Laser optoporation systems are now increasingly used for intracellular delivery. However, data on the response of cells to radiation-induced nondamaging changes in the integrity of the membrane lipid bilayer remain limited. Traditionally, confocal laser scanning microscopy and electron microscopy are used for such studies, but they have limitations for in situ experiments. The modern capabilities of atomic force microscopy (AFM) combine the resolution of electron microscopy and the possibility of noninvasive lifetime imaging of cells in vitro. Herein we used long-term AFM mapping integrated with fluorescence microscopy imaging for investigation of the whole cell cycle from irradiation time point to the total recovery to the intact cell state. For the first time we performed a comprehensive study of long-term posteffects of continuous laser and pulsed laser on the mechanical properties and the membrane recovery of HeLa cells grown on the Au nanoparticle layers of various morphologies. The set of nonpenetrating agents with various sizes ranging from 1 to 1.5 nm for propidium iodide (PI) up to 6–8 nm for 40 kDa FITC-labeled dextran was used to control the delivery efficacy. The main parameters recorded with AFM scanning of cells are Young’s modulus (YM) and the cell surface topography. We revealed that self-healing of HeLa cell from the moment of irradiation to complete restoration of the membrane integrity is lasting 22–30 h when using a continuous-wave source and 2–5 h when using a pulsed laser, respectively. The estimated time elapse was in good correspondence with the relative change in YM during the entire experiment. Our findings demonstrate the capability of AFM coupled with fluorescent microscopy for further in situ investigations of the morphological and functional state of the cells exposed to the influence of other external conditions.

The stress relaxation and creep measurements are commonly implemented in AFM indentation experiments to analyze the viscoelastic properties of a sample in the time domain. The assumption of an instantaneous step load is often applied which can lead to erroneous estimation of viscoelastic parameters from the experimental data. A viscoelastic solution for the ramp-hold indentation profile might be used but requires higher computational costs. Here, we employed simulations for the ramp-hold indentations for the power-law rheology (PLR) and standard linear solid models. The error coming from the instantaneous load assumption was analyzed and a simple empirical correction for the finite ramp time was employed as a time shift of the hold region. The correction was tested in experiments on cells, in which the data from stress relaxation, creep, and force volume experiments were compared. The correction showed good applicability for the extraction of the PLR viscoelastic parameters.