AAPM Task Group Report 311: Guidance for performance evaluation of fluorescence-guided surgery systems.

The last decade has seen a large growth in fluorescence-guided surgery (FGS) imaging and interventions. With the increasing number of clinical specialties implementing FGS, the range of systems with radically different physical designs, image processing approaches, and performance requirements is expanding. This variety of systems makes it nearly impossible to specify uniform performance goals, yet at the same time, utilization of different devices in new clinical procedures and trials indicates some need for common knowledge bases and a quality assessment paradigm to ensure that effective translation and use occurs. It is feasible to identify key fundamental image quality characteristics and corresponding objective test methods that should be determined such that there are consistent conventions across a variety of FGS devices. This report outlines test methods, tissue simulating phantoms and suggested guidelines, as well as personnel needs and professional knowledge bases that can be established. This report frames the issues with guidance and feedback from related societies and agencies having vested interest in the outcome, coming from an independent scientific group formed from academics and international federal agencies for the establishment of these professional guidelines.

Spatial frequency domain Mueller matrix imaging.

Significance: Mueller matrix polarimetry (MMP) and spatial frequency domain imaging (SFDI) are wide-field optical imaging modalities that differentiate tissue primarily by structure alignment and photon transport coefficient, respectively. Because these effects can be related, combining MMP and SFDI may enhance tissue differentiation beyond the capability of each modality alone. Aim: An instrument was developed to combine MMP and SFDI with the goal of testing whether it enhances contrast of features in reflection mode. Approach: The instrument was constructed using liquid crystal elements for polarization control, a digital light processing projector for generating sinusoidal illumination patterns, and a digital camera for imaging. A theoretical analysis shows that the SFD Mueller matrix is complex-valued and does not follow the same behavior as a regular Mueller matrix. Images were acquired from an anisotropic tissue phantom, an optical fiber bundle, and cerebellum, thalamus, and cerebrum tissues. Results: The measurement results suggest that singly scattered, few scattered, and diffusely scattered photon paths can be distinguished in some of the samples investigated. The combined imaging modality yields additional spatial frequency phase information, which highlights paths having only a few scattering events. Conclusions: The combination of MMP and SFDI offers contrast mechanisms inaccessible by each modality used alone.

Conversion of imager-specific response to tissue phantom fluorescence into system of units-traceable units.

SIGNIFICANCE: The fluorescence-guided imaging for surgical intervention community recognizes the need for performance standards for these imaging devices. Tissue phantoms are used to track an imager's performance as a fluorescence detector, but imager-specific units are of limited utility. AIM: Tissue phantoms can be calibrated to be traceable to the international system of units (SI) and in turn be used to calibrate imagers such that fluorescence measurements can be reported in universally accepted units. APPROACH: The radiometry to convert imager-specific arbitrary digital counts to SI-traceable unit of watts is described in this paper. RESULTS: An example of an imager calibration is included. CONCLUSIONS: Calibrated tissue phantoms become a tool for metrological traceability.

Performance test methods for near-infrared fluorescence imaging.

PURPOSE: Near-infrared fluorescence (NIRF) imaging using exogenous contrast has gained much attention as a technique for enhancing visualization of vasculature using untargeted agents, as well as for the detection and localization of cancer with targeted agents. In order to address the emerging need for standardization of NIRF imaging technologies, it is necessary to identify the best practices suitable for objective, quantitative testing of key image quality characteristics. Toward the development of a battery of test methods that are rigorous yet applicable to a wide variety of devices, we have evaluated techniques for phantom design, measurement, and calculation of specific performance metrics. METHODS: Using a NIRF imaging system for indocyanine green imaging, providing excitation at 780 nm and detection above 830 nm, we explored methods to evaluate uniformity, field of view, spectral crosstalk, spatial resolution, depth of field, sensitivity, linearity, and penetration depth. These measurements were performed using fluorophore-doped multiwell plate and high turbidity planar phantoms, as well as a 3D-printed multichannel phantom and a USAF 1951 resolution target. RESULTS AND CONCLUSIONS: Based on a wide range of approaches described in medical and fluorescence imaging literature, we have developed and demonstrated a cohesive battery of test methods for evaluation of fluorescence image quality in wide-field imagers. We also propose a number of key metrics that can facilitate direct, quantitative comparison of device performance. These methods have the potential to facilitate more uniform evaluation and inter-comparison of clinical and preclinical imaging systems than is typically achieved, with the long-term goal of establishing international standards for fluorescence image quality assessment.

Comparison of NIR Versus SWIR Fluorescence Image Device Performance Using Working Standards Calibrated With SI Units.

Recently, fluorescence imaging using shortwave infrared light (SWIR, 1,000-2,000 nm) has been proposed as having advantage over conventional near-infrared fluorescence (NIRF) imaging due to the reduced tissue scattering, negligible autofluorescence, comparable tissue absorption, and the discovery that indocyanine green (ICG), used clinically as a NIRF contrast agent, also has fluorescence emission in SWIR regime. Images of ICG in small animals acquired by commercial Si-based and InGaAs-based imaging cameras have been qualitatively compared, however the lack of working standards to quantify performance of these imaging systems limits quantitative comparison. Without quantification using a traceable in vitro test, clinical adoption of rapidly evolving advances in both NIRF and SWIR imaging devices will become limited. In this work, we developed an ICG based fluorescent solid working standard calibrated with SI units (mW [Formula: see text]cm [Formula: see text]sr -1) for quantification of measurement sensitivity of Si, GaAs-intensified Si, and InGaAs based camera systems, their signal-to-noise ratio (SNR), and contrast in non-clinical tests. In addition, we present small animal and large animal imaging with ICG for qualitative comparison of the same SWIR fluorescence and NIRF imaging systems. Results suggest that SWIR fluorescence imaging of ICG may have superior resolution in small animal imaging compared to NIRF imaging, but lack of measurement sensitivity, SNR, contrast, as well as water absorption limits deep penetration in large animals.

Structured illumination Mueller matrix imaging.

We perform Mueller matrix imaging (MMI) of diffusely scattering phantoms under sinusoidal irradiance of varying spatial frequency. Quantitative polarimetric sensing via MMI completely characterizes a sample's polarimetric properties, while structured illumination (SI) allows for the control of photon path length. Intralipid phantoms were measured with varying absorption and with varying depth to demonstrate photon path length control for Mueller matrix elements. We observe unpolarized intensity, linear polarization, and circular polarization to depend upon spatial frequency differently. Finally, we measured an ex vivo chicken skin sample over a bright and dark substrate to further demonstrate the sensitivity of SI-MMI to depth.

Fluorescence-guided surgery and intervention - An AAPM emerging technology blue paper.

Fluorescence-guided surgery (FGS) and other interventions are rapidly evolving as a class of technologically driven interventional approaches in which many surgical specialties visualize fluorescent molecular tracers or biomarkers through associated cameras or oculars to guide clinical decisions on pathological lesion detection and excision/ablation. The technology has been commercialized for some specific applications, but also presents technical challenges unique to optical imaging that could confound the utility of some interventional procedures where real-time decisions must be made. Accordingly, the AAPM has initiated the publication of this Blue Paper of The Emerging Technology Working Group (TETAWG) and the creation of a Task Group from the Therapy Physics Committee within the Treatment Delivery Subcommittee. In describing the relevant issues, this document outlines the key parameters, stakeholders, impacts, and outcomes of clinical FGS technology and its applications. The presentation is not intended to be conclusive, but rather to inform the field of medical physics and stimulate the discussions needed in the field with respect to a seemingly low-risk imaging technology that has high potential for significant therapeutic impact. This AAPM Task Group is working toward consensus around guidelines and standards for advancing the field safely and effectively.

Simultaneous Measurement of Dissolved Oxygen Pressure and Oxyhemoglobin Spectra in Solution.

The measurement of the spatial distribution of oxygen saturation (sO2) in superficial tissues using optical reflectance imaging has been useful in the clinical venue especially in temporally demanding applications such as monitoring tissue oxygenation during surgery. The measurement is based on relative spectrometry of oxy- and deoxyhemoglobin in tissues. We titrated deoxyhemoglobin with oxygen gas and simultaneously measured the dissolved oxygen pressure and the visible absorbance spectra to verify spectral shapes at different saturations. sO2 values derived from the measured pO2 are compared to those derived from the hemoglobin spectra at various stages of oxygenation.

Determining the Performance of Fluorescence Molecular Imaging Devices Using Traceable Working Standards With SI Units of Radiance.

To date, no emerging preclinical or clinical near-infrared fluorescence (NIRF) imaging devices for noninvasive and/or surgical guidance have their performances validated on working standards with SI units of radiance that enable comparison or quantitative quality assurance. In this work, we developed and deployed a methodology to calibrate a stable, solid phantom for emission radiance with International System of Units (SI) units of mW ·sr(-1) ·cm(-2) for use in characterizing the measurement sensitivity of ICCD and IsCMOS detection, signal-to-noise ratio, and contrast. In addition, at calibrated radiances, we assess transverse and lateral resolution of ICCD and IsCMOS camera systems. The methodology allowed demonstration of superior SNR of the ICCD over the IsCMOS technology and superior resolution of the IsCMOS over the ICCD approach. Contrast depended upon the camera settings (binning and integration time) and gain of intensifier. Finally, because the architecture of CMOS and CCD camera systems results in vastly different performance, we comment on the utility of these technologies for small animal imaging as well as clinical applications for noninvasive and surgical guidance.

Calibration and validation scheme for in vivo spectroscopic imaging of tissue oxygenation.

The determination of the level of oxygenation in optically accessible tissues using multispectral or hyperspectral imaging (HSI) of oxy- and deoxyhemoglobin has special appeal in clinical work due to its noninvasiveness, ease of use, and capability of providing molecular and anatomical information at near video rates during surgery. In this paper we refer to an example of the use of HSI in monitoring oxygenation of kidneys during partial nephrectomy. In a study using porcine models, it was found that artery-only clamping left the kidney better oxygenated, as opposed to simultaneously clamping the artery and the vein. A subsequent study correlates gradations in blood flow by partial clamping during the surgical procedure with postoperative renal function via assessment of creatinine level. We discuss the various contributions to the uncertainty of the oxygen saturation measured by this remote-sensing imaging technique in medical application.

Results of aperture area comparisons for exo-atmospheric total solar irradiance measurements.

Exo-atmospheric solar irradiance measurements made by the solar irradiance community since 1978 have incorporated limiting apertures with diameters measured by a number of metrology laboratories using a variety of techniques. Knowledge of the aperture area is a critical component in the conversion of radiant flux measurements to solar irradiance. A National Aeronautics and Space Administration (NASA) Earth Observing System (EOS) sponsored international comparison of aperture area measurements of limiting apertures provided by solar irradiance researchers was performed, the effort being executed by the National Institute of Standards and Technology (NIST) in coordination with the EOS Project Science Office. Apertures that had institutional heritage with historical solar irradiance measurements were measured using the absolute aperture measurement facility at NIST. The measurement technique employed noncontact video microscopy using high-accuracy translation stages. We have quantified the differences between the participating institutions' aperture area measurements and find no evidence to support the hypothesis that preflight aperture area measurements were the root cause of discrepancies in long-term total solar irradiance satellite measurements. Another result is the assessment of uncertainties assigned to methods used by participants. We find that uncertainties assigned to a participant's values may be underestimated.

Designing microarray phantoms for hyperspectral imaging validation.

The design and fabrication of custom-tailored microarrays for use as phantoms in the characterization of hyperspectral imaging systems is described. Corresponding analysis methods for biologically relevant samples are also discussed. An image-based phantom design was used to program a microarrayer robot to print prescribed mixtures of dyes onto microscope slides. The resulting arrays were imaged by a hyperspectral imaging microscope. The shape of the spots results in significant scattering signals, which can be used to test image analysis algorithms. Separation of the scattering signals allowed elucidation of individual dye spectra. In addition, spectral fitting of the absorbance spectra of complex dye mixtures was performed in order to determine local dye concentrations. Such microarray phantoms provide a robust testing platform for comparisons of hyperspectral imaging acquisition and analysis methods.

Algorithm validation using multicolor phantoms.

We present a framework for hyperspectral image (HSI) analysis validation, specifically abundance fraction estimation based on HSI measurements of water soluble dye mixtures printed on microarray chips. In our work we focus on the performance of two algorithms, the Least Absolute Shrinkage and Selection Operator (LASSO) and the Spatial LASSO (SPLASSO). The LASSO is a well known statistical method for simultaneously performing model estimation and variable selection. In the context of estimating abundance fractions in a HSI scene, the "sparse" representations provided by the LASSO are appropriate as not every pixel will be expected to contain every endmember. The SPLASSO is a novel approach we introduce here for HSI analysis which takes the framework of the LASSO algorithm a step further and incorporates the rich spatial information which is available in HSI to further improve the estimates of abundance. In our work here we introduce the dye mixture platform as a new benchmark data set for hyperspectral biomedical image processing and show our algorithm's improvement over the standard LASSO.

Developing digital tissue phantoms for hyperspectral imaging of ischemic wounds.

Hyperspectral imaging has the potential to achieve high spatial resolution and high functional sensitivity for non-invasive assessment of tissue oxygenation. However, clinical acceptance of hyperspectral imaging in ischemic wound assessment is hampered by its poor reproducibility, low accuracy, and misinterpreted biology. These limitations are partially caused by the lack of a traceable calibration standard. We proposed a digital tissue phantom (DTP) platform for quantitative calibration and performance evaluation of spectral wound imaging devices. The technical feasibility of such a DTP platform was demonstrated by both in vitro and in vivo experiments. The in vitro DTPs were developed based on a liquid blood phantom model. The in vivo DTPs were developed based on a porcine ischemic skin flap model. The DTPs were projected by a Hyperspectral Image Projector (HIP) with high fidelity. A wide-gap 2nd derivative oxygenation algorithm was developed to reconstruct tissue functional parameters from hyperspectral measurements. In this study, we have demonstrated not only the technical feasibility of using DTPs for quantitative calibration, evaluation, and optimization of spectral imaging devices but also its potential for ischemic wound assessment in clinical practice.

Active DLP hyperspectral illumination: a noninvasive, in vivo, system characterization visualizing tissue oxygenation at near video rates.

We report use of a novel hyperspectral imaging system utilizing digital light processing (DLP) technology to noninvasively visualize in vivo tissue oxygenation during surgical procedures. The system's novelty resides in its method of illuminating tissue with precisely predetermined continuous complex spectra. The Texas Instruments digital micromirror device, DMD, chip consisting of 768 by 1024 mirrors, each 16 µm square, can be switched between two positions at 12.5 kHz. Switching the appropriate mirrors controls the intensity of light illuminating the tissue as a function of wavelength, active spectral illumination. Meaning, the tissue can be illuminated with a different spectrum of light within 80 µs. Precisely, predetermined spectral illumination penetrates into patient tissue, its chemical composition augments the spectral properties of the light, and its reflected spectra are detected and digitized at each pixel detector of a silicon charge-coupled device, CCD. Using complex spectral illumination, digital signal processing and chemometric methods produce chemically relevant images at near video rates. Specific to this work, tissue is illuminated spectrally with light spanning the visible electromagnetic spectrum (380 to 780 nm). Spectrophotometric images are detected and processed visualizing the percentage of oxyhemoglobin at each pixel detector and presented continuously, in real time, at 3 images per second. As a proof of principle application, kidneys of four live anesthetized pigs were imaged before, during, and after renal vascular occlusion. DLP Hyperspectral Imaging with active spectral illumination detected a 64.73 ± 1.5% drop in the oxygenation of hemoglobin within 30 s of renal arterial occlusion. Producing chemically encoded images at near video rate, time-resolved hyperspectral imaging facilitates monitoring renal blood flow during animal surgery and holds considerable promise for doing the same during human surgical interventions.

Low-cost, high-throughput, automated counting of bacterial colonies.

Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called "NICE" (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods.

Characterization of renal ischemia using DLP hyperspectral imaging: a pilot study comparing artery-only occlusion versus artery and vein occlusion.

BACKGROUND AND PURPOSE: Renal artery-only (AO) occlusion, as opposed to artery and vein (AV) occlusion, has demonstrated some benefit in reducing renal insufficiency during warm ischemia. In this pilot study, we used digital light projection hyperspectral imaging (HSI) to construct a "real time" tissue oxygenation "map" to determine whether there are differences in renal tissue oxygenation during vascular occlusion with AO vs AV. MATERIALS AND METHODS: Renal vascular occlusion with either AO or AV was performed for 60 minutes in seven porcine renal units. Using HSI, the percentage of oxyhemoglobin (%HbO(2)) in the renal cortex was determined at 4-minute increments throughout the ischemic period and for 30 minutes after reperfusion. RESULTS: Average baseline %HbO(2) in all animals was approximately 70%. After vascular occlusion in both cohorts, %HbO(2) decreased by one third within 2 to 5 minutes, with a gradual decline in %HbO(2) over the remaining 55 minutes. Oxyhemoglobin profiles for AO and AV occlusion diverged significantly between 16 and 24 minutes after vascular occlusion (P = 0.0001 and 0.036, respectively), with a merging of the two curves occurring after approximately 36 minutes (P = 0.093). During reperfusion, average %HbO(2) improved to 72.4% after 25 to 30 minutes. CONCLUSION: In this pilot study, we demonstrate that renal tissue oxygenation drops rapidly after occlusion of the renal vasculature and returns to near baseline 30 minutes after reperfusion. In the porcine model, the %HbO(2) differs significantly between AO and AV occlusion for up to 35 minutes after ischemia onset, indicating a possible "ischemic window" in which AO occlusion may provide benefit over AV occlusion.

Thermodynamic-temperature determinations of the Ag and Au freezing temperatures using a detector-based radiation thermometer.

The development of a radiation thermometer calibrated for spectral radiance responsivity using cryogenic, electrical-substitution radiometry to determine the thermodynamic temperatures of the Ag- and Au-freezing temperatures is described. The absolute spectral radiance responsivity of the radiation thermometer is measured in the NIST Spectral Irradiance and Radiance Responsivity Calibrations using Uniform Sources (SIRCUS) facility with a total uncertainty of 0.15% (k=2) and is traceable to the electrical watt, and thus the thermodynamic temperature of any blackbody can be determined by using Planck radiation law and the measured optical power. The thermodynamic temperatures of the Ag- and Au-freezing temperatures are determined to be 1234.956 K (+/-0.110 K) (k=2) and 1337.344 K(+/-0.129 K) (k=2) differing from the International Temperature Scale of 1990 (ITS-90) assignments by 26 mK and 14 mK, respectively, within the stated uncertainties. The temperatures were systematically corrected for the size- of-source effect, the nonlinearity of the preamplifier and the emissivity of the blackbody. The ultimate goal of these thermodynamic temperature measurements is to disseminate temperature scales with lower uncertainties than those of the ITS-90. These results indicate that direct disseminations of thermodynamic temperature scales are possible.