The 2014 Nobel Prize in chemistry was awarded for the groundbreaking advancement of superresolution microscopy, which surpasses the fundamental diffraction limit of spatial resolution.1
Advanced Photonics Nexus, Volume. 3, Issue 1, 016001(2024)
High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy Article Video
In recent years, notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy (SIM). Nevertheless, the advancement of three-dimensional structured illumination microscopy (3DSIM) has been impeded by challenges arising from the speed and intricacy of polarization modulation. We introduce a high-speed modulation 3DSIM system, leveraging the polarization-maintaining and modulation capabilities of a digital micromirror device (DMD) in conjunction with an electro-optic modulator. The DMD-3DSIM system yields a twofold enhancement in both lateral (133 nm) and axial (300 nm) resolution compared to wide-field imaging and can acquire a data set comprising 29 sections of 1024 pixels × 1024 pixels, with 15 ms exposure time and 6.75 s per volume. The versatility of the DMD-3DSIM approach was exemplified through the imaging of various specimens, including fluorescent beads, nuclear pores, microtubules, actin filaments, and mitochondria within cells, as well as plant and animal tissues. Notably, polarized 3DSIM elucidated the orientation of actin filaments. Furthermore, the implementation of diverse deconvolution algorithms further enhances 3D resolution. The DMD-based 3DSIM system presents a rapid and reliable methodology for investigating biomedical phenomena, boasting capabilities encompassing 3D superresolution, fast temporal resolution, and polarization imaging.
1 Introduction
The 2014 Nobel Prize in chemistry was awarded for the groundbreaking advancement of superresolution microscopy, which surpasses the fundamental diffraction limit of spatial resolution.1
Various implementations have been developed to realize the structured illumination and polarization modulation of the 3DSIM system, whose crucial device includes phase grating9,19 and a ferroelectric liquid crystal spatial light modulator (FLC-SLM). Phase grating5,9,20 with a fixed linear polarizer is rotating and laterally translated to control the direction, phase, and polarization of the structured light, but the imaging speed is limited by the mechanical rotation (
The digital micromirror device (DMD) is a kind of SLM that utilizes the electromechanical rotation of micromirrors to modulate the light field reflecting off DMD. Its fast response speed, low cost, and wide availability make it a popular choice for projectors and scientific research instruments. As each micromirror is controlled in the binary form corresponding to on and off states, DMD can also become a digital reflection grating when loading with a specific pattern and thus can provide a rapid switch of structured illumination patterns for 3DSIM. Compared with FLC-SLM-based 3DSIM, DMD-based 3DSIM possesses the following advantages in the interference SIM system. First, after loading pattern images, DMD maintains a working state and does not require a refresh cycle, as FLC-SLM does, which greatly simplifies the timing control of the SIM system. Then, DMD has a higher switching speed, with the latest products capable of switching speeds up to 23 kHz/1 bit (DLP9500, TI), making it more suitable for fast imaging of live cells. In addition, due to the nature of the coating on the DMD surface, DMD can preserve the polarization state of reflected light the same as the incident light. With an electro-optic modulator (EOM) capable of switching speeds up to the order of nanoseconds, DMD enables ultrafast imaging with minimal motion artifacts. All of these aspects show the greater potential and inspired us to combine DMD and EOM to build a 3DSIM system. (Section S1 in the Supplementary Material shows the comparison of three representative SIM methods with various polarization control schemes, and Sec. S2 in the Supplementary Material shows the polarization extinction ratio of DMD-based SIM with three polarization control schemes.) Overall, SIM, with its remarkable capabilities and flexibility, combined with the distinct advantages offered by DMDs, holds tremendous potential for advancing superresolution microscopy techniques.
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In this work, we present a novel and high-speed autopolarization synchronization modulation 3DSIM system, leveraging the autopolarization-maintenance capability of DMD, in conjunction with the polarization modulation capability of EOM for the first time, we believe. By harnessing these advanced technologies, our developed system demonstrates exceptional superresolution imaging capabilities in all three dimensions
2 Methods
2.1 Optical Setup
An optical schematic for the 3DSIM setup is shown in Fig. 1(a). An excitation laser beam of 561 nm was polarized via a Glan–Taylor polarizing prism and modulated by a polarization rotator composed of an EOM and a quarter-wave plate. With linearly polarized light incident on the polarization rotator, output polarization can be rotated in azimuth at a speed up to 250 kHz by adjusting the liquid crystal retardation of the EOM. The emergent light was collimated and expanded to a diameter of 13 mm via a beam expander and then illuminated on the pattern generator DMD, a
Figure 1.Diagrams of the hardware of 3DSIM. (a) Optical setup. (b) The illumination polarization before entering the EOM remains the same (column 1), and the beam that passes through the polarization rotator exhibits different polarization states (column 2) due to the property of linear polarization rotation of the polarization rotator. Three patterns with different orientations are preloaded in the DMD (column 3). The positions of the 0 and
2.2 Data Acquisition and Time Sequence of the Synchronization of the System
The acquisition of 3D data involved the use of five pattern phases spaced evenly at intervals of
2.3 Illumination and Reconstruction Principle of 3DSIM
Because of the characteristics of the optical system, the optical transfer function (OTF) has a leaky-cone shape.9 This causes a defocus background and limitation in the
Figure 2.Principle and algorithm flow of 3DSIM. (a) Separated spectrum bands fill the leaky-cone OTF and double the 3D spectrum with the corresponding 3D insight in (b). (c) Algorithm flow of 3DSIM, including the middle spectrum results.
Different from 2D-SIM, which has 0th and 1st harmonics, the illumination light of 3DSIM can be composited into the 0th, 1st, and 2nd harmonics, and can be expressed as22
So, the 3D Fourier transform of emission light distribution
Normally, we set the three illumination angles of
The detailed algorithm flow is shown in Fig. 2(c).
2.4 Open-Source 3DSIM Hardware
Together with our recently published Open-3DSIM software,24 we have made all the hardware components and control mechanisms openly available in this study as a complementary open-source hardware counterpart. We have opened the detailed hardware devices in Sec. S5 in the Supplementary Material and the LabVIEW program in GitHub repository (available at: https://github.com/Cao-ruijie/DMD-3DSIM-hardware) to control the sequence of DMD, sCMOS camera, EOM, and piezoelectric translation platform for reproduction and development of the 3DSIM system (S6 in the Supplementary Material). Given the widespread utilization of DMD technology in digital light processing and its cost-effectiveness as a consumer product, we anticipate that our initiatives will significantly support the community in devising diverse adaptations and iterations, capitalizing on the foundation established by our open hardware approach.
3 Results
To quantify the enhanced resolution and qualify the optical sectioning capability of our 3DSIM approach, we initially imaged 100 nm fluorescent beads using a raw-frame exposure time of 15 ms and a volume time of 6.75 s (29 sections, 15 frames/section, and 125 nm sectioning step). A comparison was made between the wide-field (WF) image and 3DSIM image, as depicted in Fig. 3(a). In the magnified view of the region outlined by the blue rectangle, the 3DSIM image successfully resolves three adjacent beads that are indistinguishable in the WF image, highlighting a significant improvement in lateral resolution. Furthermore, the
Figure 3.Resolution test of DMD-based 3DSIM system and superresolution imaging of nuclear pore complex. (a) The WF and 3DSIM images of 100 nm fluorescent beads with the corresponding profiles of the intensity distribution (bottom right) along the magnified beads (top right) and the
3.1 3DSIM and Polar-3DSIM Imaging of Subcellular Organelles
We utilized 3DSIM to image more intricate subcellular structures with finely detailed 3D features encompassed mitochondrial cristae and outer membranes, microtubule proteins, and actin filaments. Figures 4(a) and 4(b) show WF and 3DSIM images showcasing the mitochondrial cristae and outer membrane, respectively. It can be seen that the 3DSIM image provides a distinct view of the intricate internal structure of the mitochondrial cristae, while WF cannot [see Fig. 4(a)]. Furthermore, the 3DSIM image of the mitochondrial outer membrane exhibits higher resolution compared to WF, with a reduced impact in the defocused background [see Fig. 4(b)]. Furthermore, we conducted imaging of two linear subcellular structures, namely actin and tubulin. Figure 4(c) shows the imaging results for tubulin in COS7, where individual microtubules can be traced and observed throughout the entire cell volume, and many parallel microtubules can be distinctly resolved as separate entities. These findings demonstrate the superior capability of 3DSIM in revealing fine details and enhancing resolution for subcellular structures. In addition, we introduce polarization dimension27
Figure 4.3DSIM reconstruction of subcellular structures. Maximum intensity projection (MIP) images of (a) mitochondrial cristae and (b) mitochondrial outer membrane with the corresponding magnified zone. (c) MIP images of tubulin in the form of a depth-color map. (d) Orientation imaging of actin filament and comparisons among WF, 3DSIM, and p3DSIM. The exposure time in (a)–(d) are 25, 20, 15, and 18 ms, respectively. (a) 29 layers. (b) 22 layers. (c) 23 layers. (d) 25 layers. Scale bar:
Figure 5.3DSIM reconstruction of plant and animal tissue samples. MIP images of (a) veins in oleander leaves, (b) the hollow structures within black algal leaves, and (c) the periodic aggregation and dispersion of structures in the root tips of corn tassels. The corresponding 3D MIP images are shown at the bottom. (d)–(f) WF, 3DSIM, and p3DSIM MIP images of actin filaments in the mouse kidney tissue slice, with the corresponding
3.2 3D-SIM Superresolution of Thick Scattering Specimen
We employed 3DSIM to image thick plant and animal tissues with a stronger defocus background. Figures 5(a)–5(c) present the WF and 3DSIM results of oleander leaves, black algal leaves, and corn tassels, respectively. In the 3DSIM images, distinct features become apparent that were not observable in conventional WF images. These include the 3D distribution of cell walls in oleander leaves, the hollow structures within black algal leaves, and the periodic aggregation and dispersion of structures in the root tips of corn tassels. In addition, we further analyzed the orientation of fluorescent dipoles within actin filaments in the mouse kidney tissue slice in Fig. 5(d). In the maximum projection image obtained from WF of the mouse kidney slices, the presence of out-of-focus scattered signals and the inability to distinguish in-focus information resulted in a less pronounced visualization of the filamentous structure. However, 3DSIM endows it with superior optical sectioning capabilities, presenting a clear representation of actin filaments, due to the higher resolution. Moreover, we utilized polar 3DSIM to investigate again the alignment of fluorescent dipoles within actin filaments in
Figure 6.Deconvolution algorithms further improve the resolution of the 3DSIM system. (a) The WF, 3DSIM, and RL3D results of 100 nm beads in the 12th layer with (b) the corresponding profile of the intensity distribution along the magnified bead, indicating the improved resolution in the
3.3 Computational 3D Deconvolution
Lastly, we use different 3D deconvolution algorithms to prove that computational superresolution technology can further improve the 3D resolution of our 3DSIM approach. We use 3D Richardson–Lucy (RL3D) deconvolution30 for the postprocessing of the 3DSIM image of 100 nm beads as shown in Fig. 6(a), finding that RL3D can further improve 3D resolution compared with 3DSIM, as the profiles in Fig. 6(b) show. We use sparse deconvolution31 to do the postprocessing of 3DSIM results of clathrin-coated pits (CCPs) in Fig. 6(c). Although 3DSIM can interpret the 3D hollow structure of CCPs, sparse deconvolution can make the hollow structure clearer. This is consistent with the physiological structure of CCPs,32 and similar results were reported with 3D single-molecule localization microscopy.33 Then, we use MRA deconvolution34 for the processing of tubulin structure of the low signal-to-noise ratio in Fig. 6(d). We can find that the artifacts of 3DSIM caused by the defocus and noise can be greatly suppressed using MRA, with an improved 3D resolution. These results show that our solution is suitable for many postprocessing algorithms, which can further improve the resolution and quality of 3DSIM reconstruction.
4 Discussion and Conclusion
First, due to the limitation of experimental conditions (we do not have a live cell incubation system in our custom-built microscope), our current DMD-based 3DSIM setup can only perform single-color imaging with a fixed specimen. But we can further introduce other lasers to incident DMD at different angles to realize multicolor imaging without change for the most part of the system, as referred to in our proposed four-color DMD-based SIM concept.35 Meanwhile, regarding the potential applications of DMD-3DSIM for real-time and dynamic live cell imaging, it is better to equip the system with a microscope body and live cell incubator for spatial and temporal stability. And the fast switch of EOM and DMD exploits the potential to conduct the imaging of fast-moving living cells with the help of these improvements.
Second, in our DMD-based 3DSIM system, we conducted tests to measure the maximal data acquisition speed for single-layer 3DSIM with varying field-of-view (FOV) sizes in synchronous readout trigger mode (Sec. S11 in the Supplementary Material). We can achieve the maximum
Third, although we have used various deconvolution algorithms to optimize the images (both sparse and MRA deconvolution use the 2D OTF without considering the complex defocus background), advanced 3D algorithms36,37 are still required to faithfully reconstruct and optimize low signal-to-noise ratio data acquired from high-speed live cell imaging.
In summary, we demonstrated our DMD-based 3DSIM system as a fast and reliable tool for subcellular structures, tissue architectures, and fluorescent dipole imaging with a 3D spatial resolution twice that of WF, and a computational superresolution algorithm can further improve the resolution of our system. Our system features the advantages of fast switching and accurate polarization modulation, opening up a new avenue for exploration and providing an invaluable tool for unraveling the complex mechanisms underlying fundamental biological processes.
5 Appendix: Supplementary Material
Yaning Li is a PhD student in the Department of Biomedical Engineering, College of Future Technology, Peking University, China. Her research interests include two-dimensional and three-dimensional structured illumination microscopy based on digital micro-mirror devices.
Ruijie Cao received his BS degree from Nanjing Normal University in 2022. Currently, he is a PhD candidate in the College of Future Technology, Peking University. His research interests include hardware and software of structure illumination microscopy.
Wei Ren received his BS degree from North University of China in 2020. Currently, he is a PhD candidate in the Department of Biomedical Engineering, Peking University. His research interests include super-resolution microscopy and mitochondrial dynamics.
Yunzhe Fu received his BS degree from Nanjing University of Science and Technology in 2021. Currently, he is a PhD student in the Department of Biomedical Engineering, Peking University. His research interests include the study of multiple subcellular organelle interactions using multimodal ultrafast low phototoxicity super-resolution microscopy.
Yiwei Hou received his BS degree from the University of Electronic Science and Technology of China in 2022. Currently, he is a PhD student in the Department of Biomedical Engineering, Peking University. His research interests include computational fluorescence imaging and structured illumination microscopy.
Suyi Zhong received her BE degree from Central South University in 2018 and her ME degree from Tsinghua University in 2021. Currently, she is a PhD student at the College of Future Technology, Peking University, Beijing, China. Her research interests include light-sheet microscopy and lensfree microscopy.
Karl Zhanghao received his BS degree from Peking University in 2012 and his PhD from Peking University and Georgia Institute of Technology. He is a research assistant professor in the Department of Biomedical Engineering, Southern University of Science and Technology. His research interests include super-resolution microscopy, multidimensional live-cell imaging, and fluorescence polarization microscopy.
Meiqi Li is an engineer at the School of Life Sciences, Peking University, China. She received her PhD from the Department of Biomedical Engineering at Peking University, and her BS degree in automation from the Harbin Institute of Technology. Her research is focused on structured illumination microscopy and fluorescence polarization microscopy.
Peng Xi is a Boya professor at the College of Future Technology, Peking University, China. His research interest is on optical super-resolution microscopy. He has been awarded the National Distinguished Young Scholar and the fellow of IAAM. He is the chief scientist of the Key R&D Plan of the Ministry of Science and Technology. He has published over 90 scientific journal papers on high impact journals including Nature, Nature Methods, etc., with > 6000 citations.
[4] L. Schermelleh et al. Super-resolution microscopy demystified. Nat. Cell Biol., 21, 72-84(2019).
[34] H. Yiwei et al. Noise-robust, physical microscopic deconvolution algorithm enabled by multi-resolution analysis regularization(2023).
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Yaning Li, Ruijie Cao, Wei Ren, Yunzhe Fu, Yiwei Hou, Suyi Zhong, Karl Zhanghao, Meiqi Li, Peng Xi, "High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy," Adv. Photon. Nexus 3, 016001 (2024)
Category: Research Articles
Received: Sep. 16, 2023
Accepted: Nov. 21, 2023
Published Online: Dec. 25, 2023
The Author Email: Meiqi Li (limeiqi@pku.edu.cn), Peng Xi (xipeng@pku.edu.cn)