Altered collagen fiber kinematics define the onset of localized ligament damage during loading

Detecting the initiation of mechanical injury to biological tissue, and not just its ultimate failure, is critical to a sensitive and specific characterization of tissue tolerance, development of quantitative relationships between macro- and microstructural tissue responses, and appropriate interpretation of physiological responses to loading. We have developed a novel methodological approach to detect the onset and spatial location of structural damage in collagenous soft tissue, before its visible rupture, via identification of atypical regional collagen fiber kinematics during loading. Our methods utilize high-speed quantitative polarized light imaging to identify the onset of tissue damage in ligament regions where mean collagen fiber rotation significantly deviates from its behavior during noninjurious loading. This technique was validated by its ability to predict the location of visible rupture (P = 0.0009). This fiber rotation-based metric of damage identifies potential facet capsular ligament injury beginning well before rupture, at 51 +/- 12% of the displacement required to produce tissue failure. Although traditional macroscale strain metrics fail to identify the location of microstructural damage, initial injury detection determined by altered fiber rotation was significantly correlated (R = 0.757, P = 0.049) with tissue yield (defined by a decrease in stiffness), supporting the capabilities of this method. Damaged regions exhibited higher variance in fiber direction than undamaged regions (P = 0.0412).     

High-speed volumetric imaging in vivo based on structured illumination microscopy with interleaved reconstruction

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies, due to its high imaging speed over large field-of-views. However, WFFM is susceptible to out-of-focus background. To overcome this problem, structured illumination microscopy (SIM) was proposed as a wide-field, optical-sectioning technique, which needs multiple raw images for image reconstruction and thus has a lower imaging speed. Here we propose SIM with interleaved reconstruction, to make SIM of lossless speed. We apply this method in volumetric imaging of neural network dynamics in brains of zebrafish larva in vivo.     

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Self-phase-modulated femtosecond laser source at 1603 nm and its application to deep-brain 3-photon microscopy in vivo

3-photon microscopy (3PM) excited at the 1700 nm window enables deep-tissue imaging in vivo, especially in brain. PC rod soliton source has previously been exclusively used as the excitation source, which is rather costly and difficult to align. Here we demonstrate a novel nonlinear optical technique to build femtosecond laser source at the 1700 nm window, based on self-phase modulation (SPM) in a short span of large-mode-area fiber. The spectral broadening experienced by the pump pulse leads to the generation of a red-shifted sidelobe at 1603 nm. After spectral filtering, this sidelobe corresponds to 170-fs, 167-nJ pulses at 1603 nm. Using this SPM source, we further demonstrate deep-brain 3 PM to a depth of 1500 μm below the mouse brain surface in vivo. Our SPM femtosecond laser source thus provides a cost effective and easy-to-align alternative excitation source to the PC rod soliton source.     

Polarization clustering of biological structures with Mueller matrix parameters

Mueller matrix imaging polarimetry (MMIP) is a promising technique for the characterization of biological tissues, including the classification of microstructures in pathological diagnosis. To expand the parameter space of Mueller matrix parameters, we propose new vector parameters (VPs) according to the Mueller matrix polar decomposition method. We measure invasive bladder cancer (IBC) with extensive necrosis and high-grade ductal carcinoma in situ (DCIS) with MMIP, and the regions of cancer cells and fibrotic stroma are classified with the VPs. Then the proposed and existing VPs are mapped on the Poincaré sphere with 3D visualization, and an indicator of spatial feature is defined based on the minimum enclosing sphere to evaluate the classification capability of the VPs. For both IBC and DCIS, the results show that the proposed VPs exhibit evident contrast between the regions of cancer cells and fibrotic stroma. This study broadens the fundamental Mueller matrix parameters and helps to improve the characterization ability of the MMIP technique.     

Determination of the diameter of simulated human capillaries using shifted position-diffuse reflectance imaging

Multiple diseases are associated with a wide spectrum of microvascular dysfunctions, microangiopathies and microcirculation disorders. Monitoring the microcirculation could thus be useful to diagnose many local and systemic circulatory disorders and to supervise critically ill patients. Many of the scores currently available to help identify the condition of a microcirculation disorder are invasive or leave scope for interpretation. Thus, the present study aims to investigate with Monte-Carlo simulations (as numerical solutions of the radiative transfer equation) whether shifted position-diffuse reflectance imaging (SP-DRI), a non-invasive diagnostic technique, reveals information on the capillary diameter to assess the state of the microcirculation. To quantify the SP-DRI signal, the modulation parameter K is introduced. It proves to correlate almost perfectly with the capillary diameter (), making it a valid parameter for reliably assessing microcirculation. SP-DRI is emerging as an important milestone on the way to early and conveniently diagnosing microcirculation associated diseases.     

Polarization-resolved SHG imaging as a fast screening method for collagen alterations during aging: Comparison with light and electron microscopy

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15-day-, 1-month- and 21-month-old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization-resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label-free method to previously fixed samples.     

Optoacoustic monitoring of water content in tissue phantoms and human skin

Skin water content monitoring is important for diagnostics and management of edema, dehydration, and other skin conditions as well as for cosmetic applications. Because optoacoustic (OA) technique has high (optical) contrast and (ultrasound) resolution and significant probing depth, it may be suitable for accurate, noninvasive water content monitoring in the skin. In this work we studied OA response from skin tissue phantoms and human wrist skin in the wavelength range from 1370 nm to 1650 nm using a novel, tunable OPO OA system. We identified optimal wavelengths for OA water content monitoring in different skin layers. The results of our study suggest that the OA technique may become a valuable, quantitative tool for accurate, high-resolution water content monitoring in the skin and other tissues and may find wide applications in dermatology, cosmetology, and tissue trauma management.     

Interferometric optical fiber sensor for optoacoustic endomicroscopy

Optical fiber sensors can offer robust and miniaturized detection of wideband ultrasound, yielding high sensitivity and immunity to electromagnetic interference. However, the lack of cost-effective manufacturing methods prevents the disseminated use of these sensors in biomedical applications. In this study, we developed and optimized a simple method to create optical cavities with high-quality mirrors for acoustic sensing based on micro-manipulation of UV-curable optical adhesives and electroless chemical silver deposition. This approach enables the manufacturing of ultrasound sensors based on Fabry-Pérot interferometers on optical fiber tips with minimal production costs. Characterization and high-resolution optoacoustic imaging experiments show that the manufacturing process yielded a fiber sensor with a small NEP () over a broad detection bandwidth (25 MHz), generally outperforming conventional piezoelectric based transducers. We discuss how the new manufacturing process leads to a high-performance acoustic detector that, due to low cost, can be used as a disposable sensor.     

Deep learning: step forward to high-resolution in vivo shortwave infrared imaging

Shortwave infrared window (SWIR: 1000–1700 nm) represents a major improvement compared to the NIR-I region (700–900 nm) in terms of temporal and spatial resolutions in depths down to 4 mm. SWIR is a fast and cheap alternative to more precise methods such as X-ray and opto-acoustic imaging. Main obstacles in SWIR imaging are the noise and scattering from tissues and skin that reduce the precision of the method. We demonstrate that the combination of SWIR in vivo imaging in the NIR-IIb region (1500–1700 nm) with advanced deep learning image analysis allows to overcome these obstacles and making a large step forward to high resolution imaging: it allows to precisely segment vessels from tissues and noise, provides morphological structure of the vessels network, with learned pseudo-3D shape, their relative position, dynamic information of blood vascularization in depth in small animals and distinguish the vessels types: artieries and veins. For demonstration we use neural network IterNet that exploits structural redundancy of the blood vessels, which provides a useful analysis tool for raw SWIR images.     

Concept of photonic hook scalpel generated by shaped fiber tip with asymmetric radiation

Structured light have made deep impacts on modern biotechnology and clinical practice, with numerous optical systems and lasers currently being used in medicine to treat disease. We demonstrate a new concept of fiber-based optical hook scalpel. The subwavelength photonic hook is obtained in the vicinity of a shaped fiber tip with asymmetric radiation. A 1550 nm continuous-wave source, commonly used for medical imaging, has been required. Photonic hook with a lateral feature size less than the half-wavelength is achieved using a hemispherical shaped fiber tip with metallic mask. This breakthrough is carried out in ambient air by using a 4-μm-diameter fiber with a shaped tip. A good correlation is observed between the computed intensity distribution of photonic hook and the tip sizes. Photonic hook generated with a shaped fiber tip, easier to manipulate, shows far-reaching benefits for potential applications such as ophthalmic laser surgery, super-resolution microscopy, photolithography and material processing.     

Optimization for continuous-wave terahertz reflection imaging for biological tissues

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuous-wave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application.     

Improved accuracy of quantitative birefringence imaging by polarization sensitive OCT with simple noise correction and its application to neuroimaging

Polarization-sensitive optical coherence tomography (PS-OCT) enables three-dimensional imaging of biological tissues based on the inherent contrast provided by scattering and polarization properties. In fibrous tissue such as the white matter of the brain, PS-OCT allows quantitative mapping of tissue birefringence. For the popular PS-OCT layout using a single circular input state, birefringence measurements are based on a straight-forward evaluation of phase retardation data. However, the accuracy of these measurements strongly depends on the signal-to-noise ratio (SNR) and is prone to mapping artifacts when the SNR is low. Here we present a simple yet effective approach for improving the accuracy of PS-OCT phase retardation and birefringence measurements. By performing a noise bias correction of the detected OCT signal amplitudes, the impact of the noise floor on retardation measurements can be markedly reduced. We present simulation data to illustrate the influence of the noise bias correction on phase retardation measurements and support our analysis with real-world PS-OCT image data.     

Near-infrared double-illumination optical-resolution photoacoustic microscopy

Label-free chemical bond imaging is of great importance in biology and medicine. Photoacoustic imaging at the third near-infrared windows (1600-1870 nm, near-infrared-III) provides a stable molecular vibrational imaging tool for lipid-rich tissue owing to the first overtone transition of the C H bond at 1.7 μm. However, lacking high-energy pulsed laser sources at 1.7 μm and the strong water absorption significantly limit the signal-to-noise ratio of the lipid imaging, especially for thin lipid tissues. To circumvent this barrier, we develop near-infrared-III double-illumination optical-resolution photoacoustic microscopy (DIOR-PAM) for improving the sensitivity of label-free lipid imaging. Using the same laser, DIOR-PAM can enhance the sensitivity by nearly 100%, which we prove in the Monte Carlo simulation. We experimentally demonstrated 50% ~ 100% sensitivity enhancements on nonbiological and biological lipid-rich samples.     

Dual‐ and single‐shot susceptibility ratio measurements with circular polarizations in second‐harmonic generation microscopy

Polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z‐axis parallel and x‐axis perpendicular to the C₆ symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P‐SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual‐shot configuration where the SHG circular anisotropy generated using incident right‐ and left‐handed circularly‐polarized light is measured; and a single‐shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes‐Mueller polarimetry. The dual‐ and single‐shot circular anisotropy measurements can be used for fast imaging that is independent of the in‐plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.     

Mesoscopic fluorescence lifetime imaging: Fundamental principles,clinical applications and future directions

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.     

Optical coherence hyperspectral microscopy with a single supercontinuum light source

In the paper, we have developed an optical coherence hyperspectral microscopy with a single supercontinuum light source. The microscopy consists of optical coherence tomography (OCT) and hyperspectral imaging (HSI), which can visualize the structural and functional characteristics of biological tissues. The 500 to 700 nm band is selected for HSI and OCT imaging, where HSI enables imaging of oxygen saturation and hemoglobin (Hb) content, while OCT acquires structural characteristics to assess the morphology of biological tissues. The system performance of the optical coherence hyperspectral microscopy is verified by normal mice ears, and the practical applications of the microscopy is further performed in 4T1 and inflammation Balb/c mice ears in vivo. The experimental results demonstrate that the microscopy has potential to provide complementary information for clinical applications.     

Development of a polarization imaging method to detect paraffin-embedded pathology tissues before applying other techniques

The present article describes the development of a technique, applied to paraffin-embedded tissues, which uses three different wavelengths of monochromatic light (λ1 = 445 nm, λ2 = 540 nm and λ3 = 660 nm) for the measures of the degree of polarization, degree of linear polarization, degree of circular polarization and birefringence, all obtained from measurements of Stokes parameters by using polarized light. The goal of this study was to detect changes in developing embryonic mouse eye when pregnant mice fed diets without folic acid for variable periods compared with a healthy control group. We present a biomedical diagnostic technique based on polarized light detection applied to paraffin-embedded tissues to visualize the structural damage to aid us in the diagnosis before applying other techniques. Through this method, we can visualize and identify which parts of the tissue were altered with respect to the control group.     

Automated thermal imaging monitors the local response to cervical cancer brachytherapy

Malignant tumors have high metabolic and perfusion rates, which result in a unique temperature distribution as compared to healthy tissues. Here, we sought to characterize the thermal response of the cervix following brachytherapy in women with advanced cervical carcinoma. Six patients underwent imaging with a thermal camera before a brachytherapy treatment session and after a 7-day follow-up period. A designated algorithm was used to calculate and store the texture parameters of the examined tissues across all time points. We used supervised machine learning classification methods (K Nearest Neighbors and Support Vector Machine) and unsupervised machine learning classification (K-means). Our algorithms demonstrated a 100% detection rate for physiological changes in cervical tumors before and after brachytherapy. Thus, we showed that thermal imaging combined with advanced feature extraction could potentially be used to detect tissue-specific changes in the cervix in response to local brachytherapy for cervical cancer.     

Histopathological correlations of bulk tissue polarimetric images: Case study

Polarimetric imaging and image analysis have gained increased interest in soft tissue analysis at the cellular level. However, polarimetric imaging has widely been tested on thin tissue sections to provide reliable information correlated with histopathological findings. Polarimetric bulk tissue analysis always offered an overall assessment of various tissue optical properties for diagnosis. In this study, the histopathological correlation of bulk tissue polarimetry images for soft tissues is discussed. The first-hand information on the use of bulk tissue Mueller polarimetry and image analysis as an alternative to tissue histopathology is presented for surgically extracted colon and breast tissues.