Simultaneous immunofluorescence and histology in pemphigus vulgaris using ex vivo confocal laser scanning microscopy

Ex vivo confocal laser scanning microscopy (ex vivo CLSM) provides rapid, high-resolution imaging and immunofluorescence examinations of the excised tissues. We aimed to evaluate the applicability of ex vivo CLSM in histomorphological and direct immunofluorescence (DIF) examination of pemphigus vulgaris (PV). 20 PV sections were stained with fluorescent-labeled anti-IgG and anti-C3 using various dilutions and incubation periods. Subsequently, the determined ideal staining protocol was applied on 20 additional PV and 20 control sections. Ex vivo CLSM identified intraepidermal blisters and acantholytic cells in 80% and 60% of PV patients, respectively. The sensitivity of ex vivo CLSM in detecting intraepidermal fluorescence was 90% both with IgG and C3. The specificity of staining for IgG and C3 was 70% and 90%, respectively. Histomorphological and immunofluorescence features of PV could be detected within the same ex vivo CSLM session showing a comparable performance to conventional histopathology and DIF microscopy.     

En‐face optical coherence tomography for the detection of cancer in prostatectomy specimens: Quantitative analysis in 20 patients

The increase histopathological evaluation of prostatectomy specimens rises the workload on pathologists. Automated histopathology systems, preferably directly on unstained specimens, would accelerate the pathology workflow. In this study, we investigate the potential of quantitative analysis of optical coherence tomography (OCT) to separate benign from malignant prostate tissue automatically. Twenty fixated prostates were cut, from which 54 slices were scanned by OCT. Quantitative OCT metrics (attenuation coefficient, residue, goodness‐of‐fit) were compared for different tissue types, annotated on the histology slides. To avoid misclassification, the poor‐quality slides, and edges of annotations were excluded. Accurate registration of OCT data with histology was achieved in 31 slices. After removing outliers, 56% of the OCT data was compared with histopathology. The quantitative data could not separate malignant from benign tissue. Logistic regression resulted in malignant detection with a sensitivity of 0.80 and a specificity of 0.34. Quantitative OCT analysis should be improved before clinical use.     

Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.     

Identification of ex‐vivo confocal laser scanning microscopic features of melanocytic lesions and their histological correlates

Ex‐vivo confocal laser scanning microscopy (CLSM) offers rapid tissue examination. Current literature shows promising results in the evaluation of non‐melanoma skin cancer but little is known about presentation of melanocytic lesions (ML). This study evaluates ML with ex‐vivo CLSM in comparison to histology and offers an overview of ex‐vivo CLSM characteristics. 31 ML were stained with acridine orange or fluorescein and examined using ex‐vivo CLSM (Vivascope2500^®; Lucid Inc; Rochester NY) in reflectance and fluorescence mode. Confocal images were correlated to histopathology. Benign and malignant features of the ML were listed and results were presented. Sensitivity and specificity were calculated using contingency tables. The ML included junctional, compound, dermal, Spitz and dysplastic nevi, as well as various melanoma subtypes. The correlation of the confocal findings with histopathology allowed the identification of different types of ML and differentiation of benign and malignant features. The study offers an overview of confocal characteristics of ML in comparison to histology. Ex‐vivo CLSM does not reproduce the typical in‐vivo horizontal mosaics but rather reflects the vertical histological presentation. Not all typical in‐vivo patterns are detectable here. These findings may help to evaluate the ex‐vivo CLSM as an adjunctive tool in the immediate intraoperative diagnosis of ML.

Superficial spreading malignant melanoma. Histopathology (H&E stain; 200×) correlated to the reflectance (RM; 830 nm) and fluorescence mode (FM; 488 nm) in the ex‐vivo CLSM (Vivablock^® by VivaScan^®, acridine orange).     

Distinguishing metastatic triple‐negative breast cancer from nonmetastatic breast cancer using second harmonic generation imaging and resonance Raman spectroscopy

Triple‐negative breast cancer (TNBC) is an aggressive subset of breast cancer that is more common in African‐American and Hispanic women. Early detection followed by intensive treatment is critical to improving poor survival rates. The current standard to diagnose TNBC from histopathology of biopsy samples is invasive and time‐consuming. Imaging methods such as mammography and magnetic resonance (MR) imaging, while covering the entire breast, lack the spatial resolution and specificity to capture the molecular features that identify TNBC. Two nonlinear optical modalities of second harmonic generation (SHG) imaging of collagen, and resonance Raman spectroscopy (RRS) potentially offer novel rapid, label‐free detection of molecular and morphological features that characterize cancerous breast tissue at subcellular resolution. In this study, we first applied MR methods to measure the whole‐tumor characteristics of metastatic TNBC (4T1) and nonmetastatic estrogen receptor positive breast cancer (67NR) models, including tumor lactate concentration and vascularity. Subsequently, we employed for the first time in vivo SHG imaging of collagen and ex vivo RRS of biomolecules to detect different microenvironmental features of these two tumor models. We achieved high sensitivity and accuracy for discrimination between these two cancer types by quantitative morphometric analysis and nonnegative matrix factorization along with support vector machine. Our study proposes a new method to combine SHG and RRS together as a promising novel photonic and optical method for early detection of TNBC.     

Telecentric design for digital-scanning-based HiLo optical sectioning endomicroscopy with an electrically tunable lens

Confocal endoscopy has been widely used to obtain fine optically sectioned images. However, confocal endomicroscopic images are formed by point-by-point scanning in both lateral and axial directions, which results in long image acquisition time. Here, an endomicroscope with telecentric configuration is presented to achieve nonmechanical and rapid axial scanning for volumetric fluorescence imaging. In our system, optical sectioning in wide-field fashion is obtained through HiLo imaging with a digital micromirror device. Axial scanning, without mechanical moving parts, is conducted by digital focus adjustment using an electrically tunable lens, offering constant magnification and contrast. We demonstrate imaging performance of our system with optically sectioned images using fluorescently labeled beads, as well as ex vivo mice cardiac tissue samples. Our system provides multiple advantages, in terms of improved scanning range, and reduced image acquisition time, which shows great potentials for three-dimensional biopsies of volumetric biological samples.     

Ex-vivo fluorescence confocal microscopy with digital staining for characterizing basal cell carcinoma on frozen sections: A comparison with histology

Ex-vivo fluorescence confocal microscopy (FCM) has been used on fresh tissue, but there is little experience on frozen sections. We evaluated the applicability of FCM on frozen sections of basal cell carcinomas (BCCs), stained with acridine orange and digitally colored to simulate hematoxylin and eosin (H&E) dyes. We compared our diagnostic accuracy in detecting and subtyping BCCs with FCM to our gold standard (H&E stained frozen sections used in 3D horizontal micrographic surgery). Fourty-six primary BCCs were analyzed for free margins as well as histological subtype with all FCM modes and conventional H&E staining. Adnexa, artifacts and diagnostic confidence were evaluated. Free margins were identified with a sensitivity and specificity of 92% and 91%. Concordance for tumor subtype was 88%. FCM may be used on both fresh tissue and frozen samples, although with reduced performance and different artifacts. The device is useful for the intraoperative diagnosis, subtyping and margin-mapping of BCCs.      

Full‐field swept‐source optical coherence tomography and neural tissue classification for deep brain imaging

Optical coherence tomography can differentiate brain regions with intrinsic contrast and at a micron scale resolution. Such a device can be particularly useful as a real‐time neurosurgical guidance tool. We present, to our knowledge, the first full‐field swept‐source optical coherence tomography system operating near a wavelength of 1310 nm. The proof‐of‐concept system was integrated with an endoscopic probe tip, which is compatible with deep brain stimulation keyhole neurosurgery. Neuroimaging experiments were performed on ex vivo brain tissues and in vivo in rat brains. Using classification algorithms involving texture features and optical attenuation, images were successfully classified into three brain tissue types.     

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.     

A novel in vivo Cerenkov luminescence image‐guided surgery on primary and metastatic colorectal cancer

Intraoperative Cerenkov luminescence imaging (CLI) can effectively improve the performance of tumor surgery. Nevertheless, the existing approaches are still unsatisfying to the clinical demands of open surgery. This study develops a novel intraoperative in vivo CLI approach to investigate the potential and value of Cerenkov luminescence (CL) image‐guided surgery. A system characterized with high sensitivity (19.61 kBq mL^{?1 18}F‐FDG) and desirable spatial resolution (88.34 μm) is developed. CL image‐guided surgery is performed on colorectal cancer (CRC) models of mice and swine. Tumor surgery is guided by the static CL images, and the resection quality is evaluated quantitatively and contrasted with other imaging modalities exemplified by bioluminescence imaging (BLI). The in vivo results demonstrated the effectiveness of the proposed intraoperative CLI approach for removing primary and metastatic CRC. Safety of performing in vivo CL image‐guided surgery is verified as well through radiation measurements of related staffs. Overall, the developed intraoperative in vivo CLI approach can efficiently improve the cancer treatment.     

Development of hyperthermia measurable fiber radiometric thermometer for thermotherapy

Temperature monitoring is extremely important during thermotherapy. Fiber‐optic temperature sensors are preferred because of their flexibility and immunity to electromagnetic interference. Although many types of fiber‐optic sensors have been developed, clinically adopting them remains challenging. Here, we report a silica fiber‐based radiometric thermometer using a low‐cost extended InGaAs detector to detect black body radiation between 1.7 and 2.4 μm. For the first time, this silica fiber‐based thermometer is capable of measuring temperatures down to 35°C, making it suitable for monitoring hyperthermia during surgery. In particular, the thermometer has potential for seamless integration with current silica fiber catheters, which are widely used in laser interstitial thermotherapy. The feasibility, capability and sensitivity of tracking tissue temperature variation were proved through ex vivo tissue studies. After further improvement, the technology has the potential to be translated into clinics for monitoring tissue temperature.     

Perspectives,potentials and trends of ex vivo and in vivo optical molecular pathology

It is pivotal for medical applications, such as noninvasive histopathologic characterization of tissue, to realize label‐free and molecule‐specific representation of morphologic and biochemical composition in real‐time with subcellular spatial resolution. This unmet clinical need requires new approaches for rapid and reliable real‐time assessment of pathologies to complement established diagnostic tools. Photonic imaging combined with digitalization offers the potential to provide the clinician the requested information both under in vivo and ex vivo conditions. This report summarizes photonic approaches and their use in combination with image processing, machine learning and augmented virtual reality that might solve current challenges in modern medicine. Details are given for pathology, intraoperative diagnosis in head and neck cancer and endoscopic diagnosis in gastroenterology.      

Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.     

Real‐time video mosaicking to guide handheld in vivo microscopy

Handheld and endoscopic optical‐sectioning microscopes are being developed for noninvasive screening and intraoperative consultation. Imaging a large extent of tissue is often desired, but miniature in vivo microscopes tend to suffer from limited fields of view. To extend the imaging field during clinical use, we have developed a real‐time video mosaicking method, which allows users to efficiently survey larger areas of tissue. Here, we modified a previous post‐processing mosaicking method so that real‐time mosaicking is possible at >30 frames/second when using a device that outputs images that are 400 × 400 pixels in size. Unlike other real‐time mosaicking methods, our strategy can accommodate image rotations and deformations that often occur during clinical use of a handheld microscope. We perform a feasibility study to demonstrate that the use of real‐time mosaicking is necessary to enable efficient sampling of a desired imaging field when using a handheld dual‐axis confocal microscope.     

Front Cover: Implantable self‐aligning fiber‐optic optomechanical devices for in vivo intraocular pressure‐sensing in artificial cornea

This work addresses an unmet clinical need, that of glaucoma monitoring through intraocular pressure (IOP) interrogation in patients with artificial corneas (keratoprost hesis). We demonstrate direct integration of a low‐drift, fiber‐optic Fabry‐Perot pressure sensor embedded in the keratoprosthesis via rapid, non‐contact micromagnetic fiber alignment. IOP interrogation is achieved by using white‐light interferometry, which provides sub‐mmHg IOP sensitivity. Further details can be found in the article by Pui‐Chuen Hui, Katia Shtyrkova, Chengxin Zhou, et al. ( e202000031 ).

     

Diagnosing and Mapping Pulmonary Emphysema on X-Ray Projection Images: Incremental Value of Grating-Based X-Ray Dark-Field Imaging

Purpose

To assess whether grating-based X-ray dark-field imaging can increase the sensitivity of X-ray projection images in the diagnosis of pulmonary emphysema and allow for a more accurate assessment of emphysema distribution.

Materials and Methods

Lungs from three mice with pulmonary emphysema and three healthy mice were imaged ex vivo using a laser-driven compact synchrotron X-ray source. Median signal intensities of transmission (T), dark-field (V) and a combined parameter (normalized scatter) were compared between emphysema and control group. To determine the diagnostic value of each parameter in differentiating between healthy and emphysematous lung tissue, a receiver-operating-characteristic (ROC) curve analysis was performed both on a per-pixel and a per-individual basis. Parametric maps of emphysema distribution were generated using transmission, dark-field and normalized scatter signal and correlated with histopathology.

Results

Transmission values relative to water were higher for emphysematous lungs than for control lungs (1.11 vs. 1.06, p<0.001). There was no difference in median dark-field signal intensities between both groups (0.66 vs. 0.66). Median normalized scatter was significantly lower in the emphysematous lungs compared to controls (4.9 vs. 10.8, p<0.001), and was the best parameter for differentiation of healthy vs. emphysematous lung tissue. In a per-pixel analysis, the area under the ROC curve (AUC) for the normalized scatter value was significantly higher than for transmission (0.86 vs. 0.78, p<0.001) and dark-field value (0.86 vs. 0.52, p<0.001) alone. Normalized scatter showed very high sensitivity for a wide range of specificity values (94% sensitivity at 75% specificity). Using the normalized scatter signal to display the regional distribution of emphysema provides color-coded parametric maps, which show the best correlation with histopathology.

Conclusion

In a murine model, the complementary information provided by X-ray transmission and dark-field images adds incremental diagnostic value in detecting pulmonary emphysema and visualizing its regional distribution as compared to conventional X-ray projections.     

The use of optical coherence tomography and convolutional neural networks to distinguish normal and abnormal oral mucosa

Incomplete surgical resection of head and neck squamous cell carcinoma (HNSCC) is the most common cause of local HNSCC recurrence. Currently, surgeons rely on preoperative imaging, direct visualization, palpation and frozen section to determine the extent of tissue resection. It has been demonstrated that optical coherence tomography (OCT), a minimally invasive, nonionizing near infrared mesoscopic imaging modality can resolve subsurface differences between normal and abnormal head and neck mucosa. Previous work has utilized two‐dimensional OCT imaging which is limited to the evaluation of small regions of interest generated frame by frame. OCT technology is capable of performing rapid volumetric imaging, but the capacity and expertise to analyze this massive amount of image data is lacking. In this study, we evaluate the ability of a retrained convolutional neural network to classify three‐dimensional OCT images of head and neck mucosa to differentiate normal and abnormal tissues with sensitivity and specificity of 100% and 70%, respectively. This method has the potential to serve as a real‐time analytic tool in the assessment of surgical margins.     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Rapid intraoperative diagnosis of gynecological cancer by ATR‐FTIR spectroscopy of fresh tissue biopsy

A rapid and reliable intraoperative diagnostic technique to support clinical decisions was developed using Fourier‐transform infrared (FTIR) spectroscopy. Twenty‐six fresh tissue samples were collected intraoperatively from patients undergoing gynecological surgeries. Frozen section (FS) histopathology aimed to discriminate between malignant and benign tumors was performed, and attenuated total reflection (ATR) FTIR spectra were collected from these samples. Digital dehydration and principal component analysis and linear discriminant analysis (PCA‐LDA) models were developed to classify samples into malignant and benign groups. Two validation schemes were employed: k‐fold and “leave one out.” FTIR absorption spectrum of a fresh tissue sample was obtained in less than 5 minutes. The fingerprint spectral region of malignant tumors was consistently different from that of benign tumors. The PCA‐LDA discrimination model correctly classified the samples into malignant and benign groups with accuracies of 96% and 93% for the k‐fold and “leave one out” validation schemes, respectively. We showed that a simple tissue preparation followed by ATR‐FTIR spectroscopy provides accurate means for very rapid tumor classification into malignant and benign gynecological tumors. With further development, the proposed method has high potential to be used as an adjunct to the intraoperative FS histopathology technique.     

Long‐term and in vivo assessment of Aβ protein‐induced brain atrophy in a zebrafish model by optical coherence tomography

In this study, a neurotoxicity model of zebrafish induced by amyloid beta (Aβ) protein was developed and evaluated in vivo by optical coherence tomography (OCT). Aβ protein and phosphate buffer saline (PBS) were separately injected into the head of two groups of adult zebrafish (n = 6 per group). Congo‐red staining results confirmed that Aβ protein had penetrated into brain tissue. All zebrafish were imaged with OCT on the 0th, 5th, 10th, 15th and 20th day postinjection. OCT images showed that PBS is not toxic to brain tissue. However, significant brain atrophy could be seen in the OCT images of zebrafish injected with Aβ‐protein that was verified by histological consequences. In addition, zebrafish in the model group showed memory decline in behavioral tests. This study verified the feasibility of in vivo long‐term assessment of Aβ protein‐induced brain atrophy in adult zebrafish by OCT that has great potential to be applied in the neurological diseases research.