Organoid Modeling of the Tumor Immune Microenvironment.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

Long noncoding RNA MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

Cancer cells collectively invade using a leader-follower organization, but the regulation of leader cells during this dynamic process is poorly understood. Using a dual double-stranded locked nucleic acid (LNA) nanobiosensor that tracks long noncoding RNA (lncRNA) dynamics in live single cells, we monitored the spatiotemporal distribution of lncRNA during collective cancer invasion. We show that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is dynamically regulated in the invading fronts of cancer cells and patient-derived spheroids. MALAT1 transcripts exhibit distinct abundance, diffusivity, and distribution between leader and follower cells. MALAT1 expression increases when a cancer cell becomes a leader and decreases when the collective migration process stops. Transient knockdown of MALAT1 prevents the formation of leader cells and abolishes the invasion of cancer cells. Taken together, our single-cell analysis suggests that MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

Combating Antimicrobial Resistance via Single-Cell Diagnostic Technologies Powered by Droplet Microfluidics.

Antimicrobial resistance is a global threat that if left unchecked could lead to 10 million annual mortalities by 2050. One factor contributing to the rise of multi-drug-resistant (MDR) pathogens is the reliance on traditional culture-based pathogen identification (ID) and antimicrobial susceptibility testing (AST) that typically takes several days. This delay of objective pathogen ID and AST information to inform clinical decision making results in clinicians treating patients empirically often using first-line, broad-spectrum antibiotics, contributing to the misuse/overuse of antibiotics. To combat the rise in MDR pathogens, there is a critical demand for rapid ID and AST technologies. Among the advances in ID and AST technologies in the past decade, single-cell diagnostic technologies powered by droplet microfluidics offer great promise due to their potential for high-sensitivity detection and rapid turnaround time. Our laboratory has been at the forefront of developing such technologies and applying them to diagnosing urinary tract infections (UTIs), one of the most common infections and a frequent reason for the prescription of antimicrobials. For pathogen ID, we first demonstrated the highly sensitive, amplification-free detection of single bacterial cells by confining them in picoliter-scale droplets and detection with fluorogenic peptide nucleic acid (PNA) probes that target their 16S rRNA (rRNA), a well-characterized marker for phylogenic classification. We subsequently improved the PNA probe design and enhanced detection sensitivity. For single-cell AST, we first employed a growth indicator dye and engineered an integrated device that allows us to detect growth from single bacterial cells under antibiotic exposure within 1 h, equivalent to two to three bacterial replications. To expand beyond testing a single antibiotic condition per device, a common limitation for droplet microfluidics, we developed an integrated programmable droplet microfluidic device for scalable single-cell AST. Using the scalable single-cell AST platform, we demonstrated the generation of up to 32 droplet groups in a single device with custom antibiotic titers and the capacity to scale up single-cell AST, and providing reliable pathogen categories beyond a binary call embodies a critical advance. Finally, we developed an integrated ID and AST platform. To this end, we developed a PNA probe panel that can identify nearly 90% of uropathogens and showed the quantitative detection of 16S rRNA from single bacterial cells in droplet-enabled AST after as little as 10 min of antibiotic exposure. This platform achieved both ID and AST from minimally processed urine samples in 30 min, representing one of the fastest turnaround times to date. In addition to tracing the development of our technologies, we compare them with contemporary research advances and offer our perspectives for future development, with the vision that single-cell ID and AST technologies powered by droplet microfluidics can indeed become a useful diagnostic tool for combating antimicrobial resistance.

Conceptual framework and documentation standards of cystoscopic media content for artificial intelligence.

BACKGROUND: The clinical documentation of cystoscopy includes visual and textual materials. However, the secondary use of visual cystoscopic data for educational and research purposes remains limited due to inefficient data management in routine clinical practice. METHODS: A conceptual framework was designed to document cystoscopy in a standardized manner with three major sections: data management, annotation management, and utilization management. A Swiss-cheese model was proposed for quality control and root cause analyses. We defined the infrastructure required to implement the framework with respect to FAIR (findable, accessible, interoperable, reusable) principles. We applied two scenarios exemplifying data sharing for research and educational projects to ensure compliance with FAIR principles. RESULTS: The framework was successfully implemented while following FAIR principles. The cystoscopy atlas produced from the framework could be presented in an educational web portal; a total of 68 full-length qualitative videos and corresponding annotation data were sharable for artificial intelligence projects covering frame classification and segmentation problems at case, lesion, and frame levels. CONCLUSION: Our study shows that the proposed framework facilitates the storage of visual documentation in a standardized manner and enables FAIR data for education and artificial intelligence research.

Utility of Blue Light Cystoscopy for Post-bacillus Calmette-Guérin Bladder Cancer Recurrence Detection: Implications for Clinical Trial Recruitment and Study Comparisons.

PURPOSE: The utility of blue light cystoscopy (BLC) in patients receiving bacillus Calmette-Guérin (BCG) during post-treatment cystoscopy is not well understood. Our objective was to determine if BLC improves recurrence detection in patients with non-muscle invasive bladder cancer (NMIBC) undergoing BCG. MATERIALS AND METHODS: Using the prospective multi-institutional Cysview® Registry (2014-2019), patients with NMIBC who received BCG within 1 year prior to BLC were identified. Primary outcomes were recurrences and whether lesions were detected on white light cystoscopy (WLC), BLC or both. We calculated the percentage of cystoscopies with recurrences that were missed with WLC alone. The cystoscopy-level BLC false-positive rate was the proportion of cystoscopies with biopsies only due to BLC suspicious lesions without recurrence. RESULTS: Of 1,703 BLCs, 282 cystoscopies were in the analytic cohort. The overall recurrence rate was 45.0% (127). With only WLC, 13% (16/127) of recurrences would have been missed as 5.7% (16/282) of cystoscopies performed had recurrence only identified with BLC. Among 16 patients with recurrence missed with WLC, 88% (14) had carcinoma in situ. The cystoscopy-level BLC false-positive rate was 5% (15). CONCLUSIONS: BLC helped detect recurrences after recent BCG that would have been missed with WLC alone. Providers should consider BLC for high-risk patients undergoing BCG and should discuss the risk of false-positives with these patients. As clinical trials of novel therapies for BCG-unresponsive disease increase and there are no clear guidelines on BLC use for post-treatment cystoscopies, it is important to consider how variable BLC use could affect enrollment in and comparisons of these studies.

Role of blue-light cystoscopy in detecting invasive bladder tumours: data from a multi-institutional registry.

OBJECTIVES: To evaluate the role of blue-light cystoscopy (BLC) in detecting invasive tumours that were not visible on white-light cystoscopy (WLC). PATIENTS AND METHODS: Using the multi-institutional Cysview registry database, patients who had at least one white-light negative (WL-)/blue-light positive (BL+) lesion with invasive pathology (≥T1) as highest stage tumour were identified. All WL-/BL+ lesions and all invasive tumours in the database were used as denominators. Relevant baseline and outcome data were collected. RESULTS: Of the 3514 lesions (1257 unique patients), 818 (23.2%) lesions were WL-/BL+, of those, 55 (7%) lesions were invasive (48 T1, seven T2; 47 unique patients) including 28/55 (51%) de novo invasive lesions (26 unique patients). In all, 21/47 (45%) patients had WL-/BL+ concommitant carcinoma in situ and/or another T1 lesions. Of 22 patients with a WL-/BL+ lesion who underwent radical cystectomy (RC), high-risk pathological features leading to RC was only visible on BLC in 18 (82%) patients. At time of RC, 11/22 (50%) patients had pathological upstaging including four (18%) with node-positive disease. CONCLUSIONS: A considerable proportion of invasive lesions are only detectable by BLC and the rate of pathological upstaging is significant. Our present findings suggest an additional benefit of BLC in the detection of invasive bladder tumours that has implications for treatment approach.

Adaptable microfluidic system for single-cell pathogen classification and antimicrobial susceptibility testing.

Infectious diseases caused by bacterial pathogens remain one of the most common causes of morbidity and mortality worldwide. Rapid microbiological analysis is required for prompt treatment of bacterial infections and to facilitate antibiotic stewardship. This study reports an adaptable microfluidic system for rapid pathogen classification and antimicrobial susceptibility testing (AST) at the single-cell level. By incorporating tunable microfluidic valves along with real-time optical detection, bacteria can be trapped and classified according to their physical shape and size for pathogen classification. By monitoring their growth in the presence of antibiotics at the single-cell level, antimicrobial susceptibility of the bacteria can be determined in as little as 30 minutes compared with days required for standard procedures. The microfluidic system is able to detect bacterial pathogens in urine, blood cultures, and whole blood and can analyze polymicrobial samples. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST.

An Efficient Framework for Video Documentation of Bladder Lesions for Cystoscopy: A Proof-of-Concept Study.

Processing full-length cystoscopy videos is challenging for documentation and research purposes. We therefore designed a surgeon-guided framework to extract short video clips with bladder lesions for more efficient content navigation and extraction. Screenshots of bladder lesions were captured during transurethral resection of bladder tumor, then manually labeled according to case identification, date, lesion location, imaging modality, and pathology. The framework used the screenshot to search for and extract a corresponding 10-seconds video clip. Each video clip included a one-second space holder with a QR barcode informing the video content. The success of the framework was measured by the secondary use of these short clips and the reduction of storage volume required for video materials. From 86 cases, the framework successfully generated 249 video clips from 230 screenshots, with 14 erroneous video clips from 8 screenshots excluded. The HIPPA-compliant barcodes provided information of video contents with a 100% data completeness. A web-based educational gallery was curated with various diagnostic categories and annotated frame sequences. Compared with the unedited videos, the informative short video clips reduced the storage volume by 99.5%. In conclusion, our framework expedites the generation of visual contents with surgeon's instruction for cystoscopy and potential incorporation of video data towards applications including clinical documentation, education, and research.

Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine.

The development of accelerated methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) for infectious diseases is necessary to facilitate evidence-based antibiotic therapy and reduce clinical overreliance on broad-spectrum antibiotics. Towards this end, droplet-based microfluidics has unlocked remarkably rapid diagnostic assays with single-cell and single-molecule resolution. Yet, droplet platforms invariably rely on testing purified bacterial samples that have been clinically isolated after lengthy (>16 h) plating. While plating-based clinical isolation is important for enriching and separating out bacteria from background in clinical samples and also facilitating buffer exchange, it creates a diagnostic bottleneck that ultimately precludes droplet-based methods from achieving significantly accelerated times-to-result. To alleviate this bottleneck, we have developed facile syringe filter-enabled strategies for bacterial separation, enrichment, and buffer exchange from urine samples. By selecting appropriately sized filter membranes, we separated bacterial cells from background particulates in urine samples and achieved up to 91% bacterial recovery after such 1-step filtration. When interfaced with droplet-based detection of bacterial cells, 1-step filtration improved the limit of detection for bacterial ID and quantification by over an order of magnitude. We also developed a facile buffer exchange strategy to prepare bacteria in urine samples for droplet-based AST that achieved up to 10-fold bacterial enrichment during buffer exchange. Our filtration strategies, can be easily integrated into droplet workflows, enable clinical isolation-free sample-to-answer ID and AST, and significantly accelerate the turnaround of standard infectious disease diagnostic workflows.

Urinary Stone Disease in Pregnancy: A Claims Based Analysis of 1.4 Million Patients.

PURPOSE: Urinary stone disease during pregnancy is poorly understood but is thought to be associated with increased maternal and fetal morbidity. We determined the prevalence of urinary stone disease in pregnancy and whether it is associated with adverse pregnancy outcomes. MATERIALS AND METHODS: We identified all pregnant women from 2003 through 2017 in the Optum® national insurance claims database. We used diagnosis claims to identify urinary stone disease and assess medical comorbidity. We established the prevalence of urinary stone disease during pregnancy stratified by week of pregnancy. We further evaluated associations among urinary stone disease, maternal complications and pregnancy outcomes in univariable and multivariable analyses. RESULTS: Urinary stone disease affects 8 per 1,000 pregnancies and is more common in white women and women with more comorbid conditions. In fully adjusted models pregnancies complicated by urinary stone disease had higher rates of adverse fetal outcomes including prematurity and spontaneous abortions. This analysis is limited by its retrospective, administrative claims design. CONCLUSIONS: The rate of urinary stone disease during pregnancy is higher than previously reported. Urinary stone disease is associated with adverse pregnancy outcomes.

Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens.

Point-of-care (POC) diagnostics for infectious diseases have the potential to improve patient care and antibiotic stewardship. Nucleic acid hybridization is at the core of many amplification-free molecular diagnostics and detection probe configuration is key to diagnostic performance. Modified nucleic acids such as peptide nucleic acid (PNA) offer advantages compared to conventional DNA probes allowing for faster hybridization, better stability and minimal sample preparation for direct detection of pathogens. Probes with tethered fluorophore and quencher allow for solution-based assays and eliminate the need for washing steps thereby facilitating integration into microfluidic devices. Here, we compared the sensitivity and specificity of double stranded PNA probes (dsPNA) and PNA molecular beacons targeting E. coli and P. aeruginosa for direct detection of bacterial pathogens. In bulk fluid assays, the dsPNAs had an overall higher fluorescent signal and better sensitivity and specificity than the PNA beacons for pathogen detection. We further designed and tested an expanded panel of dsPNA probes for detection of a wide variety of pathogenic bacteria including probes for universal detection of eubacteria, Enterobacteriaceae family, and P. mirablis. To confirm that the advantage translated to other assay types we compared the PNA beacon and dsPNA in a prototype droplet microfluidic device. Beyond the bulk fluid assay and droplet devices, use of dsPNA probes may be advantageous in a wide variety of assays that employ homogenous nucleic acid hybridization.

Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing.

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

Simple and Precise Counting of Viable Bacteria by Resazurin-Amplified Picoarray Detection.

Simple, fast, and precise counting of viable bacteria is fundamental to a variety of microbiological applications such as food quality monitoring and clinical diagnosis. To this end, agar plating, microscopy, and emerging microfluidic devices for single bacteria detection have provided useful means for counting viable bacteria, but they also have their limitations ranging from complexity, time, and inaccuracy. We present herein our new method RAPiD (Resazurin-Amplified Picoarray Detection) for addressing this important problem. In RAPiD, we employ vacuum-assisted sample loading and oil-driven sample digitization to stochastically confine single bacteria in Picoarray, a microfluidic device with picoliter-sized isolation chambers (picochambers), in <30 s with only a few minutes of hands-on time. We add AlamarBlue, a resazurin-based fluorescent dye for bacterial growth, in our assay to accelerate the detection of "microcolonies" proliferated from single bacteria within picochambers. Detecting fluorescence in picochambers as an amplified surrogate for bacterial cells allows us to count hundreds of microcolonies with a single image taken via wide-field fluorescence microscopy. We have also expanded our method to practically test multiple titrations from a single bacterial sample in parallel. Using this expanded "multi-RAPiD" strategy, we can quantify viable cells in E. coli and S. aureus samples with precision in ∼3 h, illustrating RAPiD as a promising new method for counting viable bacteria for microbiological applications.

Development of a 90-Minute Integrated Noninvasive Urinary Assay for Bladder Cancer Detection.

PURPOSE: Despite suboptimal sensitivity urine cytology is often performed as an adjunct to cystoscopy for bladder cancer diagnosis. We aimed to develop a noninvasive, fast molecular diagnostic test for bladder cancer detection with better sensitivity than urine cytology while maintaining adequate specificity. MATERIALS AND METHODS: Urine specimens were collected at 18 multinational sites from subjects prior to cystoscopy or tumor resection, and from healthy and other control subjects without evidence of bladder cancer. The levels of 10 urinary mRNAs were measured in a training cohort of 483 subjects and regression analysis was used to identify a 5-mRNA model to predict cancer status. The performance of the GeneXpert® Bladder Cancer Assay, an assay labeled for investigational use only to detect the 5 mRNAs ABL1, CRH, IGF2, ANXA10 and UPK1B, was evaluated in an independent test cohort of 450 participants. RESULTS: In the independent test cohort the assay ROC curve AUC was 0.87 (95% CI 0.81-0.92). At an example cutoff point of 0.4 overall sensitivity was 73% while specificity was 90% and 77% in the hematuria and surveillance patient populations, respectively. CONCLUSIONS: We developed a 90-minute, urine based test that is simple to perform for the detection of bladder cancer. The test can help guide physician decision making in the management of bladder cancer. Additional evaluation in a prospective study is needed to establish the clinical usefulness of this assay.

Optical and Cross-Sectional Imaging Technologies for Bladder Cancer.

Optical and cross-sectional imaging plays critical roles in bladder cancer diagnostics. White light cystoscopy remains the cornerstone for the management of non-muscle-invasive bladder cancer. In the last decade, significant technological improvements have been introduced for optical imaging to address the known shortcomings of white light cystoscopy. Enhanced cystoscopy modalities such as blue light cystoscopy and narrowband imaging survey a large area of the urothelium and provide contrast enhancement to detect additional lesions and decrease cancer recurrence. However, higher false-positive rates accompany the gain of sensitivity. Optical biopsy technologies, including confocal laser endomicroscopy and optical coherence tomography, provide cellular resolutions combined with subsurface imaging, thereby enabling optical-based cancer characterization, and may lead to real-time cancer grading and staging. Coupling of fluorescently labeled binding agents with optical imaging devices may translate into high molecular specificity, thus enabling visualization and characterization of biological processes at the molecular level. For cross-sectional imaging, upper urinary tract evaluation and assessment potential extravesical tumor extension and metastases are currently the primary roles, particularly for management of muscle-invasive bladder cancer. Multi-parametric MRI, including dynamic gadolinium-enhanced and diffusion-weighted sequences, has been investigated for primary bladder tumor detection. Ultrasmall superparamagnetic particles of iron oxide (USPIO) are a new class of contrast agents that increased the accuracy of lymph node imaging. Combination of multi-parametric MRI with positron emission tomography is on the horizon to improve accuracy rates for primary tumor diagnostics as well as lymph node evaluation. As these high-resolution optical and cross-sectional technologies emerge and develop, judicious assessment and validation await for their clinical integration toward improving the overall management of bladder cancer.

Oncologic Procedures Amenable to Fluorescence-guided Surgery.

OBJECTIVE: Although fluorescence imaging is being applied to a wide range of cancers, it remains unclear which disease populations will benefit greatest. Therefore, we review the potential of this technology to improve outcomes in surgical oncology with attention to the various surgical procedures while exploring trial endpoints that may be optimal for each tumor type. BACKGROUND: For many tumors, primary treatment is surgical resection with negative margins, which corresponds to improved survival and a reduction in subsequent adjuvant therapies. Despite unfavorable effect on patient outcomes, margin positivity rate has not changed significantly over the years. Thus, patients often experience high rates of re-excision, radical resections, and overtreatment. However, fluorescence-guided surgery (FGS) has brought forth new light by allowing detection of subclinical disease not readily visible with the naked eye. METHODS: We performed a systematic review of clinicatrials.gov using search terms "fluorescence," "image-guided surgery," and "near-infrared imaging" to identify trials utilizing FGS for those received on or before May 2016. INCLUSION CRITERIA: fluorescence surgery for tumor debulking, wide local excision, whole-organ resection, and peritoneal metastases. EXCLUSION CRITERIA: fluorescence in situ hybridization, fluorescence imaging for lymph node mapping, nonmalignant lesions, nonsurgical purposes, or image guidance without fluorescence. RESULTS: Initial search produced 844 entries, which was narrowed down to 68 trials. Review of literature and clinical trials identified 3 primary resection methods for utilizing FGS: (1) debulking, (2) wide local excision, and (3) whole organ excision. CONCLUSIONS: The use of FGS as a surgical guide enhancement has the potential to improve survival and quality of life outcomes for patients. And, as the number of clinical trials rise each year, it is apparent that FGS has great potential for a broad range of clinical applications.

Fiber-Optic Confocal Laser Endomicroscopy of Small Renal Masses: Toward Real-Time Optical Diagnostic Biopsy.

PURPOSE: The incidental detection of small renal masses is increasing. However, not all require aggressive treatments as up to 20% are benign and the majority of malignant tumors harbor indolent features. Improved preoperative diagnostics are needed to differentiate tumors requiring aggressive treatment from those more suitable for surveillance. We evaluated and compared confocal laser endomicroscopy with standard histopathology in ex vivo human kidney tumors as proof of principle towards diagnostic optical biopsy. MATERIALS AND METHODS: Patients with a solitary small renal mass scheduled for partial or radical nephrectomy were enrolled in study. Two kidneys were infused with fluorescein via intraoperative intravenous injection and 18 tumors were bathed ex vivo in dilute fluorescein prior to confocal imaging. A 2.6 mm confocal laser endomicroscopy probe was used to image tumors and surrounding parenchyma from external and en face surfaces after specimen bisection. Confocal laser endomicroscopy images were compared to standard hematoxylin and eosin analysis of corresponding areas. RESULTS: Ex vivo confocal laser endomicroscopy imaging revealed normal renal structures that correlated well with histology findings. Tumor tissue was readily distinguishable from normal parenchyma, demonstrating features unique to benign and malignant tumor subtypes. Topical fluorescein administration provided more consistent confocal laser endomicroscopy imaging than the intravenous route. Additionally, en face tumor imaging was superior to external imaging. CONCLUSIONS: We report what is to our knowledge the first feasibility study using confocal laser endomicroscopy to evaluate small renal masses ex vivo and provide a preliminary atlas of images from various renal neoplasms with corresponding histology. These findings serve as an initial and promising step toward real-time diagnostic optical biopsy of small renal masses.

Intraoperative Optical Biopsy during Robotic Assisted Radical Prostatectomy Using Confocal Endomicroscopy.

PURPOSE: Intraoperative optical biopsy technologies may aid in the identification of important anatomical landmarks and improve surgical outcomes of robotic assisted radical prostatectomy. We evaluate the feasibility of confocal laser endomicroscopy during robotic assisted radical prostatectomy. MATERIALS AND METHODS: A total of 21 patients with biopsy proven prostate cancer scheduled for robotic assisted radical prostatectomy were recruited. After intravenous administration of fluorescein 15 patients underwent in vivo intraoperative confocal laser endomicroscopy of prostatic and periprostatic structures using a 2.6 or 0.85 mm imaging probe. Standard robotic instruments were used to grasp and maneuver the confocal laser endomicroscopy probes for image acquisition. Confocal laser endomicroscopy imaging was performed ex vivo on fresh prostate specimens from 20 patients. Confocal video sequences acquired in vivo and ex vivo were reviewed and analyzed, with additional image processing using a mosaicing algorithm. Processed confocal images were compared with standard hematoxylin and eosin analysis of imaged regions. RESULTS: Confocal laser endomicroscopy was successfully integrated with robotic surgery, including co-registration of confocal video sequences with white light and probe handling with standard robotic instrumentation. Intraoperative confocal laser endomicroscopy imaging of the neurovascular bundle before and after nerve sparing dissection revealed characteristic features including dynamic vascular flow and intact axon fibers. Ex vivo confocal imaging of the prostatic parenchyma demonstrated normal prostate glands, stroma and prostatic carcinoma. CONCLUSIONS: We report the initial feasibility of optical biopsy of prostatic and periprostatic tissue during robotic assisted radical prostatectomy. Image guidance and tissue interrogation using confocal laser endomicroscopy offer a new intraoperative imaging method that has the potential to improve the functional and oncologic outcomes of prostate cancer surgery.

Rapid bladder cancer cell detection from clinical urine samples using an ultra-thin silicone membrane.

Early detection of initial onset, as well as recurrence, of cancer is paramount for improved patient prognosis and human health. Cancer screening is enhanced by rapid differentiation of cancerous from non-cancerous cells which employs the inherent differences in biophysical properties. Our preliminary testing demonstrates that cell-line derived bladder cancer cells deform our <30 nm silicone membrane within an hour and induce visually distinct wrinkle patterns while cell-line derived non-cancerous cells fail to induce these wrinkle patterns. Herein, we report a platform for the rapid detection of cancerous cells from human clinical urine samples. We performed a blinded study with cells extracted from the urine of human patients suspected to have bladder cancer alongside healthy controls. Wrinkle patterns were induced specifically by the five cancer patient samples within 12 hours and not by the healthy controls. These results were independently validated by the standard diagnostic techniques cystoscopy and cytology. Thus, our ultra-thin membrane approach for cancer diagnosis appears as accurate as standard diagnostic methods while vastly more rapid, less invasive, and requiring limited expertise.

Long-range electrothermal fluid motion in microfluidic systems.

AC electrothermal flow (ACEF) is the fluid motion created as a result of Joule heating induced temperature gradients. ACEF is capable of performing major microfluidic operations, such as pumping, mixing, concentration, separation and assay enhancement, and is effective in biological samples with a wide range of electrical conductivity. Here, we report long-range fluid motion induced by ACEF, which creates centimeter-scale vortices. The long-range fluid motion displays a strong voltage dependence and is suppressed in microchannels with a characteristic length below ~300 µm. An extended computational model of ACEF, which considers the effects of the density gradient and temperature-dependent parameters, is developed and compared experimentally by particle image velocimetry. The model captures the essence of ACEF in a wide range of channel dimensions and operating conditions. The combined experimental and computational study reveals the essential roles of buoyancy, temperature rise, and associated changes in material properties in the formation of the long-range fluid motion. Our results provide critical information for the design and modeling of ACEF based microfluidic systems toward various bioanalytical applications.