3D TIRF microscopy: Combination of the total internal reflection fluorescence microscopy and ultramicrotomy techniques

Improvement of the existing methods of ultrahigh-resolution microscopy and development of new ones, in particular, methods of three-dimensional (3D) analysis and imaging of the ultrastructure of biological objects and nanomaterials, are extremely important tasks in biomedicine, cell biology, and nanotechnology. Here, a method of 3D total internal reflection fluorescence (TIRF) microscopy is proposed that combines ultramicrotomy (successive ultrathin sections of the sample) and TIRF microscopy of the sample surface. The key feature of this combined technique is the use of a specialized diamond ultramicrotome knife both as a tool for making the sections and as a TIRF prism for obtaining layer-by-layer TIRF images within a single measurement procedure. This makes it possible to reconstruct the 3D ultrastructure of objects. © 2025 Elsevier B.V., All rights reserved.