Nonlinear Structured Illumination Microscopy Research Articles

Saturable absorption assisted nonlinear structured illumination microscopy.

We propose a novel, to the best of our knowledge, super-resolution technique, namely saturable absorption assisted nonlinear structured illumination microscopy (SAN-SIM), by exploring the saturable absorption property of a material. In the proposed technique, the incident sinusoidal excitation is converted into a nonlinear illumination by propagating through a saturable absorbing material. The effective nonlinear illumination possesses higher harmonics which multiply fold high frequency components within the passband and hence offers more than two-fold resolution improvement over the diffraction limit. The theoretical background of the technique is presented, supported by the numerical results. The simulation is performed for both symmetric as well as random samples where the raw moiré frames are processed through a blind reconstruction approach developed for the nonlinear SIM. The results demonstrate the super-resolution capability of the proposed technique.

Frequency-spatial domain joint optimization for improving super-resolution images of nonlinear structured illumination microscopy.

Introducing nonlinear fluorophore excitation into structured illumination microscopy (SIM) can further extend its spatial resolution without theoretical limitation. However, it is a great challenge to recover the weak higher-order harmonic signal and reconstruct high-fidelity super-resolution (SR) images. Here, we proposed a joint optimization strategy in both the frequency and spatial domains to reconstruct high-quality nonlinear SIM (NL-SIM) images. We demonstrate that our method can reconstruct SR images with fewer artifacts and higher fidelity on the BioSR dataset with patterned-activation NL-SIM. This method could robustly overcome one of the long-lived obstacles on NL-SIM imaging, thereby promoting its wide application in biology.

Image reconstruction approach for a high space-bandwidth product structured illumination microscopy system

Conventional structured illumination microscopy (SIM) utilizes a sinusoidal excitation pattern of frequency within the detection passband and provides a maximum of twofold resolution enhancement over the diffraction limit. A transmission approach proposed in an earlier publication [J. Phys. D 53, 044006 (2019)JPAPBE0022-372710.1088/1361-6463/ab4e68] to further improve the lateral resolution requires sequential higher frequency illumination patterns. However, the existing reconstruction algorithms fail to deliver appropriate reconstruction when the excitation frequency lies far from the passband boundary. Here, we present a correlation-based SIM reconstruction approach for sequential high-frequency illumination patterns even if the pattern frequency lies far from the passband limit. The scheme can be suitably implemented in a variety of custom-built systems where illumination frequency lies beyond the passband support (e.g., non-linear SIM and plasmonic SIM).

Nonlinear scanning structured illumination microscopy based on nonsinusoidal modulation

Structured illumination microscopy (SIM) is an essential super-resolution microscopy technique that enhances resolution. Several images are required to reconstruct a super-resolution image. However, linear SIM resolution enhancement can only increase the spatial resolution of microscopy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function. The frequency of the pattern generated by the nonlinear response in samples is not limited; therefore, nonlinear SIM (NL-SIM), in theory, has no inherent limit to the resolution. In the present study, we describe a two-photon nonlinear SIM (2P-SIM) technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern, which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process. The theoretical models of super-resolution imaging were established through our simulation, which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the resolution. The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.

Unbalanced SSFP for super-resolution in MRI.

Purpose:To achieve rapid, low specific absorption rate (SAR) super-resolution imaging by exploiting the characteristic magnetization off-resonance profile in SSFP.Theory and Methods:In the presented technique, low flip angle unbalanced SSFP imaging is used to acquire a series of images at a low nominal resolution that are then combined in a super-resolution strategy analogous to non-linear structured illumination microscopy. This is demonstrated in principle via Bloch simulations and synthetic phantoms, and the performance is quantified in terms of point-spread function (PSF) and SNR for gray and white matter from field strengths of 0.35T to 9.4T. A k-space reconstruction approach is proposed to account for B0 effects. This was applied to reconstruct super-resolution images from a test object at 9.4T.Results:Artifact-free super-resolution images were produced after incorporating sufficient preparation time for the magnetization to approach the steady state. High-resolution images of a test object were obtained at 9.4T, in the presence of considerable B0 inhomogeneity. For gray matter, the highest achievable resolution ranges from 3% of the acquired voxel dimension at 0.35T, to 9% at 9.4T. For white matter, this corresponds to 3% and 10%, respectively. Compared to an equivalent segmented gradient echo acquisition at the optimal flip angle, with a fixed TR of 8 ms, gray matter has up to 34% of the SNR at 9.4T while using a ×10 smaller flip angle. For white matter, this corresponds to 29% with a ×11 smaller flip angle.Conclusion:This approach achieves high degrees of super-resolution enhancement with minimal RF power requirements.

Upconversion Nonlinear Structured Illumination Microscopy.

Video-rate super-resolution imaging through biological tissue can visualize and track biomolecule interplays and transportations inside cellular organisms. Structured illumination microscopy allows for wide-field super resolution observation of biological samples but is limited by the strong extinction of light by biological tissues, which restricts the imaging depth and degrades its imaging resolution. Here we report a photon upconversion scheme using lanthanide-doped nanoparticles for wide-field super-resolution imaging through the biological transparent window, featured by near-infrared and low-irradiance nonlinear structured illumination. We demonstrate that the 976 nm excitation and 800 nm upconverted emission can mitigate the aberration. We found that the nonlinear response of upconversion emissions from single nanoparticles can effectively generate the required high spatial frequency components in the Fourier domain. These strategies lead to a new modality in microscopy with a resolution below 131 nm, 1/7th of the excitation wavelength, and an imaging rate of 1 Hz.

Compressive three-dimensional super-resolution microscopy with speckle-saturated fluorescence excitation

Nonlinear structured illumination microscopy (nSIM) is an effective approach for super-resolution wide-field fluorescence microscopy with a theoretically unlimited resolution. In nSIM, carefully designed, highly-contrasted illumination patterns are combined with the saturation of an optical transition to enable sub-diffraction imaging. While the technique proved useful for two-dimensional imaging, extending it to three-dimensions is challenging due to the fading of organic fluorophores under intense cycling conditions. Here, we present a compressed sensing approach that allows 3D sub-diffraction nSIM of cultured cells by saturating fluorescence excitation. Exploiting the natural orthogonality of speckles at different axial planes, 3D probing of the sample is achieved by a single two-dimensional scan. Fluorescence contrast under saturated excitation is ensured by the inherent high density of intensity minima associated with optical vortices in polarized speckle patterns. Compressed speckle microscopy is thus a simple approach that enables 3D super-resolved nSIM imaging with potentially considerably reduced acquisition time and photobleaching.

Surpassing the optical diffraction limit by matrix structured illumination microscopy with patterned excitation and patterned stimulated emission depletion

The resolution of traditional fluorescence microscopy is limited due to the optical diffraction. Various techniques have been developed to surpass the diffraction limit in recent years. Among these methods, nonlinear structured illumination microscopy (nonlinear SIM) is able to provide fast imaging speed, wide field of view and considerable resolution enhancement simultaneously. However, the current developed nonlinear SIM approaches have their own defects. We report a potential better nonlinear SIM for live-cell imaging termed ‘Matrix STED-SIM’ with patterned illumination based on both structured excitation grid and stimulated emission depletion (STED) grid. Theory and simulation analysis are presented for illustrating the feasibility of our method to achieve better super-resolution imaging than other current approaches. In addition, we demonstrate the robustness and potential practicability of this new method considering the impact of image noise and exposures.

Super-resolution Imaging by Two-photon Structured Illumination Microscopy

The application of two-photon excitation to confocal laser scanning microscope (CLSM) leads two-photon laser scanning microscopy (TPLSM) to become an important tool in biology. The simultaneous absorption of two photons can reduce the phototoxicity and scattering, which can realize better imaging depth and high quality image. However, the spatial resolution of TPLSM is limited by optical diffraction, which is a barrier for its application. Structured illumination microscopy (SIM) offers rapid imaging speed for living cells and resolution enhancement by a factor of two. In this paper, we propose a nonlinear two-photon structured illumination microscopy (NTPSIM) that combines SIM with two-photon excitation, enabling resolution in thick specimens imaging twice of the normal resolution. Our method has the ability of less scattering and better depth penetration. Through theoretical simulation, we demonstrated that NTPSIM can improve imaging resolution significantly and provide strong penetrability with rapid imaging speed.

Signal, noise and resolution in linear and nonlinear structured-illumination microscopy.

Structured-illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in-principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency-space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two-dimensional (2D) illumination patterns or sequentially rotated one-dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal-to-noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background. LAY DESCRIPTION: Structured-illumination microscopy (SIM) is a high-resolution light microscopy technique that allows imaging of fluorescence at a resolution about twice the classical diffraction limit. There are various ways that the illumination can be structured, but it is not obvious how the choice of illumination pattern affects the final image quality, especially in view of the noise. We present a detailed performance analysis considering two illumination techniques: sequential illumination with line-gratings that are shifted and rotated during image acquisition and two-dimensional (2D) illumination structures requiring only shift operations. Our analysis is based on analytical theory, supported by simulations of images considering noise. We also extend our analysis to a nonlinear variant of SIM, with which enhanced resolution can be achieved, limited only by noise. This includes nonlinear SIM based on the light-induced switching of the fluorescent molecules between a bright and a dark state. We find sequential illumination with line-gratings to be advantageous in ordinary (linear) SIM, whereas 2D patterns provides a slight signal-to-noise advantage under idealised conditions in nonlinear SIM if there is no nonswitching background.

Label-free super-resolution with coherent nonlinear structured-illumination microscopy

Structured-illumination microscopy enables up to a two-fold lateral resolution improvement by spatially modulating the intensity profile of the illumination beam. We propose a novel way to generalize the concept of structured illumination to nonlinear widefield modalities by spatially modulating, instead of field intensities, the phase of the incident field while interferometrically measuring the complex-valued scattered field. We numerically demonstrate that for second-order and third-order processes an almost four- and six-fold increase in lateral resolution is achievable, respectively. This procedure overcomes the conventional Abbe diffraction limit and provides new possibilities for label-free super-resolution microscopy.

Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength.

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

Theoretical assessment of optical resolution enhancement and background fluorescence reduction by three-dimensional nonlinear structured illumination microscopy using stimulated emission depletion

Three-dimensional structured illumination microscopy (SIM) enlarges frequency cutoff laterally and axially by a factor of two, compared with conventional microscopy. However, its optical resolution is still fundamentally limited. It is necessary to introduce nonlinearity to enlarge frequency cutoff further. We propose three-dimensional nonlinear structured illumination microscopy based on stimulated emission depletion (STED) effect, which has a structured excitation pattern and a structured STED pattern, and both three-dimensional illumination patterns have the same lateral pitch and orientation. Theoretical analysis showed that nonlinearity induced by STED effect, which causes harmonics and contributes to enlarging frequency cutoff, depends on the phase difference between two structured illuminations and that the phase difference of π is the most efficient to increase nonlinearity. We also found that undesirable background fluorescence, which degenerates the contrast of structured pattern and limits the ability of SIM, can be reduced by our method. These results revealed that optical resolution improvement and background fluorescence reduction would be compatible. The feasibility study showed that our method will be realized with commercially available laser, having 3.5 times larger frequency cutoff compared with conventional microscopy.

ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

Theoretical assessment of two-dimensional nonlinear structured illumination microscopy based on structured excitation and structured stimulated emission depletion

The performance of nonlinear structured illumination microscopy (NSIM), which has theoretically infinite optical resolution, largely depends on nonlinear phenomena. Unfortunately, nonlinear effects of excitation saturation and photoswitchable fluorophores suffer from photobleaching and slow switching time, respectively. In contrast, stimulated emission depletion (STED) is also a nonlinear phenomenon, having fast switching time and being potentially harmless. We propose NSIM based on STED, which has structured excitation light and structured STED light, and both structured illuminations have the same pitch and orientation in the sample plane. Theoretical analysis shows that two-structured illumination having the same grating vector can efficiently increase nonlinearity caused by the STED effect and improve optical resolution. We also found that nonlinearity depends on the phase difference between two-structured illumination made by excitation and STED light and that opposite phase is the most efficient to increase harmonic strength. The feasibility study shows that our method can theoretically achieve optical resolution of 1/7 of wavelength with commercially available laser over large field of view.

Full Field Nonlinear Structured Illumination Microscopy with STED

Structured illumination microscopy (SIM) allows fast full-field imaging of biological samples. Linear SIM improves the lateral resolution limit by a factor of two. Once combined with a nonlinear effect of the fluorescence emission, SIM can further suppress the resolution limit to <100 nm, as reported by Photoswitchable and excitation saturated SIM (SSIM) [1, 2]. Here, we present a novel nonlinear SIM method named STED-SIM, which utilizes the stimulated emission depletion (STED) effect to achieve 4-fold resolution enhancement [3].STED-SIM utilizes a 2D grating to generate a 2D structured illumination pattern. The grating is shifted at high speed by piezo stages. Therefore, the imaging speed of 2D STED-SIM in only limited by the speed of the camera. Compared with previously reported nonlinear SIM approaches, STED-SIM has the advantage of fast switching response, negligible stochastic noise in switching. In addition, the achievable resolution of STED-SIM in theory is unlimited.STED-SIM microscope was first tested on fluorescent beads samples and achieved full field imaging over 10×10 [μm]ˆ2 at the speed of 2s/frame with 4-fold improved resolution. Imaging experiments of biological samples are under way.1. Gustafsson M. G. L. (2005). Proc. Natl. Acad. Sci. U.S.A. 10213081-13086.2. Rego E. H., Shao L., Macklin J. J., Winoto L., Johansson G. A., Kamps-Hughes N., et al. (2012). Proc. Natl. Acad. Sci. U.S.A. 109 E135-E143.3. Zhang H., Zhao M. and Peng L. (2011). Optics Express, 19, 24783-24794.

Nanometer-scale microscopy via graphene plasmons

Using graphene plasmons (GPs), we can realize a nanometer-scale microscopy. Our scheme takes advantage of the extremely large wave number of GPs and the low loss of graphene. Comparing with conventional nonlinear structured-illumination microscopy based on high-order nonlinearity associated with high intensity light, our proposal only requires linear response. Consequently we need a very weak field, which means less damage to the sample and which may play a significantly important role in the imaging of the biological systems.

Practical structured illumination microscopy.

Structured illumination microscopy (SIM) is a method that can double the spatial resolution of wide-field fluorescence microscopy in three dimensions by using spatially structured illumination light. In this chapter, we introduce the basic principles of SIM and describe in detail several different implementations based on either a diffraction grating or liquid crystal spatial light modulators. We also describe nonlinear SIM, a method that in theory can achieve unlimited resolution. In addition, we discuss a number of key points important for high-resolution imaging.

Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device.

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.