Improving Consistency of Photobiomodulation Therapy: A Novel Flat-Top Beam Hand-Piece versus Standard Gaussian Probes on Mitochondrial Activity

Abstract

The tremendous therapeutic potential of photobiomodulation therapy in different branches of medicine has been described in the literature. One of the molecular mechanisms for this treatment implicates the mitochondrial enzyme, cytochrome C oxidase. However, the efficacy and consistency of clinical outcomes with photobiomodulation treatments has been fiercely debated. This work was motivated by this need to improve photobiomodulation devices and delivery approaches. We designed a novel hand-piece with a flat-top beam profile of irradiation. We compared the beam profile versus a standard hand-piece and a fibre probe. We utilized isolated mitochondria and performed treatments at various spots within the beam, namely, the centre, left and right edge. We examined mitochondrial activity by assessing ATP synthesis with the luciferin/luciferase chemiluminescent method as a primary endpoint, while mitochondrial damage was assessed as the secondary endpoint. We observed a uniform distribution of the power density with the flat-top prototype compared to a wide Gaussian beam profile with the standard fibre and standard hand-piece. We noted increased production of ATP in the centre of all three beams with respect to the non-treated controls (p < 0.05). Both the fibre and standard hand-piece demonstrated less increase in ATP synthesis at the edges than the centre (p < 0.05). In contrast, ATP synthesis was increased homogenously in the flat-top handpiece, both in the centre and the edges of the beam. Fibre, standard hand-piece and the flat-top hand-piece prototype have discrete beam distribution characteristics. This significantly affected the mitochondrial activity with respect to their position within the treated areas. Flat-top hand-piece enhances the uniformity of photobiomodulation treatments and can improve the rigour and reproducibility of PBM clinical outcomes.

Keywords: ATP; energetic metabolism; light therapy; light-emitting diode; low-level laser therapy; mitochondria respiratory chain; near-infrared light; phototherapy.

Figure 1

Experimental design. (A) A drop of isolated mitochondria enriched-fraction was pipetted on a coverslip, in the centre or the edge of the laser beam circular area, according to the experimental purpose. The drop was then irradiated with an 808-nm laser light (diameter 1.13 cm; spot-size 1 cm²) through a fibre, a standard hand-piece, or the flat-top hand-piece. (B) Primary and secondary endpoints were evaluated as ATP synthesis through the luciferin/luciferase chemiluminescent method. Image created with BioRender.com.

Figure 2

Schematic design of the flat-top hand-piece. (A) = fibre’s spot delivering laser light; (a,b) = collimating lens; (c,d) = couple of lens to generate an afocal optical group.

Figure 3

Characterization of the fibre (A), standard hand-piece (B) and the novel flat-top hand-piece prototype; (C) beam profile through a camera Spiricon SP928 equipped with the software BeamGage Professional. Setting the instrument for an irradiation with 1 W the main colours displayed are: purple = 0.1 W/cm²; fuchsia = 0.3 W/cm²; blue = 0.6 W/cm²; light blue = 0.9 W/cm²; green = 1 W/cm²; yellow = 1.6 W/cm²; orange = 2.0 W/cm²; red = 2.2 W/cm².

Figure 4

Schematic representation of the laser beam profile (A–C) and the drop’s area irradiated by the power density of 1 W/cm², green colour, (A’–C’) Image created with BioRender.com.

Figure 5

Effect of the photobiomodulation on mitochondrial ATP synthase activity. The isolated mitochondria were placed at the centre or edges of the laser treatment spot (1 cm²) and the treatments were performed using a fibre, flat-top hand-piece or a standard hand-piece. All samples were treated with PBM 808 nm laser in continuous wave mode with 1 W, 1 W/cm², 60 J and 60 J/cm² for 60 s. Untreated control samples were placed in a similar set-up with laser at 0 W for 60 s. Data are expressed as mean ± SD. A significant difference between the ATP production of the mitochondria in the centre vs. the edge is indicated by the symbol * and ****, respectively p < 0.05 and 0.0001, ns indicates a no-significant difference.