Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors

Chen, Youhua; Cao, Ruizhi; Liu, Wenjie; Zhu, Dan; Zhang, Zhiming; Kuang, Cuifang; Liu, Xu

doi:10.1117/1.jbo.23.4.046007

Cited by 20 publications

(10 citation statements)

References 26 publications

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“…A third way to generate the structured light is by scanning the sample with galvanometers (Figure 3). Using this approach, an entire U2OS cell with a field of view greater than 40 μm *40 μm was recorded in 4 s at 10 fps [31,32]. However, this system is more complex than the system shown in Figure 2 and requires precise adjustment [33].…”

Section: Generation Of Structured Lightmentioning

confidence: 99%

Advances in High-Speed Structured Illumination Microscopy

et al. 2021

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Super-resolution microscopy surpasses the diffraction limit to enable the observation of the fine details in sub-cellular structures and their dynamics in diverse biological processes within living cells. Structured illumination microscopy (SIM) uses a relatively low illumination light power compared with other super-resolution microscopies and has great potential to meet the demands of live-cell imaging. However, the imaging acquisition and reconstruction speeds limit its further applications. In this article, recent developments all targeted at improving the overall speed of SIM are reviewed. These comprise both hardware and software improvements, which include a reduction in the number of raw images, GPU acceleration, deep learning and the spatial domain reconstruction. We also discuss the application of these developments in live-cell imaging.

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Section: Generation Of Structured Lightmentioning

confidence: 99%

Advances in High-Speed Structured Illumination Microscopy

et al. 2021

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“…In one study, a thin homogenous layer of fluorophores in air was used to produce a SAF ring of known radius r c (air) ¼ f obj that was compared to the annular distribution of excitation light in azimuthal beam-spinning TIRF with the fluorescence filter removed (65). Two follow-up studies used a similar strategy but with either a thin layer of organic dye for TIRF-SIM calibration (45) or else with Qdots embedded in poly(methyl methacrylate) for A-TIRFM (66). In the latter case, the fluorophore layer was topped with solutions of different, known RI (previously measured with an Abbe refractometer), and the laser spot (seen when the emission filter was taken out) was steered to these known angles to attain a more precise multiangle calibration.…”

Section: Saf Applications With Aperture Filteringmentioning

confidence: 99%

Supercritical Angle Fluorescence Microscopy and Spectroscopy

Oheim

Salomon

Brunstein

2020

Biophysical Journal

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Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing, and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near) field, and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF), is comparably less established, although it achieves a similar optical sectioning as TIRF does. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission couples to the higher refractive index medium (n 2 > n 1 ) and becomes propagative. Then, most fluorescence is detectable on the side of the higher-index substrate, and a large fraction of this fluorescence is emitted into angles forbidden by Snell's law. SAF, as well as the undercritical angle fluorescence (UAF; far-field emission) components, can be collected with microscope objectives having a high-enough detection aperture (numerical aperture > n 2 ) and be separated in the back focal plane by Fourier filtering. The back focal plane image encodes information about the fluorophore radiation pattern, and it can be analyzed to yield precise information about the refractive index in which the emitters are embedded, their nanometric distance from the interface, and their orientation. A SAF microscope can retrieve this near-field information through wide-field optics in a spatially resolved manner, and this functionality can be added to an existing inverted microscope. Here, we describe the potential underpinning of SAF microscopy and spectroscopy, particularly in comparison with TIRF. We review the challenges and opportunities that SAF presents from a biophysical perspective, and we discuss areas in which we see potential.

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“…The initial I 5 S implementation used gratings to split the illumination light into three diffracted beams (order 0th and ±1st), and the interference between them produced a 3D sinusoidal pattern. In contrast to the diffraction grating, ferroelectric liquid crystal spatial light modulator [30,31] and scanning galvanometer mirror (SGM) [32][33][34] can satisfy the demands of faster and more flexible pattern generation. As a result, a combination of SGM-based I 5 S and fluorescence saturation [35] termed as saturated I 5 S or SI 5 S [36] was developed in theory and obtained near-isotropic 3D resolution below 60 nm.…”

Section: Widefield Modalitymentioning

confidence: 99%

A Review on Dual-Lens Fluorescence Microscopy for Three-Dimensional Imaging

Han

Liu

et al. 2020

Front. Phys.

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Three-dimensional (3D) imaging using dual-lens fluorescence microscopies is popular in observing fluorescently labeled biological samples, such as mammalian/model animal cells, tissues, and embryos. Specifically, dual-lens super-resolution fluorescence microscopy methods using two opposing objective lenses allow significantly higher axial resolution and better signal to noise ratio than traditional single-lens counterparts, and thus distinguish more details in 3D images of fine intracellular structures. For 3D imaging of thick tissues and entire embryos, dual-lens light-sheet fluorescence microscopy methods using two objective lenses, either orthogonal or non-orthogonal, to achieve selective plane illumination, can meet the requirements, and thus can be used to observe embryo development and structures of interest in thick tissues. This review summarizes both dual-lens fluorescence microscopy methods, including their principles, configurations, and 3D imaging applications, providing a guideline for biological laboratories with different 3D imaging needs.

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Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors

Cited by 20 publications

References 26 publications

Advances in High-Speed Structured Illumination Microscopy

Advances in High-Speed Structured Illumination Microscopy

Supercritical Angle Fluorescence Microscopy and Spectroscopy

A Review on Dual-Lens Fluorescence Microscopy for Three-Dimensional Imaging

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