Pixel-reassigned line-scanning microscopy for fast volumetric super-resolution imaging.

Super-resolution microscopy has revolutionized the field of biophotonics by revealing detailed 3D biological structures. Nonetheless, the technique is still largely limited by the low throughput and hampered by increased background signals for dense or thick biological specimens. In this paper, we present a pixel-reassigned continuous line-scanning microscope for large-scale high-speed 3D super-resolution imaging, which achieves an imaging resolution of 0.41 µm in the lateral direction, i.e., a 2× resolution enhancement from the raw images. Specifically, the recorded line images are first reassigned to the line-excitation center at each scanning position to enhance the resolution. Next, a modified HiLo algorithm is applied to reduce the background signals. Parametric models have been developed to simulate the imaging results of randomly distributed fluorescent beads. Imaging experiments were designed and performed to verify the predicted performance on various biological samples, which demonstrated an imaging speed of 3400 pixels/ms on millimeter-scale specimens. These results confirm the pixel-reassigned line-scanning microscopy is a facile and powerful method to realize large-area super-resolution imaging on thick or dense biological samples.

Compressive sensing-based multi-focus line-scanning two-photon microscopy for fast 3D imaging.

Fast 3D volume imaging methods have been playing increasingly important roles in biological studies. In this article, we present the design and characterization of a multi-focus line-scanning two-photon microscope. Specifically, a digital micromirror device (DMD) is employed to generate a randomly distributed focus array on a plane (i.e., x-z) via binary holography. Next, a galvanometric mirror scans the focus array in a direction normal to the plane (i.e., y-axis) over the imaging volume. For sparse samples, e.g., neural networks in a brain, 1-3 foci are used together with compressive sensing algorithm to achieve a volume imaging rate of 15.5 volumes/sec over 77 × 120 × 40 µm3. High-resolution optical cross-sectional images on selected planes and regions can be generated by sequentially scanning the laser focus generated on the x-z plane with good imaging speeds (e.g., 107 frames/sec over 80 × 120 × 40 µm3). In the experiments, microbeads, pollens, and mouse brain slices have been imaged to characterize the point spread function and volume image rate and quality at different sampling ratios. The results show that the multi-focus line-scanning microscope presents a fast and versatile 3D imaging platform for deep tissue imaging and dynamic live animal studies.

Scan-less 3D microscopy based on spatiotemporal encoding on a single-cavity dual-comb laser.

Dual-comb microscopy enables high-speed and high-precision optical sampling by simultaneously extracting both amplitude and phase information from the interference signals with frequency division multiplexing. In this Letter, we introduce a spatiotemporal encoding approach for dual-comb microscopy that overcomes previous limitations such as mechanical scanning, low sampling efficiency, and system complexity. By employing free-space angular-chirp-enhanced delay (FACED) and a low-noise single-cavity dual-comb laser, we achieve scan-less 3D imaging with nanometer precision and a 3D distance-imaging rate of 330 Hz, restricted only by the repetition rate difference of the dual-comb laser. Specifically, the FACED unit linearly arranges the laser beam into an array. A grating subsequently disperses this array transversely into lines, facilitating ultrafast spectroscopic applications that are 1-2 orders of magnitude quicker than traditional dual-comb methods. This spatiotemporal encoding also eases the stringent conditions on various dual-comb laser parameters, such as repetition rates, coherence, and stability. Through carefully designed experiments, we demonstrate that our scan-less system can measure 3D profiles of microfabricated structures at a rate of 7 million pixels per second. Our method significantly enhances measurement speed while maintaining high precision, using a compact light source. This advancement has the potential for broad applications, including phase imaging, surface topography, distance ranging, and spectroscopy.

Parallel two-photon lithography achieving uniform sub-200†nm features with thousands of individually controlled foci.

The limited throughput of nano-scale laser lithography has been the bottleneck for its industrial applications. Although using multiple laser foci to parallelize the lithography process is an effective and straightforward strategy to improve rate, most conventional multi-focus methods are plagued by non-uniform laser intensity distribution due to the lack of individual control for each focus, which greatly hinders the nano-scale precision. In this paper, we present a highly uniform parallel two-photon lithography method based on a digital mirror device (DMD) and microlens array (MLA), which allows the generation of thousands of femtosecond (fs) laser foci with individual on-off switching and intensity-tuning capability. In the experiments, we generated a 1,600-laser focus array for parallel fabrication. Notably, the intensity uniformity of the focus array reached 97.7%, where the intensity-tuning precision for each focus reached 0.83%. A uniform dot array structure was fabricated to demonstrate parallel fabrication of sub-diffraction limit features, i.e., below 1/4 λ or 200 nm. The multi-focus lithography method has the potential of realizing rapid fabrication of sub-diffraction, arbitrarily complex, and large-scale 3D structures with three orders of magnitude higher fabrication rate.

Enhanced polarization demosaicking network via a precise angle of polarization loss calculation method.

Many existing polarization networks reconstruct polarization information based on calculating the angle of polarization (AoP) loss. Yet, the conventional loss calculation method, which is based on a linear difference approach, compromises the reconstruction accuracy and causes additional training time when combined with learning-based methods. In this Letter, we present a new, to the best of our knowledge, method to calculate the AoP loss and apply it in an enhanced color polarization demosaicking network with a "multi-branch" structure, i.e., ePDNet. Experiments are performed to demonstrate the efficacy and superiority of the method, which improves the network convergence speed by three times as well as the output image quality. The new method may find important applications in the field of polarimetric imaging.

Self-calibration for Mueller polarimeters based on DoFP polarization imagers.

Mueller polarimeters (MPs) based on division of focal plane (DoFP) polarization imagers can achieve fast measurements and significantly improve the effectiveness of Mueller polarimetry. In this Letter, we demonstrate a unique property of the DoFP sensor-based MPs: they can be calibrated without any extra polarizing reference element. We describe a self-calibration method that only requires six image acquisitions; based on our analysis, the calibration accuracy is only limited by the noise.

Underwater image restoration via Stokes decomposition.

In this Letter, we present a Stokes imaging-based method to restore objects and enhance image contrast in turbid water. In the system, a light source illuminates the objects with two orthometric polarization states; based on a new Stokes decomposition model, the recorded images are converted to Stokes maps and subsequently restored to a clear image, free of reflections and scattered lights. A mathematical model has been developed to explain the Stokes decomposition and how the undesired reflections and scattered lights are rejected. Imaging experiments have been devised and performed on different objects, e.g., metals and plastics, under different turbidities. The results demonstrate enhanced image quality and capability to distinguish polarization differences. This new, to the best of our knowledge, method can be readily applied to practical underwater object detection and potentially realize clear vision in other scattering media.

Optimal nonlinear Stokes-Mueller polarimetry for multi-photon processes.

In this Letter, we present an optimization model for nonlinear Stokes-Mueller polarimetry (SMP) to improve the precision in estimating the nonlinear Mueller matrix (MM) for two- and three-photon processes. Although nonlinear polarimeters can measure the polarization properties of multi-photon processes or materials, existing methods are suboptimal, leading to low measurement precision. Based on the model and its solution, we have designed a new measurement strategy to substantially reduce the estimation variance of nonlinear MM coefficients by approximately 58.2% for second-harmonic generation polarimetry and 78.7% for third-harmonic generation polarimetry. The model and measurement method can be directly applied to multi-photon processes to improve the precision of SMP.

Broadband angular dispersion compensation for digital micromirror devices.

In this Letter, we present a compact broadband angular dispersion compensation method for digital micromirror devices (DMDs) and ultrashort pulse lasers, which effectively extends the conventional single-wavelength compensation design to a wide wavelength range of 300 nm. First, a parametric model was developed for the dispersion compensation unit, consisting of a transmission grating and a 4f telescope sub-unit, to guide the selection of components and parameter optimization for broadband applications. In the experiments, we designed a single slit-based metrology system to measure and quantify the compensated angular dispersion of a Ti:sapphire femtosecond laser with a pulse width of 75 fs. The results indicate that our method can reduce the angular dispersion to 0.04°, i.e., pulse widening less than 20 fs, over a wavelength range of 750-1050 nm. To demonstrate this, the DMD system was used as a multi-wavelength beam shaper to reconstruct a wavefront that contains the "CUHK" pattern and the results confirmed its ability to provide effective broadband angular dispersion compensation. This means the DMD can be used in different applications that employ a broadband light source, e.g., wavelength tunable femtosecond laser, attosecond laser, supercontinuum laser, and multi-color LED.

Single-shot optical sectioning microscopy based on structured illumination.

In this Letter, we present a single-shot 3D-resolved structured illumination microscopy (SIM) based on a digital micromirror device (DMD), a galvanometric mirror, and the HiLo algorithm. During imaging, the DMD rapidly generates sinusoidal and plane illuminations in the focal region. By synchronizing the DMD with a galvanometric scanner and exploiting the unique data readout process of the camera, the emissions from the specimen under two different illuminations, i.e., structured and uniform illumination, are projected to different regions on a camera, achieving high-resolution single-exposure optical sectioning at the camera's limiting speed, i.e., 200 Hz, without sacrificing the resolution. A model has been developed to guide the design and optimization of the optical system. Imaging experiments on pollen and mouse kidney samples have been performed to verify the predicted performance. The results show that the single-shot SIM with the HiLo algorithm achieves comparable resolution to the standard two-shot HiLo method with a twofold speed enhancement, which may find important applications in biophotonics, e.g., visualizing high-speed biological events in vivo.

Holography-based structured light illumination for temporal focusing microscopy.

In this Letter, we present a holography-based structured light illumination (SLI) method to enhance the resolution of widefield temporal focusing microscopy (TFM). In the system, a digital micromirror device is employed to simultaneously disperse the incoming femtosecond laser to induce temporal focusing at the focal plane and generate designed structured patterns via a Lee hologram. As the generated structured patterns do not contain the zeroth order beam, it improves the contrast and modulation frequency. Mathematical models have been derived to calculate the electric fields at the focal plane and to explain the effects of improved optical cross-sectioning capability. Imaging experiments have been devised and performed on fluorescent beads and mouse kidney sections; the results demonstrate enhanced axial confinement and improved suppression of out-of-focus fluorescence. The new SLI method realizes high-resolution TFM and can be readily applied to other microscopy platforms for biophotonics applications.

Light field microscopy based on structured light illumination.

In this Letter, we present the modeling, design, and characterization of a light sheet-based structured light illumination (SLI) light field microscopy (LFM) system for fast 3D imaging, where a digital micromirror device is employed to rapidly generate designed sinusoidal patterns in the imaging field. Specifically, we sequentially obtain uniformly illuminated and structured light field images, followed by post-processing with a new, to the best of our knowledge, algorithm that combines the deconvolution and HiLo algorithms. This enables fast volumetric imaging with improved optical cross-sectioning capability at a speed of 50 volumes per second over an imaging field of 250×250×80µm3 in the x, y, and z axis, respectively. Mathematical models have been derived to explain the performance enhancement due to suppressed background noises. To verify the results, imaging experiments on fluorescence beads, fern spore, and Drosophila brain samples, have been performed. The results indicate that the light sheet-based SLI-LFM presents a fast 3D imaging solution with substantially improved optical cross-sectioning capability in comparison with a standard light sheet-based LFM. The new light field imaging method may find important applications in the field of biophotonics.

Formation of nanostructures and optical analogues of massless Dirac particles via femtosecond lasers.

Subwavelength-scale surface structures have many important engineering and nanotechnology applications, e.g., superhydrophobicity and light-trapping. However, an effective and economic nanofabrication solution for general engineering materials, e.g., metals or silicon, is still not available to date. In this paper, we present an experimental and theoretical study of the nanostructure formation mechanism based on double time-delayed femtosecond laser beams and the coupled mode theory (CMT), demonstrating the use of an optical analogue of massless Dirac particles for high-throughput nanofabrication for the first time. In the experiments, a variety of complex periodic structures, including hexagonally arranged nanoholes, nano-square array, and periodic ripples, have been fabricated. The formation mechanisms of these nanostructures are explained by the CMT, where a transient plasmonic waveguide array (TPWA) is formed by the interference between the preceding laser and the induced surface plasmon polaritons (SPPs). The SPPs induced by the subsequent laser propagates through the TPWA, resulting in conical diffraction. This result shows the first practical application of the massless Dirac dynamics in nanofabrication.

Design of a multi-modality DMD-based two-photon microscope system.

We present the modular design and characterization of a multi-modality video-rate two-photon excitation (TPE) microscope based on integrating a digital micromirror device (DMD), which functions as an ultrafast beam shaper and random-access scanner, with a pair of galvanometric scanners. The TPE microscope system realizes a suite of new imaging functionalities, including (1) multi-layer imaging with 3D programmable imaging planes, (2) DMD-based wavefront correction, and (3) multi-focus optical stimulation (up to 22.7 kHz) with simultaneous TPE imaging, all in real-time. We also report the detailed optomechanical design and software development that achieves high level system automation. To verify the performance of different microscope functions, we have devised and performed imaging experiments on Drosophila brain, mouse kidney and human stem cells. The results not only show improved imaging resolution and depths via the DMD-based adaptive optics, but also demonstrate fast multi-focus stimulation for the first time. With the new imaging capabilities, e.g., tools for optogenetics, the multi-modality TPE microscope may play a critical role in the applications pertinent to neuroscience and biophotonics.

Aberration-free 3D imaging via DMD-based two-photon microscopy and sensorless adaptive optics.

In this Letter, we present a new, to our knowledge, aberration-free 3D imaging technique based on digital micromirror device (DMD)-based two-photon microscopy and sensorless adaptive optics (AO), where 3D random-access scanning and modal wavefront correction are realized using a single DMD chip at 22.7 kHz. Specifically, the DMD is simultaneously used as a deformable mirror to modulate a distorted wavefront and a fast scanner to maneuver the laser focus in a 3D space by designed binary holograms. As such, aberration-free 3D imaging is realized by superposing the wavefront correction and 3D scanning holograms. Compared with conventional AO devices and methods, the DMD system can apply optimal wavefront correction information to different imaging regions or even individual pixels without compromising the scanning speed and device resolution. In the experiments, we first focus the laser through a diffuser and apply sensorless AO to retrieve a corrected focus. After that, the DMD performs 3D scanning on a Drosophila brain labeled with green fluorescent protein. The two-photon imaging results, where optimal wavefront correction information is applied to 3×3 separate regions, demonstrate significantly improved resolution and image quality. The new DMD-based imaging solution presents a compact, low-cost, and effective solution for aberration-free two-photon deep tissue imaging, which may find important applications in the field of biophotonics.

Compressive sensing for fast 3-D and random-access two-photon microscopy.

3-D two-photon excitation (TPE) microscopy has been a critical tool for biological study since its introduction. Yet, the speed is largely limited by its point detector, e.g., photomultiplier tube (PMT), which requires a point-scanning imaging sequence. In this Letter, we present a multi-focus compressive sensing (CS) method for 3-D and random-access TPE microscopy based on a digital micromirror device (DMD). This new platform combines CS with a unique holography-based DMD random-access scanner to enhance the imaging speed by three to five times for imaging arbitrarily selected regions in 3-D specimens without sacrificing the resolution. In the experiments, 1-20 randomly selected foci are generated by modulating the wavefront of a femtosecond laser via binary holography, where the combined intensity is recorded by a PMT. By exploiting CS algorithms, 3-D images at arbitrarily selected sites can be reconstructed. Simulations and imaging experiments on different samples have been performed to verify the principle and identify the optimal processing parameters, including the number of laser foci and sampling ratios. The results show that high-resolution images can be obtained by using a 25% sampling ratio and five foci. The new CS-based TPE imaging method may find important applications in biological studies, e.g., neuronal imaging and optogenetics.

Spatially resolved random-access pump-probe microscopy based on binary holography.

In this Letter, we present a spatially resolved pump-probe microscope based on a digital micromirror device (DMD). The microscope system enables the measurements of ultrafast transient processes at arbitrarily selected regions in a 3-D specimen. To achieve random-access scanning, the wavefront of the probe beam is modulated by the DMD via binary holography. By switching the holograms stored in the DMD memory, the laser focus can be rapidly moved in space in a discrete fashion. The microscope system has a field of view of 65×130×155 µm3 in the x, y, and z axes, respectively; and a scanning speed of 8 kHz which is limited by the response time of the lock-in amplifier. To demonstrate the pump-probe system, we measured the ultrafast transient reflectivity of 2-D gold patterns on a silicon substrate and on silicon nitride cantilever beams. The results show an excellent signal-to-noise ratio and spatial-temporal resolution, as well as the 3-D random scanning capability. The new pump-probe microscope is a versatile instrument to characterize ultrafast 3-D phenomena with high spatial and temporal resolution, e.g., the propagation of localized surface plasmon resonance on curved surfaces.

Recent Advances in Surface Plasmon Resonance Imaging Sensors.

The surface plasmon resonance (SPR) sensor is an important tool widely used for studying binding kinetics between biomolecular species. The SPR approach offers unique advantages in light of its real-time and label-free sensing capabilities. Until now, nearly all established SPR instrumentation schemes are based on single- or several-channel configurations. With the emergence of drug screening and investigation of biomolecular interactions on a massive scale these days for finding more effective treatments of diseases, there is a growing demand for the development of high-throughput 2-D SPR sensor arrays based on imaging. The so-called SPR imaging (SPRi) approach has been explored intensively in recent years. This review aims to provide an up-to-date and concise summary of recent advances in SPRi. The specific focuses are on practical instrumentation designs and their respective biosensing applications in relation to molecular sensing, healthcare testing, and environmental screening.

Wood Derived Composites for High Sensitivity and Wide Linear-Range Pressure Sensing.

Natural wood possesses a unique 3D microstructure containing hierarchical interconnected channels along its growth direction. This study reports a facile processing strategy to utilize such structure to fabricate carbon/silicone composite based flexible pressure sensors. The unique contribution of the multichannel structure on the sensor performance is analyzed by comparing the pressure response of the vertically cut and horizontally cut composite structures. The results show that the horizontally cut composite based sensors exhibit much higher sensitivity (10.74 kPa-1 ) and wider linear region (100 kPa, R2 = 99%), due to their rough surface and largely deformable microstructure. Besides, the sensors also show little hysteresis and good cycle stability. The overall outstanding sensing properties of the sensors allow for accurate continuous measurement of human pulse and respiration, benefiting the real-time health signal monitoring and disease diagnoses.

Real-time multi-channel SPR sensing based on DMD-enabled angular interrogation.

This paper reports a digital micro-mirror device (DMD)-enabled real-time multi-channel biosensing system based on angular interrogation surface plasmon resonance (SPR). In the experiments, angular scanning is achieved by a DMD that facilitates SPR measurements using a single-point photodetector. In the four-channel measurement setup, real-time monitoring of bovine serum albumin (BSA) and anti-BSA binding interactions is performed at various concentration levels. The experimental results have verified that the system has a resolution of 3.54 × 10-6 RIU (refractive index unit); and a detection limit of 9 ng/mL. The new DMD-based SPR interrogation system presents a new design route for practical solid-state SPR biosensing with a user-selectable range of interrogation, enhanced signal-to-noise ratio, and fast data throughput.