Cherenkov-excited Luminescence Research Articles

Photon-limited Cherenkov imaging of radiation therapy dose.

Cherenkov imaging is a unique verification tool that could provide both dosimetric and tissue functional information during radiation therapy. However, the number of interrogated Cherenkov photons in tissue is always limited and tangled with stray radiation photons, severely frustrating the measurement the signal-to-noise ratio (SNR). As such, here, a noise-robust photon-limited imaging technique is proposed by comprehensively exploiting the physical rationale of low-flux Cherenkov measurements together with the spatial correlations of the objects. Validation experiments confirmed that the Cherenkov signal could be promisingly recovered with high SNR by irradiating at as few as one x ray pulse from a linear accelerator (10 mGy dose), and the Cherenkov excited luminescence imaging depth can be extended by >100% on average, for most concentrations of phosphorescent probe. This approach demonstrates that improved applications in radiation oncology could be seen when signal amplitude, noise robustness, and temporal resolution are comprehensively considered in the image recovery process.

Selfrec-Net: self-supervised deep learning approach for the reconstruction of Cherenkov-excited luminescence scanned tomography.

As an emerging imaging technique, Cherenkov-excited luminescence scanned tomography (CELST) can recover a high-resolution 3D distribution of quantum emission fields within tissue using X-ray excitation for deep penetrance. However, its reconstruction is an ill-posed and under-conditioned inverse problem because of the diffuse optical emission signal. Deep learning based image reconstruction has shown very good potential for solving these types of problems, however they suffer from a lack of ground-truth image data to confirm when used with experimental data. To overcome this, a self-supervised network cascaded by a 3D reconstruction network and the forward model, termed Selfrec-Net, was proposed to perform CELST reconstruction. Under this framework, the boundary measurements are input to the network to reconstruct the distribution of the quantum field and the predicted measurements are subsequently obtained by feeding the reconstructed result to the forward model. The network was trained by minimizing the loss between the input measurements and the predicted measurements rather than the reconstructed distributions and the corresponding ground truths. Comparative experiments were carried out on both numerical simulations and physical phantoms. For singular luminescent targets, the results demonstrate the effectiveness and robustness of the proposed network, and comparable performance can be attained to a state-of-the-art deep supervised learning algorithm, where the accuracy of the emission yield and localization of the objects was far superior to iterative reconstruction methods. Reconstruction of multiple objects is still reasonable with high localization accuracy, although with limits to the emission yield accuracy as the distribution becomes more complex. Overall though the reconstruction of Selfrec-Net provides a self-supervised way to recover the location and emission yield of molecular distributions in murine model tissues.

Review of Tissue Oxygenation Sensing During Radiotherapy Based Upon Cherenkov-Excited Luminescence Imaging

Review of Tissue Oxygenation Sensing During Radiotherapy Based Upon Cherenkov-Excited Luminescence Imaging

Implantable sensor for local Cherenkov-excited luminescence imaging of tumor pO2 during radiotherapy.

.Significance: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use.Aim: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue .Approach: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at concentration, allowing for local injection into tumors at the total dose of 10 nmol per animal, forming a gel at the site of injection. Cherenkov-excited luminescence imaging (CELI) was used to acquire the phosphorescence and provide intratumoral .Results: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral with the implantable gel sensors. The sensor’s performance was first evaluated in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy.Conclusions: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel–probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns.

Theoretical lateral and axial sensitivity limits and choices of molecular reporters for Cherenkov-excited luminescence in tissue during x-ray beam scanning.

.Purpose: Unlike fluorescence imaging utilizing an external excitation source, Cherenkov emissions and Cherenkov-excited luminescence occur within a medium when irradiated with high-energy x-rays. Methods to improve the understanding of the lateral spread and axial depth distribution of these emissions are needed as an initial step to improve the overall system resolution.Methods: Monte Carlo simulations were developed to investigate the lateral spread of thin sheets of high-energy sources and compared to experimental measurements of similar sources in water. Additional simulations of a multilayer skin model were used to investigate the limits of detection using both 6- and 18-MV x-ray sources with fluorescence excitation for inclusion depths up to 1 cm.Results: Simulations comparing the lateral spread of high-energy sources show approximately higher optical yield from electrons than photons, although electrons showed a larger penumbra in both the simulations and experimental measurements. Cherenkov excitation has a roughly inverse wavelength squared dependence in intensity but is largely redshifted in excitation through any distance of tissue. The calculated emission spectra in tissue were convolved with a database of luminescent compounds to produce a computational ranking of potential Cherenkov-excited luminescence molecular contrast agents.Conclusions: Models of thin x-ray and electron sources were compared with experimental measurements, showing similar trends in energy and source type. Surface detection of Cherenkov-excited luminescence appears to be limited by the mean free path of the luminescence emission, where for the given simulation only 2% of the inclusion emissions reached the surface from a depth of 7 mm in a multilayer tissue model.

High-Resolution pO2 Imaging Improves Quantification of the Hypoxic Fraction in Tumors During Radiation Therapy

The extreme microscopic heterogeneity of tumors makes it difficult to characterize tumor hypoxia. We evaluated how changes in the spatial resolution of oxygen imaging could alter measures of tumor hypoxia and their correlation to radiation therapy response. Cherenkov-Excited Luminescence Imaging in combination with an oxygen probe, Oxyphor PtG4 was used to directly image tumor pO2 distributions with 0.2 mm spatial resolution at the time of radiation delivery. These pO2 images were analyzed with variations of reduced spatial resolution from 0.2 mm to 5 mm, to investigate the influence of how reduced imaging spatial resolution would affect the observed tumor hypoxia. As an invivo validation test, mice bearing tumor xenografts were imaged for hypoxic fraction and median pO2 to examine the predictive link with tumor response to radiation therapy, while accounting for spatial resolution. In transitioning from voxel sizes of 200 μm to 3 mm, the median pO2 values increased by a few mm Hg, and the hypoxic fraction decreased by more than 50%. When looking at radiation-responsive tumors, the median pO2 values changed just a few mm Hg as a result of treatment, and the hypoxic fractions changed by as much as 50%. This latter change, however, could only be seen when sampling was performed with high spatial resolution. Median pO2 or similar quantities obtained from low resolution measurements are commonly used in clinical practice, however these parameters are much less sensitive to changes in the tumor microenvironment than the tumor hypoxic fraction obtained from high-resolution oxygen images. This study supports the hypothesis that for adequate measurements of the tumor response to radiation therapy, oxygen imaging with high spatial resolution is required to accurately characterize the hypoxic fraction.

Tracking tumor radiotherapy response in vivo with Cherenkov-excited luminescence ink imaging

This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2 mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. Ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R2 = 0.92) but not well with total tumor volume changes (R2 = 0.34). In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.

Cherenkov-excited luminescence scanned imaging using scanned beam differencing and iterative deconvolution in dynamic plan radiation delivery in a human breast phantom geometry.

The purpose of this study was to demonstrate high resolution optical luminescence sensing, referred to as Cherenkov excited luminescence scanning imaging (CELSI), could be achieved during a standard dynamic treatment plan for a whole breast radiotherapy geometry. The treatment plan beams induce Cherenkov light within tissue, and this excitation projects through the beam trajectory across the medium, inducing luminescence where there can be molecular reporter. Broad beams generally produce higher signal but low spatial resolution, yet for dynamic plans the scanning of the multileaf collimator allows for a beam-narrowing strategy by recursively temporal differencing each of the Cherenkov images and associated luminescence images. Then reconstruction from each of these size-reduced beamlets defined by the differenced Cherenkov images provides a well-conditioned matrix inversion, where the spatial frequencies are limited by the higher signal-to-noise ratio beamlets. A built-in stepwise convergence relies on stepwise beam size reduction, which is associated with a widening of the bandwidth of Cherenkov spatial frequency and resultant increase in spatial resolution. For the phantom experiments, europium nanoparticles were used as luminescent probes and embedded at depths ranging from 3 to 8 mm. An intensity modulated radiotherapy (IMRT) plan was used to test this. The Cherenkov images spatially guided where the luminescence was measured from, providing high lateral resolution, and iterative reconstruction convergence showed that optimization of the initial and stopping beamlet widths could be achieved with 15 and 4.5 mm, respectively, using a luminescence imaging frame rate of 5/s. With the IMRT breast plan, the original lateral resolution was improved 2X, that is, 0.08-0.24 mm for target depths of 3-8 mm. In comparison, a dynamic wedge (DW) plan showed an inferior image fidelity, with relative contrast recovery decreasing from 0.86 to 0.79. The methodology was applied to a three-dimensional dataset to reconstruct Cherenkov excited luminescence intensity distributions showing volumetric recovery of a 0.5 mm diameter object composed of 0.5 μM luminescent microbeads. High resolution CELSI was achieved with a clinical breast external beam radiotherapy (EBRT) plan. It is anticipated that this method can allow visualization and localization for luminescence/fluorescence tagged vasculature, lymph nodes, or superficial tagged regions with most dynamic treatment plans.

Tomographic Cherenkov-excited luminescence scanned imaging with multiple pinhole beams recovered via back-projection reconstruction.

Cherenkov-excited luminescence scanned imaging (CELSI) is achieved with a clinical linear accelerator during external beam radiotherapy to map out molecular luminescence intensity or lifetime in tissue. In order to realize a deeper imaging depth with a reasonable spatial resolution in CELSI, we optimized the original scanning procedure to complete this in a way similar to x-ray computed tomography and with image reconstruction from maximum-likelihood expectation maximization and multi-pinhole irradiation for parallelization. Resolution phantom studies showed that a 0.3mm diameter capillary tube containing 0.01nM luminescent nanospheres could be recognized at a depth of 21mm into tissue-like media. Small animal imaging with a 1mm diameter cylindrical target demonstrated that fast 3D data acquisition can be achieved by this multi-pinhole collimator approach to image high-resolution luminescence through a whole animal.

Multi-beam scan analysis with a clinical LINAC for high resolution Cherenkov-excited molecular luminescence imaging in tissue.

Cherenkov-excited luminescence scanned imaging (CELSI) is achieved with external beam radiotherapy to map out molecular luminescence intensity or lifetime in tissue. Just as in fluorescence microscopy, the choice of excitation geometry can affect the imaging time, spatial resolution and contrast recovered. In this study, the use of spatially patterned illumination was systematically studied comparing scan shapes, starting with line scan and block patterns and increasing from single beams to multiple parallel beams and then to clinically used treatment plans for radiation therapy. The image recovery was improved by a spatial-temporal modulation-demodulation method, which used the ability to capture simultaneous images of the excitation Cherenkov beam shape to deconvolve the CELSI images. Experimental studies used the multi-leaf collimator on a clinical linear accelerator (LINAC) to create the scanning patterns, and image resolution and contrast recovery were tested at different depths of tissue phantom material. As hypothesized, the smallest illumination squares achieved optimal resolution, but at the cost of lower signal and slower imaging time. Having larger excitation blocks provided superior signal but at the cost of increased radiation dose and lower resolution. Increasing the scan beams to multiple block patterns improved the performance in terms of image fidelity, lower radiation dose and faster acquisition. The spatial resolution was mostly dependent upon pixel area with an optimized side length near 38mm and a beam scan pitch of P = 0.33, and the achievable imaging depth was increased from 14mm to 18mm with sufficient resolving power for 1mm sized test objects. As a proof-of-concept, in-vivo tumor mouse imaging was performed to show 3D rendering and quantification of tissue pO2 with values of 5.6mmHg in a tumor and 77mmHg in normal tissue.

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× stronger than CEL signals. As a result, imaging signals from depths <15 mm is reasonable for Cherenkov light, and depths <3 mm 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.

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.

TH-AB-209-04: 3D Light Sheet Luminescence Imaging with Cherenkov Radiation

Purpose: To recover a three-dimensional density distribution of luminescent molecular probes located several centimeters deep within a highly scattering tissue. Methods: We developed a novel sheet beam Cherenkov-excited luminescence scanned imaging (CELSI) methodology. The sample was irradiated by a horizontally oriented, vertically scanned 6 MV X-ray sheet beam (200mm × 5mm, 0.2mm vertical step) from a radiotherapy linear accelerator. The resulting Cherenkov light emission – and thus luminescent probe excitation – occurred exclusively along the irradiation plane due to a short diffusion path of secondary particles and Cherenkov photons. Cherenkov-excited luminescence was detected orthogonally to the sheet beam by gated, intensified charge coupled device camera. Analogously to light sheet microscopy, a series of luminescence images was taken for varied axial positions (depths) of the Cherenkov light sheet in sample. Knowledge of the excitation plane position allowed a 3D image stack deconvolution and depth-variant attenuation correction. The 3D image post-processing yielded a true spatial density distribution of luminescent molecules in highly scattering tissue. Results: We recovered a three-dimensional shape and position of 400 µL lesion-mimicking phantom tubes containing 25 µM solution of PtG4 molecular probe from 3 centimeter deep tissue-like media. The high sensitivity of CELSI also allowed resolving 100 micron capillaries of test solution. Functional information of partial oxygen pressure at the site of PtG4 molecular probe was recovered from luminescence lifetime CELSI. Finally, in-vivo sheet beam CELSI localized milimeter-sized PtG4-labelled tumor phantoms in multiple biological objects (hairless mice) from single scan. Conclusion: Presented sheet beam CELSI technique greatly extended the useful depth range of luminescence molecular imaging. More importantly, the light sheet microscopy approach was successfully adapted to CELSI, providing means to recover a completely attenuation-corrected 3D image of luminescent probe distribution. Gated CELSI acquisition yielded functional information of a spatially resolved oxygen concentration map of deep lying targets. This work was supported by NIH research grant R01CA109558 and R21EB017559, as well as by Pilot Grant Funds from the Norris Cotton Cancer Center.

Comparison of Cherenkov excited fluorescence and phosphorescence molecular sensing from tissue with external beam irradiation

Ionizing radiation delivered by a medical linear accelerator (LINAC) generates Cherenkov emission within the treated tissue. A fraction of this light, in the 600–900 nm wavelength region, propagates through centimeters of tissue and can be used to excite optical probes in vivo, enabling molecular sensing of tissue analytes. The success of isolating the emission signal from this Cherenkov excitation background is dependent on key factors such as: (i) the Stokes shift of the probe spectra; (ii) the excited state lifetime; (iii) the probe concentration; (iv) the depth below the tissue surface; and (v) the radiation dose used. Previous studies have exclusively focused on imaging phosphorescent dyes, rather than fluorescent dyes. However there are only a few biologically important phosphorescent dyes and yet in comparison there are thousands of biologically relevant fluorescent dyes. So in this study the focus was a study of efficacy of Cherenkov-excited luminescence using fluorescent commercial near-infrared probes, IRDye 680RD, IRDye 700DX, and IRDye 800CW, and comparing them to the well characterized phosphorescent probe Oxyphor PtG4, an oxygen sensitive dye. Each probe was excited by Cherenkov light from a 6 MV external radiation beam, and measured in continuous wave or time-gated modes. The detection was performed by spectrally resolving the luminescence signals, and measuring them with spectrometer-based separation on an ICCD detector. The results demonstrate that IRDye 700DX and PtG4 allowed for the maximal signal to noise ratio. In the case of the phosphorescent probe, PtG4, with emission decays on the microsecond (μs) time scale, time-gated acquisition was possible, and it allowed for higher efficacy in terms of the probe concentration and detection depth. Phantoms containing the probe at 5 mm depth could be detected at concentrations down to the nanoMolar range, and at depths into the tissue simulating phantom near 3 cm. In vivo studies showed that 5 nmol of dye was readily detected with radiation doses less than 5 cGy. Since concentration, radiation dose and depth each contribute to the level of the detected signal, it may be possible to improve any of these parameters at expense of the others. This paradigm of nanoMolar sensitivity for optical reporters in vivo introduces the concept of molecular sensing of tumors during therapy or diagnostically with biologically relevant concentrations of fluorescent reporters.

TH-AB-204-01: Cherenkov- Excited Luminescence Scanned Imaging (CELSI) for High-Resolution, Deep-Tissue, in Vivo Optical Molecular Imaging with Limited Radiation Dose

Purpose: A new imaging modality Cherenkov-excited luminescence scanned imaging (CELSI) is demonstrated illustrating that LINAC beams induce Cherenkov emission within tissue, and that in-turn can excite luminescence of optical probes. This is achieved in a highly spatially confined fashion thereby allowing scanned imaging of distributions of luminescent sources. Methods: A 5 mm thick line-excitation megavoltage X-ray beam (6 MV) was collimated and moved by the multi-leaf collimator (MLC) of a linear accelerator (LINAC), to scan in 3 orthogonal directions. The scan in this demonstration was through a rat abdomen for lymph node imaging. The node was injected with oxygen-sensitive phosphor (Oxyphor PtG4), having strong luminescence lifetime, which is sensitive to the local oxygen concentration. During the scanning, Cherenkov-excited luminescence was continuously integrated by an intensified charge-coupled device (ICCD) synchronized to radiation pulses, and the position of the total signal was back-projected to the position of the scanning beam. Results: The lymph node was accurately located in the axillary region by CELSI imaging. Through lifetime measurement of the luminescence signal from within the lymph node, the pO2 was found to be reporting the true oxygenation level. Phantom studies validated that an inclusion with size below 1 mm could be reconstructed through a layer of 20 mm thick tissue equivalent phantom material. Concentrations of PtG4 down to 100 nM were detectable and capillary tubes containing the probe with diameters down to 200 µm could be resolved by CELSI, at 5 mm depth in tissue equivalent phantoms. Conclusion: CELSI is reported here for the first time, as an innovative optical molecular imaging technique by utilizing the LINAC as an optical scanning and excitation device. Potential applications of CELSI include reconstructions of interested optical probes and recoveries of local physiological information with relatively high spatial resolution.

Cherenkov-excited luminescence scanned imaging.

Ionizing radiation is commonly delivered by medical linear accelerators (LINAC) in the form of shaped beams, and it is able to induce Cherenkov emission in tissue. In fluorescence-based microscopy excitation from scanned spots, lines, or sheets can be used for fast high-resolution imaging. Here we introduce Cherenkov-excited luminescence scanned imaging (CELSI) as a new imaging methodology utilizing 2-dimensional (∼5-mm-thick) sheets of LINAC radiation to produce Cherenkov photons, which in turn excite luminescence of probes distributed in biological tissues. Imaging experiments were performed by scanning these excitation sheets in three orthogonal directions while recording Cherenkov-excited luminescence. Tissue phantom studies have shown that single luminescent inclusions ∼1 mm in diameter can be imaged within 20-mm-thick tissue-like media with minimal loss of spatial resolution. Using a phosphorescent probe for oxygen, PtG4 with the CELSI methodology, an image of partial pressure of oxygen (pO₂) was imaged in a rat lymph node, quantitatively restoring pO₂ values in differently oxygenated tissues.