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

Bokai Zhang,Han Zhang,Massimiliano Galluzzi,Lingzhi Wang,Zhiqin Chu,Xue-Feng Yu,Jiangyu Li,Yuan Lin

doi:10.3389/fmolb.2021.669361

Abstract

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 ∼40% decrease in the breakthrough force of the stained phospholipid bilayer along with a ∼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.

Highlights

Fluorescence microscopy is one of the most often used imaging techniques in biology and material science (Lichtman and Conchello, 2005; Shashkova and Leake, 2017), and this is dictated by efficient staining agents with ability to bind on target while maintaining fluorescence
The commonly integrated fluorescence microscopy, especially the confocal laser scanning microscopy (CLSM), raises the concern of photochemistry effects induced by tightly focused laser beam, in the presence of dye molecules
We revealed that the degradation of mechanical properties of model phospholipid membranes is driven by synergic presence of bounded fluorophores and focused laser irradiation

Summary

Introduction

Fluorescence microscopy is one of the most often used imaging techniques in biology and material science (Lichtman and Conchello, 2005; Shashkova and Leake, 2017), and this is dictated by efficient staining agents with ability to bind on target while maintaining fluorescence. Considering the versatility of AFM in biology, it is significant to integrate such platform with optics, the so-called correlative microscopy (Zhou et al, 2021). With such a combination, the mechanical quantities and biochemical events can be simultaneously investigated with high spatial-temporal resolution. The mechanical quantities and biochemical events can be simultaneously investigated with high spatial-temporal resolution This will provide new insights into biological processes, but previous studies suggested the used fluorophores could have huge impact on the targeted biomolecules during visualization (Luitz et al, 2017; Cosentino et al, 2019). Desirable now is to understand the underlying cross-talk between the two techniques, especially in a quantitative manner

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