Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy

Abstract

Low power laser irradiation is regarded to have a significant role in triggering cellular proliferation and in treating diseases of diverse etiologies. The present work contributes to the understanding of the mechanisms of action by studying low power laser effects in human fibroblasts. Confocal laser scanning microscopy is used for irradiation and observation of the same area of interest allowing the imaging of laser effects at the single cell level and in real time. Coverslip cultures were placed in a small incubation chamber for in vivo microscopic observation. Laser stimulation of the cells was performed using the 647 nm line of the confocal laser through the objective lens of the microscope. Mitochondrial membrane potential (delta psi(m)), intracellular pH, calcium alterations and generation of reactive oxygen species (ROS) were monitored using specific fluorescent vital probes. The induced effects were quantified using digital image processing techniques. After laser irradiation, a gradual alkalinization of the cytosolic pH and an increase in mitochondrial membrane potential were observed. Recurrent spikes of intracellular calcium concentration were also triggered by laser. Reactive oxygen species were generated as a result of biostimulation. No such effects were monitored in microscopic fields other than the irradiated ones.

Comparative analysis of different doses of coherent light (laser) and non-coherent light (light-emitting diode) on cellular necrosis and apoptosis: a study in vitro

“…A long series of discoveries have demonstrated that ATP is not only an energy currency inside cells but is also a critical signalling molecule that allows cells and tissues to communicate (Karu, 2010). Alexandratou et al (2002) found that the increased formation of ROS combined with increased intracellular Ca 2+ and mitochondrial membrane potential are mechanisms involved in necrosis following irradiation. Low doses of irradiation can stimulate increased Ca 2+ and induce mitosis increasing cellular proliferation.…”

Regulation of Cellular Molecular Signaling by Light (Invited Paper)

“…Photodamage during microscopy indeed indicates that cells can be stimulated solely by photons. Along with this idea, it was found that low energy visible light irradiation with about 0.1 mw/cm 2 power density for seconds to minutes could induce changes of intracellular Ca 2+ concentrations in macrophages [76], sperms [77][78][79], lymphocytes [80], skeletal muscle cells [81,82], human fibroblasts [83] and mast cells [84]. The cellular Ca 2+ level slowly increased during continuous laser irradiation and could maintain for minutes.…”

The effect of continuous low-intensity laser irradiation of the red spectrum on the changes in the functional activity and speed of NADPH-oxidase response of human peripheral blood neutrophils

“…Этот результат косвенно подтверждает высказанное ранее предположение о ведущей роли ионов каль-ция, высвобождаемых из внутриклеточных депо [9,10], которые, как известно, распространяются в виде волн с периодом 100 с (рис. 2) [12]. Для изучения активности фермента НАДФ-оксидазы в серии опытов в аналогичных условиях выделенные из крови доноров нейтрофилы подвер-гали лазерному освечиванию при температуре 37 °C, для чего в силиконизированную кювету со свето-изолированными стенками вносился 1 мл раствора Хенкса, содержащего 5×10 6 нейтрофилов в 1 мл.…”