Diffraction optical elements for the implementation of three-dimensional nanoscopy using rotating light fields

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Diffraction optical elements, made by contact printing on a bichrome gelatin and direct laser recording using photoresist, have been studied in order to modify the point-scattering function for the implementation of 3D ultrahigh-resolution fluorescence microscopy. It has been shown that these elements produce two-lobed, rotating light fields that can be used for 3D nanoscopy. Results of 3D subdiffractive localization of fluorescent labels, with an assessment of the accuracy of coordinate restoration, have also been presented.

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作者简介

D. Prokopova

Lebedev Physical Institute of the Russian Academy of Sciences

编辑信件的主要联系方式.
Email: prokopovadv@lebedev.ru

Branch in Samara

俄罗斯联邦, Samara

I. Eremchev

Institute of Spectroscopy of the Russian Academy of Sciences; Lebedev Physical Institute of the Russian Academy of Sciences

Email: prokopovadv@lebedev.ru

Branch in Troitsk

俄罗斯联邦, Moscow; Moscow

N. Losevsky

Lebedev Physical Institute of the Russian Academy of Sciences

Email: prokopovadv@lebedev.ru

Branch in Samara

俄罗斯联邦, Samara

D. Belousov

Institute of Automation and Electrometry of the Siberian Branch of the Russian Academy of Sciences

Email: prokopovadv@lebedev.ru
俄罗斯联邦, Novosibirsk

S. Golubtsov

Institute of Automation and Electrometry of the Siberian Branch of the Russian Academy of Sciences

Email: prokopovadv@lebedev.ru
俄罗斯联邦, Novosibirsk

S. Kotova

Lebedev Physical Institute of the Russian Academy of Sciences

Email: prokopovadv@lebedev.ru

Branch in Samara

俄罗斯联邦, Samara

А. Naumov

Lebedev Physical Institute of the Russian Academy of Sciences; Moscow State Pedagogical University

Email: prokopovadv@lebedev.ru

Branch in Troitsk

俄罗斯联邦, Moscow; Moscow

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2. Fig. 1. Phase function of the DOE optimized for operation in a three-dimensional nanoscope (a). Intensity distributions formed by the DOE manufactured by direct laser writing on photoresist at different distances near the focal plane of a lens with F = 250 mm (b). The frame side size is 2 mm.

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3. Fig. 2. Installation diagram of a three-dimensional nanoscope with a stationary reflective DOE.

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4. Fig. 3. Two-lobe images of point fluorescent labels in a 3-D nanoscope with a stationary DOE; for different positions of the emitters relative to the focal plane of the microscope δz: −2 (a1); −1 (a2); 0 (a3) ​​and 1.5 μm (a4). Dependence of the rotation angle of the two-lobe image α on the distance δz (b). Dots are experimentally measured values; the red line is a linear approximation of the dependence.

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5. Fig. 4. Accuracy of axial coordinate reconstruction. Dependence of the accuracy of longitudinal coordinate reconstruction of point fluorescent labels on the number of registered photons N for the tilt angle α ~ 0° (a). The inset shows an example of the distribution of reconstructed longitudinal coordinates z; obtained in a series of measurements n = 200 and its approximation by the Gaussian function. Dependence of the longitudinal coordinate reconstruction error σz(α) on the image rotation angle for a fixed number of photons N (b).

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