Saturated Structured Illumination Microscopy Research Articles

Superresolution MRI with a Structured-Illumination Approach

Unlike optical imaging methods, where the Fourier representation of the object is created by wave diffraction, magnetic resonance imaging (MRI) scans the Fourier domain by spatially modulating the nuclear magnetization with magnetic field gradients. Therefore, the resolution of MRI is limited by signal strength rather than the wavelength of the diffracted wave. However, in several important applications, such as the imaging of water diffusion or blood oxygenation in the human brain, MRI must collect all the Fourier data during a single evolution of the signal to avoid artifacts caused by random fluctuations of its phase. The Fourier domain coverage of the ``single-shot'' MRI strategy is limited by the lifetime of the signal and results in a resolution limit analogous to the diffraction limit in optical microscopy. Consequently, recently developed optical superresolution methods may appear helpful. This work employs the recently developed method of phaseless encoding, which implements the idea of linear structured illumination microscopy (SIM) to enable multishot data acquisition and triple the MRI resolution regardless of the presence of spin phase fluctuations, and develops it further to enhance the resolution in two dimensions and to push its gain above a factor of 3 using ``illumination patterns'' with multiple harmonics, a concept borrowed from saturated SIM (SSIM) methodology. Results obtained with enhanced phaseless encoding on a 3-Tesla clinical scanner in phantoms and human brain are presented. This method will be beneficial when conventional multishot MRI is impeded by signal instability due to motion or other factors.

Bloch Surface Wave Assisted Structured Illumination Microscopy for Sub-100 nm Resolution

Structured illumination microscopy (SIM) is one of the most powerful and versatile super-resolution methods due to its live-cell imaging ability of subcellular structures with high speed. In this paper, we propose an alternative SIM assisted by Bloch surface wave (BSW). Through replacing the conventional laser interference fringes with sub-diffraction BSW counterparts with higher spatial frequency, the imaging resolution of SIM could be enhanced greatly and the super-resolution imaging capability down to 80 nm could be achieved. Compared with traditional wide-field fluorescence microscopy, this BSW SIM demonstrates 2.78 times enhancement in imaging resolution, which surpasses general SIM with only 2 times improvement. Moreover, the structured illumination intensity could be boosted drastically, which is beneficial to nonlinear super-resolution imaging techniques, such as saturated SIM. This BSW SIM would provide a super-resolution, wide field of view approach for surface super-resolution fluorescent imaging while maintaining high imaging speed and good bio-compatibility.

Laser scanning saturated structured illumination microscopy based on phase modulation

Wide-field saturated structured illumination microscopy has not been widely used due to the requirement of high laser power. We propose a novel method called laser scanning saturated structured illumination microscopy (LS-SSIM), which introduces high order of harmonics frequency and greatly reduces the required laser power for SSIM imaging. To accomplish that, an excitation PSF with two peaks is generated and scanned along different directions on the sample. Raw images are recorded cumulatively by a CCD detector and then reconstructed to form a high-resolution image with extended optical transfer function (OTF). Our theoretical analysis and simulation results show that LS-SSIM method reaches a resolution of 0.16 λ, equivalent to 2.7-fold resolution than conventional wide-field microscopy. In addition, LS-SSIM greatly improves the optical sectioning capability of conventional wide-field illumination system by diminishing our-of-focus light. Furthermore, this modality has the advantage of implementation in multi-photon microscopy with point scanning excitation to image samples in greater depths.

Sub-diffraction-limited microscopy via Rabi gradient excitation

We propose a scheme for microscopy with resolution far beyond the diffraction limit by using coherent Rabi oscillations. When the sample is illuminated by a gradient laser field, Rabi oscillations will be induced which can lead to a sinusoidal excitation in the sample. This is similar to the structured illumination microscopy (SIM) which can recover high-spatial-frequency components in the far field. However, different from linear SIM, the sinusoidal pattern here can have a spatial frequency much higher than that of the linear standing wave. Due to this property, we can achieve extremely high resolution, as in the nonlinear saturated SIM, but keep the reconstruction algorithm as simple as the linear one.

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.

Widely accessible method for superresolution fluorescence imaging of living systems

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.

Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.

Nonlinear structured illumination microscopy by surface plasmon enhanced stimulated emission depletion

Nonlinear structured illumination microscopy (SIM) in theory has unlimited resolution over a full field of view. However under a realistic signal-to-noise ratio and a limited photon budget, the performance of nonlinear SIM strongly depends on the behavior of the nonlinear effect. Saturated SIM (SSIM) is not ideal in biological applications due to its strong photobleaching. Stimulated emission depletion (STED) SIM will have high sensitivity, higher resolution and less photo toxicity than SSIM. However, the laser power necessary to support a strong full-field STED effect is not attainable with current laser technology. We experimentally proved that surface plasmon resonance enhances (SPR) near surface STED effect by a factor of 8, and therefore STED-SIM is feasible in the total internal reflection microscopy mode with SPR enhancement. Simulation analysis predicts that SPR enhanced 2D STED is strong enough for nonlinear SIM to achieve high-speed imaging at 30-nm resolution and single molecule sensitivity. The STED-SIM superresolution microscopy method would provide a solution for observing single molecule processes in vitro or on the basal membrane of live cells.

Review of Super-Resolution Fluorescence Microscopy for Biology

Several methodologies have been developed over the past several years for super-resolution fluorescence microscopy including saturated structured-illumination microscopy (SSIM), stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). While they have shown great promise for biological research, these techniques all have individual strengths and weaknesses. This review will describe the basic principles for achieving super resolution, demonstrate some applications in biology, and provide an overview of technical considerations for implementing these methods.