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(a) Wide-field image. (b) Wide-field HiLo image. (c) Single-scan HiLo image. (a1)–(c1) Enlarged views of the area I corresponding with (a)–(c). (a2)–(c2) Enlarged views of the area II corresponding with (a)–(c). (d)–(e) Normalized intensity profiles along yellow dash lines corresponding with Figs. (a1–c1) and (a2–c2), respectively. Scale bars in (a)–(c): 100 µm. Scale bars in (a1)–(c1) and (a2)–(c2): 20 µm.
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Optical sectioning has been widely employed for inhibiting out-of-focus backgrounds in three-dimensional (3D) imaging of biological samples. However, point scanning imaging or multiple acquisitions for wide-field optical sectioning in epi-illumination microscopy remains time-consuming for large-scale imaging. In this paper, we propose a single-scan...
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Fourier ptychographic microscopy (FPM) can tackle the trade-off between the high resolution and the large field of view. However, the long capturing time limits its application. We propose a self-adapting search algorithm for FPM, termed SAS–FPM, which improves the data acquisition efficiency. Here the sparse arrangement is verified via simulations...
Citations
... In order to further eliminate the artifacts caused by inaccurate estimation of B ASLM−D and subtle differences in the distribution of B ASLM−D and background of raw images from ASLM, I ASLM , we convert the background image into local contrast, C , instead of subtracting B ASLM−D directly from I ASLM . Since the background is only present in the low-frequency part, the high-frequency part of the original ASLM images is completely preserved; we reconstruct high-contrast isotropic high-resolution images, I SD−LSM , by processing the high and low frequencies separately similar to HiLo [38][39][40][41]. The process can be written as ...
The image quality of light-sheet microscopy degrades due to the system misalignment or opacity of the sample. In this work, we proposed to synchronously detect the fluorescence from both the illumination and detection light path of axially swept light-sheet microscopy (SD-LSM) to realize the full exploitation of the excited fluorescence. We adopted spatially variable multi-view deconvolution to fuse images from the detection and illumination objective of SD-LSM to improve the resolution degradation caused by the nonlinearity of scanning devices. We proposed the fusion of images from the detection and illumination objective of SD-LSM based on background estimation to improve the signal-to-background ratio (SBR). We separately demonstrated that the spatial resolution and the SBR can be largely boosted by SD-LSM for various biological samples, after the fusion of images from the illumination and detection path. Compared with the images only from the detection path, images from SD-LSM showed the axial resolution recovery by up to 14.6 times when axial scanning devices work at high speed with large nonlinearity, and SBR enhancement by up to 8.2 dB when imaging a highly scattered sample. SD-LSM could boost the image quality without any additional time consumption for image acquisition or photon budget for the sample at a cost of a simple addition of a camera in the illumination path, compared with conventional axially swept light-sheet microscopy.
... In addition, the clarity of cryo-MOST images is suboptimal due to the use of wide-field imaging. We have previously developed a series of optical sectioning methods based on line scanning to suppress background signals and improve the optical-sectioning ability of high-throughput microscopic imaging [76][77][78][79]. We will introduce these technologies into cryo-MOST to achieve optical-sectioning imaging. ...
Rationale: Mesoscopic visualization of the main anatomical structures of the whole kidney in vivo plays an important role in the pathological diagnosis and exploration of the etiology of hydronephrosis. However, traditional imaging methods cannot achieve whole-kidney imaging with micron resolution under conditions representing in vivo perfusion.
Methods: We used in vivo cryofixation (IVCF) to fix acute obstructive hydronephrosis (unilateral ureteral obstruction, UUO), chronic spontaneous hydronephrosis (db/db mice), and their control mouse kidneys for cryo-micro-optical sectioning tomography (cryo-MOST) autofluorescence imaging. We quantitatively assessed the kidney-wide pathological changes in the main anatomical structures, including hydronephrosis, renal subregions, arteries, veins, glomeruli, renal tubules, and peritubular functional capillaries.
Results: By comparison with microcomputed tomography imaging, we confirmed that IVCF can maintain the status of the kidney in vivo. Cryo-MOST autofluorescence imaging can display the main renal anatomical structures with a cellular resolution without contrast agents. The hydronephrosis volume reached 26.11 ± 6.00 mm³ and 13.01 ± 3.74 mm³ in 3 days after UUO and in 15-week-old db/db mouse kidneys, respectively. The volume of the cortex and inner stripe of the outer medulla (ISOM) increased while that of the inner medulla (IM) decreased in UUO mouse kidneys. Db/db mice also showed an increase in the volume of the cortex and ISOM volume but no atrophy in the IM. The diameter of the proximal convoluted tubule and proximal straight tubule increased in both UUO and db/db mouse kidneys, indicating that proximal tubules were damaged. However, some renal tubules showed abnormal central bulge highlighting in the UUO mice, but the morphology of renal tubules was normal in the db/db mice, suggesting differences in the pathology and severity of hydronephrosis between the two models. UUO mouse kidneys also showed vascular damage, including segmental artery and vein atrophy and arcuate vein dilation, and the density of peritubular functional capillaries in the cortex and IM was reduced by 37.2% and 49.5%, respectively, suggesting renal hypoxia. In contrast, db/db mouse kidneys showed a normal vascular morphology and peritubular functional capillary density. Finally, we found that the db/db mice displayed vesicoureteral reflux and bladder overactivity, which may be the cause of hydronephrosis formation.
Conclusions: We observed and compared main renal structural changes in hydronephrosis under conditions representing in vivo perfusion in UUO, db/db, and control mice through cryo-MOST autofluorescence imaging. The results indicate that cryo-MOST with IVCF can serve as a simple and powerful tool to quantitatively evaluate the in vivo pathological changes in three dimensions, especially the distribution of body fluids in the whole kidney. This method is potentially applicable to the three-dimensional visualization of other tissues, organs, and even the whole body, which may provide new insights into pathological changes in diseases.
... In a picoSIM system (Fig. 5c), three individual light patterns are encoded in a single polarized illumination light distribution, allowing the acquisition of all SIM data needed for the computational reconstruction of a sectioned image in a single exposure. Recently, several improved technologies have been proposed for optical section imaging of uncleared thick tissues, including linescanning SIM 40 , HiLo endomicroscopy 41 , and single-scan HiLo 42 . Among them, a single-scan HiLo method can obtain a wide-field image and its HiLo image in a single scan and is faster than the previous two methods for acquiring multiple thick tissue images. ...
Structured illumination microscopy (SIM) has become the standard for next-generation wide-field microscopy, offering ultrahigh imaging speed, superresolution, a large field-of-view, and long-term imaging. Over the past decade, SIM hardware and software have flourished, leading to successful applications in various biological questions. However, unlocking the full potential of SIM system hardware requires the development of advanced reconstruction algorithms. Here, we introduce the basic theory of two SIM algorithms, namely, optical sectioning SIM (OS-SIM) and superresolution SIM (SR-SIM), and summarize their implementation modalities. We then provide a brief overview of existing OS-SIM processing algorithms and review the development of SR-SIM reconstruction algorithms, focusing primarily on 2D-SIM, 3D-SIM, and blind-SIM. To showcase the state-of-the-art development of SIM systems and assist users in selecting a commercial SIM system for a specific application, we compare the features of representative off-the-shelf SIM systems. Finally, we provide perspectives on the potential future developments of SIM.
... However, the construction of modulating illumination by multiple scans is a time-consuming process. Given this problem, our group proposes a series of single-scan SIM approach to avoid repetitive data acquisition [17][18][19]. Although the methods mentioned above enhance the optical sectioning ability and imaging speed, SIM algorithms, such as square law detection [11] and high and low-frequency filtering [12], don't improve spatial resolution. ...
... The number N is equal to the number of camera exposure lines. The PSF of TDI h TDI can be written as [17]: ...
Line confocal (LC) microscopy is a fast 3D imaging technique, but its asymmetric detection slit limits resolution and optical sectioning. To address this, we propose the differential synthetic illumination (DSI) method based on multi-line detection to enhance the spatial resolution and optical sectioning capability of the LC system. The DSI method allows the imaging process to simultaneously accomplish on a single camera, which ensures the rapidity and stability of the imaging process. DSI-LC improves X- and Z-axis resolution by 1.28 and 1.26 times, respectively, and optical sectioning by 2.6 times compared to LC. Furthermore, the spatially resolved power and contrast are also demonstrated by imaging pollen, microtubule, and the fiber of the GFP fluorescence-labeled mouse brain. Finally, Video-rate imaging of zebrafish larval heart beating in a 665.6 × 332.8 µm² field-of-view is achieved. DSI-LC provides a promising approach for 3D large-scale and functional imaging in vivo with improved resolution, contrast, and robustness.
... As a result, the image quality degrades rapidly with increasing imaging depth. 15 In this paper, we present a confocal rescan structured illumination microscope (CR-SIM) to address the limitation of existing SR-SIM techniques in terms of background rejection, noise suppression, and imaging speed. An image rescan technique 16 originally developed for a point-scan system is adapted to the line-scan configuration and combined with SIM to obtain superresolution in two orthogonal directions. ...
... By distinguishing the parts with and without gold nanoposts, it was confirmed that the resolutionenhanced image obtained using structured illumination had a higher dynamic range than the wide-field image. To quantitatively compare the dynamic ranges of the resolution-enhanced and wide-field images, the signal-to-background ratio (SBR) [38,39] between the signals from a gold nanopost and a substrate between the nanoposts was calculated for each profile using the following equation: ...
We developed a structured illumination-based optical inspection system to inspect metallic nanostructures in real time. To address this, we used post-image-processing techniques to enhance the image resolution. To examine the fabricated metallic nanostructures in real time, a compact and highly resolved optical inspection system was designed for practical industrial use. Structured illumination microscopy yields multiple images with various linear illumination patterns, which can be used to reconstruct resolution-enhanced images. Images of nanosized posts and complex structures reflected in the structured illumination were reconstructed into images with improved resolution. A comparison with wide-field images demonstrates that the optical inspection system exhibits high performance and is available as a real-time nanostructure inspection platform. Because it does not require special environmental conditions and enables multiple systems to be covered in arrays, the developed system is expected to provide real-time and noninvasive inspections during the production of large-area nanostructured components.
... iScience 25, 104805, August 19, 2022 3 iScience Article experimental requirements, such as digital structured modulation (DSM) (Zhong et al., 2021a), line-illumination modulation (LiMo) (Zhong et al., 2021b), hybrid illumination (HiLo) (Qiao et al., 2021), and time delay integration (TDI) . In the DSM mode, the FWHMs in the X, Y, and Z directions were 0.81 G 0.01, 0.70 G 0.03, and 3.35 G 0.03 mm, respectively, whereas those of the red channel were 0.90 G 0.01, 0.80 G 0.02, and 3.68 G 0.01 mm, respectively ( Figure S4B). ...
Optical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.
... To further increase the imaging rate, single-shot optical sectioning methods have been actively researched in recent years. Good examples include Fourier band-pass filtering [13], deep learning [14,15], and line-illumination modulation microscopy (LiMo) [16,17]. The first approach reconstructs an optical sectioning image from one raw image by shifting the in-focus signals to the +1 st order in the Fourier domain at the expense of compromised lateral resolution due to reduced high-frequency information. ...
In this Letter, we present a single-shot 3D-resolved structured illumination microscopy (SIM) based on a digital micromirror device (DMD), a galvanometric mirror, and the HiLo algorithm. During imaging, the DMD rapidly generates sinusoidal and plane illuminations in the focal region. By synchronizing the DMD with a galvanometric scanner and exploiting the unique data readout process of the camera, the emissions from the specimen under two different illuminations, i.e., structured and uniform illumination, are projected to different regions on a camera, achieving high-resolution single-exposure optical sectioning at the camera’s limiting speed, i.e., 200 Hz, without sacrificing the resolution. A model has been developed to guide the design and optimization of the optical system. Imaging experiments on pollen and mouse kidney samples have been performed to verify the predicted performance. The results show that the single-shot SIM with the HiLo algorithm achieves comparable resolution to the standard two-shot HiLo method with a twofold speed enhancement, which may find important applications in biophotonics, e.g., visualizing high-speed biological events in vivo.
... Although the high photon throughput of SiPM detectors enables high imaging frame rates, this does not directly translate into proportionally faster mosaic imaging of large specimens because at high imaging rates an increasingly large fraction of the total imaging time is spent translating the specimen between positions. To address this, previous work in light sheet, 18 confocal, 19,20 line-scan confocal, 21 fluorescence microscopy, 22 and HiLo microscopy 23 has utilized so-called "strip mode" or "push broom" scanning wherein the specimen is translated at a constant velocity through a fixed line imaging position. By setting the translation speed proportionally to the line imaging time, lines of adjacent pixels along the translation axis can be assembled into seamless frames. ...
Significance:
Two-photon and confocal microscopy can obtain high frame rates; however, mosaic imaging of large tissue specimens remains time-consuming and inefficient, with higher imaging rates leading to a larger fraction of time wasted translating between imaging locations. Strip scanning obtains faster mosaic imaging rates by translating a specimen at constant velocity through a line scanner at the expense of more complex stitching and geometric distortion due to the difficulty of translating at completely constant velocity.
Aim:
We aim to develop an approach to mosaic imaging that can obtain higher accuracy and faster imaging rates while reducing computational complexity.
Approach:
We introduce an approach based on scanner-synchronous position sampling that enables subwavelength accurate imaging of specimens moving at a nonuniform velocity, eliminating distortion.
Results:
We demonstrate that this approach increases mosaic imaging rates while reducing computational complexity, retaining high SNR, and retaining geometric accuracy.
Conclusions:
Scanner synchronous strip scanning enables accurate, high-speed mosaic imaging of large specimens by reducing acquisition and processing time.
... Recently, the HiLo technique with excellent optical sectioning ability [1, [14][15][16][17][18] based on only two captured images, a uniform illuminated image and a structured illuminated image, has been widely used in conventional wide-field microscopy [19,20] , wide-field microendoscopy [21,22] , wide-field multiphoton microscopy [23] , light-sheet microscopy [24][25][26] , light field microscopy [27] , multi-focus microscopy [28,29] and incoherent holography [30] , etc. ...
Optical sectioning technology has been widely used in various fluorescence microscopes owing to its background removing capability. Here, a virtual HiLo based on edge detection (V-HiLo-ED) is proposed to achieve wide-field optical sectioning, which requires only single wide-field image. Compared with conventional optical sectioning technologies, its imaging speed can be increased by at least twice, meanwhile maintaining nice optical sectioning performance, low cost, and excellent artifact suppression capabilities. Furthermore, the new V-HiLo-ED can also be extended to other non-fluorescence imaging fields. This simple, cost-effective and easy-to-extend method will benefit many research and application fields that needs to remove out-of-focus blurred images.
