Infrared Nanospectroscopy of Individual Extracellular Microvesicles

Abstract

Extracellular vesicles are membrane-delimited structures, involved in several inter-cellular communication processes, both physiological and pathological, since they deliver complex biological cargo. Extracellular vesicles have been identified as possible biomarkers of several pathological diseases; thus, their characterization is fundamental in order to gain a deep understanding of their function and of the related processes. Traditional approaches for the characterization of the molecular content of the vesicles require a large quantity of sample, thereby providing an average molecular profile, while their heterogeneity is typically probed by non-optical microscopies that, however, lack the chemical sensitivity to provide information of the molecular cargo. Here, we perform a study of individual microvesicles, a subclass of extracellular vesicles generated by the outward budding of the plasma membrane, released by two cultures of glial cells under different stimuli, by applying a state-of-the-art infrared nanospectroscopy technique based on the coupling of an atomic force microscope and a pulsed laser, which combines the label-free chemical sensitivity of infrared spectroscopy with the nanometric resolution of atomic force microscopy. By correlating topographic, mechanical and spectroscopic information of individual microvesicles, we identified two main populations in both families of vesicles released by the two cell cultures. Subtle differences in terms of nucleic acid content among the two families of vesicles have been found by performing a fitting procedure of the main nucleic acid vibrational peaks in the 1000-1250 cm^-1 frequency range.

Keywords: atomic force microscopy; extracellular vesicles; infrared nanoscale spectroscopy.

Figure 1

(a) Topography map of MVs deposited on ultraflat gold substrate measured by AFM. The image is representative of sample areas where MVs are not contaminated by salt crystals and other residues of the drop-casting process. (b) Topography map of MVs deposited on ultraflat gold substrate contaminated by salt crystals. (c) Topography map of a single MV deposited on ultraflat gold substrate. (d) Line-scan profile taken along the MV from which the height and the lateral size are determined. (e) The height vs. lateral size plot for all MVs measured by AFM. (f–i) Histograms of all measured topographic values, with average and standard deviation indicated in the boxes.

Figure 2

(a) AFM-IR spectrum obtained on a representative circular lens-shaped bzATP-MV; the asterisk in the map of panel (b) marks the point where the spectrum has been acquired. (b) Top: AFM-IR maps acquired at 1135 cm⁻¹ (left) and at 1660 cm⁻¹ (right) on the bzATP-MV. Bottom left: map of the mechanical resonance frequency f. Bottom right: topography line-scan profile (black curve) and AFM-IR signal line-scan acquired at 1135 cm⁻¹ (green curve), taken along the green dashed line in the upper-left sub-panel. (c,d) Same as (a,b) for a representative circular lens-shaped GO-MV.

Figure 3

(a) AFM-IR spectrum obtained on a representative bzATP-MV featuring heterogeneous mechanical, morphological and spectroscopic properties; the asterisk in the map of panel (b) marks the point where the spectrum has been acquired. (b) Top: AFM-IR maps acquired at 1135 cm⁻¹ (left) and at 1660 cm⁻¹ (right) on the bzATP-MV. Bottom left: map of the mechanical resonance frequency f. Bottom right: line-scan profiles taken along the MV in the topography map (black curve) and along the AFM-IR map acquired at 1135 cm⁻¹ (green curve) and at 1660 cm⁻¹ (violet curve). (c,d) Same as (a,b) for a representative GO-MV featuring heterogeneous mechanical, morphological and spectroscopic properties. The vertical dotted lines in panel (d) (bottom right) indicate a lateral resolution of around 40 nm.

Figure 4

(a) AFM-IR spectra (black curves) of 7 individual bzATP-MVs deposited on ultraflat gold substrate with the relative fitting curves (red lines). (b) Same as (a) for 8 individual GO-MVs. (c) Graph of the ratio between the spectral weights of component centered at 1135 cm⁻¹ (A2) and that centered at 1085 cm⁻¹ (A1).