Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research

Abstract

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

Keywords: AFM; IVF; SNOM; blastocysts; embryos; oocyte; ovary; spermatozoa.

Figure 1

Schematic illustration of the atomic force microscopy (AFM) (A) and aperture scanning near-field optical microscopy (SNOM) (B) instrumentation with the basic working elements of the two techniques. Force-distance curve (magenta approach and pink retraction trace) showing how the cantilever is brought into contact with the sample, bends and the tip located at the end “pushes” on the sample, thereby enabling one to apply force or indent the sample. When the cantilever is retracted, in the “pulling” phase, interaction forces between tip and sample can be measured, up to molecular level (C). Zoomed view of the SNOM probe: the end part of the optical fiber close to the sample surface, where the small aperture of the optical fiber and the local light interaction create the near-field used to reach high resolution (D).

Figure 2

AFM indentation measurements with a micrometer bead on an oocyte: a scheme, a bright field image (top view), and a representative force-indentation curve obtained on a human oocyte (blue line) and a rigid surface (gray line) displayed for comparison (A). AFM stress-relaxation measurements with a macrocantilever (square 300 × 300 μm²) on an oocyte: a scheme, a bright field image (side-view), and a representative stress-relaxation curve obtained from a human oocyte (B). Scale bar 50 μm.

Figure 3

SNOM topography (A) reflection (B) and transmission (C) images of human spermatozoa with normal features, as modified from [95] with permission. The arrow in (A) indicates the sperm region in which the mitochondria’s helicoidally arrangement can be discerned in the corresponding SNOM transmission image (C).

Figure 4

SNOM topography (A,E) and fluorescence (B–D) images of PNT2 epithelial cells with dual-labeling, which, thanks to the use of two separated filters, enables one to identify the location of the tight junction protein ZO-1 (in green) and E-cadherin (in red). The region included in the box in (A) was scanned at a higher resolution to generate the detailed E-cadherin fluorescence image (D) with the corresponding topography in (E). Circled regions in (C) and the arrow/arrowhead in (D) highlight E-cadherin clusters. The picture was published with permission [100].