Fluorescence optical nanotomography: combining the techniques of total internal reflection fluorescence microscopy and ultramicrotomy

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Abstract

A method of 3D-TIRF microscopy is proposed, based on combination of ultramicrotomy and total internal reflection fluorescence microscopy techniques of the sample surface after cutting, which allows reconstructing the three-dimensional ultrastructure of objects.

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About the authors

K. E. Mochalov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: antefimov@gmail.com
ORCID iD: 0000-0001-8157-9613

Doct. of Sci. (Physics and Mathematics), Senior Researcher

Russian Federation, Moscow

O. I. Agapova

Shumakov National Medical Research Center of Transplantology and Artificial Organs of Ministry of Health of the Russian Federation

Email: antefimov@gmail.com
ORCID iD: 0000-0002-4507-1852

Cand. of Sci. (Biology), Senior Researcher

Russian Federation, Moscow

I. I. Agapov

Shumakov National Medical Research Center of Transplantology and Artificial Organs of Ministry of Health of the Russian Federation

Email: antefimov@gmail.com
ORCID iD: 0000-0002-0273-4601

Doct. of Sci. (Biology), Prof., Head of Laboratory

Russian Federation, Moscow

D. S. Korzhov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS; National Research Nuclear University MEPhI

Email: antefimov@gmail.com
ORCID iD: 0009-0006-2626-1278

Technician

Russian Federation, Moscow; Moscow

D. O. Solovyeva

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: antefimov@gmail.com
ORCID iD: 0000-0002-2590-4204

Cand. of Sci. (Biology), Researcher

Russian Federation, Moscow

S. V. Sizova

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: antefimov@gmail.com
ORCID iD: 0000-0003-0846-4670

Cand. of Sci. (Chemistry), Researcher

Russian Federation, Moscow

M. S. Shestopalova

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS; National Research Nuclear University MEPhI

Email: antefimov@gmail.com
ORCID iD: 0000-0001-6543-4289

Engineer Researcher

Russian Federation, Moscow; Moscow

V. A. Oleinikov

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS; National Research Nuclear University MEPhI

Email: antefimov@gmail.com
ORCID iD: 0000-0003-4623-4913

Doct. of Sci. (Physics and Mathematics), Prof., Head of Department

Russian Federation, Moscow; Moscow

A. E. Efimov

Shumakov National Medical Research Center of Transplantology and Artificial Organs of Ministry of Health of the Russian Federation

Author for correspondence.
Email: antefimov@gmail.com
ORCID iD: 0000-0002-0769-301X

Doct. of Sci. (Biology), Chief Researcher

Russian Federation, Moscow

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Supplementary files

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2. Fig.1. Model of a specialized upright fluorescence microscope for implementing the 3D-TIRF technique. a – side view; b – back view. Designations: 1 – objective lens, 2 – precise angular positioner, 3 – axial micro- positioner for precise focusing, 4 – cube with fluorescent filters, 5 – tube lens, 6 – LED for illumination in bright field mode, 7 – precise motorized vertical microscope positioner, 8 – precise motorized horizontal microscope positioner ("forward – backward"), 9 – mounting optical panel with precise horizontal movement ("right – left"), 10 – diamond knife of the ultramicrotome

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3. Fig.2. Experimental setup for 3D-TIRF microscopy: 1 – diamond knife of ultramicrotome, 2 – fluorescence microscope objective, 3 – XYZ-piezoscanner mounted on the movable arm of ultramicrotome, 4 – sample

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4. Fig.3. Comparison of the experimental (green) and theoretical (purple) dependences of evanescent waves on the distance from the knife edge along the Z axis

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5. Fig.4. Left panel: a 2D TIRF image of a sample of polystyrene microspheres (PSMs) with an optically thin (several nanometers) surface layer of CdSe/ZnS core/shell quantum dots (QDs) fluorescing at a wavelength of about 530 nm. The image size is 25.6×25.6 μm. Right: a 3D reconstruction of the PSM–QD sample based on 33 sequential 2D TIRF images. The section thickness is 150 nm; the reconstruction size is 25.6×25.6×4.95 μm. Scale bars, 5 μm

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Copyright (c) 2024 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.