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

Remy Avila,Elisa Tamariz,Norma Medina-Villalobos,María Marsal,Jordi Andilla,Pablo Loza-Alvarez

doi:10.1038/s41598-018-36010-1

Abstract

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.

Highlights

Plasma membrane dynamics is fundamental in cell secretion, signaling, movement, cell shape changes, cytokinesis, among other cellular processes
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, the fluorescence is much higher in the cell membrane than in the media, selectively staining cell surface membrane exposed to the dye[26]
We report previously unknown effects of the near infrared (NIR) laser focussed on the cell membrane: An increase in the fluorescence intensity of the membrane exposed to FM 4-64 at the location of the NIR laser spot, and an increase of the fluorescent recovery after photobleaching (FRAP)

Summary

Introduction

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 extent[1,2] 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 membrane[3,4,5]. Our findings indicate that the fluorescence rise is due to an increase of FM-dye molecules incorporation into the cell membrane, and points towards an increase in membrane dynamic presumably driven by exocytosis events

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