Optical Biopsy Research Articles

Confocal Laser Endomicroscopy as a method for assessing endometriosis: A pilot study

ObjectivesTo validate probe-based Confocal Laser Endomicroscopy (pCLE) as a method in assessing endometriosis during conventional and robot-assisted laparoscopy. Study DesignPilot study, including five women in Hospital Moriah, São Paulo, Brazil. During laparoscopic procedures, pCLE was used to assess endometriosis lesions, complemented by subsequent histological evaluations. 214 optical biopsies were performed. These assessments contributed to the development of a comparative atlas, which was used by surgeons to respond to a questionnaire to identify specific cellular structures related to endometriosis in selected confocal images. ResultsThe patients had an average age of 35, exhibiting predominant symptoms like dysmenorrhea and dyspareunia. Despite varied familiarity with pCLE, surgeons demonstrated positive acceptance of the method, with 63.1% recommending its use for intraoperative diagnosis. The technique was particularly noted for its ability to provide real-time, detailed images aiding the identification of endometriosis and associated structures like adipocytes and vascularization, with average evaluation scores exceeding 8 of 10. ConclusionsThis study underscores pCLE’s potential as a transformative diagnostic tool in minimally invasive gynecological surgery. It highlights the feasibility and initial acceptance among surgeons, emphasizing the need for further improvements in device durability and cost-effectiveness. pCLE promises significant advancements in the diagnosis and management of endometriosis, suggesting a future direction for clinical applications and technological refinement.

Jehlová konfokální laserová endomikroskopie v diferenciální diagnostice cystických lézí pankreatu

Summary: Background: Pancreatic cystic lesions are relatively common findings, and their prevalence continues to rise, which is also related to the expansion of imaging methods. Up to 15% of pancreatic cancers arise from mucinous cystic tumours of the pancreas, primarily from intraductal papillary mucinous neoplasia (IPMN). Needle-based confocal laser endomicroscopy (nCLE) represents an advanced diagnostic method with the potential to improve the accuracy of diagnosing focal pancreatic lesions. Aim: The aim of the work is to provide a recent overview of the position of nCLE in the diagnosis of pancreatic cystic lesions and some of our own experiences. The nCLE method is based on so-called real-time optical biopsy, which brings better diagnostic results compared to traditional methods such as CT, MRI/MRCP, and EUS, as confirmed by the authors‘ own data. Although nCLE offers certain advantages, its wider use in clinical practice is limited by the high costs of equipment, technical difficulties during examination, and the need for thorough training of endoscopists. The integration of all diagnostic procedures (EUS, cytology, nCLE, and next-generation sequencing) into artificial intelligence algorithms could minimize the disadvantages of individual methods and increase the overall efficiency of diagnosing pancreatic cystic lesions. Conclusion: nCLE represents an innovative method for diagnosing pancreatic cystic lesions, which, in combination with other procedures (microforceps biopsy, next-generation sequencing, assessment of nCLE findings, and overall malignancy risk using artificial intelligence), can further enhance its diagnostic accuracy in this context. Key words: needle-based confocal laser endomicroscopy – pancreatic cystic lesion – optical biopsy – endoscopic ultrasound – artificial intelligence

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia.

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. Traditionally, the diagnosis of OSSN relies on histopathological confirmation followed by a full-thickness biopsy. However, in the past two decades, the therapeutic approach to OSSN has shifted from surgical intervention to topical chemotherapy regimens in clinical settings. This shift emphasizes the need for less invasive or non-invasive methods to diagnose ocular surface pathologies. Among various imaging devices, commercially available high-resolution optical coherence tomography (HR-OCT) has emerged as a powerful tool for the characterization of OSSN. HR-OCT provides an in vivo, cross-sectional view of ocular surface lesions, offering an "optical biopsy" for OSSN with high sensitivity and specificity. It provides valuable information in differentiating intraepithelial or invasive OSSN from other benign lesions. Additionally, HR-OCT can be used to monitor the response to topical chemotherapy and to detect subclinical OSSN during follow-up visits. In this article, the scanning protocol for image acquisition is presented, and image interpretation for OSSN is outlined. This standardized, practical, and reproducible approach is recommended in clinical workflows and is expected to assist clinicians in the management of OSSN.

(Invited) Designing Quantum Dots for Multiplexed Deep-Tissue Imaging in the NIR-II

The combination of bright, tunable semiconductor quantum dots (QDs) emitting in the shortwave infrared or second near infrared window (SWIR/NIR-II, 1000 – 1600 nm) and InGaAs camera-equipped preclinical imagers opens the door to non-invasive, multiplexed, deep tissue imaging in live mice. While imaging in the NIR-I is like imaging through fog due to highly scattering tissue, imaging in the SWIR/NIR-II is more like imaging through pond water, enabling deeper imaging with much improved resolution. We synthesized multiple PbS/CdS core/shell QDs with emission wavelengths spanning 1000-1600 nm. These particles have been encapsulated in lipid-PEG micelles and up to three colors (so far) dosed into nude mice for imaging on a Photon Etc IR VIVO imager equipped with a hyperspectral Bragg grating and 6 bandpass filters, enabling demixing of the discrete colors. We have obtained exceptionally clear images of lymphatic drainage and the vasculature system, including visualization of lymphatic valve opening in videos. Interestingly, following intravascular injection, the liver and spleen are very visible and distinct, enabling the non-invasive visualization of a key nanomedicine drug delivery parameter (i.e., are the nanoparticles accumulating in the liver or the spleen?). Image processing investigations are expanding our ability to demix colors as well as to determine the average depth of the contrast agent, based on the wavelength-dependent attenuation of the photoluminescence based on water absorption. In addition to the photophysical advantages of using QDs for NIR-II, elemental analysis of blood and tissue samples enables quantitative biodistribution and pharmacokinetic studies. Ongoing work involves the application of this imaging approach to assess the impact nanomaterial surface properties and route of delivery on their pharmacokinetics and the use of deep tissue paired agent imaging for non-invasive “optical biopsy” of orthotopic tumors. Current data are already visually stunning, and ongoing imaging highlights new opportunities for research with every experiment! Figure 1

A pilot study of optical coherence tomography-guided transbronchial biopsy in peripheral pulmonary lesions

ABSTRACT Background The diagnosis of peripheral pulmonary lesions (PPLs) remains challenging. Despite advancements in guided transbronchial biopsy (TBB) techniques, diagnostic yields haven’t reached ideal levels. Optical coherence tomography (OCT) has been developed for application in pulmonary diseases, yet no data existed evaluating effectiveness in diagnosing PPLs. Research design and methods This study included patients who underwent OCT and radial endobronchial ultrasound (R-EBUS)-guided TBB. OCT and R-EBUS imaging features were analyzed to differentiate between benign and malignant PPLs and subtypes of lung cancer. Results A total of 89 patients were included in this study. The diagnostic yield of OCT-guided TBB stood at 56.18%, R-EBUS-guided TBB was 83.15% (P<0.01). The accuracy of OCT to judge the nature of lesions was 92.59%, while R-EBUS was 77.92%. The accuracy of OCT in predicting squamous carcinoma (SCC) and adenocarcinoma were both 91.30%. Conclusions Although the diagnostic yield of OCT-guided TBB fell short of that achieved by R-EBUS, OCT possessed the capability to judge the nature of lesions and guide the pathological classification of malignant lesions. Further extensive prospective studies are necessary to thoroughly assess the characteristics of this procedure. Clinical trial registration https://register.clinicaltrials.gov/ identifier is NCT06419114

Alterations in Intestinal Mucosal Barrier Visualized by Confocal Laser Endomicroscopy in Liver Cirrhosis: A Pilot Trial (AMBIC).

Chronic liver disease occurs throughout the world irrespective of region, age, sex, or race, and it is caused by a variety of liver conditions. One of the most frequent infectious complications in liver cirrhosis that severely reduces the median survival is spontaneous bacterial peritonitis. Current guidelines recommend a paracentesis before starting an antibiotic prophylaxis for this complication. Selective intestinal decontamination significantly lowers the rate of first or recurrent SBP in cirrhotic patients, so in this study we aimed to investigate and quantify the intestinal integrity of patients with liver cirrhosis and correlate a pathologically increased permeability with the incidence of SPB. We included 14 patients who met the inclusion criteria. No patient was excluded. For the CLE investigation, we use probe based confocal laser endomicroscopy techniques from Mauna Kea (Cellvizio), enabling in vivo surface imaging. The images (optical biopsies) were analyzed for functional and structural barrier defects after the procedure using Mauna Kea software (version 1.0.09). Because of the small number of included patients and healthy controls, most results are lacking statistical relevance. We found that the CLE investigation showed an increased intestinal permeability in patients with liver cirrhosis, in concordance with previous published data, based on other assessment methods. This study confirms that previously published permeability scores can be applied for patients with liver cirrhosis and is, to our knowledge, the first to investigate the intestinal permeability in vivo in patients with liver cirrhosis. Further data are needed to identify patients at risk and help develop new and less invasive diagnostic criteria for cirrhotic patients who may profit from a prophylactic antibiotic treatment.

The use of hysteroscopy to identify and treat reproductive tract abnormalities in the mare

SummaryHysteroscopy is a complementary tool to the traditional transrectal palpation and ultrasonography commonly performed by equine practitioners. Once mastered, it is a quick and useful diagnostic tool for the general and targeted evaluation of the endometrium and provides real time information about potential causes of subfertility. Samples of intrauterine fluid and the endometrium can be taken via transendoscopic catheters or infusion pipettes or via the use of traditional endometrial biopsy forceps. In addition to being a useful diagnostic tool, treatments can often be performed at the same time the pathologies are identified, providing mare owners with information and resolution of problems. With the development of additional technologies, hysteroscopy allows for optical biopsies to be performed, which will likely increase in use as the software moves from the research laboratories to clinical practice. Hysteroscopy is an easily learned, useful clinical skill for the identification of abnormalities in the mares' reproductive tract.

In vivo and in situ robust quantitative optical biopsy of hepatocellular carcinoma and metastasis based on two-photon autofluorescence imaging.

Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.

Combined Rotating Ultra-High-Resolution Spectral Domain OCT and Scheimpflug Imaging for In Vivo Corneal Optical Biopsy.

This article introduces the Pentacam® Cornea OCT (optical coherence tomography). This advanced corneal imaging system combines rotating ultra-high-resolution spectral domain OCT with sub- 2-micron axial resolution and Scheimpflug photography. The purpose of this study is to present the first experience with the instrument and its potential for corneal diagnostics, including optical biopsy. In this prospective study, the Pentacam® Cornea OCT was used to image the corneas of seven patients. The novel wide-angle pericentric scan system enables optimal OCT imaging performance for the corneal layer structure over the entire width of the cornea, including the limbal regions. A detailed analysis of the resulting images assessed the synergism between the OCT and Scheimpflug photography. The Pentacam® Cornea OCT demonstrated significantly improved image resolution and ability to individualize corneal layers with high quality. There is a synergism between the OCT high-definition signal to individualize details on the cornea and Scheimpflug photography to detect and quantify corneal scattering. The noncontact exam was proven safe, user-friendly, and effective for enabling optical biopsy. Pentacam® Cornea OCT is an advancement in corneal imaging technology. The ultra-high-resolution spectral domain OCT and Scheimpflug photography provide unprecedented detail and resolution, enabling optical biopsy and improving the understanding of corneal pathology. Further studies are necessary to compare and analyze the tomographic reconstructions of the cornea with the different wavelengths, which may provide helpful information for diagnosing and managing corneal diseases.

Deep learning classification of ex vivo human colon tissues using spectroscopic optical coherence tomography.

Screening for colorectal cancer (CRC) with colonoscopy has improved patient outcomes; however, it remains the third leading cause of cancer-related mortality, novel strategies to improve screening are needed. Here, we propose an optical biopsy technique based on spectroscopic optical coherence tomography (OCT). Depth resolved OCT images are analyzed as a function of wavelength to measure optical tissue properties and used as input to machine learning algorithms. Previously, we used this approach to analyze mouse colon polyps. Here, we extend the approach to examine human biopsied colonic epithelial tissue samples ex vivo. Optical properties are used as input to a novel deep learning architecture, producing accuracy of up to 97.9% in discriminating tissue type. SOCT parameters are used to create false colored en face OCT images and deep learning classifications are used to enable visual classification by tissue type. This study advances SOCT toward clinical utility for analysis of colonic epithelium.

Temporal fiber-optic fluorescence study of FAD-NADH interactions in phantoms for potential applications in rapid liquid optical biopsy procedures

Body fluids carry several biomarkers and genetic material that enable the detection of carcinogenesis and tumor metastasis. Owing to this, liquid biopsy is gaining importance and entering clinical practice as an adjunct modality to the current standard of practice in cancer detection. Liquid biopsy involving specimens such as urine is non-invasive and has biomarkers reflecting the tumor microenvironment. We investigate the normal and altered levels of cellular metabolic by-products in liquid phantoms and demonstrate their photon-induced excited-state interaction behaviour for potential diagnostic applications. Short-span temporal analysis of metabolic by-products FAD and NADH in liquid phantoms with fiber optic fluorescence spectroscopy is carried out. The mixed liquid phantom of FAD and NADH, showed excited state interactions that manifested as simultaneous inverse variation in the fluorescence emission from FAD and NADH in varying pH environments. Linear discriminant analysis of the time-varying integrated fluorescence intensity resulted in a 100% accuracy in classifying liquid samples between high and low metabolite concentrations. An extension of application of the experiment model for analysis of body fluids such as urine is also discussed.

Multimodal Multiphoton Tomography with a Compact Femtosecond Fiber Laser

Multiphoton tomography (MPT) based on near infrared (NIR) femtosecond laser technology has become a versatile high-resolution clinical and research imaging tool. We report on multimodal MPT with the air-cooled fiber laser tomograph MPTcompact. The ultracompact passively mode-locked erbium-doped 50/80 MHz laser operating at 780 nm is directly integrated into the 360° imaging head mounted on a flexible mechanical arm. The tomograph provides optical biopsies with subcellular resolution and optical metabolic imaging capability based on two-photon autofluorescence (AF), second harmonic generation, fluorescence lifetime imaging (FLIM) by time-correlated single photon counting with 250 ps temporal resolution, reflectance confocal microscopy, and white LED light CMOS camera imaging. For the first time, NIR femtosecond laser pulses have been used on human skin to realize simultaneous one-photon confocal imaging and two-photon imaging. Most useful information is provided by the two-photon excited intratissue AF of the coenzymes NAD(P)H and flavins. The signals of both types of coenzymes can be separated by FLIM. Furthermore, the free and protein-bound forms can be distinguished by time-resolved AF detection because the protein-bound NADH has one order higher AF lifetime than free (non-bound) NADH. The tomograph contains onboard storage batteries so that it can operate for up to a few hours independently from external power supply. Applications include in vivo cancer detection and in situ evaluation of anti-aging drugs and pharmaceuticals. The tomograph MPTcompact has been successfully tested in a clinical multicenter study for the diagnosis of malignant melanoma on 100 patients with suspicious pigmented lesions. MPT has the potential to realize non-invasive high-resolution label-free in vivo histology within minutes and, therefore, to reduce the number of physically taken biopsies.

Feasibility of Optical Biopsy During Endoscopic Sinus Surgery With Confocal Laser Endomicroscopy: A Pilot Study.

Confocal laser endomicroscopy (CLE) is an optical imaging technique that allows in vivo, real-time, microscope-like assessment of superficial lesions. Although there is substantial data on CLE use in the upper GI tract, there is limited information regarding its application in the nasal cavity and paranasal sinuses. This study aims to assess the feasibility and diagnostic metrics of CLE in the nasal cavity and paranasal sinuses regarding differentiation between healthy/benign and malignant tissue. These structures show, however, a wider variety of frequent and concomitant benign and malignant pathologies, which could pose an increased challenge for optical biopsy by CLE. We performed CLE on a case series of six patients with various findings in the nose (three chronic rhinosinusitis, adenocarcinoma, meningoenzephalozele, esthesionneuroblastoma). Forty-two sequences (3792 images) from various structures in the nasal cavity and/or paranasal sinuses were acquired. Biopsies were taken at corresponding locations and analyzed in hematoxylin and eosin staining as a standard of reference. Three independent examiners blinded to the histopathology assessed the sequences. Healthy and inflamed mucosa could be distinguished from malignant lesions with an accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of 84.1%, 85.4%, 83.1%, 72.5%, and 92.1%, respectively, with a substantial agreement between raters (Fleiss κ = 0.62). This technique shows, despite its limitations, potential as an adjunctive imaging technique during sinus surgery; however, the creation of a scoring system based on reproducible and defined characteristics in a larger more diverse population should be the focus of further research to improve its diagnostic value and clinical utility. NA Laryngoscope, 134:4217-4224, 2024.

Highly-selective optical filter for NADH fluorescence detection in multiphoton microscopy

Colorectal cancer (CRC) is a pressing global health concern, emphasizing the need for early detection tools. In this study an optical filter for precise detection of nicotinamide adenine dinucleotide (NADH) fluorescence via two-photon excitation fluorescence (TPEF) was developed. Fabricated with silicon dioxide and titanium dioxide thin films in a Fabry-Perot structure, the filter achieved a peak transmittance of about 95% at 483 nm, with a 12 nm full-width at half maximum. TPEF measurements using a tailored setup and NADH liquid phantoms underscored the filter's significance in selectively capturing NADH fluorescence while mitigating interference from other fluorophores. This work marks a substantial stride towards integrating multiphoton microscopy into conventional colonoscopy, enabling non-invasive, objective optical biopsy for colorectal tissue analysis. Further refinements of the experimental setup are imperative to advance tissue differentiation and enhance CRC diagnosis.

Analysis of Skin Neoplasms’ Raman Spectra Using the Lorentz Approximation Method: Pilot Studies

Confocal Raman microspectroscopy provides the ability to diagnose cancer by quantitatively analyzing spectral features and identifying underlying biochemical changes. The differentiation of malignant skin neoplasms (basal cell carcinoma, squamous cell carcinoma), benign skin neoplasms (papilloma) and healthy skin was carried out by obtaining Raman spectra in vitro with excitation wavelengths of 532 and 785 nm. We present a new method for analyzing the parameters of spectral bands, based on the calculation of the second derivative and Lorentz approximation of the spectra. Using this method on a small selection of skin tumors, we have demonstrated that processes in skin tumors can cause deformation of the proteins’ secondary structure, leading to degradation and shift of the corresponding bands (972, 1655 cm–1) to the lower frequency. Bands corresponding to lipids in skin neoplasms either broaden and increase or split into two peaks (bands 1061, 1127, 1297, 1439, 1745 cm–1). The disruption of lipid structure, also indicated in several bands as a shift to lower wavenumbers, is possibly due to increased cell membrane fluidity in tumors. The results of the study may be useful for the development of optical biopsy methods for early diagnosis of tumors.

Endoscopic Ultrasound-Guided Needle-Based Confocal Endomicroscopy as a Diagnostic Imaging Biomarker for Intraductal Papillary Mucinous Neoplasms.

Pancreatic cancer is on track to become the second leading cause of cancer-related deaths by 2030, yet there is a lack of accurate diagnostic tests for early detection. Intraductal papillary mucinous neoplasms (IPMNs) are precursors to pancreatic cancer and are increasingly being detected. Despite the development and refinement of multiple guidelines, diagnosing high-grade dysplasia or cancer in IPMNs using clinical, radiologic, endosonographic, and cyst fluid features still falls short in terms of accuracy, leading to both under- and overtreatment. EUS-guided needle-based confocal laser endomicroscopy (nCLE) is a novel technology that allows real-time optical biopsies of pancreatic cystic lesions. Emerging data has demonstrated that EUS-nCLE can diagnose and risk stratify IPMNs more accurately than conventional diagnostic tools. Implementing EUS-nCLE in clinical practice can potentially improve early diagnosis of pancreatic cancer, reduce unnecessary surgeries of IPMNs with low-grade dysplasia, and advance the field of digital pathomics. In this review, we summarize the current evidence that supports using EUS-nCLE as a diagnostic imaging biomarker for diagnosing IPMNs and for risk stratifying their degree of neoplasia. Moreover, we will present emerging data on the role of adding artificial intelligence (AI) algorithms to nCLE and integrating novel fluid biomarkers into nCLE.

As how artificial intelligence is revolutionizing endoscopy.

With incessant advances in information technology and its implications in all domains of our lives, artificial intelligence (AI) has emerged as a requirement for improved machine performance. This brings forth the query of how this can benefit endoscopists and improve both diagnostic and therapeutic endoscopy in each part of the gastrointestinal tract. Additionally, it also raises the question of the recent benefits and clinical usefulness of this new technology in daily endoscopic practice. There are two main categories of AI systems: computer-assisted detection (CADe) for lesion detection and computer-assisted diagnosis (CADx) for optical biopsy and lesion characterization. Quality assurance is the next step in the complete monitoring of high-quality colonoscopies. In all cases, computer-aided endoscopy is used, as the overall results rely on the physician. Video capsule endoscopy is a unique example in which a computer operates a device, stores multiple images, and performs an accurate diagnosis. While there are many expectations, we need to standardize and assess various software packages. It is important for healthcare providers to support this new development and make its use an obligation in daily clinical practice. In summary, AI represents a breakthrough in digestive endoscopy. Screening for gastric and colonic cancer detection should be improved, particularly outside expert centers. Prospective and multicenter trials are mandatory before introducing new software into clinical practice.

Feasibility of new-generation endocytoscopy for the real-time diagnosis of ampullary lesions: A pilot study (with video).

An early and accurate diagnosis of ampullary neoplasia is crucial; however, sampling bias is still a major concern. New-generation endocytoscopy enables real-time visualization of cellular structures and enables an accurate pathological prediction; however, its feasibility for small ampullary lesions has never been investigated. We developed a novel endocytoscopic (EC) classification system for ampullary lesions after an expert review and agreement from five experienced endoscopists and one pathologist. We then consecutively enrolled a total of 43 patients with an enlarged ampulla (<3cm), all of whom received an endocytoscopic examination. The feasibility of endocytoscopy was evaluated, and the performance of the EC classification system was then correlated with the final histopathology. In five cases (11.6%), the endocytoscope could not approach the ampulla, and these cases were defined as technical failure. Among the remaining 38 patients, 8 had histopathology-confirmed adenocarcinoma, 15 had adenoma, and 15 had non-neoplastic lesions. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the EC classification system to diagnose ampullary neoplasias were 95.7%, 86.7%, 91.7%, 92.9%, and 92.1%, respectively. Moreover, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the EC classification to diagnose ampullary cancer were 62.5%, 100%, 100%, 90.9%, and 92.1%, respectively. One case with intra-ampullary papillary-tubular carcinoma was classified as having a non-neoplastic lesion by endocytoscopy. Endocytoscopy and the novel EC classification system demonstrated good feasibility to discriminate ampullary neoplasias from non-neoplastic lesions and may be useful for optical biopsies of clinically suspicious ampullary lesions.

A Novel Flexible Endomicroscopic Scanning Instrument with Visual Servoing Control

Optical biopsy using probe‐based confocal laser endomicroscopy (pCLE) and optical coherence tomography (OCT) technologies performs in vivo tissue scanning of organ surfaces and plays a crucial role in early cancer diagnosis. Herein, a flexible endomicroscopic scanning instrument based on a 3‐degree‐of‐freedom flexible hinge structure and visual servoing control is presented. The instrument integrates OCT and pCLE. OCT information compensates for the imaging distance of pCLE, which can combine the advantages of the two technologies. The mechanism allows for tissue surface scanning with a wire‐driven active bending part and dynamic adjustment of the probe‐tissue distance using a combination of wire and spring‐driven, which satisfy large‐area scanning while maintaining consistence tissue contacts. Local optimization‐based visual servoing control is proposed to optimize the tissue scanning. Through the scanning experiments, high‐quality mosaics of uneven surfaces are obtained with an effective area that is larger than 4 mm2. In vivo animal trial further verifies the feasibility and clinical potential of the instrument.

Detection of NADH and NADPH levels in vivo identifies shift of glucose metabolism in cancer to energy production.

Profound changes in the metabolism of cancer cells have been known for almost 100 years, and many aspects of these changes have continued to be actively studied and discussed. Differences in the results of various studies can be explained by the diversity of tumours, which have differing processes of energy metabolism, and by limitations in the methods used. Here, using fluorescence lifetime needle optical biopsy in a hepatocellular carcinoma (HCC) mouse model and patients with HCC, we measured reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in control liver, and in HCC tumours and their adjacent regions. We found that NADH level (mostly responsible for energy metabolism) is increased in tumours but also in adjacent regions of the same liver. NADPH level is significantly decreased in the tumours of patients but increased in the HCC mouse model. However, in the ex vivo tumour slices of mouse HCC, reactive oxygen species production and glutathione level (both dependent on NADPH) were significantly suppressed. Thus, glucose-dependent NADH and NADPH production in tumours changed but with a more pronounced shift to energy production (NADH), rather than NADPH synthesis for redox balance.