Multi-harmonic structured illumination-based optical diffraction tomography.

Imaging speed and spatial resolution are key factors in optical diffraction tomography (ODT), while they are mutually exclusive in 3D refractive index imaging. This paper presents a multi-harmonic structured illumination-based optical diffraction tomography (MHSI-ODT) to acquire 3D refractive index (RI) maps of transparent samples. MHSI-ODT utilizes a digital micromirror device (DMD) to generate structured illumination containing multiple harmonics. For each structured illumination orientation, four spherical spectral crowns are solved from five phase-shifted holograms, meaning that the acquisition of each spectral crown costs 1.25 raw images. Compared to conventional SI-ODT, which retrieves two spectral crowns from three phase-shifted raw images, MHSI-ODT enhances the imaging speed by 16.7% in 3D RI imaging. Meanwhile, MHSI-ODT exploits both the 1st-order and the 2nd-order harmonics; therefore, it has a better intensity utilization of structured illumination. We demonstrated the performance of MHSI-ODT by rendering the 3D RI distributions of 5 µm polystyrene (PS) microspheres and biological samples.

Characterization of the angular memory effect of dynamic turbid media.

The optical angular memory effect (AME) is a basic feature of turbid media and defines the correlation of speckles when the incident light is tilted. AME based imaging through solid scattering media such as ground glass and biomedical tissue has been recently developed. However, in the case of liquid media such as turbid water or blood, the speckle pattern exhibits dynamic time-varying characteristics, which introduces several challenges. The AME of the thick volume dynamic media is particularly different from the layer scatterers. In practice, there are more parameters, e.g., scattering particle size, shape, density, or even the illuminating beam aperture that can influence the AME range. Experimental demonstration of AME phenomenon in liquid dynamic media and confirm the distinctions will contribution to complete the AME theory. In this paper, a dual-polarization speckle detection setup was developed to characterize the AME of dynamic turbid media, where two orthogonal polarized beams were employed for simultaneous detection by a single CCD. The AME of turbid water, milk and blood were measured. The influence of thickness, concentration, particle size and shape, and beam diameter were analyzed. The AME increasement of upon the decrease of beam diameter was tested and verified. The results demonstrate the feasibility of this method for investigating the AME phenomenon and provide guidance for AME based imaging through scattering media.

Phase-type diffractive micro-optics elements in sulfur-based polymeric glass by femtosecond laser direct writing.

Sulfur-based polymeric glasses are promising alternative low-cost IR materials due to their profoundly high IR transparency. In this Letter, femtosecond-laser-induced refractive index change (RIC) was investigated in one typical sulfur-based polymeric glass material, poly(S-r-DIB), for the first time, to the best of our knowledge. The RIC in the laser-engineered region was quantitively characterized, which laid a foundation for phase-type optical element design. By the integration of RIC traces, embedded phase-type micro-optics elements, including Fresnel zone plates, and a Dammann grating were fabricated in bulk poly(S-r-DIB) polymeric glass substrate via the femtosecond laser direct writing technique. The imaging and beam shaping performance were demoed in the near-infrared (NIR) region.

Axial resolution enhancement for planar Airy beam light-sheet microscopy via the complementary beam subtraction method.

Airy beam light-sheet illumination can extend the field of view (FOV) of light-sheet fluorescence microscopy due to the unique propagation properties of non-diffraction and self-acceleration. However, the side lobes create undesirable out-of-focus background, leading to poor axial resolution and low image contrast. Here, we propose an Airy complementary beam subtraction (ACBS) method to improve the axial resolution while keeping the extended FOV. By scanning the optimized designed complementary beam that has two main lobes (TML), the generated complementary light-sheet has almost identical intensity distribution to that of the planar Airy light-sheet except for the central lobe. Subtraction of the two images acquired by double exposure respectively using the planar Airy light-sheet and the planar TML light-sheet can effectively suppress the influence of the out-of-focus background. The axial resolution improves from ∼4µm to 1.2 µm. The imaging performance was demonstrated by imaging specimens of aspergillus conidiophores and GFP labeled mouse brain section. The results show that the ACBS method enables the Airy beam light-sheet fluorescence microscopy to obtain better imaging quality.

Rapid Image Reconstruction of Structured Illumination Microscopy Directly in the Spatial Domain.

Super-resolution structured illumination microscopy (SIM) routinely performs image reconstruction in the frequency domain using an approach termed frequency-domain reconstruction (FDR). Due to multiple Fourier transforms between the spatial and frequency domains, SIM suffers from low reconstruction speed, constraining its applications in real-time, dynamic imaging. To overcome this limitation, we developed a new method for SIM image reconstruction, termed spatial domain reconstruction (SDR). SDR is intrinsically simpler than FDR, does not require Fourier transforms and the theory predicts it to be a rapid image reconstruction method. Results show that SDR reconstructs a super-resolution image 7-fold faster than FDR, producing images that are equal to either FDR or the widely-used FairSIM. We provide a proof-of-principle using mobile fluorescent beads to demonstrate the utility of SDR in imaging moving objects. Consequently, replacement of the FDR approach with SDR significantly enhances SIM as the desired method for live-cell, instant super-resolution imaging. This means that SDR-SIM is a "What You See Is What You Get" approach to super-resolution imaging.

Extended field of view of light-sheet fluorescence microscopy by scanning multiple focus-shifted Gaussian beam arrays.

Light-sheet fluorescence microscopy (LSFM) facilitates high temporal-spatial resolution, low photobleaching and phototoxicity for long-term volumetric imaging. However, when a high axial resolution or optical sectioning capability is required, the field of view (FOV) is limited. Here, we propose to generate a large FOV of light-sheet by scanning multiple focus-shifted Gaussian beam arrays (MGBA) while keeping the high axial resolution. The positions of the beam waists of the multiple Gaussian beam arrays are shifted in both axial and lateral directions in an optimized arranged pattern, and then scanned along the direction perpendicular to the propagation axis to form an extended FOV of light-sheet. Complementary beam subtraction method is also adopted to further improve axial resolution. Compared with the single Gaussian light-sheet method, the proposed method extends the FOV from 12 µm to 200 µm while sustaining the axial resolution of 0.73 µm. Both numerical simulation and experiment on samples are performed to verify the effectiveness of the method.

Full-color optically-sectioned imaging by wide-field microscopy via deep-learning.

Wide-field microscopy (WFM) is broadly used in experimental studies of biological specimens. However, combining the out-of-focus signals with the in-focus plane reduces the signal-to-noise ratio (SNR) and axial resolution of the image. Therefore, structured illumination microscopy (SIM) with white light illumination has been used to obtain full-color 3D images, which can capture high SNR optically-sectioned images with improved axial resolution and natural specimen colors. Nevertheless, this full-color SIM (FC-SIM) has a data acquisition burden for 3D-image reconstruction with a shortened depth-of-field, especially for thick samples such as insects and large-scale 3D imaging using stitching techniques. In this paper, we propose a deep-learning-based method for full-color WFM, i.e., FC-WFM-Deep, which can reconstruct high-quality full-color 3D images with an extended optical sectioning capability directly from the FC-WFM z-stack data. Case studies of different specimens with a specific imaging system are used to illustrate this method. Consequently, the image quality achievable with this FC-WFM-Deep method is comparable to the FC-SIM method in terms of 3D information and spatial resolution, while the reconstruction data size is 21-fold smaller and the in-focus depth is doubled. This technique significantly reduces the 3D data acquisition requirements without losing detail and improves the 3D imaging speed by extracting the optical sectioning in the depth-of-field. This cost-effective and convenient method offers a promising tool to observe high-precision color 3D spatial distributions of biological samples.

Full-polarization wavefront shaping for imaging through scattering media.

The scattering effect occurring when light passes through inhomogeneous-refractive-index media such as atmosphere or biological tissues will scramble the light wavefront into speckles and impede optical imaging. Wavefront shaping is an emerging technique for imaging through scattering media that works by addressing correction of the disturbed wavefront. In addition to the phase and amplitude, the polarization of the output scattered light will also become spatially randomized in some cases. The recovered image quality and fidelity benefit from correcting as much distortion of the scattered light as possible. Liquid-crystal spatial light modulators (LC-SLMs) are widely used in the wavefront shaping technique, since they can provide a great number of controlled modes and thereby high-precision wavefront correction. However, due to the working principle of LC-SLMs, the wavefront correction is restricted to only one certain linear polarization state, resulting in retrieved image information in only the right polarization, while the information in the orthogonal polarization is lost. In this paper, we describe a full-polarization wavefront correction system for shaping the scattered light wavefront in two orthogonal polarizations with a single LC-SLM. The light speckles in both polarizations are corrected for retrieval of the full polarization information and faithful images of objects. As demonstrated in the experiments, the focusing intensity can be increased by full-polarization wavefront correction, images of objects in arbitrary polarization states can be retrieved, and the polarization state of the object's light can also be recognized.

Robust contrast-transfer-function phase retrieval via flexible deep learning networks.

By exploiting the total variation (TV) regularization scheme and the contrast transfer function (CTF), a phase map can be retrieved from single-distance coherent diffraction images via the sparsity of the investigated object. However, the CTF-TV phase retrieval algorithm often struggles in the presence of strong noise, since it is based on the traditional compressive sensing optimization problem. Here, convolutional neural networks, a powerful tool from machine learning, are used to regularize the CTF-based phase retrieval problems and improve the recovery performance. This proposed method, the CTF-Deep phase retrieval algorithm, was tested both via simulations and experiments. The results show that it is robust to noise and fast enough for high-resolution applications, such as in optical, x-ray, or terahertz imaging.

Robust contrast-transfer-function phase retrieval via flexible deep learning networks: publisher's note.

This publisher's note contains corrections to Opt. Lett.44, 5141 (2019)OPLEDP0146-959210.1364/OL.44.005141.

Quantitative phase imaging of cells in a flow cytometry arrangement utilizing Michelson interferometer-based off-axis digital holographic microscopy.

We combined Michelson-interferometer-based off-axis digital holographic microscopy (DHM) with a common flow cytometry (FCM) arrangement. Utilizing object recognition procedures and holographic autofocusing during the numerical reconstruction of the acquired off-axis holograms, sharply focused quantitative phase images of suspended cells in flow were retrieved without labeling, from which biophysical cellular features of distinct cells, such as cell radius, refractive index and dry mass, can be subsequently retrieved in an automated manner. The performance of the proposed concept was first characterized by investigations on microspheres that were utilized as test standards. Then, we analyzed two types of pancreatic tumor cells with different morphology to further verify the applicability of the proposed method for quantitative live cell imaging. The retrieved biophysical datasets from cells in flow are found in good agreement with results from comparative investigations with previously developed DHM methods under static conditions, which demonstrates the effectiveness and reliability of our approach. Our results contribute to the establishment of DHM in imaging FCM and prospect to broaden the application spectrum of FCM by providing complementary quantitative imaging as well as additional biophysical cell parameters which are not accessible in current high-throughput FCM measurements.

Rapid wide-field imaging through scattering media by digital holographic wavefront correction.

Imaging through scattering media has been a long standing challenge in many disciplines. One of the promising solutions to address the challenge is the wavefront shaping technique, in which the phase distortion due to a scattering medium is corrected by a phase modulation device such as a spatial light modulator (SLM). However, the wide-field imaging speed is limited either by the feedback-based optimization to search the correction phase or by the update rate of SLMs. In this report, we introduce a new method called digital holographic wavefront correction, in which the correction phase is determined by a single-shot off-axis holography. The correction phase establishes the so-called "scattering lens", which allows any objects to be imaged through scattering media; in our case, the "scattering lens" is a digital one established through computational methods. As no SLM is involved in the imaging process, the imaging speed is significantly improved. We have demonstrated that moving objects behind scattering media can be recorded at the speed of 2.8 fps with each frame corrected by the updated correction phase while the image contrast is maintained as high as 0.9. The image speed can potentially reach the video rate if the computing power is sufficiently high. We have also demonstrated that the digital wavefront correction method also works when the light intensity is low, which implicates its potential usefulness in imaging dynamic processes in biological tissues.

Compressed Blind Deconvolution and Denoising for Complementary Beam Subtraction Light-Sheet Fluorescence Microscopy.

OBJECTIVE: The side-lobes of a Bessel beam (BB) create a severe out-of-focus background in scanning light-sheet fluorescence microscopy, thereby extremely limiting the axial resolution. The complementary beam subtraction (CBS) method can significantly reduce the out-of-focus background by double scanning a BB and its complementary beam. However, the blurring and noise caused by the system instability during the double scanning and subtraction operations degrade the image quality significantly. Therefore, we propose a compressed blind deconvolution and denoising (CBDD) method that solves this problem. METHODS: We use a unified formulation that comprehensively takes advantage of multiple compressed sensing reconstructions and blind sparse representation. RESULTS: The simulations and experiments were performed using the microbeads and model organisms to verify the effectiveness of the proposed method. Compared with the CBS light-sheet method, the proposed CBDD algorithm achieved the gain improvement in the axial and lateral resolution of about 1.81 and 2.22 times, respectively, while the average signal-to-noise ratio (SNR) was increased by about 3 dB. CONCLUSION: Accordingly, the proposed method can suppress the noise level, enhance the SNR, and recover the degraded resolution simultaneously. SIGNIFICANCE: The obtained results demonstrate the proposed CBDD algorithm is well suited to improve the imaging performance of the CBS light-sheet fluorescence microscopy.

Optical thickness measurement with single-shot dual-wavelength in-line digital holography.

An algorithm for quantitative reconstruction of the optical thickness distribution of objects is proposed based on single-shot dual-wavelength in-line digital holography. Two single-wavelength holograms can be extracted from a single-shot recorded dual-wavelength in-line hologram. The quantitative optical thickness distribution of the specimen can be reconstructed directly without calculations of the phase images at every single wavelength. Thus, off-axis recording and phase-shifting operation are not required, enabling a fast and high-resolution measurement. The effectiveness and accuracy of the proposed method are verified by both numerical simulations and experimental results.

Subwavelength resolution Fourier ptychography with hemispherical digital condensers.

Fourier ptychography (FP) is a promising computational imaging technique that overcomes the physical space-bandwidth product (SBP) limit of a conventional microscope by applying angular-varied illuminations. However, to date, the effective imaging numerical aperture (NA) achievable with a commercial LED board is still limited to the range of 0.3-0.7 with a 4 × /0.1NA objective due to the geometric constraint with the declined illumination intensities and attenuated signal-to-noise ratio (SNR). Thus the highest achievable half-pitch resolution is usually constrained between 500-1000 nm, which cannot meet the requirements of high-resolution biomedical imaging applications. Although it is possible to improve the resolution by using a high-NA objective lens, the FP approach is less appealing as the decrease of field-of-view (FOV) will far exceed the improvement of spatial resolution in this case. In this paper, we initially present a subwavelength resolution Fourier ptychography (SRFP) platform with a hemispherical digital condenser to provide high-angle programmable plane-wave illuminations of 0.95NA, attaining a 4 × /0.1NA objective with the final effective imaging performance of 1.05NA at a half-pitch resolution of 244 nm with the incident wavelength of 465 nm across a wide FOV of 14.60 mm2, corresponding to a SBP of 245 megapixels. Our work provides an essential step of FP towards high-throughput imaging applications.

Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy.

We present a simple and fast phase aberration compensation method in digital holographic microscopy (DHM) for quantitative phase imaging of living cells. By analyzing the frequency spectrum of an off-axis hologram, phase aberrations can be compensated for automatically without fitting or pre-knowledge of the setup and/or the object. Simple and effective computation makes the method suitable for quantitative online monitoring with highly variable DHM systems. Results from automated quantitative phase imaging of living NIH-3T3 mouse fibroblasts demonstrate the effectiveness and the feasibility of the method.

Interleaved segment correction achieves higher improvement factors in using genetic algorithm to optimize light focusing through scattering media.

Focusing and imaging through scattering media has been proved possible with high resolution wavefront shaping. A completely scrambled scattering field can be corrected by applying a correction phase mask on a phase only spatial light modulator (SLM) and thereby the focusing quality can be improved. The correction phase is often found by global searching algorithms, among which Genetic Algorithm (GA) stands out for its parallel optimization process and high performance in noisy environment. However, the convergence of GA slows down gradually with the progression of optimization, causing the improvement factor of optimization to reach a plateau eventually. In this report, we propose an interleaved segment correction (ISC) method that can significantly boost the improvement factor with the same number of iterations comparing with the conventional all segment correction (ASC) method. In the ISC method, all the phase segments are divided into a number of interleaved groups; GA optimization procedures are performed individually and sequentially among each group of segments. The final correction phase mask is formed by applying correction phases of all interleaved groups together on the SLM. The ISC method has been proved significantly useful in practice because of its ability to achieve better improvement factors when noise is present in the system. We have also demonstrated that the imaging quality is improved as better correction phases are found and applied on the SLM. Additionally, the ISC method lowers the demand of dynamic ranges of detection devices. The proposed method holds potential in applications, such as high-resolution imaging in deep tissue.

Full-color structured illumination optical sectioning microscopy.

In merits of super-resolved resolution and fast speed of three-dimensional (3D) optical sectioning capability, structured illumination microscopy (SIM) has found variety of applications in biomedical imaging. So far, most SIM systems use monochrome CCD or CMOS cameras to acquire images and discard the natural color information of the specimens. Although multicolor integration scheme are employed, multiple excitation sources and detectors are required and the spectral information is limited to a few of wavelengths. Here, we report a new method for full-color SIM with a color digital camera. A data processing algorithm based on HSV (Hue, Saturation, and Value) color space is proposed, in which the recorded color raw images are processed in the Hue, Saturation, Value color channels, and then reconstructed to a 3D image with full color. We demonstrated some 3D optical sectioning results on samples such as mixed pollen grains, insects, micro-chips and the surface of coins. The presented technique is applicable to some circumstance where color information plays crucial roles, such as in materials science and surface morphology.

Double-exposure optical sectioning structured illumination microscopy based on Hilbert transform reconstruction.

Structured illumination microscopy (SIM) with axially optical sectioning capability has found widespread applications in three-dimensional live cell imaging in recent years, since it combines high sensitivity, short image acquisition time, and high spatial resolution. To obtain one sectioned slice, three raw images with a fixed phase-shift, normally 2π/3, are generally required. In this paper, we report a data processing algorithm based on the one-dimensional Hilbert transform, which needs only two raw images with arbitrary phase-shift for each single slice. The proposed algorithm is different from the previous two-dimensional Hilbert spiral transform algorithm in theory. The presented algorithm has the advantages of simpler data processing procedure, faster computation speed and better reconstructed image quality. The validity of the scheme is verified by imaging biological samples in our developed DMD-based LED-illumination SIM system.

Intrinsic optical torque of cylindrical vector beams on Rayleigh absorptive spherical particles.

The intrinsic optical torque of a focused cylindrical vector beam on a Rayleigh absorptive spherical particle is calculated via the corrected dipole approximation. Numerical results show that, for the radially polarized input field, the torque is distributed in the focal plane strictly along the azimuthal direction anywhere except at the focus. This shows a completely different property from what is observed in the focusing of a circularly polarized beam, where a strong axial torque component arises. For other cylindrically polarized input fields, the torque tends to align itself along the radial direction, as the polarization angle (the angle between the electric vector and the radial direction) changes from 0° to 90°. When limited to considering the torque at the equilibrium position, we find that only for those input fields with polarization angles larger than 50°, the particle experiences a nonzero torque at its equilibrium position. This is verified by showing quantitatively the effects of the polarization angle on the magnitude and orientation of the torque at the equilibrium position.