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Glioblastoma is a highly aggressive and common brain cancer in adults with a grave prognosis, and aggressive radio and chemotherapy provide only a 15 months median survival. We evaluated the tolerability, and efficacy of the Ruthenium-based photosensitizer TLD-1433 in the formulation with apo-Transferrin (Rutheriin®) in the RG2, rat glioblastoma model. The specific tumour uptake ratio, PDT threshold, of the RG2 rat glioblastoma models and normal brain in vivo were determined as well as the survival post-PDT and the extent of CD8+T cell infiltration post-PDT. Results were compared with those obtained by 5-ALA-induced PpIX mediated PDT in the same animal model. As both photosensitizers have different photophysical properties, the number of absorbed photons required to achieve an equal cell kill is compared during in-vitro and in vivo studies. A significantly lower absorbed energy was enough to achieve LD50 with Rutherrin® versus -PpIX mediated PDT. Rutherrin® provides higher selective uptake ratio (SUR>20) in RG2 tumours compared to normal brain, whereas the SUR for ALA-induced PpIX was 10.6 in the same tumour model. To evaluate the short-term tissue response in vivo enhanced T2-weighted MR images provided the spatial extent of edema, which is twice post PpIX-PDT versus Rutherrin®-PDT suggesting reduced non-specific damage typically associated with a secondary wave of neuronal damage. A significant survival increase was observed in Rutherrin® treated rats bearing RG2 versus PpIX-PDT for the selected treatment conditions, associated with an increased CD8+T cell infiltration in the tumours. Rutherrin®-PDT was well tolerated providing safe and effective treatment of RG2 -induced glioblastoma.

While the prevalence of central bronchial tumours is declining, that of peripheral lung tumours is increasing. Peripheral lung tumours present either as individual index lesion or as field cancerization, requiring for the former targeting of particular confined volumes of lung tissue versus a therapy for an entire lung or particular lobes thereof. Using FullMonte, a Monte Carlo code; the ability to achieve a tumour selective PDT by transbronchial light source placement was simulated for 525, 665 and 808 nm wavelength. Simulations were executed utilizing in silica models with up to 10 generations of the bronchial tree, tissue photosensitizer concentrations taken from literature or measure in preclinical model systems and tissue optical properties measured with alive ex vivo pig and human lungs perfused with either blood or a transparent low cellular (STEEN) fluid. The measured effective attenuation coefficients [cm-1] at the three wavelengths for ventilated lungs with either blood 1.26±1.07, 1.93±0.534, 1.09±0.93 or STEEN fluid 1.01±0.873, 0.901±0.318, 0.641±0.31 used as perfusate. When modelling the PDT dose distribution in the lung’s the bronchial air ducts up to the eight generations perturb the fluence considerably. In all simulations, a dose sufficient to cause necrosis in 98% of the target volume placement of 3 source fibres albeit with various extent of normal lung tumour damage. Full coverage of an entire lung lobe with only three source fibres placed does not provide for effective coverage of the diffuse disease unless a very high selective uptake of the photosensitizer in malignant tissues can be achieved.

Even though a patient has a good immune system, tumors are shielded from it, because tumors grow by suppressing the host’s immune-response by various mechanisms. They are keeping their local microenvironment immune suppressed by producing immune suppressive cytokines like IL-10 and TGF-b, express immune checkpoint ligands like Programmed Death Ligand 1 (PDL1), and harbor immune suppressive cells like Tregs and MDSCs. To overcome these barriers a stronger anti-tumour immune-response is essential. We evaluated a whole cell vaccine with extracorporeal Rutherrin®-PDT treated cancer cells (RuVaCareTM) to break the suppressive barrier in the RG2-glioblastoma model. Rutherrin®-PDT induced strong immunogenic cell death (ICD) in glioblastoma cells in-vitro. RuVaCareTM supernatants showed significantly higher level of extracellular ATP, which is known to induce recruitment of antigen presenting cells (APCs) and their activation by eliciting an effective anti-tumour immune-response. Extracellular calreticulin (CRT) is one of the hallmarks of ICD; its expression went up in more than 85% cells undergoing Rutherrin®-PDT mediated cell death. There was a close to 10 times increase in expression of HSP 70 in RuVaCareTM. Immunostimulatory cytokines IFNa, IL-1b and GMCSF expression is high in the RuVaCareTM. In-vivo efficacy of the RuVaCare™ was evaluated in orthotopic RG2 rat glioblastoma model. There was a significant increase (~43% with 2-time vaccine and 87% in the 6-time vaccine) in survival in the RuVaCare™ vaccinated groups compared to unvaccinated controls. Increased intratumoral CD8+T-cell numbers are shown to be correlated with increased survival in glioblastoma rats, with RuVaCare™ there was a significant increase in the number of CD8+T-cells.

For patients failing standard Bacillus Calmette-Guerin based immunotherapy for Non-Muscle Invasive Bladder Cancer (NMIBC), PDT may delay or prevent cystectomy. A Phase IB clinical trial evaluated the feasibility and safety of TLD1433, a novel Ruthenium coordination-complex as photosensitizer (PS) for PDT. The clinical trial combined PS instillation for one hour and the use of strongly attenuated green (525nm) light to reduce PDT caused damage to the bladder wall. The low and high PS doses were defined as 0.35mg and 0.7mg TLD1433 per cm^2 bladder surface (N=3 each) and 90+/-9J/cm^2 as target radiant exposure on the bladder wall. The PS concentration in the urine and blood at 24hrs post instillation was below 1ng/ml indicating rapid drug clearing. In all patients, the average target radiant exposure was attained as verified by irradiance sensors in the bladder. The average measured irradiance was ~ 15mW/cm^2, never exceeding 35mW/cm2 at the sensor positions. At 30 days post-treatment, all patients receiving the low PS dose tolerated the procedure well with no grade 3, 4 or 5 AEs. Three patients were then treated at the Therapeutic Dose, again with no grade 3, 4 or 5 AEs, and an identical pharmacokinetic profile to the half dose. At half dose, all patients had recurrent, but no progressive NMIBC noted at the 180-day cystoscopy. At therapeutic dose, 2 of 3 patients were tumour-free at the 180-day cystoscopy. Moderate bladder irritability was reported at full dose which primarily resolved within 90 days.

Recurrent Non-Muscle Invasive Bladder Cancer (NMIBC) is a diffuse disease, and patients have failed standard BCG therapy face prophylactic cystectomy. PDT fell out of favour due to its variable outcome, and high morbidity. To overcome PDT associate toxicity to the bladder’s muscle layer, the use of shorter wavelength and instillation of the photosensitizer were suggested. While either approach was shown to improve the outcome in animal models they have not previously combined in human studies. Additionally, the effects of highly variable tissue optical properties of the bladder and its shape have not been studied. Here, we present surface dose histograms derived from light propagation simulation in 6 human bladders using CT images for anatomical detail and the FullMonte software package. The ability of a single light sensor versus 3 or 12 light sensors to measure the average irradiance on the bladder surface was evaluated as a function of the bladder wall’s tissue optical properties. Results show that the irradiance in non-spherical bladders can vary over an order of magnitude, but the irradiance histograms are affected little by displacement of the emitter inside the bladder void. As the surface area monitored by a single sensor depends strongly on the bladder shape, the responsivity of a single sensor to the average bladder irradiance can vary equally. Twelve light sensors monitor the entire bladder surface almost complete and hence their average responsivity is constant to the average irradiance on the bladder largely independent of shape. The dependency of the sensor’s response on the tissue optical properties is also lower.

The utility to perform treatment planning for transbronchial light delivery is investigated using Monte Carlo simulations. Optical properties of pig and human lungs were determined, and dose volume histograms determined. These dose volume histograms indicate for example the minimum photosensitizer specific uptake ratio required to achieve selective tumour destruction.

Determining the light propagation in heterogeneous media is a challenging task which can only be approximated by solving the Boltzmann transport equation via diffusion theory. However, diffusion theory becomes very inaccurate at interfaces, boundaries, sources, and sinks, which are present in heterogeneous media. Monte Carlo methods are able to converge to the correct solution by simulating a sufficiently high number of photons, at the cost of increased runtime. Therefore, it is important to optimize the Monte Carlo simulator, thereby allowing more photons to be simulated and a more accurate solution within a given runtime. FullMonte is a full-featured simulator that uses processor-optimized operations to achieve the highest performance of any 3D tetrahedral Monte Carlo light propagation software to date. This paper presents two medical use cases which benefit from FullMonte, highlights new features and explains the optimizations that lead to its high performance.

While Photofrin mediated PDT for bladder cancer was the first approved indication for this technique, it failed to attract the confidence of urologists as a treatment option, primarily due to the high incidence of incontinence linked to PDT damage to the bladder muscle. To mitigate this hazard a phase I clinical trial using instillation of the Ru(II) coordination complex TLD1433 and 530 nm activation light was initiated. To achieve the intended drug doses of 0.35 and 0.7 mg/cm2 and a radiant exposure of 90 J/cm2 the concentration of the instillation was adjusted to each patients' bladder volume and the irradiance was measured at up to 12 positions in the bladder. Irradiance monitoring proved helpful in adjusting the irradiation time to the bladder wall albedo and also for increased light scattering and absorption due to turbidity built up in the bladder void. The initial multiplication factors of the bladders (n=6) ranged from 1.1 to 2.8. Monte Carlo simulations based on CT-scans from all 6 participants approximate the range of irradiances observed during these studies. Nevertheless, a fraction of the surface can see a multiple of the average irradiance whereas other regions (typically less than 5% of the surface area) see significantly less than the average irradiance. These variations are due to the actual bladder shape and are somewhat independent of the position of the spherical emitter. Fitting of the measured surface irradiance to the simulated dose surface histograms enables extraction of the bladder wall and bladder void’s optical properties.

Disruption of cell respiration and metabolic changes accompanies many tissue disorders including neurodegenerative diseases and cancers. Fluorescence lifetime imaging microscopy (FLIM), a non-invasive and information-rich technique, can be invoked to reveal these changes. The method allows for monitoring of fluorescent intrinsic metabolic coenzymes, first of all, NADH (nicotinamide adenine dinucleotide), on the level of single cells and can be applied to living tissue. The ratio between protein-bound and free NADH gives information about the balance between oxidative phosphorylation and glycolysis in cells. There is a correlation between cellular metabolic activity, redox ratio and fluorescence lifetime of NADH. In combination with laser scanning microscopy, the time-correlated single photon counting (TCSPC) technology enables FLIM NADH mapping of the biomedical samples. On the other hand, TCSPC provides quantification of the phosphorescence lifetime of oxygen-sensing molecules. Accordingly, the oxygendependent quenching of phosphorescence of compounds such as transition metal complexes can be employed for evaluation of oxygen partial pressure (pO2) by PLIM (phosphorescence lifetime imaging microscopy). We demonstrate correlated FLIM/PLIM imaging, which provides simultaneous mapping of NADH and oxygen in living cells. Continuous FLIM/PLIM imaging enables to monitor changes in oxygen levels and cell metabolic status dynamically during PDT and provides new opportunities in theranostics.

Treatment planning is of utmost importance in interstitial photodynamic therapy, as it predicts the required light delivery to the target volume in an upcoming treatment. However, planning remains a major challenge due to several uncertainties such as the tissue optical properties and the concentrations of the photosensitizer and oxygen. Any difference in these parameters from the assumed values during planning could significantly affect the outcome of the actual treatment. This work introduces PDT-SPACE, a PDT light source power allocation using a convex optimization engine to minimize damage to organs-at-risk (OAR) with robustness against variation in tissue optical properties. Three power allocation methods are proposed and compared with respect to the resulting standard deviation in the damage to organs-at-risk and their runtime. The proposed approaches are demonstrated for ALA induced PpIX as photosensitizer in a virtual brain tumor that models a glioblastoma multiforme case. Results show that choosing a power allocation to minimize the OAR damage standard deviation under optical property variation tends to also minimize the tumor coverage as there is only one degree of freedom to optimize upon. This motivates simultaneous source position and power allocation optimization.

The majority of denovo cancers are today being diagnosed in low and middle-income countries, which often lack resources and a range of therapeutic options. Minimally invasive therapies such as Photodynamic Therapy (PDT) and photobiomodulation (PBM) could become treatment options, albeit widespread acceptance is hindered by multiple factors ranging from training of surgeons in optical therapeutic techniques, lack of easily usable treatment optimizing tools and prediction of the anticipated treatment outcome. Based on the publicly available FullMonte software in combination with other open source image processing tools, a work plan is proposed that allows for personalized treatment planning. Starting with, generating 3D in silico models, execution of the Monte Carlo simulation and presentation of the 3D fluence rate distribution a treatment procedure is presented. Calculation of the forward solution of photon transport in biological tissues is executed in less than a minute for 3D models comprising 106 tetrahedral elements. The ability of the program to find optimal source placements was demonstrated for in silico brain tumour models for solid tumours. In hollow organs the impact of non-isotropic cavities is demonstrated on bladder cancer patient data. For photodynamic therapy treatment optimization, the process considers the selective uptake ratio of the photosensitizer between the target, host tissues and organs at risk and establish PDT sensitivities of these tissues based on the photodynamic threshold values. Tumours are assigned only a minimum required dose, whereas host and organs at risk a maximum permissible dose. For PBM the target and the host tissue are assigned minimum and maximum permissible dose due to the well documented biphasic response effect in PBM. For PDT sources of errors are uncertainties in the contouring whereas for PBM the depth of the actual target in the tissue is unknown and need often to be estimated based on body mass Index, and other morphometric parameters. Both photo therapeutic applications suffer from unknown tissue optical properties. Hence, the proposed workflow includes a perturbation of the planning tissue optical properties, uncertainties in the photon source placement and contouring errors, to validate the invariance of the attained solution against these unknowns. This requires also the need to determine the patients actual tissue optical properties at the onset of therapy, which in turn can only be achieved when the appropriate placement of invasive or diffuse reflective sensors is provided for. Hence, the planning process needs to include also identification of the most responsive positions for these sensors in the planning volume.

Non-muscle invasive bladder cancer remains one of the costliest cancer to treat, and while a cystectomy will reduce a patient’s risk of developing metastatic disease it reduced the patient’s quality of life. While Photofrin mediate Photodynamic Therapy was approved already in 1993, poor control over the photon density and drug accumulation in of target tissue resulted in overdosing the bladder muscle layers, causing permanent volume shrinkage and incontinence. For an ongoing Phase Ib clinical trial, evaluating the safety of TLD1433 ([Ru(II)(4,4'-dimethyl-2,2'-bipyridine(dmb))2(2-(2',2'':5'',2'''-terthiophene)-imidazo[4,5-f][1,10]phenanthroline)]2+) a Ru(II) coordination complex, significant deviations from the previous studies are implemented. The photosensitizer is instilled, to reduce the sensitization of the muscle layer, 525 nm light is used to limit the light penetration into the bladder and the photon density is measured in each patient at up to 12 positions. Improved tumour selectivity is provided by this photosensitizer as it is block by the Urothelium from entering healthy tissue, whereas it enters tumour cells, supposing via the transferrin receptor, as demonstrated in in-vitro and in vivo studies. The Ru(II) coordination complex stains tissues where the urothelium is damaged very strongly in an orange-rust colour, visible under white light illumination. Preclinical in vivo studies showed the destruction of tumours up to 1 mm in depth following 1 hr of drug instillation, followed by 3 washes and the delivery of 90 Jcm-2 of 525nm light in the wistar rat Ay-27 tumour model. Histology showed very limited muscle damage and in general intact urothelium layers, with a moderate infiltration of macrophages. To achieve the prescribed target radiant exposure of 90 Jcm-2, independent of the bladder tissue diffuse reflectivity and shape, an optical dosimetry system was developed which can be deployed via a cystoscope. The optical dose monitoring device allows the treating physician to adjust the source position to achieve the target optical dose for the 12 sensor positions. The optical radiation was delivered via a 0.8 mm diameter spherical diffuser at up to 2.5 W power. The attainable photon density and the anticipated PDT dose are simulated for each patient using a photon propagation engine and the patient’s anatomical information. During these simulations, a range of tissue optical properties is simulated and compared to the initial photon density measurements to advise the physician further about the ability to achieve a homogenous illumination in each patient. The multiplication factor of the irradiance inside the bladder, calculated based on the spherical volume equivalent size of the bladder, the delivered power and measured irradiance, varied between patients (from 1.1 to 2.5) and also over the course of the treatment. The changes over the course of the treatment were predominantly due to light diffusing proteinaceous material floating in the bladder. Expanding prior work by the Rotterdam group on light propagation in the bladder we determined that the tissue albedo varied from 0.87 to 0.92 for 525 nm in this patient population. The estimated average effective attenuation coefficient for this population was approximately 1 mm adequate for the treatment of non-muscle invasive bladder cancer after transurethral resection of the large tumours. Monte Carlo modelling demonstrated that the fluence as function of depth into the tissue is determined by the exposure of bladder wall elements to the remaining bladder surface and can vary by factor of more than 2 even when assuming homogenous tissue optical properties across the bladder wall surface. These studies and analysis demonstrate the need for accurate light dosimetry in hollow organs when variations in the tissue albedo and the shape of the organ can influence the photodynamic dose significantly.

Background: The majority of cancers upregulate their transferrin receptor (Tf-R) to satisfy their higher Fe3+ requirements for proliferation. TLD-1433 can bind to transferrin to form Rutherrin, which is a promising photosensitizer with stable chemical structure and higher tissue selectivity. Methods: To investigate the effect of Rutherrin®-mediated photodynamic treatment (PDT), we used non-small lung cancer cell lines H2170, A549, and H460. Subcutaneous tumors were treated with Rutherrin-mediated PDT, 4hrs post intravenous administration. The treatment parameters10 mg/kg Rutherrin and 600 Jcm-2 808 nm radiation. In an orthotopic A549 tumor model, the presence of tumor after inoculation in lungs was confirmed by microCT. Tissue samples were collected for Inductively Coupled Mass Spectrometry to quantify the Rutherrin concentrations via a Ru isotope in tumor and normal lung tissue. Results: Evaluation of TfR expression by flow cytometric and western blotting showed that almost all cancer cells express TfR. In in-vitro cytotoxicity assay, all cancer cell lines showed high cell kill by PDT at 100nM Rutherrin concentrations. In the subcutaneous tumor model, PDT after Rutherrin injection significantly inhibited the tumor growth and histopathology showed extensive necrosis at 24 hrs, which was confirmed with lowered Ki67 staining. In an orthotopic model, the lung lobe with tumor retained more Rutherrin than the contralateral lung, showing specific tumor uptake. Conclusion: These results support the hypothesis that safe and efficient Rutherrin-mediated PDT is feasible due to improved photosensitizer localization to lung tumors tissue. Selective irradiation of the cancer lesions by strategic placement of the light source remains a requirement.

A common property during tumor development is altered energy metabolism, which could lead to a switch from oxidative phosphorylation and glycolysis. The impact of this switch for theranostic applications could be significant. Interestingly altered metabolism could be correlated with a change in the fluorescence lifetimes of both NAD(P)H and FAD. However, as observed in a variety of investigations, the situation is complex and the result is influenced by parameters like oxidative stress, pH or viscosity. Besides metabolism, oxygen levels and consumption has to be taken into account in order to understand treatment responses. For this, correlated imaging of phosphorescence and fluorescence lifetime parameters has been investigated by us and used to observe metabolic markers simultaneously with oxygen concentrations. The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.

Ultrafast `pulsetrain-burst' machining has proven to deliver qualitative advantages for the processing of hard materials. This approach to tailoring the delivery of radiant exposure uses microsecond bursts of picosecond or femtosecond pulses. The mixed-timescale mode reaps the rewards of providing laser-material interactions at ultrashort timescales, while allowing for some control of heat and stress dissipation over longer timescales. As a result, our group has been able to ablate hard materials such as aluminum [1], as well as optically transparent materials like fused silica [2,3] beyond the capabilities of more conventional methods that employ slower repetition rates. It is well known that gross differences in the end results of materials-processing are the result of the manner in which the laser fluence is delivered - wavelength, continuous-wave vs. pulsed, pulse shape, and the pulse duration itself all have significant impact, and each parameter-value has significant advantages. For brittle materials, where limited tolerance to heat and tensile stress sets limits on the etch-depth possible for multi-kHz repetition rates, 'pulsetrain-burst' machining has enabled us to drill deeper and more cleanly, while eliminating damage due to thermal cycling and over-pressure shocks.

Background: The majority of de novo cancers are diagnosed in low and middle-income countries, which often lack the resources to provide adequate therapeutic options. None or minimally invasive therapies such as Photodynamic Therapy (PDT) or photothermal therapies could become part of the overall treatment options in these countries. However, widespread acceptance is hindered by the current empirical training of surgeons in these optical techniques and a lack of easily usable treatment optimizing tools. Methods: Based on image processing programs, ITK-SNAP, and the publicly available FullMonte light propagation software, a work plan is proposed that allows for personalized PDT treatment planning. Starting with, contoured clinical CT or MRI images, the generation of 3D tetrahedral models in silico, execution of the Monte Carlo simulation and presentation of the 3D fluence rate, Φ, [mWcm -2 ] distribution a treatment plan optimizing photon source placement is developed. Results: Permitting 1-2 days for the installation of the required programs, novices can generate their first fluence, H [Jcm -2 ] or Φ distribution in a matter of hours. This is reduced to 10th of minutes with some training. Executing the photon simulation calculations is rapid and not the performance limiting process. Largest sources of errors are uncertainties in the contouring and unknown tissue optical properties. Conclusions: The presented FullMonte simulation is the fastest tetrahedral based photon propagation program and provides the basis for PDT treatment planning processes, enabling a faster proliferation of low cost, minimal invasive personalized cancer therapies.

Background: In excess of 60% of all cancers are detected in low and middle-income countries, with breast cancer (BC) the dominant malignancy for women. Incidence rates continue to climb, most noticeably in the less than 50-year-old population. Expansion of mammography infrastructure and resources is lacking, resulting in over 60% of women diagnosed with stage III/IV BC in the majority of these countries. Optical Breast Spectroscopy (OBS) was shown to correlate well with mammographic breast density (MBD). OBS could aid breast screening programs in low- and middle-income countries by lowering the number of mammographs required for complete population coverage. However, its performance needs to be tested in large population trails to ensure high sensitivity and acceptable specificity. Methods: For the planned studies in low- and middle-income countries in different continents, online methods need to be implemented to monitor the performance and data collection by these devices, operated by trained nurses. Based on existing datasets, procedures were developed to validate an individual woman's data integrity and to identify operator errors versus system malfunctions. Results: Using a dataset comprising spectra from 360 women collected by 2 instruments in different locations and with 3 different trained operators, automated methods were developed to identify 100% of the source or photodetector malfunctions as well as incorrect calibrations and 96% of instances of insufficient tissue contact. Conclusions: Implementing the dataset validation locally in each instrument and tethered to a cloud database will allow the planned clinical trials to proceed.

Brain tumors are characterized with morphological changes at cellular level such as enlarged, non-spherical nuclei, microcalcifications, cysts, etc., and are highly vascularized. In this study, two research-grade optical coherence tomography (OCT) systems operating at ~800 nm and ~1060 nm with axial resolution of 0.95 µm and 3.5 µm in biological tissue respectively, were used to image in vivo and ex vivo the structure of brain tumours in rats. Female Fischer 344 rats were used for this study, which has received ethics clearance by the Animal Research Ethics Committees of the University of Waterloo and the University Health Network, Toronto. Brain tumours were induced by injection of rat brain cancer cell line (RG2 glioma) through a small craniotomy. Presence of brain tumours was verified by MRI imaging on day 7 post tumour cells injection. The in vivo OCT imaging session was conducted on day 14 of the study with the 1060 nm OCT system and both morphological OCT, Doppler OCT and OMAG images were acquired from the brain tumour and the surrounding healthy brain tissue. After completion of the imaging procedure, the brains were harvested, fixed in formalin and reimaged after 2 weeks with the 800 nm OCT system. The in vivo and ex vivo OCT morphological images were correlated with H and E histology. Results from this study demonstrate that UHR-OCT can distinguish between healthy and cancerous brain tissue based on differences in structural and vascular pattern.

A portable, spatially resolved diffuse reflectance (SRDR) lensless imaging technique based on the charge coupled device (CCD), or complementary metal-oxide semiconductor (CMOS) sensor directly coupled with fiber optic bundle can be proposed for visualization of subsurface structures such as intrapapillary capillary loops (IPCLs). In this article, we discuss an experimental method for emulating a lensless imaging setup via raster scanning a single fiberoptic cable (where image is relayed onto the sensor surface through a fiber-optic cable equivalent to coupling a fiber optic conduit directly onto the sensor surface without any lenses) over a microfluidic phantom containing periodic hemoglobin absorption contrast. For mimicking scattering properties of turbid media, a diffusive layer formed of polydimethylsiloxane (PDMS) and titanium dioxide (TiO2) was placed atop of the microfluidic phantom. Thickness of the layers ranged from 0.2‒0.7mm, and the µs value of the layers were in the range of 0.85 mm-1 ‒ 4.25mm-1. The results demonstrate that a fiber-optic bundle/plate coupled lensless imaging setup has a high potential to recover intensity modulations from the subsurface patterns. Decreasing of the interrogation volumes leads to enhanced spatial resolution of diffuse reflectance imaging, and hence, can potentially overcome the scattering caused blurring.

Photodynamic therapy (PDT) delivers a localized cytotoxic dose that is a function of tissue oxygen availability, photosensitive drug concentration, and light fluence. Providing safe and effective PDT requires an understanding of all three elements and the physiological response to the radicals generated. Interstitial PDT (IPDT) for solid tumours poses particular challenges due to complex organ geometries and the associated limitations for diffusion theory based fluence rate prediction, in addition to restricted access for light delivery and dose monitoring. As a first step towards enabling a complete prospective IPDT treatment-planning platform, we demonstrate use of our previously developed FullMonte tetrahedral Monte Carlo simulation engine for modeling of the interstitial fluence field due to intravesicular insertion of brief light sources. The goal is to enable a complete treatment planning and monitoring work flow analogous to that used in ionizing radiation therapy, including plan evaluation through dose-volume histograms and algorithmic treatment plan optimization. FullMonte is to our knowledge the fastest open-source tetrahedral MC light propagation software. Using custom hardware acceleration, we achieve 4x faster computing with 67x better power efficiency for limited-size meshes compared to the software. Ongoing work will improve the performance advantage to 16x with unlimited mesh size, enabling algorithmic plan optimization in reasonable time. Using FullMonte, we demonstrate significant new plan-evaluation capabilities including fluence field visualization, generation of organ dose-volume histograms, and rendering of isofluence surfaces for a representative bladder cancer mesh from a real patient. We also discuss the advantages of MC simulations for dose-volume histogram generation and the need for online personalized fluence-rate monitoring.

Emerging clinical applications including bioluminescence imaging require fast and accurate modelling of light propagation through turbid media with complex geometries. Monte Carlo simulations are widely recognized as the standard for high-quality modelling of light propagation in turbid media, albeit with high computational requirements. We present FullMonte: a flexible, extensible software framework for Monte Carlo modelling of light transport from extended sources through general 3D turbid media including anisotropic scattering and refractive index changes. The problem geometry is expressed using a tetrahedral mesh, giving accurate surface normals and avoiding artifacts introduced by voxel approaches. The software uses multithreading, Intel SSE vector instructions, and optimized data structures. It incorporates novel hardware-friendly performance optimizations that are also useful for software implementations. Results and performance are compared against existing implementations. We present a discussion of current state-of-the-art algorithms and accelerated implementations of the modelling problem. A new parameter permitting accuracy-performance tradeoffs is also shown which has significant implications including performance gains of over 25% for real applications. The advantages and limitations of both CPU and GPU implementations are discussed, with observations important to future advances. We also point the way towards custom hardware implementations with potentially large gains in performance and energy efficiency.

Up regulation of iNOS gene expression is playing a role in the initiation of the anti-inflammatory and tissue protective mechanisms related to nitric oxide (NO) for continuous wave red and infrared as well as 905nm superpulsed laser therapy (SPLT). The iNOS expression before and after laser therapy was evaluated in a zymosan-induced acute arthritis model, in knee joints of young ( 15 weeks and 35 weeks) FVB/N-Tg (iNOS-luc) mice by bioluminescence imaging.

Low Level Light Therapy (LLLT) has been implicated to stimulate tissue, promoting healing and reducing pain. One of the potential pathways stimulated by LLLT relates to the electron transport chain, where photon quantum energy can induce a change in the biochemical reactions within the cell. The aim of this study is to assess the feasibility to exploit light additionally as a diagnostic tool to determine tissue physiological states, particularly in quantifying the changes in redox states of Cytochrome C as a result of induced LLLT biochemical reactions.

Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.

This paper presents a basic computational framework for real-time, 3-D light dosimetry on graphics processing unit (GPU) clusters. The GPU-based approach offers a direct solution to overcome the long computation time preventing Monte Carlo simulations from being used in complex optimization problems such as treatment planning, particularly if simulated annealing is employed as the optimization algorithm. The current multi- GPU implementation is validated using a commercial light modelling software (ASAP from Breault Research Organization). It also supports the latest Fermi GPU architecture and features an interactive 3-D visualization interface. The software is available for download at http://code.google.com/p/gpu3d.

Fluorescent multi-sensor fiber-optic probes for spatially resolved monitoring of Interstitial Photodynamic Therapy were absolute calibrated using an integrating cylinder. The dynamic response was evaluated and showed linear responsivity in the test range from 1 – 60 mW·cm−2. Sensor-probe and optoelectronic readout provides a accuracy of better than 10%.

An integrating cylinder composed of high-density polyurethane with a measured multiplication factor (M) of 38 has been developed for absolute calibration of fluence rate probes designed for monitoring of photodynamic therapy (PDT) and other light-based therapies.

Recent improvements in the computing power and programmability of graphics processing units (GPUs) have enabled the possibility of using GPUs for the acceleration of scientific applications, including time-consuming simulations in biomedical optics. This paper describes the acceleration of a standard code for the Monte Carlo (MC) simulation of photons on GPUs. A faster means for performing MC simulations would enable the use of MC-based models for light dose computation in iterative optimization problems such as PDT treatment planning. We describe the computation and how it is mapped onto the many parallel computational units now available on the NVIDIA GTX 200 series GPUs. For a 5 layer skin model simulation, a speedup of 277x was achieved on a single GTX280 GPU over the code executed on an Intel Xeon 5160 processor using 1 CPU core. This approach can be scaled by employing multiple GPUs in a single computer — a 1052x speedup was obtained using 4 GPUs for the same simulation.

Photodynamic Therapy (PDT) is a minimally invasive treatment that uses a photosensitive drug into convert triplet state oxygen ( 3 O2) to singlet oxygen ( 1 O2) to destroy malignant tissue. A fiber-optic system based on frequency domain detection of phosphorescence quenching by 3 O2 is described which optically measures the distribution of 3 O2 in the treatment volume during PDT to permit adjustments of treatment parameters to improve outcome. A specially designed fiber optic probe containing phosphorescent sensors embedded along its length permit spatially resolved measurements. Each sensor is composed of a phosphorescent metalloporphyrin compound that emits a characteristic spectrum. Four candidate sensors with high absorption at the excitation wavelength of 405nm and emission in the 650nm to 700nm region are considered. The dependence of phosphorescence lifetime (τ) on 3 O2 concentration is described by the linearized Stern-Volmer relationship as being inversely proportional. Determination of τ, and hence 3 O2 concentration, is accomplished in the frequency domain by means of phase-modulation detection of the phosphorescence signal due to an amplitude modulated excitation. The τ’s of each sensor are recovered by performing global non-linear least squares fit on the measured phase and modulation index over a range of frequencies and wavelengths. With the τ of each sensor known, the oxygen concentration at each sensor’s location can be determined with the Stern-Volmer relationship.

Colonoscopy is the gold standard for screening for inflammatory bowel disease and colorectal cancer. Flexible endoscopes are difficult to manipulate, especially in the distensible and tortuous colon, sometimes leading to disorientation during the procedure and missed diagnosis of lesions. Our goal is to design a navigational aid to guide colonoscopies, presenting a three dimensional representation of the endoscope in real-time. Therefore, a flexible sensor that can track the position and shape of the entire length of the endoscope is needed. We describe a novel shape-tracking technology utilizing a single modified optical fiber. By embedding fluorophores in the buffer of the fiber, we demonstrated a relationship between fluorescence intensity and fiber curvature. As much as a 40% increase in fluorescence intensity was achieved when the fiber’s local be nd radius decreased from 58 mm to 11 mm. This approach allows for the construction of a three-dimensional shape tracker that is small enough to be easily inserted into the biopsy channel of current endoscopes. Keywords: Colonoscopy, shape-tracking, optical fiber, fluorophores, navigation

Interstitial Photodynamic therapy (IPDT) has been under intense investigation in recent years, with multiple clinical trials underway. This effort has demanded the development of optimization strategies that determine the best locations and output powers for light sources (cylindrical or point diffusers) to achieve an optimal light delivery. Furthermore, we have recently introduced cylindrical diffusers with customizable emission profiles, placing additional requirements on the optimization algorithms, particularly in terms of the stability of the inverse problem. Here, we present a general class of linear feasibility algorithms and their properties. Moreover, we compare two particular instances of these algorithms, which are been used in the context of IPDT: the Cimmino algorithm and a weighted gradient descent (WGD) algorithm. The algorithms were compared in terms of their convergence properties, the cost function they minimize in the infeasible case, their ability to regularize the inverse problem, and the resulting optimal light dose distributions. Our results show that the WGD algorithm overall performs slightly better than the Cimmino algorithm and that it converges to a minimizer of a clinically relevant cost function in the infeasible case. Interestingly however, treatment plans resulting from either algorithms were very similar in terms of the resulting fluence maps and dose volume histograms, once the diffuser powers adjusted to achieve equal prostate coverage.

Multi-sensory fiber-optic probes using nanocrystals as the sensing materials are shown to overcome most limitations imposed by those using organic dyes as the fluence-rate sensing materials. These nanocrystals are shown to be excitable by a wide range of wavelengths covering the entire tissue transparent window utilized by various light-enabled treatment modalities. The optical response of the sensors made from the nanocrystals is shown to be linear without any sign of photobleaching and sensor crosstalk over a wide range of irradiance and fluence. Fiber probes using the nanocrystals are promising in offering high spatial resolution in fluence-rate dosimetry for photodynamic therapy and photothermal treatment.

Tailored light diffusers offer the flexibility of shaping the delivered light dose (fluence rate) distribution, potentially leading to conformal light delivery. Because of scattering and absorption, tissue acts as a spatial low pass filter of the diffuser's emission profile, and therefore some dose distributions with high spatial frequencies cannot be delivered. We characterize the set of attainable light dose distributions in terms of the spatial frequency of the emission profile and identify regimes where such distributions are less sensitive to changes in optical properties. Furthermore, we contrast two different algorithms to solve the inverse problem: Simulated Annealing (SA) and Non-negative Least Squares (NNLS). SA is plagued by superimposed high frequency components that do not contribute significantly to the cost. We present an iterative low pass filter that smooths the emission profile without considerably increasing the cost. A non-negative least square (NNLS) algorithm is also tested. We conclude that non-negative least squares (NNLS) is superior to simulated annealing (SA) in terms of time performance and cost minimization.

While Low-level Light Therapy (LLLT) has demonstrated efficacy for certain indications, some aspects of the technology are still controversial. Clinical studies on LLLT range from low quality anecdotal studies to blinded, randomized, control clinical studies. These have used a variety of wavelengths, optical powers and variations in other laser parameters. While these studies show a large range in treatment outcome, comparison of treatment efficacy between these studies with respect to light dose is all but impossible since the light dose characterization in the LLLT field has not been properly defined and is not standardized. Surface irradiance is typically used in the LLLT field as the light dose parameter, ignoring factors such as tissue optical properties, beam divergence, pulsing of the source and tissue thickness to the organ or joint of interest. Drawing on experience with light dosimetry for photodynamic and photothermal therapy, we will provide an overview of light transport and dosimetry in tissue and its implications for LLLT dosimetry. In particular, we suggest that the proper measure of dose is the light fluence rate delivered to the organ or tissue of interest, usually several millimeters below the tissue surface. We have developed a technique that provides an estimate of the subsurface fluence rate based on the diffuse reflectance measured at the tissue surface. Using Monte Carlo simulations and measurements on tissue simulating phantoms, we demonstrate that this technique can be used to predict the subsurface fluence rate to within 30% of the actual value at 3-10 mm below the tissue surface.

Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize fundamental processes related to cancer development and response at cellular and molecular levels. This technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9Lluc cells). Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at administered pO2 levels below about 5%, falling by approximately 50% at 0.2% pO2. Additional experiments showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic period reducing the ATP concentration to limiting levels for BLI.

Energy absorption and heat transfer are important factors for regulating the effects of ablation of biological tissues. Heat transfer to surrounding material may be desirable when ablating hard tissue, such as teeth or bone, since melting can produce helpful material modifications. However, when ablating soft tissue it is important to minimize heat transfer to avoid damage to healthy tissue - for example, in eye refractive surgery (e.g., Lasik), nanosecond pulses produce gross absorption and heating in tissue, leading to shockwaves, which kill and thin the non-replicating epithelial cells on the inside of the cornea; ultrafast pulses are recognized to reduce this effect. Using a laser system that delivers 1ps pulses in 10μs pulsetrains at 133MHz we have studied a range of heat- and energy-transfer effects on hard and soft tissue. We describe the ablation of tooth dentin and enamel under various conditions to determine the ablation rate and chemical changes that occur. Furthermore, we characterize the impact of pulsetrain-burst treatment of collagen-based tissue to determine more efficient methods of energy transfer to soft tissues. By studying the optical science of laser tissue interaction we hope to be able to make qualitative improvements to medical treatments using lasers.

Purpose: To determine the effect of low intensity laser therapy (LILT) on healing of infected skin wounds in the rat. Methods: Wounds on the dorsum of Sprague-Dawley rats (14 per group) were inoculated or sham-inoculated with P. aeruginosa . Wounds were irradiated or sham-irradiated three times weekly from Day 1-19 using 635nm or 808nm diode lasers at radiant exposure of 1 or 20 J/cm 2 delivered in continuous wave (CW) or at an intensity modulation frequency of 3800Hz. Wound area and bacterial growth were evaluated three times weekly. Results: CW 808 nm (1 and 20 J/cm 2 ) irradiation generally delayed healing in acute wounds. However, from Day 10 onwards CW 808 nm (1 J/cm 2 and 20 J/cm 2 ) and 808 nm 3800 Hz (1 J/cm 2 ) irradiation improved healing in inoculated wounds. Healing in acute wounds improved using 635 nm irradiation at low radiant exposure (1 J/cm 2 ); however, using 635 nm irradiation at high radiant exposure (20 J/cm 2 ) delayed healing. Bacterial balance in wounds was significantly altered using 635 nm (20 J/cm 2 ) and CW 808 nm irradiation (1 and 20 J/cm 2 ). Conclusion: Clearing wounds of normal flora was not associated with improved healing. Proliferation of staphylococcal species in wounds was associated with delayed healing.

This study investigates the efficacy of low level laser therapy (LLLT) in modulating inducible nitric oxide synthase (iNOS) expression as molecular marker of the inflammation signaling pathway. LLLT was mediated by different therapeutic wavelengths using transgenic animals with the luciferase gene under control of the iNOS gene expression. Inflammation in 30 transgenic mice (iNOS-luc mice, from FVB strain) was induced by intra-articular injection of Zymosan-A in both knee joints. Four experimental groups were treated with one of four different wavelengths (λ=635, 785, 808 and 905nm) and one not laser-irradiated control group. Laser treatment (25 mW cm -2 , 5 J cm -2 ) was applied to the knees 15 minutes after inflammation induction. Measurements of iNOS expression were performed at multiple times (0, 3, 5, 7, 9 and 24h) post-LLLT by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The responsivity of BLI was sufficient to demonstrate a significant increase in bioluminescence signals after laser irradiation of 635nm when compared to non-irradiated animals and the other LLLT treated groups, showing the wavelength-dependence of LLLT on iNOS expression during the acute inflammatory process.

Enhanced green fluorescent protein (EGFP)-expressing cells are customarily used in a variety of in vitro and in vivo studies and assays to ease visualization and localization. Nonetheless, the effects of EGFP expression on cellular responsivity to Photodynamic therapy (PDT), a combination th erapy combining a photoactive drug and light, have yet to be characterized. To address this effect, rat astrocytoma cells (CNS-1), a lentivirus-transduced EGFP variant (CNS-1 GFP), human glioblastoma (U-87), and the transfected EGFP variant (U-87 GFP) are analyzed in terms of cell survival following PDT mediated by two different photoactive drugs. Cell survival is quantified via colony forming assays and Alamar blue assays, as a function of light dose, using the photosensitizers Photofrin (1ug ml -1 for 24h) and ALA (200ug ml for 5h). Furthermore, effect of GFP expression on the responsivity to Cisplatin, a DNA-binding chemotherapeutic agent is determined for these cell lines. Our results show that EGFP ex pression does not affect the responsivity of Photofrin-PDT in comparison to parental cell lines (non GFP expressing cells), but does alter that of ALA-PDT. No change in responsivity is observed for Cisplatin treatment for either cell line. These results can be explained by oxidative stress induced by EGFP expression. This work will establish under which circumstances it is appropriate to use EGFP-expressing cell lines in the context of PDT preclinical research in vivo and in vitro . Keywords: Photodynamic therap y, Green Fluorescent Protein, transduction, transfection.

A technique to produce fluorescent cell phantom standards based on calcium alginate microspheres with encapsulated fluorescein-labeled dextrans is presented. An electrostatic ionotropic gelation method is used to create the microspheres which are then exposed to an encapsulation method using poly-l-lysine to trap the dextrans inside. Both procedures were examined in detail to find the optimal parameters producing cell phantoms meeting our requirements. Size distributions favoring 10-20 microns microspheres were obtained by varying the high voltage and needle size parameters. Typical size distributions of the samples were centered at 150 μm diameter. Neither the molecular weight nor the charge of the dextrans had a significant effect on their retention in the microspheres, though anionic dextrans were chosen to help in future capillary electrophoresis work. Increasing the exposure time of the microspheres to the poly-l-lysine solution decreased the leakage rates of fluorescein-labeled dextrans.

Photodynamic therapy (PDT) as a non-radiative treatment has been applied successfully in various cancers. PDT may be a useful adjunct in the treatment of vertebral metastases. PDT efficacy requires the administration of a photosensitiser drug followed by subsequent drug activation by wavelength specific light. The study purpose was to establish the pharmacokinetic profiles for 2 photosensitisers, BPD-MA and 5-ALA induced PpIX, to determine the optimal drug-light interval for vertebral PDT.

Tissue undergoing transformation into a state that is more favourable for tumor growth may present itself with different tissue optical properties and contain different amounts of the major tissue chromophores. Here, we decomposed transillumination spectra obtain in women from various risk levels of developing breast cancer.

Optical technology holds considerable promise to improve early detection, diagnosis and risk assessment of breast cancer. Unlike current clinical risk assessment tools such as the Gail model, the most widely accepted risk assessment tool, optical risk assessment technology can be applied to the entire female population of all ages. This study is investigating the use of optical reflectance spectroscopy (ORS) as a possible breast tissue development monitoring tool for adolescent girls. Changes in breast development due to proliferation of mammary gland and the surrounding stroma are reflected in changes in breast tissue density and composition which can be interrogated optically. Modifications of development influenced by micronutrients and hormonal status from exposures (e.g. toxins), lifestyle and diet effects, may ultimately be tracked. Preliminary data suggests that ORS has the ability to detect differences in bulk tissue properties in the developing breast of adolescent girls when compared to developmental stages assessed by Tanner, regional variation within breast tissue structure and asymmetries between left and right breast size and shape. Spectral comparison of unilateral breast development permits adjusting the optode separation as function of developmental breast size to minimize optical sampling of pectoral muscle.