Effects of near infrared focused laser on the fluorescence of labelled cell membrane

www.nature.com/scientificreports OPEN Received: 10 July 2018 Accepted: 9 November 2018 Published: xx xx xxxx Effects of near infrared focused laser on the fluorescence of labelled cell membrane Remy Avila 1,2, Elisa Tamariz3, Norma Medina-Villalobos2,3, Jordi Andilla2, María Marsal2 & Pablo Loza-Alvarez2 Near infrared (NIR) laser light can have important reactions on live cells. For example, in a macroscopic scale, it is used therapeutically to reduce inflammation and in a single-cell scale, NIR lasers have been experimentally used to guide neuronal growth. However, little is known about how NIR lasers produce such behaviours on cells. In this paper we report effects of focussing a continuous wave 810-nm wavelength laser on in vivo 3T3 cells plasma membrane. Cell membranes were labelled with FM 4-64, a dye that fluoresces when associated to membrane lipids. Confocal microscopy was used to image cell membranes and perform fluorescence recovery after photobleaching (FRAP) experiments. We found that the NIR laser produces an increase of the fluorescence intensity at the location of laser spot. This intensity boost vanishes once the laser is turned off. The mean fluorescence increase, calculated over 75 independent measurements, equals 19%. The experiments reveal that the fluorescence rise is a growing function of the laser power. This dependence is well fitted with a square root function. The FRAP, when the NIR laser is acting on the cell, is twice as large as when the NIR laser is off, and the recovery time is 5 times longer. Based on the experimental evidence and a linear fluorescence model, it is shown that the NIR laser provokes a rise in the number of molecular associations dye-lipid. The results reported here may be a consequence of a combination of induced increments in membrane fluidity and exocytosis. Plasma membrane dynamics is fundamental in cell secretion, signaling, movement, cell shape changes, cytokinesis, among other cellular processes. Cell membrane is in constant motion due to its characteristic fluid mosaic conformation, where the bilayer of amphiphilic phospholipids diffuse along the membrane plane into specific and heterogeneous membrane domains by translation, rotational motions around the axis perpendicular to the membrane, and by trans-bilayer diffusion in a less extent1,2. Cells continually adjust their membrane content and composition by two fundamental processes, the uptake of cell surface membrane called endocytosis, with which fluids or macromolecules may be introduced to the cell, and the fusion of vesicles at the cytoplasmic side of the membrane, called exocytosis, involved in secretion and expression of proteins on the cell membrane3–5. Endocytosis and exocytosis are fundamental events in cell protrusion and migration6–9. The interaction of a near infrared (NIR) laser beam with cells has been addressed from many different view angles. From a macroscopic perspective, low levels of NIR laser light are used to reduce pain, inflammation, nerve injuries and to promote tissue regeneration10–12. Studies of the influence of low-power helium-neon laser irradiation on selected peripheral blood cells have revealed the importance of photodynamic reactions on the ability of blood to transport oxygen and on immunomodulatory effects on leukocytes13. More recently it has been reported that low-intensity NIR laser radiation induces free radical generation and changes in enzymatic and anti-oxidative activities of cellular components14. Tightly focused NIR laser radiation has also been used as an attractant of cell projections. This has been described for fibroblasts15–17 and neurons18–25, opening a new perspective for a possible guidance cue. Although different cellular mechanisms have been suggested to take part in those phenomena, no model has yet gain consensus and in general, the biochemical and biophysical processes that take place when NIR light radiates cells are still poorly understood. 1 Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México (UNAM), A. P. 1-1010, Juriquilla, 76000, Querétaro, Mexico. 2ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860, Castelldefels, Barcelona, Spain. 3Instituto de Ciencias de la Salud, Universidad Veracruzana, Avenicda Luis Castelazo Ayala s/n, Xalapa, 91190, Veracruz, Mexico. Correspondence and requests for materials should be addressed to R.A. (email: ) Scientific Reports | (2018) 8:17674 | DOI:10.1038/s41598-018-36010-1 1 www.nature.com/scientificreports/ Figure 1. Effect of a 810-nm continuous wave focused laser on fluorescence intensity. (a) Confocal image of a 3T3 cell previously stained with FM 4-64, with the presence of dye molecules in the medium. NIR laser is turned off in this image. (b) Same as in (a) but with the 810-nm laser focused on the centre of the yellow circle which represents the ROI where intensity average is computed. An intensity rise is clearly seen by comparing images in the two magnified regions shown. Cyan circles indicate the regions used to estimate background mean intensity. (c) Difference of images b minus a. (d) Blue line represents the mean relative intensity (Irel, Equation 1) inside the ROI delimited by the yellow circle. Red line indicates the NIR laser power at the exit of the microscope objective. (e) Irel measured outside the cell (orange line) and on a cell without staining (blue line). (f) Same as in (c), but on a sample whose medium is label-free (blue line) and on a fixed cell immersed in medium that contained FM4-46 dye at 1, 1 μM. For the intensity scales in frames (c), (d) and (e) to be comparable among each other, a second normalization is performed on the relative intensity such that plotted values are Irel(〈ICELL − IBG〉/〈IROI − IBG〉) where 〈〉 operator represents a temporal average. See equation (1) for the definition of Irel. In this paper, we analyze the effect of focused 810-nm laser stimulation on 3T3 fibroblast membrane dynamics. We use FM 4-64, a plasma membrane marker of the family of fluorescent amphiphilic styryl dyes, with a long hydrophobic tail able to interact into the lipid bilayer, whereas the positively charged head group of the molecule prevents the complete insertion into the membrane26. Between head and tail, two aromatic rings create the fluorophore. Its quantum yield strongly depends on the solvent polarity, such that in a polar medium like water, the fluorescence is more than two orders of magnitude dimmer than in a non-polar environment, therefore the fluorescence is much higher in the cell membrane than in the media, selectively staining cell surface membrane exposed to the dye26. FM dyes have been extensively used to study endo and exocytosis processes in different cell types26–29. Here we report previously unknown effects of the NIR laser focussed on the cell membrane: An increase in the fluorescence intensity of the membrane exposed to FM 4-64 at the (...truncated)