Design and validation of an open-source modular Microplate Photoirradiation System for high-throughput photobiology experiments

Abstract

Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle-but not frequency-in a pulsed irradiance regimen.

Fig 1. Components of the MPS.

Photograph of the MPS, featuring the main device comprising an acrylic base mounting the LED Control Board and LED Platform using aluminum standoffs. The system is powered by standard ATX power supply/ATX breakout board through a pluggable screw terminal with flat cable.

Fig 2. LED control board.

Photograph of the MPS LED Control Board, with key components and connectors identified. Only solder points are visible for labeled header sockets as the sockets are on the other side of the board.

Fig 3. Photographs of the 24-well and 96-well LED platforms and boards.

24-well LED platform with (A) and without (B) isolation plate and 96-well LED platform with (C) and without (D) isolation plate.

Fig 4. Screenshots of MPS software GUIs and MPS functional diagram.

(A) Screenshot of HTML5-based GUI for generating light program JSON files to be uploaded to MPS (B) Screenshot of MPS Control GUI, which is loaded from the ESP8266 Wi-Fi module webserver and allows user to control MPS and upload and run light programs (C) Functional block diagram of the MPS software components, with all major interconnections between software and hardware components shown.

Fig 5. Biological validation of the Microplate Photoirradiation System (MPS) and a microplate fluorescence assay to detect ALA-induced PpIX photobleaching in live adherent wildtype (WT) fibroblasts.

(A) A diagram of the heme biosynthesis pathway and PpIX accumulation in an in vitro model of exogenous ALA addition. Abbreviations: ALA = Aminolevulinic acid, PpIX = Protoporphyrin IX, PBG = Porphobilinogen, HMB = Hydroxymethylbilane, Uro’gen = Uroporphyrinogen, Copro’gen = Coproporphyrinogen, Proto’gen = Protoporphyrinogen, Ferroch = Ferrochelatase (B) Biophysical rationale for photobleaching of PpIX, a type II photosensitizer. (C) Experimental validation of PpIX accumulation and photobleaching in 24 and 96-well microplates irradiated with their respective MPS LED boards at fixed duration of 20 minutes and increasing irradiances of 0.75, 1.5, 3, 4.5, and 6 mW/cm2 (resulting fluence values of 0.9, 1.8, 3.6, 5.4, and 7.2 J/cm², respectively). Experimental validation of the photobleaching effect at 6h (D), 24h (E) and 36h (F) ALA incubations using 96-well LED board and microplate to treat cells with 405nm light at a fixed irradiance of 1.5mW/cm² with increasing durations of 150, 300, 600, and 1200 seconds (resulting in fluence values of 0.1, 0.2, 0.5, 0.9, and 1.8 J/cm², respectively). (E) WT fibroblasts treated with a fixed irradiance of 4.5mW/cm2² and fixed fluence of 1.35 J/cm², while varying duty-cycle (25%, 33.3%, 50%, or 100%) and frequencies (0.5, 1, 3.33, 5, 10, 25 Hz). All conditions include data from 3 or more independent experiments (microplate batches) with 4 or more wells per condition in 96-well plates and 3 or more wells per condition in the 24-well plate (before outlier removal). Visual layout of the microplate and the plate configuration for the experimental parameters used in the light treatment, as well as plate ID’s of the microplate batches used in the experiments, are outlined in S3 File. Details regarding the number of samples used in each experimental condition, the means and SDs for each condition used to plot the data on the graphs, and the respective microplate batches utilized for each graph, are presented in S1 Data.

Fig 6. Photobleaching of PpIX in live adherent wildtype fibroblasts as a function of 405nm radiant exposure.

(A, B, C) Comparison of the PpIX photobleaching kinetics as a function of fluence for each irradiance condition, for the 6h, 24h, and 36h ALA Incubation time-points. (D) Biexponential decay model fitted to PpIX photobleaching, represented as percent loss in fluorescence from initial value as function of delivered fluence (points = raw data, lines = fitted model) for 6, 24 and 36h ALA incubations as well as the model fitted to the combined data. (E) Coefficients for biexponential fit of data in the previous graph, according to the equation described in the text. The “half-fluence”, denoted as hf, is a transformation of the exponential decay constant to represent the fluence required to reduce fluorescence by half (analog of half-life). All conditions include data from 3 or more independent experiments (microplate batches) with 4 or more wells per condition in 96-well plates (before outlier removal). Visual layout of the microplate and the plate configuration for the experimental parameters used in the light treatment, as well as plate ID’s of the microplate batches used in the experiments, are outlined in S3 File. Details regarding the number of samples used in each experimental condition, the means and SDs for each condition used to plot the data on the graphs, and and the respective microplate batches utilized for each graph, are presented in S1 Data.