of this Because lamp a are large no parthotter of thanthe heat the topis directed of the lamp. towards In fact, the samplesthe window under seems test, tothis be kindMeasurements Figurethe hottest of 12lampc showspart will of of a havethe side lamp.ambient a view greater The of temperature the reasonimpact UVA is on LEDthat sampleinside a lamp. fluorescent temperaturethe The incubator bottom tube radiatesthan part (Figure both of thelight 11)LED-based lamp, in do all which reflectdirections lamps, actsthe differenceequally,aswhich a heatsink, direct and in theremostpower is considerably isof usually consumptionthe heat a towards hotterreflec oftor thanboth the behind theback LED acrylic the of la thetubemps. glasslamp, toHowever, reflect cover. away the Thisfrom the light means fluorescentthe that samples. thatis radiated atube-based large away part of lampfrom the shouldthegenerated target, have heatback had is towards about dissipated the the same to target. the effect ambient, Unfortunately, as the away UVA from lamp.such the reflectorsThis samples discrepancy underreflect test. all can the be heat attributed towards to the largewindowThe heatsink and UVB the lampof targetboth has LED as a well. much lamps, Since smaller which the temperaturemetal each partsweigh of effect1 the kg lampand on samples have conduct a considerable under heat testquite than well,heat the capacity.all UVA the metal lamp As apartsbecause result, of thethe of the lampambient diffusor, seem temperature to which have isa basicallyconsiderably rises slowly a second comparuniform sheeted temperature. to of the acrylic VL-6LC glass, The lamp, providingglass because window, a second it ontakes the layer a other long of timehand,thermal to does heat insulation. not up conductthe heatsink IR images heat which very of the well, in UVB turn which lampheats is areupwh they shown the ambient IR in images Figure air. canThe 13. revealdelayed which heating of the effect two makes tubes theis currently LEDFigure lamps 14in operation.shows much IRbetter images Because suited of a thefor large VL-6LCexperiment part of lamp thes on afterheat temperature 60is directed min of operation.sensitive towards biologicalthe This samples lamp samples. is under noticeably test, thishotter kind than of thelamp LED will lamps. have Pleasea greater note, impact the images on sample in Figure temperature 14 were than taken both later LED-based in the year lamps, when 4.5.whichambient Cell direct Culture temperatures most Tests of the were heat higher, towards which the isback why of their the lamp, colour away scale from is offset the bysamples. 5 ◦C. Figure 14a shows the lampThe uniform from the irradiation top and Figure is supposed 14b from to thegenerate side. Thea comparable left side of biological the glass windowresponse is all noticeably over the seededhotter than microtiter the right plate. side We of the selected window two and cell the lines rest frequently of the enclosure. used in The toxicity hottest bioassays, spot was and measured exposed to them to irradiation, resulting in a roughly 20% reduction of the viability signal. The doses of UVA (50000 J/m2, 966 s) and UVB (1000 J/m2, 666 s) were determined empirically by pretesting (data not shown). The extent of metabolic conversion of PrestoBlue® viability substrate to a fluorescent product was taken as a measure of viability signal proportional to cell number. The microtiter well positions on both lamps and the corresponding irradiance are shown in Figure 15.

(a) (b)

Figure 15. Positioning of the microtiter wells on the UVA (a) and UVB (b) lamp and position- dependent irradiance. The irradiance scale ranges only from 90% to 100%. The white background shows where the irradiance drops below 90%.

The fluorescence viability signal readings from the control plates show high uniformity with only a slight reduction in the border wells when compared to the plate average (Figure 16a,16b). This indicates well-controlled growth conditions in all wells, including the border ones. The plates irradiated with UVA and UVB exhibited a reduced viability signal when compared to the control plate. The viability signal was measured 24 h after irradiation to intensify the effect as shown by Khalila and Shebabya [33]. The overall variability of the viability signal in individual microplate positions in each series of experiments with UV exposure increased compared to both control series. In the UVA irradiated plates, the viability signal decreased over the whole plate. In the Jurkat cells,

Electronics 2019, 8, x FOR PEER REVIEW 14 of 18

Electronics 2019, 8, 343 14 of 18 be 53 ◦C. The sides of this lamp are no hotter than the top of the lamp. In fact, the window seems to be the hottest part of the lamp. The reason is that a fluorescent tube radiates light in all directions equally, and there is usually a reflector behind the tube to reflect the light that is radiated away from the target, back towards the target. Unfortunately, such reflectors reflect all the heat towards the window and the target as well. Since the metal parts of the lamp conduct heat quite well, all the metal parts of the lamp seem to have a considerably uniform temperature. The glass window, on the other hand, does not conduct heat very well, which is why(a) the IR images can reveal which(b) of the two tubes is currently in operation.Figure 14. Because Infrared a largeimages part of the of fluorescent the heat is tube-b directedased towardslamp after the 60 min samples of operation: under test,(a) top this view; kind of lamp(b will) side have view. a greater impact on sample temperature than both LED-based lamps, which direct most of the heat towards the back of the lamp, away from the samples. Measurements ofof the the ambient ambient temperature temperature inside inside the incubator the incubator (Figure (Figure 11) do reflect11) do the reflect difference the differencein power consumption in power consumption of both LED lamps.of both However,LED lamps. the fluorescentHowever, the tube-based fluorescent lamp tube-based should have lamp had shouldabout the have same had effect about as the UVAsame lamp. effect Thisas the discrepancy UVA lamp. can This be attributed discrepancy to the can large be attributed heatsink of to both the largeLED lamps,heatsink which of both each LED weigh lamps, 1 kg which and haveeach aweig considerableh 1 kg and heat have capacity. a considerable As a result, heat capacity. the ambient As atemperature result, the ambient rises slowly temperature compared rises to slowly the VL-6LC compar lamp,ed to because the VL-6LC it takes lamp, a long because time toit takes heat upa long the timeheatsink to heat which up the in turnheatsink heats which up the in ambientturn heats air. up The the ambient delayed air. heating The delayed effect makes heating the effect LED makes lamps themuch LED better lamps suited much for better experiments suited for on experiment temperatures on sensitive temperature biological sensitive samples. biological samples.

4.5. Cell Cell Culture Culture Tests The uniform irradiation is supposed to generate a comparable biological response all over the seeded microtiter plate. We We selected two cell lines frequently used in toxicity bioassays, and exposed them to irradiation, resulting resulting in in a a roughly 20% reduction of of the viability signal. The The doses doses of of UVA UVA 2 2 (50000 J/m2, ,966 966 s) s) and and UVB UVB (1000 (1000 J/m J/m2, ,666 666 s) s) were were determined determined empiri empiricallycally by by pretesting pretesting (data (data not ® shown). The The extent extent of of metabolic metabolic conversion conversion of PrestoBlue® viabilityviability substrate substrate to to a a fluorescent ﬂuorescent product product was taken asas aa measuremeasure ofof viabilityviability signal signal proportional proportional to to cell cell number. number. The The microtiter microtiter well well positions positions on onboth both lamps lamps and and the the corresponding corresponding irradiance irradiance are are shown shown in Figurein Figure 15. 15.

(a) (b)

Figure 15.15. PositioningPositioning of of the the microtiter microtiter wells wells on the on UVA the (UVAa) and (a UVB) and (b )UVB lamp ( andb) lamp position-dependent and position- dependentirradiance. irradiance. The irradiance The scaleirradiance ranges scale only ranges from 90% only to from 100%. 90% The to white 100%. background The white shows background where showsthe irradiance where the drops irradiance below 90%.drops below 90%.

The ﬂuorescencefluorescence viabilityviability signal signal readings readings from from the the control control plates plates show show high high uniformity uniformity with with only onlya slight a slight reduction reduction in the in border the border wells wells when when compared compared to the plateto the average plate average (Figure (Figure 16a,b). 16a,16b). This indicates This indicateswell-controlled well-controlled growth conditions growth conditions in all wells, in including all wells, the including border ones. the Theborder plates ones. irradiated The plates with irradiatedUVA and UVBwith exhibitedUVA and a UVB reduced exhibited viability a reduced signal when viability compared signal to when the control compared plate. to The theviability control plate.signal The was measuredviability signal 24 h after was irradiation measured to24 intensify h after theirradiation effect as to shown intensify by Khalila the effect and Shebabyaas shown [ 33by]. KhalilaThe overall and variabilityShebabya [33]. of the The viability overall signalvariability in individual of the viability microplate signal positions in individual in each microplate series of positionsexperiments in each with series UV exposure of experiments increased with compared UV exposure to both increased control series. compared In the to UVA both irradiated control series. plates, Inthe the viability UVA irradiated signal decreased plates, the over viability the whole signal plate. decreased In the Jurkatover the cells, whole the plat decreasee. In the of theJurkat viability cells, signal was uniform with only a slightly heightened effect in the border wells (Figure 16c). In the adherent HEK-293 cells, the decrease of the viability signal in the border wells was more pronounced Electronics 2019, 8, x FOR PEER REVIEW 15 of 18

the decrease of the viability signal was uniform with only a slightly heightened effect in the border wells (Figure 16c). In the adherent HEK-293 cells, the decrease of the viability signal in the border wells was more pronounced (Figure 16d). In the UVB irradiated plates, the viability signal was also decreased over the whole plate. However, variabilities were higher than in the UVA experiments. In the Jurkat cells, an area of higher viability signal loss was observed in the central part of the mictotiter plate (Figure 16e). The same pattern could be observed in three independent repetitions of the experiment; however, it could not be directly attributed to the measured differences in UVB irradiance. In the HEK-293 cells, a similar, but much less pronounced, position-dependent effect was observed (Figure 16f). The irradiance distributions of the UVA and UVB lamps are quite uniform. Our ambition was to demonstrate a similar uniformity of a physiological response in correspondence with the irradiance. We obtained a uniform reduction in the viability signal after irradiation with the UVA lamp in the Jurkat cell line (Figure 16c) and after irradiation with the UVB lamp in the HEK-293 cell line (Figure 16 f), but a slightly higher difference between the centre and the periphery of the plates in the other two cases (Figure 16d,16e). We reason that these irregularities cannot be attributed to a lack of irradiation uniformity, as such differences would most likely be reflected in both cell lines for each lamp. The difference could be partly explained with the increased overall variability in irradiated plates and might partly be related to other, not strictly controlled Electronicsconditions2019 , like8, 343 temperature. Jurkat cells are known for their sensitivity to temperature and their15 of 18 ability to show pro-apoptotic signalling after an increase in temperature to 42 °C and above [34]. Our temperature measurements showed a slightly higher temperature increase in the central wells (Figure 16d). In the UVB irradiated plates, the viability signal was also decreased over the whole plate. compared to the peripheral wells during UV irradiation, which could potentially influence the However, variabilities were higher than in the UVA experiments. In the Jurkat cells, an area of higher results. The temperature increase was not high enough to reduce the viability signal on its own. viabilityAccording signal to lossthe temperature was observed measurements in the central inside part ofmicrotiter the mictotiter wells plateshown (Figure in Figure 16e). 11a, The the same patterntemperature could beof the observed cells illuminated in three independent with the UVA repetitions lamp did ofnot the increase experiment; by more however, than 2.5 it°C could in the not be966 directly s of illumination attributed and to the the measured temperature differences of the cells in illuminated UVB irradiance. with the In UVB theHEK-293 lamp did cells,not increase a similar, butby muchmore than less 1 pronounced, °C in 666 s of position-dependent illumination. A synergistic effect effect was observedbetween the (Figure UV illumination 16f). The irradianceand the distributionstemperature of or the any UVA other and unforeseen UVB lamps influence are quite is, however, uniform. possible.

(a) (b)

Electronics 2019, 8, x FOR PEER REVIEW 16 of 18 (c) (d)

(e) (f)

FigureFigure 16. 16.Position-dependent Position-dependent viabilityviability signalssignals in a 96-well microtiter microtiter plate: plate: (a ()a Jurkat) Jurkat control control signal; signal; (b)(b HEK-293) HEK-293 control control signal; signal; ( c(c)) Jurkat Jurkat irradiatedirradiated by by UVA UVA lamp; lamp; (d (d) )HEK-293 HEK-293 irradiated irradiated byby UVA UVA lamp; lamp; (e)( Jurkate) Jurkat irradiated irradiated by by UVB UVB lamp; lamp; ( f()f) HEK-293 HEK-293 irradiatedirradiated by UVB UVB lamp. lamp.

5. ConclusionsOur ambition was to demonstrate a similar uniformity of a physiological response in correspondenceUVA and withUVB theLED-based irradiance. lamps We were obtained designed, a uniform built reductionand tested. in The the viabilitymeasured signal average after irradiationirradiance with at a distance the UVA of lamp 30 mm in theabove Jurkat the LEDs cell line produced (Figure by 16 thec) andUVA after lamp irradiation was 70.1 W/m with2, thewith UVB a lampmaximum in the HEK-293deviation cellof 4.9% line (Figureover an 16areaf), of but 148 a slightlymm × 66 higher mm. For difference the UVB between lamp, an the additional centre and thediffusor periphery was ofrequired the plates to achieve in the othersatisfactory two cases resu (Figurelts, due 16to d,e).the more We reasoncomplex that radiation these irregularities pattern of cannotUVB LEDs. be attributed The final to average a lack irradiance of irradiation produced uniformity, by the UVB as such lamp differences was 3.1 W/m would2 with mosta maximum likely be reﬂecteddeviation in bothof 4.8% cell over lines an for area each of lamp. 120 mm The × difference 60 mm. The could irradiance be partly homogeneities explained with of both the increased lamps overallcompare variability favourably in irradiatedto the stated plates inhomogeneity and might of partly ±7% of be the related Vilber to Bio-Sun other, notUV strictlyilluminator controlled [26]. conditionsAn added like bonus temperature. compared to Jurkat the Vilber cells areBio-Sun known is the for possibility their sensitivity of using to these temperature LED lamps and for their abilitybottom-up to show or pro-apoptotictop-down illumination, signalling which after ancan increase make it in easier temperature to deliver to 42the◦ Cexact and aboverequired [34 ]. Ourirradiation temperature dose measurementsto the samples. showed a slightly higher temperature increase in the central wells The mechanical design for the effective thermal management of LED-based lamps indeed proved to be effective, since the influence of the LED-based lamps on sample temperatures in an incubator was much lower than the influence of the fluorescent tube-based handheld lamp, despite all three lamps having had almost identical electrical power consumption. The UVA LED lamp was able to illuminate samples for 40 min, the UVB LED lamp for 67 min and the fluorescent tube-based lamp for only 20 min, before the temperature of the samples increased by more than 5 °C. In experiments with real cell cultures the illumination doses were tailored to generate a suitable physiological response of the tested cell cultures, which required significantly shorter illumination times of only 966 s and 666 s for the UVA and UVB lamp, respectively. During these illumination periods, the temperature effects were on a much more acceptable level of less than 2.5 °C and 1 °C for the UVA and UVB lamps, respectively. The tests on cell cultures confirmed the general suitability of the design for research purposes with regard to ease of use, installation into an incubator, the correct positioning of samples under test and so forth. The test results also confirmed uniformity of physiological responses to irradiation with both lamps in general. Slight border to centre differences in viability signals did occur in the HEK- 293 cell culture irradiated by the UVA lamp and the Jurkat cell culture irradiated by the UVB lamp. There could be many reasons for these deviations, including an inhomogeneity of the lamps. However, since the results for the HEK-293 cell culture irradiated by the UVB lamp and the Jurkat cell culture irradiated by the UVA lamp exhibit very good uniformity, it can be concluded that both lamps produce sufficiently homogenous irradiance. The presented solution is a careful balance between the desired specifications, cost, and simplicity of installation and use. Only one flat cable connection is required, which does not prevent closure of the incubator door. Should additional or more stringent requirements arise it would still be possible to improve the performance of the lamps: homogeneity could be improved with even more LEDs or an additional diffusor, heat dissipation within the measurement chamber could be further reduced by relocating the LED driving ICs to the control module at the expense of having more wires connecting the LED and the control module, etc.

Electronics 2019, 8, 343 16 of 18 compared to the peripheral wells during UV irradiation, which could potentially inﬂuence the results. The temperature increase was not high enough to reduce the viability signal on its own. According to the temperature measurements inside microtiter wells shown in Figure 11a, the temperature of the cells illuminated with the UVA lamp did not increase by more than 2.5 ◦C in the 966 s of illumination and the temperature of the cells illuminated with the UVB lamp did not increase by more than 1 ◦C in 666 s of illumination. A synergistic effect between the UV illumination and the temperature or any other unforeseen inﬂuence is, however, possible.

5. Conclusions UVA and UVB LED-based lamps were designed, built and tested. The measured average irradiance at a distance of 30 mm above the LEDs produced by the UVA lamp was 70.1 W/m2, with a maximum deviation of 4.9% over an area of 148 mm × 66 mm. For the UVB lamp, an additional diffusor was required to achieve satisfactory results, due to the more complex radiation pattern of UVB LEDs. The final average irradiance produced by the UVB lamp was 3.1 W/m2 with a maximum deviation of 4.8% over an area of 120 mm × 60 mm. The irradiance homogeneities of both lamps compare favourably to the stated inhomogeneity of ±7% of the Vilber Bio-Sun UV illuminator [26]. An added bonus compared to the Vilber Bio-Sun is the possibility of using these LED lamps for bottom-up or top-down illumination, which can make it easier to deliver the exact required irradiation dose to the samples. The mechanical design for the effective thermal management of LED-based lamps indeed proved to be effective, since the influence of the LED-based lamps on sample temperatures in an incubator was much lower than the influence of the fluorescent tube-based handheld lamp, despite all three lamps having had almost identical electrical power consumption. The UVA LED lamp was able to illuminate samples for 40 min, the UVB LED lamp for 67 min and the fluorescent tube-based lamp for only 20 min, before the temperature of the samples increased by more than 5 ◦C. In experiments with real cell cultures the illumination doses were tailored to generate a suitable physiological response of the tested cell cultures, which required significantly shorter illumination times of only 966 s and 666 s for the UVA and UVB lamp, respectively. During these illumination periods, the temperature effects were on a much more acceptable level of less than 2.5 ◦C and 1 ◦C for the UVA and UVB lamps, respectively. The tests on cell cultures confirmed the general suitability of the design for research purposes with regard to ease of use, installation into an incubator, the correct positioning of samples under test and so forth. The test results also confirmed uniformity of physiological responses to irradiation with both lamps in general. Slight border to centre differences in viability signals did occur in the HEK-293 cell culture irradiated by the UVA lamp and the Jurkat cell culture irradiated by the UVB lamp. There could be many reasons for these deviations, including an inhomogeneity of the lamps. However, since the results for the HEK-293 cell culture irradiated by the UVB lamp and the Jurkat cell culture irradiated by the UVA lamp exhibit very good uniformity, it can be concluded that both lamps produce sufficiently homogenous irradiance. The presented solution is a careful balance between the desired specifications, cost, and simplicity of installation and use. Only one flat cable connection is required, which does not prevent closure of the incubator door. Should additional or more stringent requirements arise it would still be possible to improve the performance of the lamps: homogeneity could be improved with even more LEDs or an additional diffusor, heat dissipation within the measurement chamber could be further reduced by relocating the LED driving ICs to the control module at the expense of having more wires connecting the LED and the control module, etc.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-9292/8/3/343/s1, Electronic schematics, Source code, Android application. Author Contributions: Conceptualisation, P.F. and M.P.; methodology, M.P.; software, M.P.; validation, S.C. and M.P.; formal analysis, S.C. and M.P.; resources, M.T. and S.C.; writing—original draft preparation, M.P., S.C. and P.F.; writing—review and editing, M.T.; visualization, M.P.; project administration, M.T.; funding acquisition, M.T. Electronics 2019, 8, 343 17 of 18

Funding: This research was funded by the Slovenian Research Agency ARRS, grant number P2-0197. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

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