We propose a chiral imaging modality based on optical chirality engineering, fluorescence-detected circular dichroism and structured illumination microscopy. In this method, the optical chirality of the illumination is structured and the circular dichroism dependent fluorescence is detected. With image reconstruction, the spatial distribution of chiral domains can be obtained at sub-diffraction limited resolution. We theoretically demonstrate this method and discuss the feasibility using an optical chirality engineering approach based on far-field optics.
Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the achievable resolution both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field detection. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5S microscopy, have improved the axial resolution beyond that of the lat…
Phase contrast techniques, such as differential interference contrast (DIC) microscopy, are widely used to provide morphological images of unstained biological samples. The trans-illumination geometry required for these techniques has restricted their application to thin samples. We introduce oblique back-illumination microscopy (OBM), a method of collecting en face phase gradient images of thick scattering samples, enabling near video-rate in vivo phase imaging with a miniaturized probe suitable for endoscopy.
We present a simple and compact approach of structured illumination microscopy by using three $2\times2$ fiber couplers and one $1\times4$ MEMS optics switch. One uniform and three fringe illumination patterns were produced by placing seven output fiber tips at the conjugate Fourier plane of the illumination path. Stable and relatively high-speed illumination switching was achieved by the optics switch. Super-resolution and optical sectioned information was reconstructed from 4-frame data by using algorithms based on a joint Richardson-Lucy deconvolution method and a Hilbert transform method. …
We propose to enhance the performance of localized plasmon structured illumination microscopy (LP-SIM) via intensity correlations. LP-SIM uses sub-wavelength illumination patterns to encode high spatial frequency information. It can enhance the resolution up to three-fold before gaps in the OTF support arise. For blinking fluorophores or for quantum antibunching an intensity correlation analysis induces higher harmonics of the illumination pattern and enlarges the effective OTF. This enables ultrahigh resolutions without gaps in the OTF support, and thus a fully deterministic imaging scheme. W…
Structured illumination microscopy (SIM) has emerged as an essential technique for 3D and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualisation and image processin…
Recently, chiral structured illumination microscopy has been proposed to image fluorescent chiral domains at sub-wavelength resolution. Chiral structured illumination microscopy is based on the combination of structured illumination microscopy, fluorescence-detected circular dichroism, and optical chirality engineering. Since circular dichroism of natural chiral molecules is typically weak, the differential fluorescence is also weak and can be easily buried by the noise, hampering the fidelity of the reconstructed images. In this work, we systematically study the impact of the noise on the qua…
Current optoacoustic microscopy configurations often have narrow focal ranges that limit their use for fast volumetric imaging applications. Here, the focal range of optoacoustic microscopes is extended by matching the elongated optical illumination profile of a Bessel beam with the pencil beam acoustic sensitivity profile of a broadband axicon detector. An inverted optoacoustic microscope was developed with interchangeable optical illumination and acoustic detection units to assess the imaging depths and resolutions retained with several combinations of illumination and detection profiles. Ma…
High-content biological microscopy targets high-resolution imaging across large fields-of-view (FOVs). Recent works have demonstrated that computational imaging can provide efficient solutions for high-content microscopy. Here, we use speckle structured illumination microscopy (SIM) as a robust and cost-effective solution for high-content fluorescence microscopy with simultaneous high-content quantitative phase (QP). This multi-modal compatibility is essential for studies requiring cross-correlative biological analysis. Our method uses laterally-translated Scotch tape to generate high-resoluti…
The structured illumination (SI) architecture can be adopted in a digital holographic (DH) microscope to enhance the spatial resolution. In this paper, we propose and demonstrate a compact and simple SI method in a self-referencing common-path configuration of DH microscopy. The combination of SI pattern generated by a compact disk and common-path off-axis geometry formed by Fresnel biprism introduces a low-cost and highly stable system to image both amplitude and phase objects with a twofold improvement of the spatial resolution. Experimental results for the case of a standard test sample val…
In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. In this paper, using a combination of theoretical and experimental techniques, we show that Selective Plane Illumination Microscopy (SPIM)…
Quantitative phase microscopy (QPM) is often based on recording an object-reference interference pattern and its further phase demodulation. We propose Pseudo Hilbert Phase Microscopy (PHPM) where we combine pseudo thermal light source illumination and Hilbert spiral transform phase demodulation to achieve hybrid hardware-software-driven noise robustness and increase in resolution of single-shot coherent QPM. Those advantageous features stem from physically altering the laser spatial coherence and numerically restoring spectrally overlapped object spatial frequencies. Capabilities of the PHPM …
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 absorption and scattering of light by biological tissues, which 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 n…
Plasmonic hyperbolic metasurfaces have emerged as an effective platform for manipulating the propagation of light. Here, confined modes on arrays of silver nanoridges that exhibit hyperbolic dispersion are used to demonstrate and model a super-resolution imaging technique based on structured illumination microscopy. A spatial resolution of ~75 nm at 458 nm is demonstrated, which is 3.1 times better than an equivalent diffraction limited image. This work emphasizes the ability to engineer the properties of confined optical modes and to leverage those characteristics for applications in imaging.…
This Letter presents the first demonstration of multi-tile stitching for large scale 3D imaging in single objective light-sheet microscopy. We show undistorted 3D imaging spanning complete zebrafish larvae, and over 1 mm3 volumes for thick mouse brain sections. We use remote galvo scanning for light-sheet creation and develop a processing pipeline for 3D tiling across different axes. With the improved one photon (1p) tilt-invariant scanned oblique plane illumination (SOPi) microscope presented here, we demonstrate cellular resolution imaging at depths exceeding 330 um in optically scattering m…
A method is proposed for assessing the temporal resolution of Structured Illumination Microscopy (SIM), by tracking the amplitude of different spatial frequency components over time, and comparing them to a temporally-oscillating ground-truth. This method is used to gain insight into the performance limits of SIM, along with alternative reconstruction techniques (termed 'rolling SIM') that claim to improve temporal resolution. Results show that the temporal resolution of SIM varies considerably between low and high spatial frequencies, and that, despite being used in several high profile paper…
Structured illumination microscopy (SIM) provides images of fluorescent objects at an enhanced resolution greater than that of conventional epifluorescence wide-field microscopy. Initially demonstrated in 1999 to enhance the lateral resolution two-fold, it has since been extended to enhance axial resolution two-fold (2008), applied to live-cell imaging (2009) and combined with myriad other techniques, including interferometric detection (2008), confocal microscopy (2010) and light sheet illumination (2012). Despite these impressive developments, SIM remains, perhaps, the most poorly understood…
The performance of light-field microscopy is improved by selectively illuminating the relevant subvolume of the specimen with a second objective lens [1-3]. Here we advance this approach to a single-objective geometry, using an oblique one-photon illumination path or two-photon illumination to accomplish selective-volume excitation. The elimination of the second orthogonally oriented objective to selectively excite the volume of interest simplifies specimen mounting; yet, this single-objective approach still reduces out-of-volume background, resulting in improvements in image contrast, effecti…
We propose to use intensity correlation microscopy in combination with structured illumination to image quantum emitters that exhibit antibunching with a spatial resolution reaching far beyond the Rayleigh limit. Combining intensity measurements and intensity auto correlations up to order $m$ creates an effective PSF with FWHM shrunk by the factor $\sqrt{m}$. Structured Illumination microscopy on the other hand introduces a resolution improvement of factor 2 by use of the principle of moiré fringes. Here, we show that for linear low-intensity excitation and linear optical detection the simulta…
Structured illumination microscopy (SIM) achieves doubled spatial resolution by exciting the specimen with a high-contrast, high-frequency sinusoidal pattern. Such an excitation pattern can be generated by interference between multiple laser beams, which are diffracted from a grating. In SIM, 2D imaging requires 9 patterns and 3D imaging requires 15 patterns. Compared to mechanical movement of gratings, opti-electro devices provide rapid switch of the excitation patterns, in which Digital Micro-mirror Device (DMD) is most common in industry. Here we model DMD as the blazed grating and report a…
Structured illumination microscopy (SIM) can double the resolution beyond the light diffraction limit, but it comes at the cost of multiple camera exposures and the heavy computation burden of multiple Fourier transforms. In this paper, we report a novel technique termed single-shot SIM, to overcome these limitations. A multi-task joint deep-learning strategy is proposed. Generative adversative networks (GAN) are employed to generate five structured illumination images based on the single-shot structured illumination image. U-Net is employed to reconstruct the super-resolution image from these…
Intensity correlation microscopy (ICM), which is prominently known through antibunching microscopy or super-resolution optical fluctuation imaging (SOFI), provides superresolution through a correlation analysis of antibunching of independent quantum emitters or temporal fluctuations of blinking fluorophores. For correlation order $m$ the PSF in the signal is effectively taken to the $m$th power, and is thus directly shrunk by the factor $\sqrt{m}$. Combined with deconvolution a close to linear resolution improvement of factor $m$ can be obtained. Yet, analysis of high correlation orders is cha…
Differential phase contrast microscopy (DPC) provides high-resolution quantitative phase distribution of thin transparent samples under multi-axis asymmetric illuminations. Typically, illumination in DPC microscopic systems is designed with 2-axis half-circle amplitude patterns, which, however, result in a non-isotropic phase contrast transfer function (PTF). Efforts have been made to achieve isotropic DPC by replacing the conventional half-circle illumination aperture with radially asymmetric patterns with 3-axis illumination or gradient amplitude patterns with 2-axis illumination. Neverthele…
University of California, Berkeley（加州大学伯克利分校）；Boston University（波士顿大学）
Structured illumination microscopy (SIM) improves resolution by down-modulating high-frequency information of an object to fit within the passband of the optical system. Generally, the reconstruction process requires prior knowledge of the illumination patterns, which implies a well-calibrated and aberration-free system. Here, we propose a new \textit{algorithmic self-calibration} strategy for SIM that does not need to know the exact patterns {\it a priori}, but only their covariance. The algorithm, termed PE-SIMS, includes a Pattern-Estimation (PE) step requiring the uniformity of the sum of …
Illumination uniformity is a critical parameter for excitation and data extraction quality in widefield biological imaging applications. However, typical imaging systems suffer from spatial and spectral non-uniformity due to non-ideal optical elements, thus require complex solutions for illumination corrections. We present Effective Uniform Color-Light Integration Device (EUCLID), a simple and cost-effective illumination source for uniformity corrections. EUCLID employs a diffuse-reflective, adjustable hollow cavity that allows for uniform mixing of light from discrete light sources and modifi…
In wide-field nonlinear microscopy, wavefront modulation by means of phase-only spatial light modulators (SLMs) allows achieving simultaneous two-photon excitation and fluorescence emission from specific region-of-interests (ROIs) of biological specimens. This is basically accomplished at the illumination path of the microscope by the reconstruction of computer generated holograms (CGHs) onto the sample plane. However, as two-photon absorption (TPA) is inherently an intensity-square dependent process and iterative Fourier transform algorithms (IFTAs) can only approximate the illumination of se…
For quantitative phase imaging (QPI) based on transport-of-intensity equation (TIE), partially coherent illumination provides speckle-free imaging, compatibility with brightfield microscopy, and transverse resolution beyond coherent diffraction limit. Unfortunately, in a conventional microscope with circular illumination aperture, partial coherence tends to diminish the phase contrast, exacerbating the inherent noise-to-resolution tradeoff in TIE imaging, resulting in strong low-frequency artifacts and compromised imaging resolution. Here, we demonstrate how these issues can be effectively add…
In this paper, a method for increasing the temporal resolution of a temporal imaging system has been developed. Analogously to the conventional spatial imaging systems in which resolution limit is due to the finite aperture of the lens, in a temporal imaging system, finite temporal aperture of the time lens is responsible for limited temporal resolution. Based on the method used in spatial structured illumination super-resolution microscopy in spatial optics, we have utilized time prisms, which are temporal versions of conventional prisms, to shift the illusive frequency components of the inpu…
Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles, which plays a vital role in studying the molecular structure and dynamics of bio-complex. However, it is difficult to resolve the dipole assemblies on the subcellular structure and their dynamics in living cells with super-resolution. Here we report polarized structured illumination microscopy (pSIM), which decouples the entangled spatial and angular structured illumination through interpreting the dipoles in spatio-angular hyperspace. We demonstrate its application on a series of biological …
In recent years there has been great interest in using deep neural networks (DNN) for super-resolution image reconstruction including for structured illumination microscopy (SIM). While these methods have shown very promising results, they all rely on data-driven, supervised training strategies that need a large number of ground truth images, which is experimentally difficult to realize. For SIM imaging, there exists a need for a flexible, general, and open-source reconstruction method that can be readily adapted to different forms of structured illumination. We demonstrate that we can combine…
Structured Illumination Microscopy (SIM) is an imaging technique for achieving both super-resolution (SR) and optical sectioning (OS) in wide-field microscopy. It consists in illuminating the sample with periodic patterns at different orientations and positions. The resulting images are then processed to reconstruct the observed object with SR and/or OS. In this work, we present BOSSA-SIM, a general-purpose SIM reconstruction method, applicable to moving objects such as encountered in in vivo retinal imaging, that enables SR and OS jointly in a fully unsupervised Bayesian framework. By modelin…
Modern optical microscopy methods have been advanced to provide super resolution at high imaging speed, but not chirality discriminative. We recently proposed chiral structured-illumination microscopy (SIM) method to image chiral fluorescent domains at sub-wavelength resolution. Chiral SIM is suitable for imaging chiral domains at sub-wavelength resolution but loses the high spatial frequency of the achiral ones. In order to obtain the full picture of all fluorescent domains at sub-wavelength resolution, we propose an advanced version of chiral SIM, termed double SIM, which enables simultaneou…
Sub-diffraction resolution, gentle sample illumination, and the possibility to image in multiple colors make Structured Illumination Microscopy (SIM) an imaging technique which is particularly well suited for live cell observations. Here, we present Machine learning Assisted Interferometric-SIM (MAI-SIM), an easy-to-implement method for high speed SIM imaging in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible,…
We present experimental demonstration of tilt-mirror assisted transmission structured illumination microscopy (tSIM) that offers a large field of view super resolution imaging. An assembly of custom-designed tilt-mirrors are employed as the illumination module where the sample is excited with the interference of two beams reflected from the opposite pair of mirror facets. Tunable frequency structured patterns are generated by changing the mirror-tilt angle and the hexagonal-symmetric arrangement is considered for the isotropic resolution in three orientations. Utilizing high numerical aperture…
A phase-sensitive microscopy technique is proposed and demonstrated that employs the momentum correlations inherent in spontaneous parametric down-conversion. One photon from a correlated pair is focused onto a microscopic target while the other is measured in the Fourier plane. This provides knowledge of the position and angle of illumination for every photon striking the target, allowing full post-production control of the illumination angle used to form an image. The versatility of this approach is showcased with asymmetric illumination and differential phase contrast imaging, without any b…
Many optical microscopy techniques rely on structured illumination by way of a projected image (eg. structured illumination microscopy) or a tailored angular distribution of light (eg. ptychography). Electro-optical equipment such as spatial light modulators and light emitting diode (LED) arrays are commonly used to sculpt light for these imaging schemes. However, these high-tech devices are not a requirement for light crafting. Patterned diffusely reflecting surfaces also imprint a spatio-angular structure onto reflected light. We demonstrate that paper patterned with a standard commercial pr…
In this work, we present a highly efficient quantitative phase imaging (QPI) approach using programmable annular LED illumination based on traditional bright-field microscope. As a new type of coded illumination, the LED array provides a flexible and compatible way to realize QPI. The proposed method modulates the transfer function of system by changing the LED illumination pattern, which achieves twice resolution of objective NA and gives noise-robust response of transfer function. The phase of a sample could be recovered from the intensity images with the inversion of transfer function. More…
This work presents the development of a flexible microscopic chemical imaging platform for cultural heritage that utilizes wavelength-tunable oblique illumination from a point source to obtain per-pixel reflectance spectra in the VIS-NIR range. The microscope light source can be adjusted on two axes allowing for a hemisphere of possible illumination directions. The synthesis of multiple illumination angles allows for the calculation of surface normal vectors, similar to phase gradients, and axial optical sectioning. The extraction of spectral reflectance images with high spatial resolutions fr…
