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Real-time reconstruction using electro-optics modulator-based structured illumination microscopy

Fanghui Xu, Jiachen Zhang, Dongdong Ding, Wenjie Liu, Chi Zheng, Sijia Zhou, Youhua Chen, and Cuifang Kuang

Open Access Open Access

Abstract

Structured illumination microscopy (SIM), a super-resolution technology, has a wide range of applications in life sciences. In this study, we present an electro-optic high-speed phase-shift super-resolution microscopy imaging system including 2D SIM, total internal reflection fluorescence-SIM, and 3D SIM modes. This system uses galvanometers and an electro-optic modulator to flexibly and quickly control the phase and direction of structured illumination patterns. Moreover, its design consists of precise timing for improved acquisition speed and software architecture for real-time reconstruction. The highest acquisition rate achieved was 151 frames/s, while the highest real-time super-resolution reconstruction frame rate achieved was over 25 frames/s.

© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement

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Supplementary Material (1)

NameDescription
Visualization 1       Real-time SIM imaging results. We used our SIM system to display the super-resolution effect of huFIB cells. Imaging was performed at 10 fps with 30 ms exposure time and 512*512 pixel raw image size.

Data availability

Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.

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Figures (8)
Fig. 1.
Fig. 1. Diagram of high-speed SIM setup. (a) Optical setup. BC, beam collimator; HWP1-HWP4, half-wave plate; PBS1-PBS2, polarized beam splitter; M1-M8, reflecting mirror; EOM1-EOM2, electro-optical modulator; LS, light stick; GM1-GM2, scanning galvanometer; BS, beam splitter; PHWP, pizza-type half-wave plate; QWP, quarter-wave plate; WP, wedge-shaped plate; L1-L5, lens; S, shutter; BCP, beam combining plate; FC, filter cube; TL, tube lens. (b) The polarization and place of the light beams in the three pattern orientations (0°, 120°, 240°) on the BFP of the objective lens. (c) The polarization change caused by the incident light passing through the pizza-type half-wave plate. (d) The spatial frequency components of the illumination intensity for the 2D SIM (left) and 3D SIM (right).

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Fig. 2.
Fig. 2. Timing diagram and overview of achievable frame rates. (a) 2D SIM timing diagram of all components, including camera, laser, EOM1, GM(x) (Galvo mirrors that control the movement of light along the x-axis in GM1 and GM2), GM(y) (Galvo mirrors that control the movement of light along the y-axis in GM1 and GM2); (b) 3D SIM timing diagram of all components, including camera, laser, EOM1, EOM2, GM(x), GM(y), Axial position (microscope stand).

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Fig. 3.
Fig. 3. Overview of software system. The software structure is shown in the left panel, including the control module and the reconstruction module. The image data transmission is shown in the right panel, illustrating how the data is transferred in real-time reconstruction mode. The meanings of the different colored lines and shapes are shown below the panels.

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Fig. 4.
Fig. 4. Diagram of software at real-time 2D SIM reconstruction mode. Real-time SIM imaging results at different times (6s, 9s, 28s, 33s) from video-recording (see Visualization 1) are shown in diagram. We used our SIM system to display the super-resolution effect on microtubules of huFIB cells. Imaging was performed at 10 frames/s with 30 ms exposure time and 512 × 512 pixel raw image size.

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Fig. 5.
Fig. 5. Nanoscale rulers TIRF-SIM imaging results. (a) TIRF-SIM reconstruction image of 120 nm nanoscale ruler; (b) 120 nm nanoscale ruler wide-field image; (c) 90 nm nanoscale ruler TIRF-SIM reconstruction image; (d) 90 nm nanoscale ruler wide-field image; (e) Figures (a) and (b) are the intensity distribution image at the position of the solid yellow line; (f) Figures (c) and (d) are the intensity distribution image at the position of the solid yellow line. Scale bar: 5 $\mathrm{\mu}\textrm{m}$ for overall image, 0.5 $\mathrm{\mu}\textrm{m}$ for magnified area.

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Fig. 6.
Fig. 6. Mitochondria of huFIB cells imaging results. (a) 2D SIM reconstruction image; (b) 2D SIM wide-field image; (c) TIRF-SIM reconstruction image; (d) TIRF-SIM wide-field image; (e) The intensity distribution image at the position of the solid yellow line for Figures (a) and (b); (f) the intensity distribution image at the position of the solid yellow line for Figure (c) and Figure (d). Scale bar: 5$\mathrm{\mu}\textrm{m}$ for overall image, 1$\mathrm{\mu}\textrm{m}$ for magnified area.

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Fig. 7.
Fig. 7. Mitochondrial 3D SIM imaging results. (a) SIM reconstruction image of xy plane; (b) wide-field image of xy plane; (c) SIM reconstruction image of xz plane at the position of the white horizontal line in (a); (d) wide-field reconstruction image of xz plane at the position of the white horizontal line in (b); (e) left: the spectrum of (a), right: the spectrum of (b); (f) The intensity distribution at the position of the blue solid line of the picture (a) and the picture (b); (g) the intensity distribution at the position of the blue solid line in (c) and (d). Scale bar: 5 µm in (a) and (b), 1 µm in the enlarged section in (a) and (b), and 1 µm in (c) and (d).

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Fig. 8.
Fig. 8. Microtubules of huFIB cells 3D SIM imaging results. (a, b, c) SIM reconstruction image (right) and wide-filed image (left) at axial positions of z = 0 µm, 1.25 µm and 3.125 µm ; (d) Axial slices cut through the green line in (a); (e) Axial slices cut through the blue line in (a). Scale bars, 5 µm in (a, b, c), 1 µm in (d, e).

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Tables (1)

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Table 1. Highest achievable frame rates for different region of interest and different exposure time

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