Unveiling a Hidden Event in Fluorescence Correlative Microscopy by AFM Nanomechanical Analysis

Fluorescent imaging combined with atomic force microscopy (AFM), namely AFM-fluorescence correlative microscopy, is a popular technology in life science. However, the influence of involved fluorophores on obtained mechanical information is normally underestimated, and such subtle changes are still challenging to detect. Herein, we combined AFM with laser light excitation to perform a mechanical quantitative analysis of a model membrane system labeled with a commonly used fluorophore. Mechanical quantification was additionally validated by finite element simulations. Upon staining, we noticed fluorophores forming a diffuse weakly organized overlayer on phospholipid supported membrane, easily detected by AFM mechanics. The laser was found to cause a degradation of mechanical stability of the membrane synergically with presence of fluorophore. In particular, a 30 min laser irradiation, with intensity similar to that in typical confocal scanning microscopy experiment, was found to result in a similar to 40% decrease in the breakthrough force of the stained phospholipid bilayer along with a similar to 30% reduction in its apparent elastic modulus. The findings highlight the significance of analytical power provided by AFM, which will allow us to see the unseen in correlative microscopy, as well as the necessity to consider photothermal effects when using fluorescent dyes to investigate, for example, the deformability and permeability of phospholipid membranes.

Automated summary

The paper introduces the popular AFM-fluorescence correlative microscopy technology in life sciences, highlighting the impact of fluorophores on mechanical information and the degradation of mechanical stability caused by light exposure. The study performed mechanical quantification of a labeled membrane system using AFM mechanics and finite element simulations, emphasizing the necessity to consider photothermal effects when investigating deformability and permeability of phospholipid membranes using fluorescent dyes.