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.
Highlights
Advances in photobiology, especially in the fields of optogenetics and photomedicine, have widened interest in studying the effects of isolated spectra of light on cells in a controlled environment
The type II reaction—in part due to the low energy gap between this tetrapyrrole and molecular oxygen and in part due to the short diffusion distance of the cytotoxic singlet oxygen [47]—is the reason why Protoporphyrin IX (PpIX) photosensitization can be utilized in the destruction of malignant and aberrant tissue types with aminolevulinic acid (ALA)-based photodynamic therapy (PDT)
We demonstrate that our assay can effectively detect the percentage of PpIX photobleaching occuring in WT fibroblasts incubated with ALA and that this assay can be done comparably in either the 24 or 96-well microplate formats
Summary
Especially in the fields of optogenetics and photomedicine, have widened interest in studying the effects of isolated spectra of light on cells in a controlled environment. Available lamps, laser, LED, and microscope systems used as irradiation sources in these experiments are typically not designed for in vitro cell culture format They are often expensive, cumbersome to use, have fixed spectral components, and lack the ability to irradiate individual cell culture wells in standard microplates with independently controllable timing and irradiance. Several groups have attempted to address some of these issues by developing custom LED arrays for irradiating cells in multiwell plates [1,2,3], while more recent attempts have resulted in systems capable of managing individual or grouped wells through a computer interface [4,5,6,7] None of these systems contain all the key features listed below necessary for a truly highthroughput, reproducible and highly-configurable photoirradiation system suitable for a wide range of photobiology experiments in standard microplates:
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