Nanoscale topography of cells and vesicles in adhesion revealed by quantitative Total Internal Reflection Fluorescence Microscopy
Authors: Dos Santos Marcelina Cardoso , Vézy Cyrille , Jaffiol Rodolphe 
Published: December 2016

Total Internal Reflection Fluorescence (TIRF) microscopy is becoming a widespread technique to study cellular processes occurring near the contact region with the glass substrate [1]. The characteristics of TIRF microscopy are directly related to the singular properties of evanescent waves, such as the exponential decay of the electric field along the z direction. This providing a selective excitation of fluorescent molecules close to the interface. Determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process [2]. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters. The first one is obviously the distance z, which separates the dye molecules from the surface, as the emitted fluorescence signal is mainly governed by the exponential decay of the evanescent wave. But TIRF images contrast is also affected by unknown parameters such as the local concentration of dyes, their dipole moment orientation and consequences on their related angular emission pattern (which influences the detection efficiency η
d
), and also their absorption cross section (σ
abs
) and their fluorescence lifetime (τ). Moreover, these last three parameters (η
d
, σ
abs
, τ) can be strongly altered as a function of z near the surface [3].

To get around this problem, we propose two strategies allowing us to map the membrane‐substrate separation distance with a nanometric resolution (typically 10 nm). The first one is dedicated to study the adhesion of Giant Unilamellar Vesicles (GUVs), which are often used as a biomimetic system to reproduce cells spreading. This approach, called normalized TIRF, is based on dual observation, which combine epi‐fluorescence microscopy and TIRF microscopy: TIRF images are normalized by epi‐fluorescence ones [4, 5]. Figure 1 shows an example of a negatively charged GUV completely spread on a thin layer of a positively charged polyelectrolyte (PDDA) recovering the coverslip. The second technique is devoted to explore the adhesion of living cells. This last is called variable‐angle TIRF (vaTIRF) microscopy. vaTIRF is an old technique introduced in the middle of 80s and quickly forgot due to the high complexity of the first experimental setup used. We propose an improved straightforward version of vaTIRF microscopy adapted to modern TIRF setup. This technique involves the recording of a stack of several TIRF images, by gradually increasing the incident angle θ on the sample. We developed a comprehensive theory to extract the membrane/substrate separation distance from fluorescently labeled cell membranes [6], as illustrated in figure 2 for a MDA‐MB‐231 cell in adhesion on fibronectin. Finally, we demonstrate that these two techniques (nTIRF and vaTIRF) are useful to quantify the adhesion of vesicles and cells from weak to strong membrane‐surface interactions, achieved on various functionalized substrates with polymers or proteins, such as collagen and fibronectin.