Study and optimization of the photobiomodulation effects induced on mitochondrial metabolic activity of human cardiomyocytes for different radiometric and spectral conditions.

Photobiomodulation (PBM) represents a promising and powerful approach for non-invasive therapeutic interventions. This emerging field of research has gained a considerable attention due to its potential for multiple disciplines, including medicine, neuroscience, and sports medicine. While PBM has shown the ability to stimulate various cellular processes in numerous medical applications, the fine-tuning of treatment parameters, such as wavelength, irradiance, treatment duration, and illumination geometry, remains an ongoing challenge. Furthermore, additional research is necessary to unveil the specific mechanisms of action and establish standardized protocols for diverse clinical applications. Given the widely accepted understanding that mitochondria play a pivotal role in the PBM mechanisms, our study delves into a multitude of PBM illumination parameters while assessing the PBM's effects on the basis of endpoints reflecting the mitochondrial metabolism of human cardiac myocytes (HCM), that are known for their high mitochondrial density. These endpoints include: i) the endogenous production of protoporphyrin IX (PpIX), ii) changes in mitochondrial potential monitored by Rhodamine 123 (Rhod 123), iii) changes in the HCM's oxygen consumption, iv) the fluorescence lifetime of Rhod 123 in mitochondria, and v) alterations of the mitochondrial morphology. The good correlation observed between these different methods to assess PBM effects underscores that monitoring the endogenous PpIX production offers interesting indirect insights into the mitochondrial metabolic activity. This conclusion is important since many approved therapeutics and cancer detection approaches are based on the use of PpIX. Finally, this correlation strongly suggests that the PBM effects mentioned above have a common "fundamental" mechanistic origin.

Identification of excimer delayed fluorescence by Protoporphyrin IX: A novel access to local chromophore concentration?

Protoporphyrin IX (PpIX) is a molecule produced in the mitochondria following the administration of its approved precursor, aminolevulinic acid (ALA). Strong light absorber at different wavelengths in the visible range, PpIX is extensively used as a photosensitizer (PS) for Photodynamic Therapy (PDT). PpIX is also an ideal molecular probe for the quantification of the tissue oxygen partial pressure (pO2), as its delayed fluorescence (DF) is quenched by oxygen, creating a direct relationship between the DF lifetime and the pO2. A limitation of both techniques is the ignorance of the PpIX concentration in tissues when the pO2 is measured or during PDT. In this study, the prompt (PF) and delayed fluorescence of PpIX dissolved in DiMethylFormamide (DMF) were acquired, in absence of oxygen, at different PpIX concentrations. Measurements of the PpIX emission for different excitation energies and temperatures, as well as spectral considerations led to the conclusion that E-type (thermal) DF was the dominant DF mechanism at low PpIX excited states concentrations (density of absorbed energy Hε[PpIX] < 1 µJ. cm-3, H:excitation radiant exposure per pulse, ε: molar extinction coefficient at excitation wavelength) while P-type (Triplet Triplet Annihilation) DF took place at higher excited states concentrations (Hε[PpIX] > 10 µJ. cm-3). The gradual development of a strong, red-shifted structureless DF peak at 670 nm, invisible in the PF and absorption spectra, strongly points towards the first observation of PpIX excimer DF (EDF). It appears that, similarly to other aromatic molecules, PpIX excimers can be formed either by the encounter of two molecules in the first excited triplet state T1, or by the reaction of an excited singlet S1 with a triplet T1. Excimer DF could be beneficially used to determine the local concentration of PpIX, as the initial DF intensity ratio I0670/I0630 is linearly correlated with the local PpIX concentration, and thus rises up to the challenge of PpIX based pO2 measurement and PDT. This work could also pave the way for a fine comprehension of the production, diffusion and catabolization of PpIX in biological tissues.

Stimulation and homogenization of the protoporphyrin IX endogenous production by photobiomodulation to increase the potency of photodynamic therapy.

Protoporphyrin IX (PpIX) is produced in the mitochondria and used as fluorescent contrast agent or photosensitizer after exogenous 5-aminolevulinic acid (ALA) delivery in cancer photodynamic detection and therapy (PDT). Although routinely used in the clinics, the stimulated production of PpIX is often insufficient and/or heterogeneous within the lesions, thereby limiting the PDT performances. Since photobiomodulation, which is based on the illumination of the tissues with sub-thermal radiometric conditions in the red or near-infrared, is known to stimulate the cell metabolism, we have optimized these conditions in vitro. Some of them lead to the homogenization and strong stimulation of the PpIX endogenous production. Interestingly, combined sequentially, PBM enhanced significantly the potency of PpIX-based PDT in vitro and in vivo in tumors grown on the chicken embryo chorioallantoic membrane. These results are in excellent agreement with other assays based on measurements of the cell survival/death, the production of reactive oxygen species, including singlet oxygen, and the mitochondrial membrane potential.

A general framework for non-exponential delayed fluorescence and phosphorescence decay analysis, illustrated on Protoporphyrin IX.

Delayed fluorescence (DF) is a long-lived luminescence process used in a variety of applications ranging from oxygen sensing in biological tissues to organic Light Emitting Diodes. In common cases, DF results from the de-excitation of the first excited triplet state via the first excited singlet state of the chromophore, which produces a mono-exponential light signal whose amplitude and lifetime give an insight into the probed environment. However, non-linear de-excitation reactions such as triplet-triplet annihilation, which can cause decays to lose their mono-exponential nature, are often neglected. In this work, we derive a global framework to properly interpret decays resulting from a combination of linear and non-linear de-excitation processes. We show why the standard method of using multi-exponential models when decays are not mono-exponential is not always relevant, nor accurate. First, we explain why the triplet de-excitation and light production processes should be analyzed individually: we introduce novel concepts to precisely describe these two processes, namely the deactivation pathway - the reaction which mainly contributes to the triplet state de-excitation - and the measurement pathway - the reaction which is responsible for light production. We derive explicit fitting functions which allow the experimenter to estimate the reaction rates and excited state concentrations in the system. To validate our formalism, we analyze the in vitro Transient Triplet Absorption and DF of Protoporphyrin IX, a well-known biological aromatic molecule used in photodynamic therapy, cancer photodetection and oxygen sensing, which produces DF through various mechanisms depending on concentration and excitation intensity. We also identify the precise assumptions necessary to conclude that triplet-triplet annihilation DF should follow a mono-exponential decay with a lifetime of half the triplet state lifetime. Finally, we describe why the commonly used definitions of triplet / DF lifetime are ill-defined in the case where second-order reactions contribute to the deactivation process, and why the fitting of precise mixed-orders DF kinetics should be preferred in this case. This work could allow the correct interpretation of various long-lived luminescence processes and facilitate their understanding.

Measurements of the optical coefficients of the protoporphyrin IX endogenously producing yeast-based model in the visible and NIR.

Models mimicking the endogenous production of protoporphyrin IX (PpIX), as well as its fluorescence, are of high interest for applied and fundamental studies in the fields of cancer detection by fluorescence imaging, photodynamic therapy (PDT), and photobiomodulation (PBM). Here, we present and describe optical properties of the yeast-based models able to produce PpIX endogenously after the administration of 5-aminolevulinic acid (ALA) and/or 2,2'-bipyridyl. As their optical properties have an important impact on the spatial distribution of the fluence rate in these liquid models, their absorption and reduced scattering coefficients were determined to be between 400 and 808 nm for two yeast solutions previously described by our group. These coefficients were derived from measurements of the total reflectance and light penetration depth using a dedicated Monte Carlo simulation. We observed that absorption and scattering coefficients were smaller than those of soft tissues at all wavelengths. This work will enable the production of a low-cost optical phantom loaded with appropriate amounts of light-absorbing and -scattering particles to mimic tumors containing PpIX, offering a useful tool to optimize the spectral and radiometric design of certain cancer photodetection setups.

Measurement of pO2 by luminescence lifetime spectroscopy: A comparative study of the phototoxicity and sensitivity of [Ru(Phen)3 ]2+ and PdTCPP in vivo.

Dysfunctions in tissue metabolism can be detected at early stages by oxygen partial pressure (pO2 ) measurement. The measurement of emission lifetimes offers very promising and non-invasive approach to estimate pO2 in vivo. This study compares two extensively used oxygen sensors and assesses their in vivo oxygen sensitivity and phototoxic effect. Luminescence lifetime of Ru-polypyridyl complex and of Pd-porphyrin is measured in the Chick's Chorioallantoic Membrane (CAM) model with a dedicated optical fiber-based, time-resolved spectrometer. The Pd-porphyrin luminescence lifetimes measured in the CAM model exposed to different pO2 levels are longer and have a broader dynamic range (10-100 µs) than those of Ru-polypyridyl complex (0.6-1 µs). The combined statistical analysis based on an estimate of the kurtosis and skewness, bootstrapping method and routine normality tests is performed. The indicators of the averages and signal to noise ratio stability are also calculated. The combination of several data processing allows selection of the better sensor for a given application. In particular, it is found that the advantage of Ru-polypyridyl complex over Pd-porphyrin is two-fold: i) Ru-polypyridyl complex datasets have consistently better statistical characteristics, ii) Ru-polypyridyl exhibits lower cytotoxicity.

Optimization and characterization of the endogenous production of protoporphyrin IX in a yeast model.

The availability of reproducible, convenient, and inexpensive model organisms able to generate predictable levels of endogenous porphyrins, including protoporphyrin IX (PpIX), is essential in photomedicine research. Saccharomyces cerevisiae produces endogenous PpIX and was used as a model organism for this study with the aim to maximize endogenous PpIX fluorescence intensity. It was found that PpIX fluorescence was significantly enhanced by administration of 5-aminolevulinic acid (ALA) and 2,2?-bipyridyl. Fluorescence intensity and spectroscopy of PpIX produced endogenously were measured in diluted yeast solutions under various conditions. The optimal protocol was: 5 ?? ? M ALA and 1 mM 2,2?-bipyridyl administered synchronously at 32°C. After 3 h, PpIX in yeast demonstrated similar steady-state and time-resolved spectroscopy as that of PpIX in DMSO. Moreover, under hypoxic conditions, the reciprocal lifetime of PpIX delayed fluorescence measured in real time was correlated to the partial pressure of oxygen ( pO 2 ) measured concomitantly with a commercially available pO 2 probe. These data show that yeast can, in optimal conditions, reproducibly generate PpIX. This is of interest in various fields such as photodiagnosis, photodynamic therapy, and photobiomodulation. Use of this model organism focuses on essential mechanisms, without the complexity of a multicellular organism.

Effect of PpIX photoproducts formation on pO2 measurement by time-resolved delayed fluorescence spectroscopy of PpIX in solution and in vivo.

The measurement of Protoporphyrin IX delayed fluorescence lifetime is a minimally invasive method for monitoring the levels of oxygen in cells and tissues. The excitation of Protoporphyrin IX during this measurement can lead to the formation of photoproducts in vitro and in vivo. The influence of their luminescence on the measured Protoporphyrin IX delayed fluorescence lifetimes was studied in solution and in vivo on the Chick's chorioallantoic membrane (CAM) model under various oxygen enriched air conditions (0mmHg, 37mmHg and 155mmHg). The presence of photoproducts disturbs such measurements since the delayed fluorescence emission of some of them spectrally overlaps with that of Protoporphyrin IX. One possible way to avoid this obstacle is to detect Protoporphyrin IX's delayed fluorescence lifetime in a very specific spectral range (620-640nm). Another possibility is to excite Protoporphyrin IX with light doses much lower than 10J/cm2, quite possibly as low as a fraction 1J/cm2 at 405nm. This leads to an increased accuracy of pO2 detection. Furthermore, this method allows combination of diagnosis and therapy in one step. This helps to improve detection systems and real-time identification of tissue respiration, which is tuned for the detection of PpIX luminescence and not its photoproducts.

Nanoantenna-induced fringe splitting of Fabry-Perot interferometer: a model study of plasmonic/photonic coupling.

In this paper, we present a simple approach to study the coupling mechanisms between a plasmonic system consisting of bowtie nanoantennas and a photonic structure based on a Fabry-Perot interferometer. The nanoantenna array is represented by an equivalent homogeneous layer placed at the interferometer surface and yielding the effective dielectric function of the NA resonance. A phase matching model based on thin film interference is developed to describe the multi-layer interferences in the device and to analyze the fringe variations induced by the introduction of the plasmonic layer. The general model is validated by an experimental system consisting of a bowtie nanoantenna array and a porous-silicon-based interferometer. The optical response of this hybrid device exhibits both the enhancement induced by the nanoantenna resonance and the fringe pattern of the interferometer. Using the phase matching model, we demonstrate that strong coupling can occur in such a system, leading to fringe splitting. A study of the splitting strength and of the coupling behavior is given. The model study performed in this work enables to gain deeper understanding of the optical behavior of plasmonic/photonic hybrid devices.