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 Ltd

Authors
Mochalov K.E. , Agapova O.I. , Agapov I.I. , Korzhov D.S. , Solovyeva D.O. , Sizova S.V. , Shestopalova M.S. , Oleinikov V.A. , Efimov A.E.
Journal
Optics and Lasers in Engineering
Publisher
Elsevier Ltd
Language
English
Status
Published
Link
External link
DOI
10.1016/J.OPTLASENG.2025.108891
Number
108891
Volume
188
Year
2025
Organizations
  • 1 Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ulitsa Miklukho-Maklaya 16/10, Moscow, 117997, Russian Federation
  • 2 RUDN University, Ulitsa Miklukho-Maklaya 6, Moscow, 117198, Russian Federation
  • 3 Academician V.I. Shumakov National Medical Research Center of Transplantology and Artificial Organs, Ministry of Health of the Russian Federation, Schukinskaya St., 1, Moscow, 123182, Russian Federation
  • 4 National Research Nuclear University “MEPhI”(Moscow Engineering Physics Institute), Kashirskoe highway 31, Moscow, 115409, Russian Federation
Keywords
3D Imaging; 3D Total internal reflection fluorescence; Axial resolution; Diffraction limit; Ultramicrotomy
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