Biocompatibility evaluation of bioprinted decellularized collagen sheet implanted in vivo cornea using swept‐source optical coherence tomography

Corneal transplantation by full‐thickness penetrating keratoplasty with human donor tissue is a widely accepted treatment for damaged or diseased corneas. Although corneal transplantation has a high success rate, a shortage of high‐quality donor tissue is a considerable limitation. Therefore, bioengineered corneas could be an effective solution for this limitation, and a decellularized extracellular matrix comprises a promising scaffold for their fabrication. In this study, three‐dimensional bioprinted decellularized collagen sheets were implanted into the stromal layer of the cornea of five rabbits. We performed in vivo noninvasive monitoring of the rabbit corneas using swept‐source optical coherence tomography (OCT) after implanting the collagen sheets. Anterior segment OCT images and averaged amplitude‐scans were acquired biweekly to monitor corneal thickness after implantation for 1 month. The averaged cornea thickness in the control images was 430.3 ± 5.9 μm, while the averaged thickness after corneal implantation was 598.5 ± 11.8 μm and 564.5 ± 12.5 μm at 2 and 4 weeks, respectively. The corneal thickness reduction of 34 μm confirmed the biocompatibility through the image analysis of the depth‐intensity profile base. Moreover, hematoxylin and eosin staining supported the biocompatibility evaluation of the bioprinted decellularized collagen sheet implantation. Hence, the developed bioprinted decellularized collagen sheets could become an alternative solution to human corneal donor tissue, and the proposed image analysis procedure could be beneficial to confirm the success of the surgery.      

Quantitative assessment of optical clearing methods in various intact mouse organs

Various tissue optical clearing techniques have sprung up for large volume imaging. However, there are few methods showed clearing and imaging data on different organs while most of them were focused on mouse brain, and as a result, it is difficult to select the suitable method for organs in practical applications due to lack of quantitative evaluation and comprehensive comparison. Therefore, it is necessary to evaluate and compare the performances of clearing methods for different organs. In this paper, several typical optical clearing methods were applied, including 3DISCO, uDISCO, SeeDB, FRUIT, CUBIC, ScaleS and PACT to clear intact brain, heart, kidney, liver, spleen, stomach, lung, small intestine, skin and muscle. The clearing efficiency, sample deformation, fluorescence preservation and imaging depth of these methods were quantitatively evaluated. Finally, based on the systemic evaluation of various parameters described above, the appropriate clearing method for specific organ including kidney or intestine was screened out. This paper will provide important references for selection of appropriate clearing methods in related researches.      

Optical coherence tomography to detect acute esophageal radiation‐induced damage in mice: A validation study

Radiation therapy for patients with non‐small‐cell lung cancer is hampered by acute radiation‐induced toxicity in the esophagus. This study aims to validate that optical coherence tomography (OCT), a minimally invasive imaging technique with high resolution (~10 μm), is able to visualize and monitor acute radiation‐induced esophageal damage (ARIED) in mice. We compare our findings with histopathology as the gold standard. Irradiated mice receive a single dose of 40 Gy at proximal and distal spots of the esophagus of 10.0 mm in diameter. We scan mice using OCT at two, three, and seven days post‐irradiation. In OCT analysis, we define ARIED as a presence of distorted esophageal layering, change in backscattering signal properties, or change in the esophageal wall thickness. The average esophageal wall thickness is 0.53 mm larger on OCT when ARIED is present based on histopathology. The overall sensitivity and specificity of OCT to detect ARIED compared to histopathology are 94% and 47%, respectively. However, the overall sensitivity of OCT to assess ARIED is 100% seven days post‐irradiation. We validate the capability of OCT to detect ARIED induced by high doses in mice. Nevertheless, clinical studies are required to assess the potential role of OCT to visualize ARIED in humans.      

Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air‐puff optical coherence tomography and corneal hysteresis

Application of the air‐puff swept source optical coherence tomography (SS‐OCT) instrument to determine the influence of viscoelasticity on the relation between overall the air‐puff force and corneal apex displacement of porcine corneas ex vivo is demonstrated. Simultaneous recording of time‐evolution of the tissue displacement and air pulse stimulus allows obtaining valuable information related in part to the mechanical properties of the cornea. A novel approach based on quantitative analysis of the corneal hysteresis of OCT data is presented. The corneal response to the air pulse is assessed for different well‐controlled intraocular pressure (IOP) levels and for the progression of cross‐linking‐induced stiffness of the cornea. Micrometer resolution, fast acquisition and noncontact character of the air‐puff SS‐OCT measurements have potential to improve the in vivo assessment of mechanical properties of the human corneas.      

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.      

Wide‐field optical property mapping and structured light imaging of the esophagus with spatial frequency domain imaging

As the incidence of esophageal adenocarcinoma continues to rise, there is a need for improved imaging technologies with contrast to abnormal esophageal tissues. To inform the design of optical technologies that meet this need, we characterize the spatial distribution of the scattering and absorption properties from 471 to 851 nm of eight resected human esophagi tissues using Spatial Frequency Domain Imaging. Histopathology was used to categorize tissue types, including normal, inflammation, fibrotic, ulceration, Barrett's Esophagus and squamous cell carcinoma. Average absorption and reduced scattering coefficients of normal tissues were 0.211 ± 0.051 and 1.20 ± 0.18 mm^?1, respectively at 471 nm, and both values decreased monotonically with increasing wavelength. Fibrotic tissue exhibited at least 68% larger scattering signal across all wavelengths, while squamous cell carcinoma exhibited a 36% decrease in scattering at 471 nm. We additionally image the esophagus with high spatial frequencies up to 0.5 mm^?1 and show strong reflectance contrast to tissue treated with radiation. Lastly, we observe that esophageal absorption and scattering values change by an average of 9.4% and 2.7% respectively over a 30 minute duration post‐resection. These results may guide system design for the diagnosis, prevention and monitoring of esophageal pathologies.      

In vivo detection of endotracheal tube biofilms in intubated critical care patients using catheter‐based optical coherence tomography

The formation of biofilms in the endotracheal tubes (ETTs) of intubated patients on mechanical ventilation is associated with a greater risk of ventilator‐associated pneumonia and death. New technologies are needed to detect and monitor ETTs in vivo for the presence of these biofilms. Longitudinal OCT imaging was performed in mechanically ventilated subjects at 24‐hour intervals until extubation to detect the formation and temporal changes of in vivo ETT biofilms. OCT‐derived attenuation coefficient images were used to differentiate between mucus and biofilm. Extubated ETTs were examined with optical and electron microscopy, and all imaging results were correlated with standard‐of‐care clinical test reports. OCT and attenuation coefficient images from four subjects were positive for ETT biofilms and were negative for two subjects. The processed and stained extubated ETTs and clinical reports confirmed the presence/absence of biofilms in all subjects. Our findings confirm that OCT can detect and differentiate between biofilm‐positive and biofilm‐negative groups (P < 10^?5). OCT image‐based features may serve as biomarkers for direct in vivo detection of ETT biofilms and help drive investigation of new management strategies to reduce the incidence of VAP.      

Analyzing the feasibility of discriminating between collagen types I and II using polarization‐resolved second harmonic generation

According to previous studies, the nonlinear susceptibility tensor ratio χ₃₃/χ₃₁ obtained from polarization‐resolved second harmonic generation (P‐SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P‐SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ₃₃/χ₃₁‐ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.      

Angular range,sampling and noise considerations for inverse light scattering analysis of nuclear morphology

In recent years, significant work has been devoted to the use of angle‐resolved elastic scattering for the extraction of nuclear morphology in tissue. By treating the nucleus as a Mie scattering object, techniques such as angle‐resolved low‐coherence interferometry (a/LCI) have demonstrated substantial success in identifying nuclear alterations associated with dysplasia. Because optical biopsies are inherently noninvasive, only a small, discretized portion of the 4π scattering field can be collected from tissue, limiting the amount of information available for diagnostic purposes. In this work, we comprehensively characterize the diagnostic impact of variations in angular sampling, range and noise for inverse light scattering analysis of nuclear morphology, using a previously reported dataset from 40 patients undergoing a/LCI optical biopsy for cervical dysplasia. The results from this analysis are applied to a benchtop scanning a/LCI system which compromises angular range for wide‐area scanning capability. This work will inform the design of next‐generation optical biopsy probes by directing optical design towards parameters which offer the most diagnostic utility.      

Label‐free bioanalysis of Leishmania infantum using refractive index tomography with partially coherent illumination

In this work, we report the use of refractive index (RI) tomography for quantitative analysis of unstained DH82 cell line infected with Leishmania infantum. The cell RI is reconstructed by using a modality of optical diffraction tomography technique that employs partially coherent illumination, thus enabling inherent compatibility with conventional wide‐field microscopes. The experimental results demonstrate that the cell dry mass concentration (DMC) obtained from the RI allows for reliable detection and quantitative characterization of the infection and its temporal evolution. The RI provides important insight for studying morphological changes, particularly membrane blebbing linked to an apoptosis (cell death) process induced by the disease. Moreover, the results evidence that infected DH82 cells exhibit a higher DMC than healthy samples. These findings open up promising perspectives for clinical diagnosis of Leishmania.