SHG fiberscopy assessment of collagen morphology and its potential for breast cancer optical histology.

OBJECTIVE: this study is to investigate the feasibility of our recently developed nonlinear fiberscope for label-free in situ breast tumor detection and lymph node status assessment based on second harmonic generation (SHG) imaging of fibrillar collagen matrix with histological details. The long-term goal is to improve the current biopsy-based cancer paradigm with reduced sampling errors. METHODS: in this pilot study we undertook retrospective SHG imaging study of ex vivo invasive ductal carcinoma human biopsy tissue samples, and carried out quantitative image analysis to search for collagen structural signatures that are associated with the malignance of breast cancer. RESULTS: SHG fiberscopy image-based quantitative assessment of collagen fiber morphology reveals that: 1) cancerous tissues contain generally less extracellular collagen fibers compared with tumor-adjacent normal tissues, and 2) collagen fibers in lymph node positive biopsies are more aligned than lymph node negative counterparts. CONCLUSION/SIGNIFICANCE: the results demonstrate the promising potential of our SHG fiberscope for in situ breast tumor detection and lymph node involvement assessment and for offering real-time guidance during ongoing tissue biopsy.

Comprehensive analysis of FOXM1 immune infiltrates, m6a, glycolysis and ceRNA network in human hepatocellular carcinoma.

Background: Forkhead box M1 (FOXM1) is a member of the Forkhead box (Fox) transcription factor family. It regulates cell mitosis, cell proliferation, and genome stability. However, the relationship between the expression of FOXM1 and the levels of m6a modification, immune infiltration, glycolysis, and ketone body metabolism in HCC has yet to be fully elucidated. Methods: Transcriptome and somatic mutation profiles of HCC were downloaded from the TCGA database. Somatic mutations were analyzed by maftools R package and visualized in oncoplots. GO, KEGG and GSEA function enrichment was performed on FOXM1 co-expression using R. We used Cox regression and machine learning algorithms (CIBERSORT, LASSO, random forest, and SVM-RFE) to study the prognostic value of FOXM1 and immune infiltrating characteristic immune cells in HCC. The relationship between FOXM1 and m6A modification, glycolysis, and ketone body metabolism were analyzed by RNA-seq and CHIP-seq. The competing endogenous RNA (ceRNA) network construction relies on the multiMiR R package, ENCORI, and miRNET platforms. Results: FOXM1 is highly expressed in HCC and is associated with a poorer prognosis. At the same time, the expression level of FOXM1 is significantly related to the T, N, and stage. Subsequently, based on the machine learning strategies, we found that the infiltration level of T follicular helper cells (Tfh) was a risk factor affecting the prognosis of HCC patients. The high infiltration of Tfh was significantly related to the poor overall survival rate of HCC. Besides, the CHIP-seq demonstrated that FOXM1 regulates m6a modification by binding to the promoter of IGF2BP3 and affects the glycolytic process by initiating the transcription of HK2 and PKM in HCC. A ceRNA network was successfully obtained, including FOXM1 - has-miR-125-5p - DANCR/MIR4435-2HG ceRNA network related to the prognosis of HCC. Conclusion: Our study implicates that the aberrant infiltration of Tfh associated with FOXM1 is a crucial prognostic factor for HCC patients. FOXM1 regulates genes related to m6a modification and glycolysis at the transcriptional level. Furthermore, the specific ceRNA network can be used as a potential therapeutic target for HCC.

High-speed light-sheet microscopy for the in-situ acquisition of volumetric histological images of living tissue.

Histological examinations typically require the excision of tissue, followed by its fixation, slicing, staining, mounting and imaging, with timeframes ranging from minutes to days. This process may remove functional tissue, may miss abnormalities through under-sampling, prevents rapid decision-making, and increases costs. Here, we report the feasibility of microscopes based on swept confocally aligned planar excitation technology for the volumetric histological imaging of intact living tissue in real time. The systems' single-objective, light-sheet geometry and 3D imaging speeds enable roving image acquisition, which combined with 3D stitching permits the contiguous analysis of large tissue areas, as well as the dynamic assessment of tissue perfusion and function. Implemented in benchtop and miniaturized form factors, the microscopes also have high sensitivity, even for weak intrinsic fluorescence, allowing for the label-free imaging of diagnostically relevant histoarchitectural structures, as we show for pancreatic disease in living mice, for chronic kidney disease in fresh human kidney tissues, and for oral mucosa in a healthy volunteer. Miniaturized high-speed light-sheet microscopes for in-situ volumetric histological imaging may facilitate the point-of-care detection of diverse cellular-level biomarkers.

Multicolor fiber-optic two-photon endomicroscopy for brain imaging.

Visualizing activity patterns of distinct cell types during complex behaviors is essential to understand complex neural networks. It remains challenging to excite multiple fluorophores simultaneously so that different types of neurons can be imaged. In this Letter, we report a multicolor fiber-optic two-photon endomicroscopy platform in which two pulses from a Ti:sapphire laser and an optical parametric oscillator were synchronized and delivered through a single customized double-clad fiber to excite multiple chromophores. A third virtual wavelength could also be generated by spatial-temporal overlapping of the two pulses. The performance of the fiber-optic multicolor two-photon endomicroscope was demonstrated by in vivo imaging of a mouse cerebral cortex with "Brainbow" labeling.

Throughput-Speed Product Augmentation for Scanning Fiber-Optic Two-Photon Endomicroscopy.

Compactness, among several others, is one unique and very attractive feature of a scanning fiber-optic two-photon endomicroscope. To increase the scanning area and the total number of resolvable pixels (i.e., the imaging throughput), it typically requires a longer cantilever which, however, leads to a much undesired, reduced scanning speed (and thus imaging frame rate). Herein we introduce a new design strategy for a fiber-optic scanning endomicroscope, where the overall numerical aperture (NA) or beam focusing power is distributed over two stages: 1) a mode-field focuser engineered at the tip of a double-clad fiber (DCF) cantilever to pre-amplify the single-mode core NA, and 2) a micro objective of a lower magnification (i.e.,  âˆ¼ 2× in this design) to achieve final tight beam focusing. This new design enables either an ~9-fold increase in imaging area (throughput) or an ~3-fold improvement in imaging frame rate when compared to traditional fiber-optic endomicroscope designs. The performance of an as-designed endomicroscope of an enhanced throughput-speed product was demonstrated by two representative applications: (1) high-resolution imaging of an internal organ (i.e., mouse kidney) in vivo over a large field of view without using any fluorescent contrast agents, and (2) real-time neural imaging by visualizing dendritic calcium dynamics in vivo with sub-second temporal resolution in GCaMP6m-expressing mouse brain. This cascaded NA amplification strategy is universal and can be readily adapted to other types of fiber-optic scanners in compact linear or nonlinear endomicroscopes.

Low-cost, ultracompact handheld optical coherence tomography probe for in vivo oral maxillofacial tissue imaging.

SIGNIFICANCE: Optical coherence tomography (OCT) has proven useful for detecting various oral maxillofacial abnormalities. To apply it to clinical applications including biopsy guidance and routine screening, a handheld imaging probe is indispensable. OCT probes reported for oral maxillofacial imaging were either based on a bulky galvanometric mirror pair (not compact or long enough) or a distal-end microelectromechanical systems (MEMS) scanner (raised safety concerns), or adapted from fiber-optic catheters (ill-suited for oral cavity geometry). AIM: To develop a handheld probe featuring great compactness and excellent maneuverability for oral maxillofacial tissue imaging. APPROACH: A dual-axis MEMS scanner was deployed at the proximal end of the probe and the scanned beam was relayed to the distal end through a 4f configuration. Such design provides both a perfect dual-axis telecentric scan and excellent compactness. RESULTS: A handheld probe with a rigid part 70 mm in length and 7 mm in diameter and weighing 25 g in total was demonstrated through both ex vivo and in vivo experiments, including structural visualization of various oral maxillofacial tissues and monitoring the recovery process of an oral mucosa canker sore. CONCLUSIONS: The proposed probe exhibits excellent maneuverability and imaging performance showing great potential in clinical applications.

Robust, accurate depth-resolved attenuation characterization in optical coherence tomography.

Depth-resolved optical attenuation coefficient is a valuable tissue parameter that complements the intensity-based structural information in optical coherent tomography (OCT) imaging. Herein we systematically analyzed the under- and over-estimation bias of existing depth-resolved methods when applied to real biological tissues, and then proposed a new algorithm that remedies these issues and accommodates general OCT data that contain incomplete decay and noise floor, thereby affording consistent estimation accuracy for practical biological samples of different scattering properties. Compared with other algorithms, our method demonstrates remarkably improved estimation accuracy and numerical robustness, as validated via numerical simulations and on experimental OCT data obtained from both silicone-TiO2 phantoms and human ventral tongue leukoplakia samples.

Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0.

The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes per second and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving Caenorhabditis elegans, and analyze real-time blood flow and calcium dynamics in the beating zebrafish heart. The same system also permits high-throughput structural imaging of mounted, intact, cleared and expanded samples. SCAPE 2.0's significantly lower photodamage compared to point-scanning techniques is also confirmed. Our results demonstrate that SCAPE 2.0 is a powerful, yet accessible imaging platform for myriad emerging high-speed dynamic and high-throughput volumetric microscopy applications.

A biopsy-needle compatible varifocal multiphoton rigid probe for depth-resolved optical biopsy.

In this work, we report a biopsy-needle compatible rigid probe, capable of performing three-dimensional (3D) two-photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge-14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two-dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high-numerical aperture micro-objective, the miniature rigid probe offers a high two-photon resolution (0.833 × 6.11 µm, lateral × axial), a lateral field of view of 120 µm, and an axial focus tuning range of 200 µm. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first-of-its-kind depth-resolved two-photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.

Super-achromatic optical coherence tomography capsule for ultrahigh-resolution imaging of esophagus.

Endoscopic optical coherence tomography (OCT) is a noninvasive technology allowing for imaging of tissue microanatomies of luminal organs in real time. Conventional endoscopic OCT operates at 1300 nm wavelength region with a suboptimal axial resolution limited to 8-20 µm. In this paper, we present the first ultrahigh-resolution tethered OCT capsule operating at 800 nm and offering about 3- to 4-fold improvement of axial resolution (plus enhanced imaging contrast). The capsule uses diffractive optics to manage chromatic aberration over a full ~200 nm spectral bandwidth centering around 830 nm, enabling to achieve super-achromaticity and an axial resolution of ~2.6 µm in air. The performance of the OCT capsule is demonstrated by volumetric imaging of swine esophagus ex vivo and sheep esophagus in vivo, where fine anatomic structures including the sub-epithelial layers are clearly identified. The ultrahigh resolution and excellent imaging contrast at 800 nm of the tethered capsule suggest the potential of the technology as an enabling tool for surveillance of early esophageal diseases on awake patients without the need for sedation.

High-speed, ultrahigh-resolution distal scanning OCT endoscopy at 800 nm for in vivo imaging of colon tumorigenesis on murine models.

We present the first, most compact, ultrahigh-resolution, high-speed, distal scanning optical coherence tomography (OCT) endoscope operating at 800 nm. Achieving high speed imaging while maintaining an ultrahigh axial resolution is one of the most significant challenges with endoscopic OCT at 800 nm. Maintaining an ultrahigh axial resolution requires preservation of the broad spectral bandwidth of the light source throughout the OCT system. To overcome this critical limitation we implemented a distal scanning endoscope with diffractive optics to minimize loss in spectral throughput. In this paper, we employed a customized miniature 900 µm diameter DC micromotor fitted with a micro reflector to scan the imaging beam. We integrated a customized diffractive microlens into the imaging optics to reduce chromatic focal shift over the broad spectral bandwidth of the Ti:Sapphire laser of an approximately 150 nm 3dB bandwidth, affording a measured axial resolution of 2.4 µm (in air). The imaging capability of this high-speed, ultrahigh-resolution distal scanning endoscope was validated by performing 3D volumetric imaging of mouse colon in vivo at 50 frames-per-second (limited only by the A-scan rate of linear CCD array in the spectral-domain OCT system and sampling requirements). The results demonstrated that fine microstructures of colon could be clearly visualized, including the boundary between the absorptive cell layer and colonic mucosa as well the crypt patterns. Furthermore, this endoscope was employed to visualize morphological changes in an enterotoxigenic Bacteriodes fragilis (ETBF) induced colon tumor model. We present the results of our feasibility studies and suggest the potential of this system for visualizing time dependent morphological changes associated with tumorigenesis on murine models in vivo.

Signal-to-noise ratio analysis and improvement for fluorescence tomography imaging.

CCD-based fluorescence tomography is widely used for small animal whole-body imaging. In this report, systematic signal-to-noise ratio (SNR) analyses of a fluorescence tomography imaging (FTI) system were performed, resulting in an easy-to-follow strategy to optimize hardware configurations and operational conditions for acquiring high-quality imaging data and for improving the overall system performance. Phantom experiments were conducted to demonstrate the performance improvement by these optimizations. The improved performance was further verified by imaging a tumor-bearing mouse in vivo. This report provides general and practical guidelines for setting up a high-performance electron multiplying charge coupled device based FTI system to achieve an optimized SNR, which can be useful for future FTI technology development.

Spectro-temporal dispersion management of femtosecond pulses for fiber-optic two-photon endomicroscopy.

The emerging fiber-optic two-photon endomicroscopy technology holds a strong promise for enabling translational applications of nonlinear optical imaging. Effective femtosecond pulse dispersion management is critical for achieving high-quality imaging. Here we report systematic analyses and performance characterization of a dual-fiber spectro-temporal dispersion management scheme involving a grating pair as the pulse stretcher. Compared with conventional linear-only compensation, the grating-based spectro-temporal compensation also takes into account nonlinear effects and enhances the two-photon signal by ~3-fold as experimentally demonstrated. Numerical simulations were carried out to systematically investigate the influence of several key design parameters on the overall compensation efficacy. Furthermore, comprehensive performance comparison with an ideal grism-pair counterpart reveals that a grating-pair stretcher affords much higher power throughput and thus is preferable for portable endomicroscopy systems with limited laser source power.

Fitting-free algorithm for efficient quantification of collagen fiber alignment in SHG imaging applications.

Collagen fiber alignment derived from second harmonic generation (SHG) microscopy images can be important for disease diagnostics. Image processing algorithms are needed to robustly quantify the alignment in images with high sensitivity and reliability. Fourier transform (FT) magnitude, 2D power spectrum, and image autocorrelation have previously been used to extract fiber information from images by assuming a certain mathematical model (e.g. Gaussian distribution of the fiber-related parameters) and fitting. The fitting process is slow and fails to converge when the data is not Gaussian. Herein we present an efficient constant-time deterministic algorithm which characterizes the symmetricity of the FT magnitude image in terms of a single parameter, named the fiber alignment anisotropy R ranging from 0 (randomized fibers) to 1 (perfect alignment). This represents an important improvement of the technology and may bring us one step closer to utilizing the technology for various applications in real time. In addition, we present a digital image phantom-based framework for characterizing and validating the algorithm, as well as assessing the robustness of the algorithm against different perturbations.

Endoscopic forward-viewing optical coherence tomography and angiography with MHz swept source.

Endoscopic optical coherence tomography (OCT) instruments are mostly side viewing and rely on at least one proximal scan, thus limiting accuracy of volumetric imaging and en face visualization. Previous forward-viewing OCT devices had limited axial scan speeds. We report a forward-viewing fiber scanning 3D-OCT probe with 900 µm field of view and 5 µm transverse resolution, imaging at 1 MHz axial scan rate in the human gastrointestinal tract. The probe is 3.3 mm diameter and 20 mm rigid length, thus enabling passage through the endoscopic channel. The scanner has 1.8 kHz resonant frequency, and each volumetric acquisition takes 0.17 s with 2 volumes/s display. 3D-OCT and angiography imaging of the colon was performed during surveillance colonoscopy.

Focus scanning with feedback-control for fiber-optic nonlinear endomicroscopy.

Fiber-optic endomicroscopes open new avenues for the application of non-linear optics to novel in vivo applications. To achieve focus scanning in vivo, shape memory alloy (SMA) wires have been used to move optical elements in miniature endomicroscopes. However, this method has various limitations, making it difficult to achieve accurate and reliable depth scanning. Here we present a feedback-controlled SMA depth scanner. With a Hall effect sensor, contraction of the SMA wire can be tracked in real time, rendering accurate and robust control of motion. The SMA depth scanner can achieve up to 490 µm travel and with open-loop operation, it can move more than 350 µm within one second. With the feedback loop engaged, submicron positioning accuracy was achieved along with superior positioning stability. The high-precision positioning capability of the SMA depth scanner was verified by depth-resolved nonlinear endomicroscopic imaging of mouse brain samples.

Automatic and robust segmentation of endoscopic OCT images and optical staining.

We report a generic method for automatic segmentation of endoscopic optical coherence tomography (OCT) images. In this method, OCT images are first processed with L1 -L0 norm minimization based de-noising and smoothing algorithms to increase the signal-to-noise ratio (SNR) and enhance the contrast between adjacent layers. The smoothed images are then formulated into cost graphs based on their vertical gradients. After that, tissue-layer segmentation is performed with the shortest path search algorithm. The efficacy and capability of this method are demonstrated by automatically and robustly identifying all five interested layers of guinea pig esophagus from in vivo endoscopic OCT images. Furthermore, thanks to the ultrahigh resolution, high SNR of endoscopic OCT images and the high segmentation accuracy, this method permits in vivo optical staining histology and facilitates quantitative analysis of tissue geometric properties, which can be very useful for studying tissue pathologies and potentially aiding clinical diagnosis in real time.

Robust and fast characterization of OCT-based optical attenuation using a novel frequency-domain algorithm for brain cancer detection.

Cancer is known to alter the local optical properties of tissues. The detection of OCT-based optical attenuation provides a quantitative method to efficiently differentiate cancer from non-cancer tissues. In particular, the intraoperative use of quantitative OCT is able to provide a direct visual guidance in real time for accurate identification of cancer tissues, especially these without any obvious structural layers, such as brain cancer. However, current methods are suboptimal in providing high-speed and accurate OCT attenuation mapping for intraoperative brain cancer detection. In this paper, we report a novel frequency-domain (FD) algorithm to enable robust and fast characterization of optical attenuation as derived from OCT intensity images. The performance of this FD algorithm was compared with traditional fitting methods by analyzing datasets containing images from freshly resected human brain cancer and from a silica phantom acquired by a 1310 nm swept-source OCT (SS-OCT) system. With graphics processing unit (GPU)-based CUDA C/C++ implementation, this new attenuation mapping algorithm can offer robust and accurate quantitative interpretation of OCT images in real time during brain surgery.

Nonlinear optical endomicroscopy for label-free functional histology in vivo.

This manuscript reports on the first two-photon, label-free, metabolic imaging of biological tissues in vivo at histological resolution on an extremely compact, fiber-optic endomicroscopy platform. This system provides new opportunities for performing non-invasive and functional histological imaging of internal organs in vivo, in situ and in real time. As a routine clinical procedure, traditional histology has made significant impacts on medicine. However, the procedure is invasive and time consuming, suffers random sampling errors, and cannot provide in vivo functional information. The technology reported here features an extremely compact and flexible fiber-optic probe ~2 mm in diameter, enabling direct access to internal organs. Unprecedented two-photon imaging quality comparable to a large bench-top laser scanning microscope was achieved through technological innovations in double-clad fiber optics and miniature objective lenses (among many others). In addition to real-time label-free visualization of biological tissues in situ with subcellular histological detail, we demonstrated for the first time in vivo two-photon endomicroscopic metabolic imaging on a functioning mouse kidney model. Such breakthroughs in nonlinear endoscopic imaging capability present numerous promising opportunities for paradigm-shifting applications in both clinical diagnosis and basic research.

Optimal operational conditions for supercontinuum-based ultrahigh-resolution endoscopic OCT imaging.

We investigated the optimal operational conditions for utilizing a broadband supercontinuum (SC) source in a portable 800 nm spectral-domain (SD) endoscopic OCT system to enable high resolution, high-sensitivity, and high-speed imaging in vivo. A SC source with a 3-dB bandwidth of ∼246 nm was employed to obtain an axial resolution of ∼2.7 µm (in air) and an optimal detection sensitivity of ∼-107 dB with an imaging speed up to 35 frames/s (at 70 k A-scans/s). The performance of the SC-based SD-OCT endoscopy system was demonstrated by imaging guinea pig esophagus in vivo, achieving image quality comparable to that acquired with a broadband home-built Ti:sapphire laser.