Imaging peritoneal blood vessels through optical coherence tomography angiography for laparoscopic surgery.

Laparoscopic surgery presents challenges in identifying blood vessels due to lack of tactile feedback. The image-guided laparoscopic surgical tool (IGLaST) integrated with optical coherence tomography (OCT) has potential for in vivo blood vessel imaging; however, distinguishing vessels from surrounding tissue remains a challenge. In this study, we propose utilizing an inter-A-line intensity differentiation-based OCT angiography (OCTA) to improve visualization of blood vessels. By evaluating a tissue phantom with varying flow speeds, we optimized the system's blood flow imaging capabilities in terms of minimum detectable flow and contrast-to-noise ratio. In vivo experiments on rat and porcine models, successfully visualized previously unidentified blood vessels and concealed blood flows beneath the 1 mm depth peritoneum. Qualitative comparison of various OCTA algorithms indicated that the intensity differentiation-based algorithm performed best for our application. We believe that implementing IGLaST with OCTA can enhance surgical outcomes and reduce procedure time in laparoscopic surgeries.

Lateral image reconstruction of optical coherence tomography using one-dimensional deep deconvolution network.

BACKGROUND AND OBJECTIVES: Optical coherence tomography (OCT) is a cross-sectional imaging method utilizing a low coherence interferometry. The lateral resolution of the OCT is limited by the numerical aperture (NA) of the imaging lens. Using a high NA lens improves the lateral resolution but reduces the depth of focus (DOF). In this study, we propose a method to improve the lateral resolution of OCT images by end-to-end training of a deep 1-D deconvolution network without use of high-resolution images. MATERIALS AND METHODS: To improve the lateral resolution of the OCT, we trained the 1-D deconvolution network using lateral profiles of OCT images and the beam spot size. We used our image-guided laparoscopic surgical tool (IGLaST) to acquire OCT images of nonbiological and biological samples ex vivo. The OCT images were then blurred by applying Gaussian functions with various full width half maximums ranging from 40 to 160 µm. The network was trained using the blurred OCT images as input and the non-blurred original OCT images as output. We quantitatively evaluated the developed network in terms of similarity and signal-to-ratio (SNR), using in-vivo images of mesenteric tissue from a porcine model that was not used for training. In addition, we performed knife-edge tests and qualitative evaluation of the network to show the lateral resolution improvement of ex-vivo and in-vivo OCT images. RESULTS: The proposed method showed an improvement of image quality on both blurred images and non-blurred images. When the proposed deconvolution network was applied, the similarity to the non-blurred image was improved by 1.29 times, and the SNR was improved by 1.76 dB compared to the artificially blurred images, which was superior to the conventional deconvolution method. The knife-edge tests at distances at 200 to 1000 µm from the imaging probe showed an approximately 1.2 times improvement in lateral resolution. In addition, through qualitative evaluation, it was found that the image quality of both ex-vivo and in-vivo tissue images was improved with clear structure and less noise. CONCLUSIONS: This study showed the ability of the 1-D deconvolution network to improve the image quality of OCT images with variable lateral resolution. We were able to train the network with a small amount of data by constraining the network in 1-D. The quantitative evaluation showed better results than conventional deconvolution methods for various amount of blurring. Qualitative evaluation showed analogous results with quantitative results. This simple yet powerful image restoration method provides improved lateral resolution and suppresses background noise, making it applicable to a variety of OCT imaging applications.

Real-time cancer diagnosis of breast cancer using fluorescence lifetime endoscopy based on the pH.

A biopsy is often performed for the diagnosis of cancer during a surgical operation. In addition, pathological biopsy is required to discriminate the margin between cancer tissues and normal tissues in surgical specimens. In this study, we presented a novel method for discriminating between tumor and normal tissues using fluorescence lifetime endoscopy (FLE). We demonstrated the relationship between the fluorescence lifetime and pH in fluorescein using the proposed fluorescence lifetime measurement system. We also showed that cancer could be diagnosed based on this relationship by assessing differences in pH based fluorescence lifetime between cancer and normal tissues using two different types of tumor such as breast tumors (MDA-MB-361) and skin tumors (A375), where cancer tissues have ranged in pH from 4.5 to 7.0 and normal tissues have ranged in pH from 7.0 to 7.4. To support this approach, we performed hematoxylin and eosin (H&E) staining test of normal and cancer tissues within a certain area. From these results, we showed the ability to diagnose a cancer using FLE technique, which were consistent with the diagnosis of a cancer with H&E staining test. In summary, the proposed pH-based FLE technique could provide a real time, in vivo, and in-situ clinical diagnostic method for the cancer surgical and could be presented as an alternative to biopsy procedures.

Rapid Molecular Diagnostic Sensor Based on Ball-Lensed Optical Fibers.

Given the fatal health conditions caused by emerging infectious pathogens, such as severe acute respiratory syndrome coronavirus 2, their rapid diagnosis is required for preventing secondary infections and guiding correct treatments. Although various molecular diagnostic methods based on nucleic acid amplification have been suggested as gold standards for identifying different species, these methods are not suitable for the rapid diagnosis of pathogens owing to their long result acquisition times and complexity. In this study, we developed a rapid bio-optical sensor that uses a ball-lensed optical fiber (BLOF) probe and an automatic analysis platform to precisely diagnose infectious pathogens. The BLOF probe is easy to align and has a high optical sensing sensitivity (1.5-fold) and a large detection range (1.2-fold) for an automatic optical sensing system. Automatic signal processing of up to 250 copies/reaction of DNA of Q-fever-causing Coxiella burnetii was achieved within 8 min. The clinical utility of this system was demonstrated with 18 clinical specimens (9 Q-fever and 9 other febrile disease samples) by measuring the resonant wavelength shift of positive or negative samples for Coxiella burnetii DNA. The results from the system revealed the stable and automatic optical signal measurement of DNA with 100% accuracy. We envision that this BLOF probe-based sensor would be a practical tool for the rapid, simple, and sensitive diagnosis of emerging infectious pathogens.

Moxifloxacin based fluorescence imaging of intestinal goblet cells.

Goblet cells (GCs) in the intestine are specialized epithelial cells that secrete mucins to form the protective mucous layer. GCs are important in maintaining intestinal homeostasis, and the alteration of GCs is observed in inflammatory bowel diseases (IBDs) and neoplastic lesions. In the Barrett's esophagus, the presence of GCs is used as a marker of specialized intestinal metaplasia. Various endomicroscopic imaging methods have been used for imaging intestinal GCs, but high-speed and high-contrast GC imaging has been still difficult. In this study, we developed a high-contrast endoscopic GC imaging method: fluorescence endomicroscopy using moxifloxacin as a GC labeling agent. Moxifloxacin based fluorescence imaging of GCs was verified by using two-photon microscopy (TPM) in the normal mouse colon. Label-free TPM, which could visualize GCs in a negative contrast, was used as the reference. High-speed GC imaging was demonstrated by using confocal microscopy and endomicroscopy in the normal mouse colon. Confocal microscopy was applied to dextran sulfate sodium (DSS) induced colitis mouse models for the detection of GC depletion. Moxifloxacin based GC imaging was demonstrated not only by 3D microscopies but also by wide-field fluorescence microscopy, and intestinal GCs in the superficial region were imaged. Moxifloxacin based endomicroscopy has a potential for the application to human subjects by using FDA approved moxifloxacin.

Rapid histologic diagnosis using quick fluorescence staining and tissue confocal microscopy.

With the development of advanced and minimally invasive surgical techniques, and in view of the functional and cosmetic aspects, the need for rapid and accurate diagnosis during surgery is increasing. This study was conducted to develop a tissue diagnosis method using confocal microscopy after simple tissue staining that does not require freezing and slicing. At present, fluorescence staining with confocal microscopy is not generalized for real-time diagnosis during surgery. In this paper, we propose a fluorescence staining method using Hoechst 33342 and Eosin that does not require tissue freezing and slicing. The proposed method can be used as part of a rapid tissue diagnosis method that is suitable for use in the operating room, although further research is required before it can be applied in clinical practice.

A Digital Shade-Matching Device for Dental Color Determination Using the Support Vector Machine Algorithm.

In this study, we developed a digital shade-matching device for dental color determination using the support vector machine (SVM) algorithm. Shade-matching was performed using shade tabs. For the hardware, the typically used intraoral camera was modified to apply the cross-polarization scheme and block the light from outside, which can lead to shade-matching errors. For reliable experiments, a precise robot arm with ±0.1 mm position repeatability and a specially designed jig to fix the position of the VITA 3D-master (3D) shade tabs were used. For consistent color performance, color calibration was performed with five standard colors having color values as the mean color values of the five shade tabs of the 3D. By using the SVM algorithm, hyperplanes and support vectors for 3D shade tabs were obtained with a database organized using five developed devices. Subsequently, shade matching was performed by measuring 3D shade tabs, as opposed to real teeth, with three additional devices. On average, more than 90% matching accuracy and a less than 1% failure rate were achieved with all devices for 10 measurements. In addition, we compared the classification algorithm with other classification algorithms, such as logistic regression, random forest, and k-nearest neighbors, using the leave-pair-out cross-validation method to verify the classification performance of the SVM algorithm. Our proposed scheme can be an optimum solution for the quantitative measurement of tooth color with high accuracy.

High-speed dual-layer scanning photoacoustic microscopy using focus tunable lens modulation at resonant frequency.

The range of imaging depth in optical resolution photoacoustic microscopy (PAM) is limited by the short depth of focus of high-numerical aperture objective lenses. In this paper, focus tunable lens modulation has been employed at the resonant frequency of focus tunable lenses in order to enhance both the range of imaging depth and the scanning speed. By electrically controlling the focal length in the axial direction of the sample, the range of imaging depth was extended approximately 1.22 times and the scanning speed was enhanced by approximately 7.40 times, in comparison to corresponding values of conventional PAM systems.

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method.

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection.

Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo.

Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection.

Optimum phase shift for quantitative phase microscopy in volume measurement.

Volume measurement of a phase object is one of the most distinctive capabilities of quantitative phase microscopy (QPM). However, the accuracy of a measured volume is limited by the different noises of a measurement system and the finite bandpass filter used in the phase extraction algorithm. In this paper, we analyze the inherent errors in volume measurement with QPM and propose the optimum condition that can minimize these errors. We find that phase information of a sample in the frequency domain nonlinearly oscillates as a function of the phase shift corresponding to the sample and its medium, and that the phase information of a sample inside the bandpass filter can be maximized by a proper phase shift. Through numerical simulations and actual experiments, we demonstrate that the error in phase volume measurement can be effectively reduced by the enhancement of the phase signal inside the bandpass region using an optimum amount of phase, which can be controlled by changing either the medium index or the wavelength of illumination.

Detrended fluctuation analysis of membrane flickering in discocyte and spherocyte red blood cells using quantitative phase microscopy.

Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.

Dynamic analysis of pathogen-infected host cells using quantitative phase microscopy.

We present the real-time quantitative analysis of Vibrio vulnificus-infected host cells using quantitative phase microscopy (QPM) based on interferometric techniques. This provides the ability to retrieve the phase or optical path-length distribution over the cell with nanometer path-length sensitivity from a single interferogram image. We have used QPM to study dynamic cell morphologic changes and to noninvasively quantify the cell volumes of rat basophilic leukemia RBL-2H3 cells infected with V. vulnificus strains: wild type (MO6-24∕O) and RtxA1 toxin mutant (CMM770). During the process of V. vulnificus infection in RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes, and areas were observed in real time using QPM. In contrast, dramatic changes were not detected in RBL-2H3 cells infected with the noncytotoxic RtxA1 toxin mutant. The results showed good correlation between QPM analysis and biochemical assays, such as lactate dehydrogenase assay or ß-hexosaminidase release assay. We suggest that QPM is a powerful quantitative method to study the dynamic process of host cells infected with pathogens in a noninvasive manner.

Silver nanoparticle-induced degranulation observed with quantitative phase microscopy.

Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 microg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca(2+)](i)) and histamine with fluorescent methods.

Autofocusing and edge detection schemes in cell volume measurements with quantitative phase microscopy.

We have proposed and demonstrated a very sensitive volume measurement scheme for a live cell with a quantitative phase microscopy (QPM) utilizing auto-focusing and numerical edge detection schemes. An auto-focusing technique with two different focus measures is applied to find the focus dependent errors in our live cell volume measurement system. The volume of a polystyrene bead sample with 3 mum diameter has been measured for the validity test of our proposed method. We have shown that a small displacement of an object from its focusing position can cause a large volume error. A numerical edge detection technique is also used to accurately resolve the boundary between a cell and its suspension medium. We have applied this method to effectively suppress errors by the surrounding medium of a single red blood cell (RBC).