New planar light source for the induction and monitoring of photodynamic processes in vitro

Abstract

We recently developed a new light source that allows for the continuous monitoring of light-induced changes using common spectrophotometric devices adapted for microplate analyses. This source was designed primarily to induce photodynamic processes in cell models. Modern light components, such as LED chips, were used to improve the irradiance homogeneity. In addition, this source forms a small hermetic chamber and thus allows for the regulation of the surrounding atmosphere, which plays a significant role in these light-dependent reactions. The efficacy of the new light source was proven via kinetic measurements of reactive oxygen species generated during the photodynamic reaction of chloroaluminium phthalocyanine disulfonate (ClAlPcS₂) in three cell lines: human melanoma cells (G361), human breast adenocarcinoma cells (MCF7), and human fibroblasts (BJ).

Introduction

Many workplaces collect their measurements in spectrophotometric microplate readers because they allow for the rapid analysis of hundreds of samples. The significant disadvantage of these instruments is the use of a dim and broad excitation light source. This type of light source contrasts with the need for a variety of fluorescent probes. When these probes are used for the continuous monitoring of light-induced changes, these devices must be equipped with another light source. Photodynamic therapy (PDT) is an example of studying the effect of light on biological systems. This type of therapy represents a combined effect of four components: a photosensitive compound (sensitizer), molecular oxygen, light, and a substrate (most often biological tissue) [1]. The resulting products are different forms of reactive oxygen, an oxidized substrate, and a regenerated sensitizer. This approach finds its main use in photodynamic therapy, in which sensitizers are preferably trapped in pathologically changed cells. After the irradiation of cells with visible radiation, for which the wavelength is identical to the absorption maximum of the sensitizer, the subsequent generation of singlet oxygen and other radicals causes the destruction of the cells. The lifetime of these products in the cellular environment is very short (10⁻⁵–10⁻⁷ s) because they react very rapidly with surrounding biomolecules [2]. The significance of these reactions depends on the molecular structure and function of the reactants. The extent of damage can trigger a series of cellular processes that lead to cell death [3]. The sensitizer is non-toxic without light activation and has a characteristic fluorescence that is also used for diagnostics. It is possible to observe the induced damage of many cell processes via various light-activated probes and the detection of fluorescence by means of a spectrophotometric reader.

Efficacy studies of this type of phototherapy use various tumor and non-tumor cell lines, which are usually cultivated in Petri dishes or assay microplates. Lasers or light-emitting diodes are most often used as the light source for sensitizer activation. The efficacy and the toxicity associated with PDT are significantly linked to the quantity of light delivered/absorbed by sensitizer-bearing tissues. Thus, for PDT to be effective, the light delivery must be homogeneous and sufficient throughout the target tissue volume [4]. To form a wider irradiation area, an optic diffuser is often placed between a light source and the object. For light that originates from LED diodes, the irradiation field is formed with different arrangements of several LED diodes that generate different levels of light flow homogeneity for applications both in vivo [5–9] and in vitro [10–14]. The arrangement of diodes in irradiators is either random or in rows with the same or different mutual distances. In addition, several LED devices designed for phototherapy have already been patented. However, few of these devices addressed the issue of homogeneity in the light field. For example, in patent device CZ302829 [15], the LED diodes are arranged in a hexagonal shape at a constant distance from each other and are bonded to a pad above a bed. A utility model described in the document CN203247266 [16] shows that homogeneity is achieved via the movement of a plate with bonded LED elements and via diffusion on supplementary diffusing glass. In addition, it has been also reported that light sources made from woven light-emitting fabrics can be characterized by a higher fluence rate, a high homogeneity of light delivery, and good flexibility [17].

Sample analysis is conducted via an instrument in which the signal detection unit is often placed above or below the sample. However, this arrangement only allows the researcher to measure the final effect of the photodynamic therapy (e.g., to determine the total amount of oxidized compound after irradiation). In this case, a cell permeable, chemical marker is the key probe that becomes fluorescent after oxidation. This signal can be measured with commercial spectrofluorometers or plate readers. It is best to perform the measurement continuously because of the fast photophysical-chemical changes that are connected to the formation of ROS and the autophotooxidation of the detection marker itself. The device described in patent document CZ302084 [18] provides one type of solution to the problem of continuous monitoring. The device was designed for relatively small samples for which the irradiation is delivered from the side. However, whether this design achieves the same homogeneity as the direct irradiation approach described above remains unclear.

The aim of this paper is to introduce a planar irradiation source that is structurally simple and provides light with a relatively uniform flow. The device should also enable the continuous monitoring of the products formed during PDT via commercially accessible microplate spectrofluorometers or readers. This irradiation source should allow the user to change the composition and pressure of the atmosphere surrounding the analyzed sample.

Materials and methods

Planar light source

A source with homogeneous irradiance was constructed primarily for the induction and monitoring of photodynamic effects in a commercially standardized analytical 40 mm Petri dish. The Petri dish containing a sample (e.g., cells) is placed in a bed formed with a sealable body that is procured from above via an opened central chamber and a removable semitransparent thin-walled irradiation glass plate. This plate is procured with LED chips that are placed in rows with a mutual hexagonal arrangement, and the LED chips are parallel and connected with a bus that follows the perimeter of the irradiation plate and connects to an external power supply. The LED chips are placed in the irradiation plate such that their light beams are directed straight down toward the bed (sample) (Fig. 1). In addition, the body is procured with a lid on whose inner face is a central groove whose shape and size corresponds to the shape and size of the upper frame of the central chamber of the body. On the upper frame is a peripheral seal that enables the hermetical closure of the central chamber. Furthermore, the body is equipped with two built-in sealable passages that are led into the central chamber from the side or from the front to fill the chamber with a gas. There is also a side port that enables the connection of electric conductors that extend from the bus of the irradiation plate to a power supply. In an optimal design, the central groove of the lid and the bottom of the central chamber are procured with transparent windows that enable the monitoring of processes inside the central chamber.

Fig. 1.

Axonometric view of the new planar transparent light source in the assembled (A) and exploded (B) states. Because of the small size of the entire system, it is possible to analyze various photodynamic processes through the optically transparent windows using commonly available, commercial microplate analyzers. The closing valves that exchange the sample atmosphere and the power connector for the LED chip plates are located on the side-wall of the source. The irradiation plates are bonded with diode chips and are easily removable. It is possible to push the plates in/out and exchange them for plates with different irradiation characteristics

The chip arrangement was chosen to achieve maximum uniformity of the light field and to ensure that the effect of partial shading was relatively independent of the sample location within the Petri dish. The individual LED chips are thereby located in a hexagonal arrangement (Fig. 2A). The surface density of the chips directly determines the degree of homogeneity and transparency of the light source. More homogeneity results in less transparency and vice versa. When studying photodynamic processes, the issue of homogeneity is more important. The efficacy and functionality of the planar irradiation source has been tested for contemporary, commercially produced highly illuminative C4L-H12T5 LED chips (Chips 4 Light GmbH, Germany), which have a size of 0.313 × 0.313 mm. The irradiation performance of one LED chip is up to 50 mW, and the emission wavelength of radiation is in the range of 650–672.5 nm. For a field arrangement with 195 chips, we obtained a homogeneous irradiated surface area at the bottom of Petri dish with an inner diameter of approximately 34 mm. To achieve higher homogeneity, it would be optimal to increase the number of chips. For example, if we consider this type of LED chip and neglect electric connections, then the number of chips can be increased to 9267 chips for a surface area with a diameter of 34 mm (π × 17²/0.313²) in order to maintain at least 50% permeability for the chip plate.

Fig. 2.

Top view of the LED chip plate (A) and conventional plate made of 5 mm LEDs (B)

Measurement of irradiance homogeneity

The irradiance homogeneity of the new transparent planar light source was determined using the IL 1705 radiometer system equipped with an SED033 sensor (International Light Technologies, USA) covered by a shield with a 1 mm pinhole aperture in the center. The sensor was attached to a mechanical microscope stage, allowing for fine shifts in the longitudinal and lateral directions. The irradiance was measured 12 mm above the LED chip plate, which corresponds to the real distance between the sample (bottom of the Petri dish) and the light source. The data reading was performed at a shift of 0.5 mm in both the longitudinal and lateral directions. For a comparative study, this investigation was completed with irradiance measurements of a red light source made of conventional 5 mm rounded LEDs (L53SRC-F, Kingbright Electronic, Taiwan) (Fig. 2B). The electronically stabilized Volfcraft VLP-2403 Pro power supply (Conrad Electronic SE, Germany) was used as a power supply.

Determination of heating properties

Four milliliters of PBS-G were exposed to the light source made of 195 C4L-H12T5 LED chips. The current values were set to 5 and 10 mA per chip. The resulting temperature changes in the buffer were monitored with a Voltcraft PL-125-T2USB thermometer (Conrad Electronic SE, Germany) equipped with a 1.5 mm K type thermoelement (Greisinger Electronic, Germany), which was immersed in the buffer. The maximal temperature changes inside the LED chip plate at a constant current value of 5 mA per LED chip were monitored simultaneously with an infrared FLIR E4 camera (FLIR Systems, USA).

Cell lines, sensitizer and irradiation conditions

Human melanoma (G361) cells, human breast adenocarcinoma cells (MCF7), and human fibroblasts (BJ) (1 × 10⁵ per 40 mm Petri dish) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 0 (control) and 10 μM ClAlPcS₂ and incubated in an incubator at 37 °C with 5% CO₂ for 24 h. After incubation and before measurement, the cells were washed three times with PBS buffer containing 5 mM glucose (PBS-G). The photodynamic treatment was performed inside the sealed planar light source equipped with 195 LED chips at 19 mW.cm⁻² (5 mA per chip) for 5 min.

Estimation of the reactive oxygen species

The products of the type I photodynamic reaction (i.e., hydrogen peroxide and its downstream products) [1] were determined by using the fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) (Molecular Probes by Life Technologies) [19]. Upon crossing the membrane, the compound undergoes deacetylation by intracellular esterases to produce the non-fluorescent product CM-H₂DCF, which reacts quantitatively with the oxygen species inside the cell to produce the highly fluorescent dye CM-DCF. Another fluorescence probe – the singlet oxygen sensor green reagent (SOSG, Molecular Probes by Life Technologies) was used to estimate the type II photodynamic reaction. This probe is connected to the production of singlet oxygen [1]. After washing the cells, 2 ml of fresh PBS-G supplemented with 10 μM CM-H₂DCFDA or 2 μM SOSG were added to the cells and incubated for 20 min in the dark. The formation of the fluorescent products during PDT was monitored continuously inside the transparent planar light source by recording the fluorescence intensities of the products directly with a Tecan Infinite 200pro reader (Tecan Group, Switzerland). The excitation wavelengths for CM-DCF and SOSG were 480 nm and 500 nm, respectively. The emission wavelength was set to 530 nm for both probes.

Data analysis

The presented data represent the means ± standard errors for 3 independent experiments. The estimation of ROS with the Tecan Infinite 200pro microplate reader was achieved by reading a 96-well microplate template in which only 4 wells in the center of each Petri dish were considered (i.e., approximately 15% of the total sample area).

Results

Measurement of irradiance homogeneity

To evaluate the irradiance homogeneity of the new planar light source, we examined the intensities of the light falling on the 1 mm diameter surface at a distance of 12 mm. These values were compared with those obtained from a light source formed by a dense field of 5 mm LED diodes (Fig. 3). These measurements showed that the light source made of LED chips provided higher intensity values when the same electric current values passed through the given light element (Fig. 3, right graphs). The last two data points for the LED chip source, which are not connected to an increase in light intensity, are caused by a current limiter. This limiter was added to each parallel branch of the electrical circuit. However, the main feature of the LED chip source in comparison to the conventional diode source is the homogeneity of the surface irradiance. The difference between the lowest and highest irradiated spot was less than 9% for the LED chip source but reached almost 250% for the LED diode source.

Fig. 3.

Surface distribution of the light intensities for the new planar LED chip source (A) and the conventional light source made of 5 mm LEDs (B) at a 12 mm distance (left graphs). The right graphs plot the dependence of the light intensity averaged over the whole surface of the SED033 sensor (approximately 25 mm in diameter) on the electric current passing through one light element

Determination of heating properties

Although LEDs are cold light sources, we studied the effect of the new light source on the temperature changes of a PBS solution placed in the sample bed. We found an increase in temperature of ~ 5.5 °C or 12 °C with continuous irradiation and a constant current of 5 or 10 mA, respectively (Fig. 4A). These changes likely occur because the light elements are warmed by the electric currents, although this response proceeded markedly faster (Fig. 4B). However, the main result related to these temperature measurements is that the irradiance of the new light source did not reflect the enormous and rapid increase of the inner temperature and remained relatively constant (Fig. 4B).

Fig. 4.

Heating properties of the new planar LED chip source. This source induced time-dependent temperature changes in 4 ml of PBS buffer placed in the sample bed with a continuous power supply at constant electric currents of 5 (A, filled circles) and 10 mA (A, open circles) per chip (A). Measurements of the maximal temperature changes inside the new planar light source body (B, filled circles) and its irradiance at a distance of 12 mm (B, open circles) during continuous connection to a constant electric current of 5 mA are shown in the graph on the right (B)

Estimation of the reactive oxygen species

The photodynamic effect of the sensitizer ClAlPcS₂ on ROS formation in the MCF7, G361 and BJ cell lines was monitored continuously during 5 min of irradiance by the new planar LED chip source. The production of H₂O₂, ¹O₂ and their downstream products was determined using CM-H₂DCFDA and SOSG, respectively (Fig. 5). The results of three independent measurements revealed statistically significant differences in ROS production between the control samples (irradiated cells in the absence of the sensitizer or non-irradiated cells in the presence of the sensitizer) and photodynamically treated cells.

Fig. 5.

Production of ROS in the MCF7 (A), G361 (B), and BJ (C) cell lines during PDT. The formation of hydrogen peroxide and its downstream products is expressed as an increase in the CM-DCF fluorescence intensity (left graphs, filled diamonds). A similar increase in the SOSG fluorescence intensity reflects the formation of singlet oxygen (right graphs, filled diamonds). Negligible changes are observed in control cell lines (i.e., in irradiated cells without sensitizer (open circles) and in non-irradiated cells with accumulated sensitizer (filled squares))

Conclusions

During the past 40 years, dramatic developments have occurred in the field of semiconductor light sources. These light sources are currently available in wavelengths that cover the entire visible region, as well as the near ultraviolet (UV) and near infrared (NIR) regions [20]. The main utility of such light sources is in photochemical and biophysical investigations, as well as in new bioanalytical instruments.

With the use of modern spot lighting elements (LED chips), we have developed a new transparent flat light source as an add-on for microplate readers. We determined the planar irradiation homogeneity of this light source. We successfully verified the utility of this new light source in a PDT study and we examined the kinetics of ROS generation in different cell lines. The cells were continuously exposed to light in the presence of ClAlPcS₂. A removable semitransparent thin-walled irradiation glass plate can be very easily replaced by another with different radiation characteristics and used with other photosensitive substances. Moreover, the analyzed area and the irradiation plate are placed inside of the source body, which can be hermetically sealed and filled with gas. This approach can modify the composition of the surrounding atmosphere and thereby affect photodynamic processes.

These processes also depend on the irradiation dose; therefore, the homogeneity of the emitted light is critical. We found excellent homogeneity for the light source made of LED chips. The difference in the irradiation dose is less than 9%, whereas for a conventional planar source consisting of 5 mm LEDs, this difference reaches more than 250%, even if the diodes are tightly arranged.

Pieslinger and coworkers [21] previously published data showing significantly higher homogeneity for a similar LED source (4.040 ± 0.292 mW.cm⁻²); however, this result was obtained when the authors investigated homogeneity using a very broad evaluation pattern (1.18 × 1.18 cm; in comparison to a circle of 1 mm in diameter in our system). Other authors [22] reported another LED source that was composed of 10 × 7 LEDs and designed primarily for PDT studies in microplates. The heterogeneity of this source was determined to be 15%. However, similar to the previously mentioned case, this parameter was determined with a 1.9 cm diameter circular sensor. Thus, the obtained results may not accurately reflect the heterogeneity of that light system. Obviously, when microplates with more wells are used, the irradiance heterogeneity among the individual wells may be significantly higher.

The second important feature of a light source is potential heating, especially if long-term processes are studied. When 4 ml of a salt solution were irradiated by our new red LED chip source at a continuous irradiance of 19 mW.cm⁻², the temperature increased by 0.37 °C.min⁻¹. This relatively higher increment likely occurred because our new light source forms a small and closed system with a short distance from the solution (sample) to the LED chip plate. Although this light source was designed primarily to induce photodynamic processes in cell models, we believe that it may also be useful for studying the effects of light in other fields of research.

Aknowledgments

This work was supported by the grant from European Regional Development Fund CZ.02.1.01/0.0/0.0/16_019/0000868. We would also like to thank American manuscript editors (http://americanmanuscripteditors.com) for proofreading our manuscript.

Abbreviations

¹O₂

Singlet oxygen

BJ

Human fibroblast cell line

ClAlPcS₂

Chloraluminium phtalocyanine disulfonate

CM-H₂DCFDA

5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate

DMEM

Dullbecco’s modified Eagle’s medium

G361

Human melanoma cell line

LED

Light emitting diode

MCF7

Human breast adenocarcinoma cell line

MMP

Mitochondrial membrane potential

MTT, PBS

Phosphate-buffered saline

PBS-G

PBS supplemented with 5 mM glucose

PDT

Photodynamic therapy

ROS

Reactive oxygen species

SOSG

Singlet oxygen sensor green reagent

Compliance with ethical standards

Conflict of interest

The authors declare that they have are no conflicts of interest.