Ethan LaRochelle

The microcirculation image quality score: Development and preliminary evaluation of a proposed approach to grading quality of image acquisition for bedside videomicroscopy☆

PURPOSEnSide-stream dark-field microscopy is currently used to directly visualize sublingual microcirculation at the bedside. Our experience has found inherent technical challenges in the image acquisition process. This article presents and assesses a quality assurance method to rate image acquisition quality before analysis.nnnMATERIALS AND METHODSnWe identified 6 common image capture and analysis problem areas in sublingual side-stream dark-field videos: illumination, duration, focus, content, stability, and pressure. We created the Microcirculation Image Quality Score by assigning a score of optimal (0 points), suboptimal but acceptable (1 point), or unacceptable (10 points) to each category (for further details, go to http://www.MicroscanAnalysis.blogspot.com). We evaluated 59 videos from a convenience sample of 34 unselected, noncritically ill emergency department patients to create a test set. Two raters, blinded to each other, implemented the score. Any video with a cumulative score of 10 or higher (range, 0-60) was considered unacceptable for further analysis.nnnRESULTSnWe created the Microcirculation Image Quality Score and applied it to 59 videos. For this particular set of 59 videos, the mean (SD) passing quality score was 1.68 (0.90), and the mean (SD) failing quality score was 15.74 (6.19), with 27 of 59 passing the quality score less than 10. Highest failure occurred from pressure artifact. The interrater agreement for acceptability was assessed using Cohen κ for each category: illumination (κ = 1.0), duration (κ = 1.0), focus (κ = 0.91), content (κ = 0.76), stability (κ = 0.71), and pressure (κ = 0.82) and overall pass-fail rates (score >10) (κ = 0.66).nnnCONCLUSIONnOur Microcirculation Image Quality Score addresses many of the common areas where video quality can degrade. The criteria introduced are an objective way to assess the quality of image acquisition, with the goal of selecting videos of adequate quality for analysis. The interrater reliability results in our preliminary study suggest that the Microcirculation Image Quality Score is reasonably repeatable between reviewers. Further assessment is warranted.

Assessing daylight & low-dose rate photodynamic therapy efficacy, using biomarkers of photophysical, biochemical and biological damage metrics in situ

BACKGROUNDnSunlight can activate photodynamic therapy (PDT), and this is a proven strategy to reduce pain caused byconventional PDT treatment, but assessment of this and other alternative low dose rate light sources, and their efficacy, has not been studied in an objective, controlled pre-clinical setting. This study used three objective assays to assess the efficacy of different PDT treatment regimens, using PpIX fluorescence as a photophysical measure, STAT3 cross-linking as a photochemical measure, and keratinocyte damage as a photobiological measure.nnnMETHODSnNude mouse skin was used along with in vivo measures of photosensitizer fluorescence, keratinocyte nucleus damage from pathology, and STAT3 cross-linking from Western blot analysis. Light sources compared included a low fluence rate red LED panel, compact fluorescent bulbs, halogen bulbs and direct sunlight, as compared to traditional PDT delivery with conventional and fractionated high fluence rate red LED light delivery.nnnRESULTSnOf the three biomarkers, two had strong correlation to the PpIX-weighted light dose, which is calculated as the product of the treatment light dose (J/cm2) and the normalized PpIX absorption spectra. Comparison of STAT3 cross-linking to PpIX-weighted light dose had an R=0.74, and comparison of keratinocyte nuclear damage R=0.70. There was little correlation to PpIX fluorescence. These assays indicate most of the low fluence rate treatment modalities were as effective as conventional PDT, while fractionated PDT showed the most damage.nnnCONCLUSIONSnDaylight or artificial light PDT provides an alternative schedule for delivery of drug-light treatment, and this pre-clinical assay demonstrated that in vivo assays of damage could be used to objectively predict a clinical outcome in this altered delivery process.

Comparison of Blue and White Lamp Light with Sunlight for Daylight-Mediated, 5-ALA Photodynamic Therapy, in vivo

Daylight‐mediated photodynamic therapy (d‐PDT) as a treatment for actinic keratosis (AK) is an increasingly common technique due to a significant reduction in pain, leading to better patient tolerability. While past studies have looked at different light sources and delivery methods, this study strives to provide equivalent PpIX‐weighted light doses with the hypothesis that artificial light sources could be equally as effective as natural sunlight if their PpIX‐weighted fluences were equalized. Normal mouse skin was used as the model to compare blue LED light, metal halide white light and natural sunlight, with minimal incubation time between topical ALA application and the onset of light delivery. A total PpIX‐weighted fluence of 20 Jeff cm−2 was delivered over 2 h, and the efficacy of response was quantified using three acute bioassays for PDT damage: PpIX photobleaching, Stat3 crosslinking and quantitative histopathology. These bioassays indicated blue light was slightly inferior to both sunlight and white light, but that the latter two were not significantly different. The results suggest that metal halide white light could be a reasonable alternative to daylight PDT, which should allow a more controlled treatment that is independent of weather and yet should have similar response rates with limited pain during treatment.

Maps of in vivo oxygen pressure with submillimetre resolution and nanomolar sensitivity enabled by Cherenkov-excited luminescence scanned imaging

Low signal-to-noise ratios and limited imaging depths restrict the ability of optical-imaging modalities to detect and accurately quantify molecular emissions from tissue. Here, by using a scanning external X-ray beam from a clinical linear accelerator to induce Cherenkov excitation of luminescence in tissue, we demonstrate in vivo mapping of the oxygenation of tumours at depths of several millimetres, with submillimetre resolution and nanomolar sensitivity. This was achieved by scanning thin sheets of the X-ray beam orthogonally to the emission-detection plane, and by detecting the signal via a time-gated CCD camera synchronized to the radiation pulse. We also show with experiments using phantoms and with simulations that the performance of Cherenkov-excited luminescence scanned imaging (CELSI) is limited by beam size, scan geometry, probe concentration, radiation dose and tissue depth. CELSI might provide the highest sensitivity and resolution in the optical imaging of molecular tracers in vivo.A method that uses thin sheets of X-ray radiation to generate emissions of Cherenkov luminescence can image an oxygen-sensitive molecular probe to map oxygenation in tumours in vivo at submillimetre resolution and nanomolar sensitivity.

Implementation of Multicolor Melt Curve Analysis for High-Risk Human Papilloma Virus Detection in Low- and Middle-Income Countries: A Pilot Study for Expanded Cervical Cancer Screening in Honduras

Purpose Cervical cancer is a leading cause of cancer-related mortality in low- and middle-income countries (LMICs) and screening in LMICs is extremely limited. We aimed to implement on-site high-risk human papillomavirus (hrHPV) DNA testing in cohorts of women from an urban factory and from a rural village. Methods A total of 802 women were recruited for this study in partnership with La Liga Contra el Cancer through the establishment of women’s health resource fairs at two locations in Honduras: a textile factory (n = 401) in the city of San Pedro Sula and the rural village of El Rosario (n = 401) in Yoro. Participants received a routine cervical examination during which three sterile cytobrushes were used to collect cervical samples for testing. hrHPV genotyping was performed using a hrHPV genotyping assay and a real-time polymerase chain reaction instrument. Results hrHPV status across all participants at both sites was 13% hrHPV positive and 67% hrHPV negative. When hrHPV status was compared across all three testing sites, hrHPV-positive rates were approximately equal among the factory (13%), village (12%), and confirmatory testing at Dartmouth-Hitchcock Medical Center (Lebanon, NH; 14%). hrHPV genotype was compared across sites, with HPV16 showing the highest infection rate (15%), followed by HPV59 (12%), and HPV68 (11%). There was a low prevalence of HPV18 observed in both populations compared with the hrHPV-positive population in the United States. Conclusion In collaboration with oncologists and pathologists from La Liga Contra el Cancer, we were able to provide a continuum of care once health-fair testing was performed. We established a method and implementation plan for hrHPV testing that is sustainable in LMICs.

Application of Fluorescence-Guided Surgery to Subsurface Cancers Requiring Wide Local Excision: Literature Review and Novel Developments Toward Indirect Visualization

The excision of tumors by wide local excision is challenging because the mass must be removed entirely without ever viewing it directly. Positive margin rates in sarcoma resection remain in the range of 20% to 35% and are associated with increased recurrence and decreased survival. Fluorescence-guided surgery (FGS) may improve surgical accuracy and has been utilized in other surgical specialties. ABY-029, an anti-epidermal growth factor receptor Affibody molecule covalently bound to the near-infrared fluorophore IRDye 800CW, is an excellent candidate for future FGS applications in sarcoma resection; however, conventional methods with direct surface tumor visualization are not immediately applicable. A novel technique involving imaging through a margin of normal tissue is needed. We review the past and present applications of FGS and present a novel concept of indirect FGS for visualizing tumor through a margin of normal tissue and aiding in excising the entire lesion as a single, complete mass with tumor-free margins.

Signal intensity analysis and optimization for in vivo imaging of Cherenkov and excited luminescence

During external beam radiotherapy (EBRT), in vivo Cherenkov optical emissions can be used as a dosimetry tool or to excite luminescence, termed Cherenkov-excited luminescence (CEL) with microsecond-level time-gated cameras. The goal of this work was to develop a complete theoretical foundation for the detectable signal strength, in order to provide guidance on optimization of the limits of detection and how to optimize near real time imaging. The key parameters affecting photon production, propagation and detection were considered and experimental validation with both tissue phantoms and a murine model are shown. Both the theoretical analysis and experimental data indicate that the detection level is near a single photon-per-pixel for the detection geometry and frame rates commonly used, with the strongest factor being the signal decrease with the square of distance from tissue to camera. Experimental data demonstrates how the SNR improves with increasing integration time, but only up to the point where the dominance of camera read noise is overcome by stray photon noise that cannot be suppressed. For the current camera in a fixed geometry, the signal to background ratio limits the detection of light signals, and the observed in vivo Cherenkov emission is on the order of 100×u2009u2009stronger than CEL signals. As a result, imaging signals from depthsu2009u2009<15u2009mm is reasonable for Cherenkov light, and depthsu2009u2009<3u2009mm is reasonable for CEL imaging. The current investigation modeled Cherenkov and CEL imaging of two oxygen sensing phosphorescent compounds, but the modularity of the code allows for easy comparison of different agents or alternative cameras, geometries or tissues.

Evaluating the efficacy of continuous, low irradiance photodynamic therapy in vivo: artificial light versus natural sunlight (Conference Presentation)

Introduction: Topical photodynamic therapy (PDT) is a popular treatment for many non-melanoma skin cancers including actinic keratosis, Bowen’s disease, and some basal cell carcinomas. A chief complaint among patients is the pain that sometimes accompanies the procedure. This has led to a surge of interest in continuous, low-fluence rate PDT which is thought to be less painful. There is myriad evidence in the literature to suggest that natural sunlight can be an effective photo-activator of PpIX, the cytotoxic intermediate. However, there is some concern among the dermatological community regarding the degree to which this method of light delivery may be controlled. It is in this vein that we seek to compare the efficacy of natural sunlight with artificial light. nPurpose: The present study compares three low-fluence rate light sources in normal mouse skin, combined with ALA-PDT. The light sources of interest are natural sunlight, a 415 nm LED bulb, and a broad spectrum, white light, horticultural bulb.nMethods: A PpIX weighted fluence of 20 J/cm^2 was given to each light group, delivered over ~ 2 hours. Acute indicators of PDT efficacy were assayed via PpIX dosimetry, interrogation of Stat3 crosslinking, and analysis of epidermal keratinocytes for morphological changes.

Structural Cherenkov luminescence imaging with Hadamard-patterned field illumination (Conference Presentation)

Cherenkov-excited luminescence scanned imaging (CELSI) has been proposed for radiation-dose determination in medical physics due to its high spatial-resolution over centimeters of tissue. However, dense line-scanning illumination in typical CELSI is time-cost owing to the mechanical movement of the leaves in multi leaf collimator (MLC), resulting into increased radiation exposure. As a result, a scanningless Cherenkov luminescence imaging modality is herein proposed through structuring epi-illumination with MLC-based Hadamard-patterns, which utilizes a reduced radiation does by limiting illumination patterns, extremely shortening the sampling process. In order to effectively reconstruct unknowns from the resultant underdetermined linear system with sparse samplings, a compressed sensing-based reconstruction methodology with l1-norm regularization is adopted. Numerical and phantom experiments show that the proposed methodology achieves the same image quality as the traditional CELSI does.

Single photon detection imaging of Cherenkov light emitted during radiation therapy

Cherenkov imaging during radiation therapy has been developed as a tool for dosimetry, which could have applications in patient delivery verification or in regular quality audit. The cameras used are intensified imaging sensors, either ICCD or ICMOS cameras, which allow important features of imaging, including: (1) nanosecond time gating, (2) amplification by 103-104, which together allow for imaging which has (1) real time capture at 10-30 frames per second, (2) sensitivity at the level of single photon event level, and (3) ability to suppress background light from the ambient room. However, the capability to achieve single photon imaging has not been fully analyzed to date, and as such was the focus of this study. The ability to quantitatively characterize how a single photon event appears in amplified camera imaging from the Cherenkov images was analyzed with image processing. The signal seen at normal gain levels appears to be a blur of about 90 counts in the CCD detector, after going through the chain of photocathode detection, amplification through a microchannel plate PMT, excitation onto a phosphor screen and then imaged on the CCD. The analysis of single photon events requires careful interpretation of the fixed pattern noise, statistical quantum noise distributions, and the spatial spread of each pulse through the ICCD.