Role of reflectance confocal microscopy for in vivo investigation of oral disorders: White, red and pigmented lesions.

In vivo reflectance confocal microscopy (RCM) is poorly investigated in oral pathology due to the peculiar anatomical and topographical oral mucosa features. A dedicated handheld confocal microscope with an intra-oral probe was developed for oral mucosa imaging. The main objective was to describe the healthy oral mucosa and the cytoarchitectural findings detectable in different oral disorders by means of the newly designed handheld confocal microscope. Secondary aim was to identify the main RCM criteria that differentiate oral lesions in order to provide algorithm for a rapid non-invasive evaluation. This observational retrospective study included all consecutive patients with oral disorders and volunteers with healthy oral mucosa who underwent RCM examination in our outpatient clinic from September 2018 to December 2021. Three different investigators examined together the RCM images to detect the key features and secondary criteria for each type of oral lesion collected. The study population included 110 patients affected by oral lesions and seven volunteers with healthy oral mucosae. A total of 15 oral disorders were imaged and divided in three main groups: white, red and pigmented lesions. Key features and secondary criteria were identified for every single type of oral disease. RCM permits a cytoarchitectural evaluation of the oral mucosae affected by inflammatory, dysplastic and neoplastic diseases, thus orienting the clinicians towards non-invasive diagnosis and enhancing the diagnostic management. The "tree diagrams" proposed allow a schematic and simplified view of confocal features for each type of oral disease, thus drastically reducing the diagnostic timing.

Novel Sources of Wheat Head Blast Resistance in Modern Breeding Lines and Wheat Wild Relatives.

Wheat head blast (WHB), caused by the fungus Magnaporthe oryzae pathotype triticum, is a devastating disease affecting South America and South Asia. Despite 30 years of intensive effort, the 2NVS translocation from Aegilops ventricosa contains the only useful source of resistance to WHB effective against M. oryzae triticum isolates. The objective of this study was to identify non-2NVS sources of resistance to WHB among elite cultivars, breeding lines, landraces, and wild-relative accessions. Over 780 accessions were evaluated under field and greenhouse conditions in Bolivia, greenhouse conditions in Brazil, and at two biosafety level-3 laboratories in the United States. The M. oryzae triticum isolates B-71 (2012), 008 (2015), and 16MoT001 (2016) were used for controlled experiments, while isolate 008 was used for field experiments. Resistant and susceptible checks were included in all experiments. Under field conditions, susceptible spreaders were inoculated at the tillering stage to guarantee sufficient inoculum. Disease incidence and severity were evaluated as the average rating for each 1-m-row plot. Under controlled conditions, heads were inoculated after full emergence and individually rated for percentage of diseased spikelets. The diagnostic marker Ventriup-LN2 was used to test for the presence of the 2NVS translocation. Four non-2NVS spring wheat International Maize and Wheat Improvement Center breeding lines (CM22, CM49, CM52, and CM61) and four wheat wild-relatives (A. tauschii TA10142, TA1624, TA1667, and TA10140) were identified as resistant (<5% of severity) or moderately resistant (5 to <25% severity) to WHB. Experiments conducted at the seedling stage showed little correlation with disease severity at the head stage. M. oryzae triticum isolate 16MoT001 was significantly more aggressive against 2NVS-based varieties. The low frequency of WHB resistance and the increase in aggressiveness of newer M. oryzae triticum isolates highlight the threat that the disease poses to wheat production worldwide and the urgent need to identify and characterize new resistance genes that can be used in breeding for durably resistant varieties.

A Response to Gupta et al. (2019) Regarding the MoT3 Wheat Blast Diagnostic Assay.

This is a response to a recent Letter to the Editor of Phytopathology, in which Gupta et al. (2019) caution against the indiscriminate use of the MoT3 diagnostic assay that distinguishes isolates of Magnaporthe oryzae in the Triticum lineage from those that do not cause aggressive wheat blast. We confirm that the assay does reliably distinguish between wheat and rice isolates from Bangladesh and worldwide, as described in the original paper by Pieck et al. (2017) . We have been unable to reproduce the equally intense amplification of WB12 and WB12-like sequences reported in Figure 1 of the Letter. Other data presented by Gupta et al. (2019) support the specificity of the MoT3 assay. Therefore, cautions beyond those always associated with accurate reproduction of diagnostic assays are unwarranted.

Feasibility of a Video-Mosaicking Approach to Extend the Field-of-View For Reflectance Confocal Microscopy in the Oral Cavity In Vivo.

BACKGROUND: Reflectance confocal microscopy (RCM) is a developing approach for noninvasive detection of oral lesions with label-free contrast and cellular-level resolution. For access into the oral cavity, confocal microscopes are being configured with small-diameter telescopic probes and small objective lenses. However, a small probe and objective lens allows for a rather small field-of-view relative to the large areas of tissue that must be examined for diagnosis. To extend the field-of-view for intraoral RCM imaging, we are investigating a video-mosaicking approach. METHODS: A relay telescope and objective lens were adapted to an existing confocal microscope for access into the oral cavity. Imaging was performed using metal three-dimensional-printed objective lens front-end caps with coverslip windows to contact and stabilize the tissue and set depth. Four healthy volunteers (normal oral mucosa), one patient (with an amalgam tattoo) in a clinical setting, and 20 anesthetized patients (with oral squamous cell carcinoma [OSCC]) in a surgical setting were imaged. Instead of the usual still RCM images, videos were recorded and then processed into video-mosaics. Thirty video-mosaics were read and qualitatively assessed by an expert reader of RCM images of the oral mucosa. RESULTS: Whereas the objective lens' native field-of-view is 0.75 mm × 0.75 mm, the video-mosaics display larger areas, ranging from 2 mm × 2 mm to 4 mm × 2 mm, with resolution, morphologic detail, and image quality that is preserved relative to that observed in the original videos (individual images). Video-mosaics in healthy volunteers' and the patients' images showed cellular morphologic patterns in the lower epithelium and at the epithelial junction, and connective tissue along with capillary loops and blood flow in the deeper lamina propria. In OSCC, tumor nests could be observed along with normal looking mucosa in margin areas. CONCLUSIONS: Video-mosaicking is a reasonably quick and efficient approach for extending the field-of-view of RCM imaging, which can, to some extent, overcome the inherent limitation of an intraoral probe's small field-of-view. Reading video-mosaics can mimic the procedure for examining pathology: initial visualization of the spatial cellular and morphologic patterns of the tumor and the spread of tumor margins over larger areas of the lesion, followed by digitally zooming (magnifying) for closer inspection of suspicious areas. However, faster processing of videos into video-mosaics will be necessary, to allow examination of video-mosaics in real-time at the bedside. Lasers Surg. Med. 51:439-451, 2019. © 2019 Wiley Periodicals, Inc.

Wide-field imaging combined with confocal microscopy using a miniature f/5 camera integrated within a high NA objective lens.

Wide-field (WF) imaging paired with reflectance confocal microscopy can noninvasively detect skin cancer with high accuracy. However, two separate devices are required to perform each imaging procedure. We describe a new concept that integrates the two into one device: a miniature WF color camera within the objective lens used for confocal microscopy, providing simultaneous sub-surface cellular imaging and WF surface morphologic imaging. The camera, inserted between a hyperhemisphere front lens and a back lens group of the objective, commands a field of view of 4.0 mm, with a resolution better than 30 µm, while confocal optical sectioning is preserved at sharper than 2.5 µm.

Genomics-Based Marker Discovery and Diagnostic Assay Development for Wheat Blast.

Wheat blast has emerged as a major threat to wheat production in South America. Although originally restricted to Brazil, the disease has since been observed in the neighboring countries of Argentina, Bolivia, and Paraguay and recently the pathogen, Magnaporthe oryzae Triticum pathotype, was isolated from infected wheat in Bangladesh. There is growing concern that the pathogen may continue to spread to other parts of the world, including the United States, where several M. oryzae pathotypes are endemic. M. oryzae pathotypes are morphologically indistinguishable and, therefore, must be characterized genotypically. Symptoms of wheat blast include bleaching of the head, which closely resembles the symptoms of Fusarium head blight, further complicating efforts to monitor for the presence of the pathogen in the field. We used a genomics-based approach to identify molecular markers unique to the Triticum pathotype of M. oryzae. One of these markers, MoT3, was selected for the development of a polymerase chain reaction (PCR)-based diagnostic assay that was evaluated for specificity using DNA from 284 M. oryzae isolates collected from a diverse array of host species. Conventional PCR primers were designed to amplify a 361-bp product, and the protocol consistently amplified from as little as 0.1 ng of purified DNA. The specificity of the MoT3-based assay was also evaluated using Fusarium spp. DNA, from which no amplicons were detected.

The Lolium Pathotype of Magnaporthe oryzae Recovered from a Single Blasted Wheat Plant in the United States.

Wheat blast is a devastating disease that was first identified in Brazil and has subsequently spread to surrounding countries in South America. In May 2011, disease scouting in a University of Kentucky wheat trial plot in Princeton, KY identified a single plant with disease symptoms that differed from the Fusarium head blight that was present in surrounding wheat. The plant in question bore a single diseased head that was bleached yellow from a point about one-third up the rachis to the tip. A gray mycelial mass was observed at the boundary of the healthy tissue and microscopic examination of this material revealed pyriform spores consistent with a Magnaporthe sp. The pathogen was subsequently identified as Magnaporthe oryzae through amplification and sequencing of molecular markers, and genome sequencing revealed that the U.S. wheat blast isolate was most closely related to an M. oryzae strain isolated from annual ryegrass in 2002 and quite distantly related to M. oryzae strains causing wheat blast in South America. The suspect isolate was pathogenic to wheat, as indicated by growth chamber inoculation tests. We conclude that this first occurrence of wheat blast in the United States was most likely caused by a strain that evolved from an endemic Lolium-infecting pathogen and not by an exotic introduction from South America. Moreover, we show that M. oryzae strains capable of infecting wheat have existed in the United States for at least 16 years. Finally, evidence is presented that the environmental conditions in Princeton during the spring of 2011 were unusually conducive to the early production of blast inoculum.

Sugarcane Aphid (Hemiptera: Aphididae): Host Range and Sorghum Resistance Including Cross-Resistance From Greenbug Sources.

The graminous host range and sources of sorghum [Sorghum bicolor (L.) Moench.] plant resistance, including cross-resistance from greenbug, Schizaphis graminum (Rondani), were studied for the newly emerging sugarcane aphid, Melanaphis sacchari (Zehntner), in greenhouse no-choice experiments and field evaluations. The sugarcane aphid could not survive on field corn, Zea mays (L.), Teff grass, Eragrostis tef (Zucc.), proso millet, Panicum miliaceum L., barley, Hordeum vulgare L., and rye, Secale cereale L. Only sorghum genotypes served as hosts including Johnsongrass, Sorghum halepense (L.), a highly suitable noncrop host that generates high numbers of sugarcane aphid and maintains moderate phenotypic injury. The greenbug-resistant parental line RTx2783 that is resistant to greenbug biotypes C and E was resistant to sugarcane aphid in both greenhouse and field tests, while PI 55607 greenbug resistant to biotypes B, C, and E was highly susceptible. PI 55610 that is greenbug resistant to biotypes B, C, and E maintained moderate resistance to the sugarcane aphid, while greenbug-resistant PI 264453 was highly susceptible to sugarcane aphid. Two lines and two hybrids from the Texas A&M breeding program B11070, B11070, AB11055-WF1-CS1/RTx436, and AB11055-WF1-CS1/RTx437 were highly resistant to sugarcane aphid, as were parental types SC110, SC170, and South African lines Ent62/SADC, (Macia/TAM428)-LL9, (SV1*Sima/IS23250)-LG15. Tam428, a parental line that previously showed moderate resistance in South Africa and India, also showed moderate resistance in these evaluations. Overall, 9 of 20 parental sorghum entries tested for phenotypic damage in the field resulted in good resistance to the sugarcane aphid and should be utilized in breeding programs that develop agronomically acceptable sorghums for the southern regions of the United States.

Grazing and no-till cropping impacts on nitrogen retention in dryland agroecosystems.

As the world's population increases, marginal lands such as drylands are likely to become more important for food production. One proven strategy for improving crop production in drylands involves shifting from conventional tillage to no-till to increase water use efficiency, especially when this shift is coupled with more intensive crop rotations. Practices such as no-till that reduce soil disturbance and increase crop residues may promote C and N storage in soil organic matter, thus promoting N retention and reducing N losses. By sampling soils 15 yr after a N tracer addition, this study compared long-term soil N retention across several agricultural management strategies in current and converted shortgrass steppe ecosystems: grazed and ungrazed native grassland, occasionally mowed planted perennial grassland, and three cropping intensities of no-till dryland cropping. We also examined effects of the environmental variables site location and topography on N retention. Overall, the long-term soil N retention of >18% in these managed semiarid ecosystems was high compared with published values for other cropped or grassland ecosystems. Cropping practices strongly influenced long-term N retention, with planted perennial grass systems retaining >90% of N in soil compared with 30% for croplands. Grazing management, topography, and site location had smaller effects on long-term N retention. Estimated 15-yr N losses were low for intact and cropped systems. This work suggests that semiarid perennial grass ecosystems are highly N retentive and that increased intensity of semiarid land management can increase the amount of protein harvested without increasing N losses.

A Prospective Double-Blinded Comparison of Reflectance Confocal Microscopy with Conventional Histopathology for In Vivo Assessment in Oral Cancer.

PURPOSE: We investigated reflectance confocal microscopy (RCM) as a possible noninvasive approach for the diagnosis of cancer and real-time assessment of surgical margins. EXPERIMENTAL DESIGN: In a phase I study on 20 patients, we established the RCM imaging morphologic features that distinguish oral squamous cell carcinoma (OSCC) from normal tissue with a newly developed intraoral RCM probe. Our subsequent phase II prospective double-blinded study in 60 patients tested the diagnostic accuracy of RCM against histopathology. Five RCM videos from the tumor and five from normal surrounding mucosa were collected on each patient, followed by a 3-mm punch biopsy of the imaged area. An experienced RCM reader, who was blinded to biopsy location and histologic diagnosis, examined the videos from both regions and classified each as "tumor" or "not tumor" based on RCM features established in phase I. Hematoxylin and eosin slides from the biopsies were read by a pathologist who was blinded to RCM results. Using histology as the gold standard, we calculated the sensitivity and specificity of RCM. RESULTS: We report a high agreement between the blinded readers (95% for normal tissue and 81.7% for tumors), high specificity (98.3%) and negative predictive values (96.6%) for normal tissue identification, and high sensitivity (90%) and positive predictive values (88.2%) for tumor detection. CONCLUSIONS: RCM imaging is a promising technology for noninvasive in vivo diagnosis of OSCC and for real-time intraoperative evaluation of mucosal surgical margins. Its inherent constraint, however, stems from the diminished capability to evaluate structures located at more substantial depths within the tissue.

Short-term outcomes of the C3 excluder for patients with abdominal aortic aneurysms and unfavorable proximal aortic seal zones.

BACKGROUND: Endovascular abdominal aortic aneurysm repair (EVAR) in patients with unfavorable proximal seal zones remains challenging. The purpose of this study was to identify the incidence of proximal extension cuff usage for type I endoleaks in patients with abdominal aortic aneurysms and unfavorable necks treated with the C3 Excluder repositionable endoprosthesis compared with the traditional Excluder stent-graft. METHODS: This is a retrospective review of patients undergoing EVAR with unfavorable neck anatomy from January 2010 to October 2011 using the Excluder endoprosthesis on the traditional deployment system or the C3 repositionable system. Seventy-seven patients were treated with the Excluder device, with 44 (57%) having unfavorable neck anatomy defined as proximal aortic neck length of <15 mm, neck diameter of >28 mm, neck angulation of >60°, circumferential thrombus of >50% or calcification at the proximal seal zone, or a "reverse taper" on computed tomographic angiography. Of the 44 patients with unfavorable neck anatomy, 24 patients received the C3 Excluder and 20 received the traditional Excluder. RESULTS: The groups' comorbidities, aneurysm characteristics, and high-risk neck criteria were comparable. Initial success was 100% in both groups. Sixteen of the 44 patients (36%) with high-risk neck criteria required proximal extension cuffs for type I endoleaks, with 3 of the 24 patients (13%) in the C3 group compared with 13 of the 20 patients (65%) in the traditional Excluder group requiring proximal extension (P = 0.0005). Operative variables between the two groups were similar. At mean follow-up of 2 months (range: 1-6 months), there were no type I endoleaks or renal artery occlusion, and sac size regression was similar. CONCLUSIONS: The C3 Excluder endoprosthesis significantly reduces the need for proximal extension cuffs in patients with unfavorable aortic neck anatomy compared with the traditional Excluder with identical short-term clinical outcomes. Repositionable grafts could increase the number of patients who can effectively be treated with EVAR.

Endovascular repair of a para-anastomotic pseudoaneurysm after renal autotransplantation: an alternative to open reconstruction.

Renal artery anastomotic pseudoaneurysms are rare after renal transplantation. The etiology tends to be technical, infectious, or degenerative, and repair is difficult with a high postsurgical complication rate. We report the first case of a complex autotransplant renal artery pseudoaneurysm repaired with kissing covered stents. A 52-year-old woman presented with severe left lower quadrant abdominal pain 6 years after a renal autotransplant for ureteral stenosis and recurrent pyelonephritis. A computed tomographic angiography (CTA) scan revealed a bilobed aneurysm arising at the anastomosis between the renal and common iliac arteries. Kissing covered stents were placed within the common iliac artery proximally and extending into the transplant renal artery and external iliac artery. Postdeployment angiography confirmed complete exclusion of the pseudoaneurysm and excellent flow into the transplant kidney and left lower extremity. A follow-up CTA scan at 1 month revealed continued stent-graft patency and complete exclusion of the pseudoaneurysm. An endovascular approach to transplant anastomotic pseduoaneurysms using kissing covered stents is a viable option to exclude aneurysmal changes and preserve flow to the transplanted organ in carefully selected patients.

Preliminary Assessment of Resistance Among U.S. Wheat Cultivars to the Triticum Pathotype of Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of blast disease on several graminaceous plants. The M. oryzae population causing wheat blast has not been officially reported outside South America. Wheat production in the United States is at risk to this pathogen if it is introduced and established. Proactive testing of U.S. wheat cultivars for their reaction to blast and identification of resistance resources is crucial due to the national and global importance of the U.S. wheat industry. In this preliminary study, the phenotypic reaction of 85 U.S. wheat cultivars to M. oryzae (Triticum pathotype) was determined. Although there was a significant correlation in the reaction to blast at the seedling and adult plant stages, only 57% of the head reaction was explained by the seedling reaction. Because of the importance of disease development at the head stage in the field, assessment of all 85 cultivars occurred at the head stage. Among cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars Postrock, JackPot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection. No evidence of the presence of physiological races among isolates T-7, T-12, T-22, and T-25 was found.

In vivo tumor immune microenvironment phenotypes correlate with inflammation and vasculature to predict immunotherapy response.

Response to immunotherapies can be variable and unpredictable. Pathology-based phenotyping of tumors into 'hot' and 'cold' is static, relying solely on T-cell infiltration in single-time single-site biopsies, resulting in suboptimal treatment response prediction. Dynamic vascular events (tumor angiogenesis, leukocyte trafficking) within tumor immune microenvironment (TiME) also influence anti-tumor immunity and treatment response. Here, we report dynamic cellular-level TiME phenotyping in vivo that combines inflammation profiles with vascular features through non-invasive reflectance confocal microscopic imaging. In skin cancer patients, we demonstrate three main TiME phenotypes that correlate with gene and protein expression, and response to toll-like receptor agonist immune-therapy. Notably, phenotypes with high inflammation associate with immunostimulatory signatures and those with high vasculature with angiogenic and endothelial anergy signatures. Moreover, phenotypes with high inflammation and low vasculature demonstrate the best treatment response. This non-invasive in vivo phenotyping approach integrating dynamic vasculature with inflammation serves as a reliable predictor of response to topical immune-therapy in patients.

Flavonoid composition of lemon-yellow sorghum genotypes.

Flavonoid composition of lemon-yellow sorghums grown in two locations in Texas, USA was evaluated and compared with that of white and red sorghums using high performance liquid chromatography (HPLC-PDA). Sorghums from Lubbock were brighter in colour and had minimal weathering compared to those from College Station. Sorghums with red/purple secondary plant colour had the highest levels of 3-deoxyanthocyanidins (8-187µg/g) and their levels were highest in grains from College Station (39-187µg/g). Pericarp colour did not have any effect on 3-deoxyanthocyanidin levels (p>0.05). The tan plant lemon-yellow sorghum Tx2953 had the highest levels of flavones (268-362µg/g). Among the genotypes, lemon-yellow sorghums had the highest levels of flavanones (134-1780µg/g), which are located in the pericarp and their levels were increased in the grains with a bright yellow pericarp and minimal weathering. The high flavanone levels in lemon-yellow sorghums makes this sorghum genotype a good source of those compounds.

Analysis of Induction and Establishment of Dwarf Bunt of Wheat Under Marginal Climatic Conditions.

Dwarf bunt caused by Tilletia contraversa is a disease of winter wheat that has a limited geographic distribution due to specific winter climate requirements. The pathogen is listed as a quarantine organism by several countries that may have wheat production areas with inadequate or marginal climate for the disease-in particular the People's Republic of China. Field experiments were conducted in the United States in an area of Kansas that is a climatic analog to the northern winter wheat areas of China to evaluate the risk of disease introduction into such areas. The soil surface of four replicate 2.8 × 9.75 m plots, planted with a highly susceptible cultivar, was inoculated with six teliospore concentrations ranging from 0.88 to 88,400 teliospores/cm2. A single initial inoculation was done in each of three nurseries planted during separate seasons followed by examination for disease for 4 to 6 years afterward. Any diseased spikes produced were crushed and returned to the plots where they were produced. One nursery had no disease during all six seasons. In two nurseries, the disease was induced at trace levels at the three highest inoculation rates. Disease carryover to the second year occurred during one year in one nursery in plots at the highest inoculation rate, but no disease occurred the following three seasons. A duplicate nursery planted in a disease conducive area in Utah demonstrated that the highest rate of inoculum used in the experiments was sufficient to cause almost 100% infection. This study demonstrated that in an area with marginal climatic conditions it was possible to induce transient trace levels of dwarf bunt, but the disease was not established even with a highly susceptible cultivar and high levels of inoculum. Our results support the conclusions of the 1999 Agreement on U.S.-China Agricultural Cooperation which set a tolerance for teliospores in grain, and supports the Risk Assessment Model for Importation of United States Milling Wheat Containing T. contraversa.

Screening and Mapping for Head Blast Resistance in a Panel of CIMMYT and South Asian Bread Wheat Germplasm.

Wheat blast (WB) is a destructive disease in South America and its first outbreak in Bangladesh in 2016 posed a great risk to food security of South Asian countries. A genome wide association study (GWAS) was conducted on a diverse panel of 184 wheat genotypes from South Asia and CIMMYT. Phenotyping was conducted in eight field experiments in Bolivia and Bangladesh and a greenhouse experiment in the United States. Genotypic data included 11,401 SNP markers of the Illumina Infinium 15K BeadChip and four additional STS markers on the 2NS/2AS translocation region. Accessions with stable WB resistance across experiments were identified, which were all 2NS carriers. Nevertheless, a dozen moderately resistant 2AS lines were identified, exhibiting big variation among experiments. Significant marker-trait associations (MTA) were detected on chromosomes 1BS, 2AS, 6BS, and 7BL; but only MTAs on 2AS at the 2NS/2AS translocation region were consistently significant across experiments. The resistant accessions identified in this study could be used in production in South Asian countries as a preemptive strategy to prevent WB outbreak.

In vivo optical imaging-guided targeted sampling for precise diagnosis and molecular pathology.

Conventional tissue sampling can lead to misdiagnoses and repeated biopsies. Additionally, tissue processed for histopathology suffers from poor nucleic acid quality and/or quantity for downstream molecular profiling. Targeted micro-sampling of tissue can ensure accurate diagnosis and molecular profiling in the presence of spatial heterogeneity, especially in tumors, and facilitate acquisition of fresh tissue for molecular analysis. In this study, we explored the feasibility of performing 1-2 mm precision biopsies guided by high-resolution reflectance confocal microscopy (RCM) and optical coherence tomography (OCT), and reflective metallic grids for accurate spatial targeting. Accurate sampling was confirmed with either histopathology or molecular profiling through next generation sequencing (NGS) in 9 skin cancers in 7 patients. Imaging-guided 1-2 mm biopsies enabled spatial targeting for in vivo diagnosis, feature correlation and depth assessment, which were confirmed with histopathology. In vivo 1-mm targeted biopsies achieved adequate quantity and high quality of DNA for next-generation sequencing. Subsequent mutational profiling was confirmed on 1 melanoma in situ and 2 invasive melanomas, using a 505-gene mutational panel called Memorial Sloan Kettering-Integrated mutational profiling of actionable cancer targets (MSK-IMPACT). Differential mutational landscapes, in terms of number and types of mutations, were found between invasive and in situ melanomas in a single patient. Our findings demonstrate feasibility of accurate sampling of regions of interest for downstream histopathological diagnoses and molecular pathology in both in vivo and ex vivo settings with broad diagnostic, therapeutic and research potential in cutaneous diseases accessible by RCM-OCT imaging.

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.