Architecture-driven quantitative nanoscopy maps cytoskeleton remodeling.

Cytoskeleton remodeling which generates force and orchestrates signaling and trafficking to govern cell migration remains poorly understood, partly due to a lack of an investigation tool with high system flexibility, spatiotemporal resolution, and computational sensitivity. Herein, we developed a multimodal superresolution imaging system-based architecture-driven quantitative (ADQ) framework in spatiotemporal-angular hyperspace to enable both identification of the optimal imaging mode with well-balanced fidelity and phototoxicity and accurate postcharacterization of microtubule remodeling. In the ADQ framework, a pixel/voxel-wise metric reflecting heterogeneous intertubule alignment was proposed with improved sensitivity over previous efforts and further incorporated with temporal features to map dynamic microtubule rearrangements. The ADQ framework was verified by assessing microtubule remodeling in drug-induced (de)polymerization, lysosome transport, and migration. Different remodeling patterns from two migration modes were successfully revealed by the ADQ framework, with a front-rear polarization for individual directed migration and a contact site-centered polarization for cell-cell interaction-induced migration in an immune response model. Meanwhile, these migration modes were found to have consistent orientation changes, which exhibited the potential of predicting migration trajectory.

Long range topography by dispersion unmatched spectral-domain interferometry based on virtually imaged phased array modes.

We propose to realize a long range topography by dispersion unmatched spectral-domain interferometry based on virtually imaged phased array (VIPA) modes. By filtering the continuous spectrum of a supercontinuum source through a side-entrance Fabry-Perot etalon configured at two input angles, two groups of VIPA modes are generated. A method based on unmatched dispersion is proposed for non-aliasing reconstruction of the true depth from the interference spectrum under-sampled at two groups of VIPA modes. With the high spectral resolution provided by the VIPA modes instead of the grating-based spectrometer, only a 10 dB falloff in sensitivity over a range of 10 mm was demonstrated. The feasibility of the proposed method was confirmed by topography of a sample of gauge blocks and a model of three-dimensional (3D) printed tooth. The occlusal surface of the tooth model was further quantitatively evaluated, demonstrating its potential application in long range 3D topography.

Mapping port wine stain in vivo by optical coherence tomography angiography and multi-metric characterization.

Port wine stain (PWS) is a congenital cutaneous capillary malformation composed of ecstatic vessels, while the microstructure of these vessels remains largely unknown. Optical coherence tomography angiography (OCTA) serves as a non-invasive, label-free and high-resolution tool to visualize the 3D tissue microvasculature. However, even as the 3D vessel images of PWS become readily accessible, quantitative analysis algorithms for their organization have mainly remained limited to analysis of 2D images. Especially, 3D orientations of vasculature in PWS have not yet been resolved at a voxel-wise basis. In this study, we employed the inverse signal-to-noise ratio (iSNR)-decorrelation (D) OCTA (ID-OCTA) to acquire 3D blood vessel images in vivo from PWS patients, and used the mean-subtraction method for de-shadowing to correct the tail artifacts. We developed algorithms which mapped blood vessels in spatial-angular hyperspace in a 3D context, and obtained orientation-derived metrics including directional variance and waviness for the characterization of vessel alignment and crimping level, respectively. Combining with thickness and local density measures, our method served as a multi-parametric analysis platform which covered a variety of morphological and organizational characteristics at a voxel-wise basis. We found that blood vessels were thicker, denser and less aligned in lesion skin in contrast to normal skin (symmetrical parts of skin lesions on the cheek), and complementary insights from these metrics led to a classification accuracy of ∼90% in identifying PWS. An improvement in sensitivity of 3D analysis was validated over 2D analysis. Our imaging and analysis system provides a clear picture of the microstructure of blood vessels within PWS tissues, which leads to a better understanding of this capillary malformation disease and facilitates improvements in diagnosis and treatment of PWS.

Three-dimensional optical coherence digital-null deformography of multi-refractive-surface optics with nanometer sensitivity.

Knowledge of the lens deformation during the reliability test is critical for lens design and fabrication. Refractive surface distorts the optical path of probing light, and poses a great challenge to measuring the test-induced nanoscale changes of all refractive lens surfaces simultaneously. In this work, we present an optical coherence digital-null deformography (ODD). A digital null, i.e., the interference signals (including intensity and phase) of the backscattered probing light from each lens surface, was recorded prior to the test with a phase-sensitive optical coherence tomography (OCT). Then the post-test lens was physically aligned to the digital null by actuating a hexapod iteratively with a digital null alignment (DNA) method, so that the refractive distortion was matched. Finally, the changes between the aligned lens and its digital null were measured with an intensity centroid shift (ICS) at micron scale and a joint wavenumber (k)-depth (z) domain phase shift (kz-PhS) at nanoscale. We demonstrate that the proposed kz-PhS has a sensitivity of 4.15 nm and a range of 5 µm without phase wrapping; and the sensitivities of DNA are z translation 0.04 µm, x/y translation 0.24 µm, tilt 0.0003°, and rotation 0.03°. A lens drop test was performed with ODD. Circumventing refractive distortion by the null measurement, ODD can visualize the test-induced changes of all refractive surfaces non-destructively and simultaneously, and it will greatly facilitate lens design and fabrication.

Optimized number of the primary singular values for image reconstruction in reflection matrix based optical coherence tomography.

A reflection matrix based optical coherence tomography (OCT) is recently proposed and expected to extend the imaging-depth limit twice. However, the imaging depth and hence the image quality heavily depend on the number of primary singular values considered for image reconstruction. To this regard, we propose a method based on correlation between image pairs reconstructed from different number of singular values and corresponding remainders. The obtained correlation curve and another feature curve fetched from the former are then fed to a long short-term memory (LSTM) network classifier to identify the optimized number of primary singular values for image reconstruction. Simulated targets with different combinations of filling fraction and signal-to-noise ratio (SNR) are reconstructed by the developed method as well as two current adopted methods for comparison. The results demonstrate that the proposed method is robust to recover the image with satisfactory similarity close to the reference one. To our knowledge, this is the first comprehensive study on the optimized number of the primary singular values considered for image reconstruction in reflection matrix based OCT.

Identification of human ovarian cancer relying on collagen fiber coverage features by quantitative second harmonic generation imaging.

Ovarian cancer has the highest mortality rate among all gynecological cancers, containing complicated heterogeneous histotypes, each with different treatment plans and prognoses. The lack of screening test makes new perspectives for the biomarker of ovarian cancer of great significance. As the main component of extracellular matrix, collagen fibers undergo dynamic remodeling caused by neoplastic activity. Second harmonic generation (SHG) enables label-free, non-destructive imaging of collagen fibers with submicron resolution and deep sectioning. In this study, we developed a new metric named local coverage to quantify morphologically localized distribution of collagen fibers and combined it with overall density to characterize 3D SHG images of collagen fibers from normal, benign and malignant human ovarian biopsies. An overall diagnosis accuracy of 96.3% in distinguishing these tissue types made local and overall density signatures a sensitive biomarker of tumor progression. Quantitative, multi-parametric SHG imaging might serve as a potential screening test tool for ovarian cancer.

Identification of endoplasmic reticulum formation mechanism by multi-parametric, quantitative super-resolution imaging.

The endoplasmic reticulum (ER) is a highly dynamic membrane-bound organelle in eukaryotic cells which spreads throughout the whole cell and contacts and interacts with almost all organelles, yet quantitative approaches to assess ER reorganization are lacking. Herein we propose a multi-parametric, quantitative method combining pixel-wise orientation and waviness features and apply it to the time-dependent images of co-labeled ER and microtubule (MT) from U2OS cells acquired from two-dimensional structured illumination microscopy (2D SIM). Analysis results demonstrate that these morphological features are sensitive to ER reshaping and a combined use of them is a potential biomarker for ER formation. A new, to the best of our knowledge, mechanism of MT-associated ER formation, termed hooking, is identified based on distinct organizational alterations caused by interaction between ER and MT which are different from those of the other three mechanisms already known, validated by 100% discrimination accuracy in classifying four MT-associated ER formation mechanisms.

Fast simulation and design of the fiber probe with a fiber-based pupil filter for optical coherence tomography using the eigenmode expansion approach.

Fiber probes for optical coherence tomography (OCT) recently employ a short section of step-index multimode fiber (SIMMF) to generate output beams with extended depth of focus (DOF). As the focusing region of the output beam is generally close to the probe end, it is not feasible to adopt the methods for bulk-optics with spatial pupil filters to the fiber probes with fiber-based filters. On the other hand, the applicable method of the beam propagation method (BPM) to the fiber probes is computationally inefficient to perform parameter scan and exhaustive search optimization. In this paper, we propose the method which analyzes the non-Gaussian beams from the fiber probes with fiber-based filters using the eigenmode expansion (EME) method. Furthermore, we confirm the power of this method in designing fiber-based filters with increased DOF gain and uniformly focusing by introducing more and higher-order fiber modes. These results using the EME method are in good agreement with that by the BPM, while the latter takes 1-2 orders more computation time. With higher-order fiber modes involved, a novel probe design with increased DOF gain and suppressed sidelobe is proposed. Our findings reveal that the fiber probes based on SIMMFs are able to achieve about four times DOF gain at maximum with uniformly focusing under acceptable modal dispersion. The EME method enables fast and accurate simulation of fiber probes based on SIMMFs, which is important in the design of high-performance fiber-based micro-imaging systems for biomedical applications.

Uniform focusing with an extended depth range and increased working distance for optical coherence tomography by an ultrathin monolith fiber probe.

It is difficult to maintain high transverse resolution over an increased depth range using miniature probes for optical coherence tomography (OCT) due to the rapid divergence of light and the space limitation. To solve this problem, we introduce a fiber-based filter in the proposed probe to manipulate its output beam. Significant mode interference (MI) is exploited to enhance the depth of focus (DOF), and the mode phase difference is tuned to achieve a uniform axial intensity within the DOF. The magnified MI field instead of the diffracted one is adopted as the final pupil filter in the probe to increase its working distance (WD). The probe is fabricated with a diameter of 125 µm and a total length of 2.6 mm for its distal fiber optics. Compared to the conventional probe with similar minimal lateral resolution of better than 4.4 µm, the proposed probe achieves two times that of the DOF gain and 1.7 times that of the WD. Improvements in performance of the probe are demonstrated by OCT imaging using a fresh lemon and human skin. With merits of enhanced imaging quality and easy fabrication, the proposed probe poses great potential for important applications, especially for endoscopic imaging of human internal organs in vivo.

Motion correction using overlapped data correlation based on a spatial-spectral encoded parallel optical coherence tomography.

This paper presents an approach to remove motion artifacts based on a spatial-spectral encoded parallel OCT (SSE-POCT) system, where encoded rectangular illumination is employed. Motion artifacts within a B-scan are avoided due to parallel detection intrinsic to parallel OCT, while those between successive B-scans are estimated and corrected by a proposed overlapped data correlation (ODC) algorithm. To preserve axial resolution, decoded B-scan corresponding to complete spectrum is stitched from successive encoded B-scans after motion correction. Imaging is conducted on several samples under preset motion trajectories, and OCT images with unnoticed motion artifacts and well-preserved resolutions are reconstructed. The approach based on the developed SSE-POCT system and the proposed ODC algorithm for motion correction can be applicable for in vivo imaging where uncontrolled motion is usually unavoidable.

Lens-free all-fiber probe with an optimized output beam for optical coherence tomography.

A high-efficiency lensless all-fiber probe for optical coherence tomography (OCT) is presented. The probe is composed of a segment of large-core multimode fiber (MMF), a segment of tapered MMF, and a length of single-mode fiber (SMF). A controllable output beam can be designed by a simple adjustment of its probe structure parameters (PSPs), instead of the selection of fibers with different optical parameters. A side-view probe with a diameter of 340 µm and a rigid length of 6.37 mm was fabricated, which provides an effective imaging range of ∼0.6 mm with a full width at half-maximum beam diameter of less than 30 µm. The insertion loss of the probe was measured to be 0.81 dB, ensuring a high sensitivity of 102.25 dB. Satisfactory images were obtained by the probe-based OCT system, demonstrating the feasibility of the probe for endoscopic OCT applications.

Photon ensemble correlation spectroscopy enables decorrelation-rate-limited ultrafast measurement of diffusive particle dynamics.

In contrast to conventional dynamic light scattering (DLS) measurement via a single sampling volume (SV) observation over a long time span, we propose a novel technique named "photon ensemble correlation spectroscopy" for ultrafast characterization of diffusive particle dynamics through decorrelation analysis of complex-valued DLS scattering signals from an ensemble of independent SVs. We confirm that the ensemble analysis provides a decorrelation-rate-limited ultrafast measurement and demonstrates the feasibility of imaging spatially resolved particle dynamics. Moreover, the use of complex-valued signals gives additional superiority in terms of reliability.

Quasi-needle-like focus synthesized by optical coherence tomography.

It is known that lateral resolution and depth of focus (DOF) in an optical imaging system are coupled, and a compromise between them has to be made. In this Letter, we propose to resolve the trade-off between lateral resolution and the DOF by a synthetic effective point spread function in optical path length (OPL) domain. A quasi-needle-like focus is synthesized by optical coherence tomography. We demonstrate that the synthesized quasi-needle-like focus provides a four-fold extension of a conventional DOF, while maintaining a high lateral resolution of 2.5 µm over a depth range of approximately 240 µm. The focal range can be further extended with more optical path length coded beams for synthesis involved.

Resolution enhancement of saturated fluorescence emission difference microscopy.

Fluorescence emission difference microscopy (FED) obtains resolution-enhanced images by subtracting acquired solid and doughnut confocal images. Because of the mismatch of the outer contours of the two subtraction components, negative values are inevitable in the conventional FED method, giving rise to deformations. In this study, by using a saturation effect, we obtain imaging results with a profile-extended solid and center-shrunken doughnut point spread function. Owing to the nonlinear effect, two better-matched saturated images not only eliminate the deformations, but also enhance the resolving ability and signal to noise ratio compared to conventional FED. Simulations based on the saturated model of rhodamine 6G, as well as experiments on biological samples, are presented to verify the capability of the proposed concept, while experimental results show the unprecedented resolving ability of the saturated FED method.

Hybrid averaging offers high-flow contrast by cost apportionment among imaging time, axial, and lateral resolution in optical coherence tomography angiography.

The current temporal, wavelength, angular, and spatial averaging approaches trade imaging time and resolution for multiple independent measurements that improve the flow contrast in optical coherence tomography angiography (OCTA). We find that these averaging approaches are equivalent in principle, offering almost the same flow contrast enhancement as the number of averages increases. Based on this finding, we propose a hybrid averaging strategy for contrast enhancement by cost apportionment. We demonstrate that, compared with any individual approach, the hybrid averaging is able to offer a desired flow contrast without severe degradation of imaging time and resolution. Making use of the extended range of a VCSEL-based swept-source OCT, an angular averaging approach by path length encoding is also demonstrated for flow contrast enhancement.

Single-shot angular compounded optical coherence tomography angiography by splitting full-space B-scan modulation spectrum for flow contrast enhancement.

We proposed a single-shot spatial angular compounded optical coherence tomography angiography (AC-Angio-OCT) for blood flow contrast enhancement. By encoding incident angles in B-scan modulation frequencies and splitting the modulation spectrum in the spatial frequency domain, angle-resolved independent subangiograms were obtained and compounded to improve the flow contrast. A full space of the spatial frequency domain allows a wide modulation spectrum. To get access to the full space of the spatial frequency domain and avoid the complex-conjugate ambiguity of the modulation spectrum, a complex-valued OCT spectral interferogram was retrieved by removing one of the conjugate terms in the depth space. To validate the proposed concept, both flow phantom and live animal experiments were performed. The proposed AC-Angio-OCT offers a ∼50% decrease of misclassification errors, and an improved flow contrast and vessel connectivity, which contributes to the interpretation of OCT angiograms.

High-speed high-precision and ultralong-range complex spectral domain dimensional metrology.

A precise, nondestructive dimensional metrological system is crucial to manufacturing and packaging of multi-component optical system. To this end, an orthogonal dispersive spectrometer based complex spectral domain interferometric system for high-speed high-precision and ultralong-range dimensional metrology is developed. An improved complex method based on actual spectral phase shift is proposed to achieve ultrahigh suppression of artifacts. Suppression ratios of 80 dB for DC and 60 dB for mirror images are realized, the highest ratios among existing complex methods. To ensure high-precision in distance determination, an averaged spectral phase measurement algorithm is adopted. A precision of 60 nm within a measurement range of 200 mm without axial movement of the sample is demonstrated. The measurement range is readily extendable if axial movement of the sample and range cascading are involved. The system holds potential applications in various areas for real-time nondestructive testing and evaluation.

Identification of surface defects on glass by parallel spectral domain optical coherence tomography.

Defects can dramatically degrade glass quality, and automatic inspection is a trend of quality control in modern industry. One challenge in inspection in an uncontrolled environment is the misjudgment of fake defects (such as dust particles) as surface defects. Fortunately, optical changes within the periphery of a surface defect are usually introduced while those of a fake defect are not. The existence of changes within the defect peripheries can be adopted as a criterion for defect identification. However, modifications within defect peripheries can be too small to be noticeable in intensity based optical image of the glass surface, and misjudgments of modifications may occur due to the incorrectness in defect demarcation. Thus, a sensitive and reliable method for surface defect identification is demanded. To this end, a nondestructive method based on optical coherence tomography (OCT) is proposed to precisely demarcate surface defects and sensitively measure surface deformations. Suspected surface defects are demarcated using the algorithm based on complex difference from expectation. Modifications within peripheries of suspected surface defects are mapped by phase information from complex interface signal. In this way, surface defects are discriminated from fake defects using a parallel spectral domain OCT (SD-OCT) system. Both simulations and experiments are conducted, and these preliminary results demonstrate the advantage of the proposed method to identify glass surface defects.

Dual-mode super-resolution imaging with stimulated emission depletion microscopy and fluorescence emission difference microscopy.

Dual-mode super-resolution imaging system with two different super-resolution imaging methods, STED and FED, is presented. Electrical shutters controlled by the host computer are introduced to switch the two imaging modes. Principles of both methods are analyzed theoretically, and enhancements in the lateral resolution and SNR are demonstrated theoretically and experimentally. Results show that both imaging methods offered by the proposed system can break the diffraction barrier. Furthermore, the presented system provides a meaningful way to image fluorescent samples by a corresponding imaging mode according to the specific characteristics of samples analyzed for study. For samples that can endure high-power illumination, it is appropriate to use the STED mode to achieve a better resolution, while for samples that are vulnerable to high intensity, the FED method is a better choice because no high-power beam is needed, and the FED method can provide better resolution than STED when no high-power beam is allowed. The flexible switching of the two super-resolution imaging modes can help researchers to make most of the advantages of each imaging method. It is believed that the presented system has the potential to be widely used in future nanoscale investigations.

Orthogonal dispersive spectral-domain optical coherence tomography.

Ultrahigh depth range spectral domain optical coherence tomography (SDOCT) can be realized based on the orthogonal dispersive spectrometer consisted by a high spectral resolution virtually-imaged phased array (VIPA) and a low spectral resolution grating. However, two critical issues result in the challenge of obtaining desirable one-dimensional (1-D) spectra from the recorded two-dimensional (2-D) orthogonal spectra for high-quality OD-SDOCT imaging. One is the wavenumber mapping errors and the other is the periodic intensity modulations. The paper proposes a method for desirable reconstruction of 1-D spectra from the recorded 2-D orthogonal spectra. A sample etalon with identical parameters to the dispersive VIPA is used to determine the free spectrum range (FSR) of the VIPA, and spectral phases from two reflecting mirrors are further applied for broadband wavenumber calibration. The cascading of column spectra are performed from interval of four lines of column spectra, and four records of cascaded 1-D spectra are obtained and then averaged to alleviate the periodic intensity modulations. Broadband 1-D spectra are thus reconstructed with an ultrahigh spectral resolution. To demonstrate the feasibility of the proposed method, three typical samples are imaged by the OD-SDOCT system.