Light Fluence Rate Research Articles

Study of tissue oxygen supply rate in a macroscopic photodynamic therapy singlet oxygen model

An appropriate expression for the oxygen supply rate (Γ(s)) is required for the macroscopic modeling of the complex mechanisms of photodynamic therapy (PDT). It is unrealistic to model the actual heterogeneous tumor microvascular networks coupled with the PDT processes because of the large computational requirement. In this study, a theoretical microscopic model based on uniformly distributed Krogh cylinders is used to calculate Γ(s) = g (1 - [³O₂]/[³O₂]₀) that can replace the complex modeling of blood vasculature while maintaining a reasonable resemblance to reality; g is the maximum oxygen supply rate and [³O₂]/[³O₂]₀ is the volume-average tissue oxygen concentration normalized to its value prior to PDT. The model incorporates kinetic equations of oxygen diffusion and convection within capillaries and oxygen saturation from oxyhemoglobin. Oxygen supply to the tissue is via diffusion from the uniformly distributed blood vessels. Oxygen can also diffuse along the radius and the longitudinal axis of the cylinder within tissue. The relations of Γ(s) to [³O₂]/[³O₂]₀ are examined for a biologically reasonable range of the physiological parameters for the microvasculature and several light fluence rates (ϕ). The results show a linear relationship between Γ(s) and [³O₂]/[³O₂]₀, independent of ϕ and photochemical parameters; the obtained g ranges from 0.4 to 1390 μM/s.

In-vivo outcome study of HPPH mediated PDT using singlet oxygen explicit dosimetry (SOED).

Type II photodynamic therapy (PDT) is based on the use of photochemical reactions mediated through an interaction between a tumor-selective photosensitizer, photoexcitation with a specific wavelength of light, and production of reactive singlet oxygen. However, the medical application of this technique has been limited due to inaccurate PDT dosimetric methods. The goal of this study is to examine the relationship between outcome (in terms of tumor growth rate) and calculated reacted singlet oxygen concentration ([1O2]rx) after HPPH-mediated PDT to compare with other PDT dose metrics, such as PDT dose or total light fluence. Mice with radiation-induced fibrosarcoma (RIF) tumors were treated with different light fluence and fluence rate conditions. Explicit measurements of photosensitizer drug concentration and tissue optical properties via fluorescence and absorption measurement with a contact probe before and after PDT were taken to then quantify total light fluence, PDT dose, and [1O2]rx based on a macroscopic model of singlet oxygen. In addition, photobleaching of photosenitizer were measured during PDT as a second check of the model. Changes in tumor volume were tracked following treatment and compared to the three calculated dose metrics. The correlations between total light fluence, PDT dose, reacted [1O2]rx and tumor growth demonstrate that [1O2]rx serves as a better dosimetric quantity for predicting treatment outcome and a clinically relevant tumor growth endpoint.

Real-time treatment light dose guidance of Pleural PDT: an update.

The goal of this study was to develop and improve an infrared (IR) navigation system to deliver light dose uniformly during intracavitory PDT by tracking the movement of the light source and providing real-time feedback on the light fluence rate on the entire cavity surface area. In the current intrapleural PDT protocol, several detectors placed in selected locations in the pleural cavity monitor the light doses. To improve the delivery of light dose uniformity, an IR camera system is used to track the motion of the light source as well as the surface contour of the pleural cavity. Monte-Carlo simulation is used to improve the calculation algorithm for the effect of light that undergoes multiple scattering along the surface in addition to an improvement of the direct light calculation using an improved model that accounts for the anisotropy of the light from the light source.

Phototropin 1 and dim-blue light modulate the red light de-etiolation response

Light signals regulate seedling morphological changes during de-etiolation through the coordinated actions of multiple light-sensing pathways. Previously we have shown that red-light-induced hypocotyl growth inhibition can be reversed by addition of dim blue light through the action of phototropin 1 (phot1). Here we further examine the fluence-rate relationships of this blue light effect in short-term (hours) and long-term (days) hypocotyl growth assays. The red stem-growth inhibition and blue promotion is a low-fluence rate response, and blue light delays or attenuates both the red light and far-red light responses. These de-etiolation responses include blue light reversal of red or far-red induced apical hook opening. This response also requires phot1. Cryptochromes (cry1 and cry2) are activated by higher blue light fluence-rates and override phot1's influence on hypocotyl growth promotion. Exogenous application of auxin transport inhibitor naphthylphthalamic acid abolished the blue light stem growth promotion in both hypocotyl growth and hook opening. Results from the genetic tests of this blue light effect in auxin transporter mutants, as well as phytochrome kinase substrate mutants indicated that aux1 may play a role in blue light reversal of red light response. Together, the phot1-mediated adjustment of phytochrome-regulated photomorphogenic events is most robust in dim blue light conditions and is likely modulated by auxin transport through its transporters.

Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles

The phytochrome gene family, which is in Arabidopsis thaliana, consists of phytochromes A-E (phyA to phyE), regulates plant responses to ambient light environments. PhyA and phyB have been characterized in detail, but studies on phyC to phyE have reported discrepant functions. In this study, we show that phyD regulates the Arabidopsis gravitropic response by inhibiting negative gravitropism of hypocotyls under red light condition. PhyD had only a limited effect on the gravitropic response of roots in red light condition. PhyD also enhanced phyB-regulated gravitropic responses in hypocotyls. Moreover, the regulation of hypocotyl gravitropic responses by phyD was dependent upon the red light fluence rate.

SU‐E‐T‐20: A Novel Hybrid CBCT, Bioluminescence and Fluorescence Tomography System for Preclinical Radiation Research

Purpose:A novel standalone bioluminescence and fluorescence tomography (BLT and FT) system equipped with high resolution CBCT has been built in our group. In this work, we present the system calibration method and validate our system in both phantom and in vivo environment.Methods:The CBCT is acquired by rotating the animal stage while keeping the x‐ray source and detector panel static. The optical signal is reflected by the 3‐mirror system to a multispectral filter set and then delivered to the CCD camera with f/1.4 lens mounted. Nine fibers passing through the stage and in contact with the mouse skin serve as the light sources for diffuse optical tomography (DOT) and FT. The anatomical information and optical properties acquired from the CBCT and DOT, respectively, are used as the priori information to improve the BLT/FT reconstruction accuracy. Flat field correction for the optical system was acquired at multiple wavelengths. A home‐built phantom is used to register the optical and CBCT coordinates. An absolute calibration relating the CCD photon counts rate to the light fluence rate emitted at animal surface was developed to quantify the bioluminescence power or fluorophore concentration.Results:An optical inhomogeneous phantom with 2 light sources (3mm separation) imbedded is used to test the system. The optical signal is mapped onto the mesh generated from CBCT for optical reconstruction. Our preliminary results show that the center of mass can be reconstructed within 2.8mm accuracy. A live mouse with the light source imbedded is also used to validate our system. Liver or lung metastatic luminescence tumor model will be used for further testing.Conclusion:This hybrid system transforms preclinical research to a level that even sub‐palpable volume of cells can be imaged rapidly and non‐invasively, which largely extends the scope of radiobiological research.The research is supported by the NCI grant R01CA158100‐01

The design of nitric oxide donor drugs: s-nitrosothiol tDodSNO is a superior photoactivated donor in comparison to GSNO and SNAP

We have recently developed tert-dodecane S-nitrosothiol (tDodSNO) as a photoactivated nitric oxide (NO) donor. We here compare the potency of tDodSNO to S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), drugs which are also based upon the S-nitrosothiol functionality and have been extensively used for NO release studies. Photoactivation in vitro, at a clinically relevant light fluence rate (200W/m2), demonstrated that tDodSNO released much higher levels of NO than either GSNO or SNAP. When evaluated in an ex vivo aortic ring vasorelaxation assay, tDodSNO was also the only drug to exhibit a photodynamic response, with an 8 fold decrease in EC50 (8.1–1.0µM) upon irradiation. While both GSNO and SNAP induced NO dependent vasorelaxation at lower concentrations than tDodSNO (EC50׳s of 158 and 38nM respectively), this activity was due to their rapid metabolic decomposition, and could not be modulated by photoactivation. Additionally, tDodSNO׳s photodynamic response allowed vascular tone to be directly regulated by light intensity. Molecular modeling of drug properties suggested that these differences in activity could be attributed to a combination of an increase in tDodSNO׳s hydrophobicity, and substantial steric shielding of molecule׳s S-nitrosothiol group from solvent interactions. In conclusion, our study demonstrates that tDodSNO is currently the most effective known s-nitrosothiol for phototherapeutic applications.

Diffuse optical tomography using multichannel robotic platform for interstitial PDT.

In the operating room, time is extremely precious, and the speed of one's data acquisition system often determines whether the data will be taken or not. Our multichannel robotic platform addresses this issue by optimizing source and detector scanning procedures. Up to 16 fibers can be moved independently with resolution of 0.05 mm and speed of 50 mm/s using motors with position feedback. The initial fiber alignment employs a light beam/optical detector system for identical positioning of all motors. Peak and edge detection algorithms, for point and linear sources, are used with multiple fibers simultaneously for fast realignment of sources and detectors. The robotic platform is used to perform Diffuse Optical Tomography (DOT) measurements in solid prostate phantoms with both homogenous and inhomogeneous Optical Properties (OP). Correct positioning is critical for the accurate recovery of the OP. The light fluence rate distribution is determined by scanning multiple detector fibers simultaneously along lit linear sources placed throughout the phantom volume inside catheter needles. The scanning time for the entire DOT is about 10 seconds after the initial alignment. The OP distribution reconstruction is based on the steady-state light diffusion equation. The inverse interstitial DOT problem is solved using NIRFAST. The optical properties are recovered by iterative minimization of the difference between measured and calculated light fluence rates. Recovered OP agree with the actual values within 10%. The OP corrections are used to significantly improve light fluence accuracy for the entire volume of bulk tumor.

Comparative functional analysis of full‐length and N‐terminal fragments of phytochrome C, D and E in red light‐induced signaling

Phytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species. We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins inplanta. The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds. PHYE-YFP was active in the absence of phyB and phyD, and PHYE-YFP controlled responses, as well as accumulation, of the fusion protein in the nuclei, was saturated at low fluence rates of red light and did not require functional FAR-RED ELONGATED HYPOCOTYL1 (FHY-1) and FHY-1-like proteins. Our data suggest that PHYC-YFP, PHYD-YFP and PHYE-YFP fusion proteins, as well as their truncated N-terminal derivatives, are biologically active in the modulation of red light-regulated photomorphogenesis. We propose that PHYE-YFP can function as a homodimer and that low-fluence red light-induced translocation of phyE and phyA into the nuclei is mediated by different molecular mechanisms.

SU‐C‐105‐02: Real‐Time Treatment Guidance Using An IR Navigation System for Pleural PDT

Purpose: Pleural photodynamic therapy (PDT) has been used in conjunction with surgical debulking of the tumorous tissue in treatment for mesothelioma. Uniform light fluence distribution is critical for effective and predictable PDT outcome. Current treatment methods involve monitoring the light dose delivered with 8 detectors that are located at various locations inside the pleural cavity. A tracking and real‐time feedback system can be utilized to improve the uniformity of light. Methods: An infrared (IR) tracking camera to monitor the movement of the light source. The same system is used to determine the pleural contour geometry of the treatment area. This study examines the fluence (rate) delivered between measurement and calibration in phantom studies. Isotropic detectors were used for in‐vivo light dosimetry. The tracking data for the light source was used to calculate the light fluence rate delivered which was then compared with the in‐vivo measurement. The real‐time feedback software developed shows the tracking data and calculations during treatment time, enabling more uniform treatment of the pleural cavity. Results: Using the real‐time feedback system, we are able to match the calculated fluence rate with the in‐vivo measurements. Furthermore, we are able to treat more effectively and uniformly with the feedback software. The light fluence rate can have up to 50% deviation compared to the prescription in phantom experiments. With the navigation system, we are able to collect more data regarding the light delivered and the location to which the light was delivered. Conclusion: The IR camera has been used successfully during pleural PDT patient treatment to track the motion of the light source. It is possible to use the feedback system to deliver a more uniform dose of light throughout the pleural cavity.

SU‐E‐I‐88: Diffuse Optical Tomography for Characterization of Optical Properties in Interstitial PDT

Purpose: To characterize spatial distribution of optical properties (OP) for the interstitial photodynamic therapy (PDT) dosimetry using diffuse optical tomography (DOT). Methods: A spatial distribution of interstitial optical properties (absorption and reduced scattering coefficients) in a solid prostate phantom with OP heterogeneities was measured by scanning optical detector and source fibers with a multi‐channel motorized system. Several algorithms optimizing the scanning process were developed for the data acquisition system. The calculated light fluence rates were compared with measured ones to recover the heterogeneous optical properties. A mathematical phantom was used to test the reconstruction. Results: The ability to correctly separate absorption and scattering coefficients depended on the accuracy of the light fluence source measurement. The difference between measured and reconstructed fluence rates was within 10%. The DOT allowed reliable recovery of the absorption coefficients. Conclusion: The efficacy of PDT depends on the light distribution within the treated tissue. Since the spatial distribution of tissue optical properties affects the light distribution, the accurate determination of the OP spatial distribution is critical for the interstitial PDT treatment. This work is supported by the national Institute of Health (NIH) grants P01 CA87971 and R01 CA154562

WE‐C‐WAB‐05: A Novel Integrated X‐Ray and Fluorescence Tomography System for Small Animal Radiation Research Platform

Purpose: A novel, low cost, and fast acquisition fluorescence tomography (FT) system was designed and built for preclinical focal radiation research to provide soft tissue guidance as well as to support highly sensitive functional imaging. Materials & Methods: The cone beam (CB) CT/FT system was designed and tested in a standalone form. The mouse is placed prone on a rotation stage. The x‐ray source and flat panel detector for the CBCT are aligned perpendicular to the mouse anterior‐posterior axis. The anatomical information obtained from CBCT is used as prior information to enhance subsequent optical reconstruction by limiting the solution space. Prior to FT acquisition, diffuse optical tomography (DOT) is performed to reconstruct the animal optical properties. Optical fibers at fixed positions in contact with the animal deliver the source light of different wavelengths for DOT. For FT, a diode laser replaces the lamp as the excitation source. A CCD camera with f/1.4 lens is used as the optical detector. The CCD camera response readout was calibrated using an integrating sphere to provide the absolute light fluence rate at the desired wavelength. A mirror system capable of 90 degree rotation around the animal reflects the optical signal to the CCD camera. Results: To test the FT system, pre‐drilled holes in a tissue‐simulating phantom were filled with a solution of indocyanine green (ICG). Several scenarios were used to test the optical system, such as examining the spatial resolution by placing the same fluorophore at variable separations, and reconstructing the ICG and another infrared fluorophore simultaneously to simulate multiple color functional imaging. Our initial results show that FT can localize the ICG source to within 1 mm. Conclusion: The novel CBCT/FT system presents new research opportunities for image guided pre‐clinical radiation research.

Phototropins function in high-intensity blue light-induced hypocotyl phototropism in Arabidopsis by altering cytosolic calcium.

Phototropins (phot1 and phot2), the blue light receptors in plants, regulate hypocotyl phototropism in a fluence-dependent manner. Especially under high fluence rates of blue light (HBL), the redundant function mediated by both phot1 and phot2 drastically restricts the understanding of the roles of phot2. Here, systematic analysis of phototropin-related mutants and overexpression transgenic lines revealed that HBL specifically induced a transient increase in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in Arabidopsis (Arabidopsis thaliana) hypocotyls and that the increase in [Ca(2+)]cyt was primarily attributed to phot2. Pharmacological and genetic experiments illustrated that HBL-induced Ca(2+) increases were modulated differently by phot1 and phot2. Phot2 mediated the HBL-induced increase in [Ca(2+)]cyt mainly by an inner store-dependent Ca(2+)-release pathway, not by activating plasma membrane Ca(2+) channels. Further analysis showed that the increase in [Ca(2+)]cyt was possibly responsible for HBL-induced hypocotyl phototropism. An inhibitor of auxin efflux carrier exhibited significant inhibitions of both phototropism and increases in [Ca(2+)]cyt, which indicates that polar auxin transport is possibly involved in HBL-induced responses. Moreover, PHYTOCHROME KINASE SUBSTRATE1 (PKS1), the phototropin-related signaling element identified, interacted physically with phototropins, auxin efflux carrier PIN-FORMED1 and calcium-binding protein CALMODULIN4, in vitro and in vivo, respectively, and HBL-induced phototropism was impaired in pks multiple mutants, indicating the role of the PKS family in HBL-induced phototropism. Together, these results provide new insights into the functions of phototropins and highlight a potential integration point through which Ca(2+) signaling-related HBL modulates hypocotyl phototropic responses.

Feasibility of interstitial diffuse optical tomography using cylindrical diffusing fibers for prostate PDT

Interstitial diffuse optical tomography (DOT) has been used to characterize spatial distribution of optical properties for prostate photodynamic therapy (PDT) dosimetry. We have developed an interstitial DOT method using cylindrical diffuse fibers (CDFs) as light sources, so that the same light sources can be used for both DOT measurement and PDT treatment. In this novel interstitial CDF-DOT method, absolute light fluence per source strength (in unit of 1 cm−2) is used to separate absorption and scattering coefficients. A mathematical phantom and a solid prostate phantom including anomalies with known optical properties were used, respectively, to test the feasibility of reconstructing optical properties using interstitial CDF-DOT. Three dimension spatial distributions of the optical properties were reconstructed for both scenarios. Our studies show that absorption coefficient can be reliably extrapolated while there are some cross talks between absorption and scattering properties. Even with the suboptimal reduced scattering coefficients, the reconstructed light fluence rate agreed with the measured values to within ±10%, thus the proposed CDF-DOT allows greatly improved light dosimetry calculation for interstitial PDT.

Light dosimetry and dose verification for pleural PDT.

In-vivo light dosimetry for patients undergoing photodynamic therapy (PDT) is critical for predicting PDT outcome. Patients in this study are enrolled in a Phase I clinical trial of HPPH-mediated PDT for the treatment of non-small cell lung cancer with pleural effusion. They are administered 4mg per kg body weight HPPH 48 hours before the surgery and receive light therapy with a fluence of 15-45 J/cm2 at 661 and 665nm. Fluence rate (mW/cm2) and cumulative fluence (J/cm2) are monitored at 7 sites during the light treatment delivery using isotropic detectors. Light fluence (rate) delivered to patients is examined as a function of treatment time, volume and surface area. In a previous study, a correlation between the treatment time and the treatment volume and surface area was established. However, we did not include the direct light and the effect of the shape of the pleural surface on the scattered light. A real-time infrared (IR) navigation system was used to separate the contribution from the direct light. An improved expression that accurately calculates the total fluence at the cavity wall as a function of light source location, cavity geometry and optical properties is determined based on theoretical and phantom studies. The theoretical study includes an expression for light fluence rate in an elliptical geometry instead of the spheroid geometry used previously. The calculated light fluence is compared to the measured fluence in patients of different cavity geometries and optical properties. The result can be used as a clinical guideline for future pleural PDT treatment.

A robotic multi-channel platform for interstitial photodynamic therapy.

A custom-made robotic multichannel platform for interstitial photodynamic therapy (PDT) and diffuse optical tomography (DOT) was developed and tested in a phantom experiment. The system, which was compatible with the operating room (OR) environment, had 16 channels for independent positioning of light sources and/or isotropic detectors in separate catheters. Each channel's motor had an optical encoder for position feedback, with resolution of 1.5 mm, and a maximum speed of 5 cm/s. Automatic calibration of detector positions was implemented using an optical diode beam that defined the starting position of each motor, and by means of feedback algorithms controlling individual channels. As a result, the accuracy of zero position of 0.1 mm for all channels was achieved. We have also employed scanning procedures where detectors automatically covered the appropriate range around source positions. Thus, total scan time for a typical optical properties (OP) measurement throughout the phantom was about 1.5 minutes with point sources. The OP were determined based on the measured light fluence rates. These enhancements allow a tremendous improvement of treatment quality for a bulk tumor compared to the systems employed in previous clinical trials.

Blue light regulates the rhythm of diurnal vertical migration in the raphidophyte red-tide alga Chattonella antiqua

We examined the effects of photoperiod, wavelength and light fluence rate on diurnal vertical migration (DVM) cycle in a coastal raphidophyte, Chattonella antiqua. We first observed the DVM under different combinations of lightdark (LD) cycles and light spectra. Under continuous white, UV-A or blue light, DVM followed the LD cycle established during the white light pre-conditioning, for one cycle, and then became arrhythmic. Under red light, however, the DVM rhythms under the different LD regimes continued approximately as during pre-conditioning. When C. antiqua cultured under continuous red light was exposed to a 2-h pulse of blue light at the beginning or end of artificial night, the DVM was delayed or advanced, respectively. The fluence rateresponse curve indicates a blue-light threshold of 10(2) mol photons m(2) s(1) for the DVM phase shift. The equal-quantum action spectra for phase shift peaked in the UV-A/blue region (360480 nm), which is the part of the light spectrum most transmitted in its natural habitat. We show that C. antiqua can sense the weak blue component of sunlight throughout its depth range, allowing it to cue its DVM to the daynight cycle regardless of weather and transparency.

How to Avoid Local Side Effects of Bladder Photodynamic Therapy: Impact of the Fluence Rate

We studied how to avoid irritative bladder symptoms after bladder photodynamic therapy, such as urgency, frequency and pain, which are associated with the inflammation and destruction of normal urothelium. Rats bearing orthotopic bladder tumors were instilled with hexyl-aminolevulinate and illuminated with red light at a high vs low (100 vs 15 mW/cm(2)) fluence rate. Cystectomy specimens 48 hours after treatment were subjected to anatomopathological examination. Inflammatory reaction and apoptosis were evaluated. In vivo photobleaching was assessed during illumination at each fluence rate. All superficial tumors were eradicated irrespective of light dose and fluence rate. High fluence rates induced necrosis with inflammatory reaction and absent normal urothelium. Low fluence rates did not provoke inflammation and resulted in apoptotic cell death with preserved urothelial integrity. This could be attributable to faster photobleaching of the photosensitizer in normal urothelium at low fluence rates. Bladder photodynamic therapy at a low fluence rate minimizes side effects without hampering therapeutic efficacy.

Contributions of green light to plant growth and development

Light passing through or reflected from adjacent foliage provides a developing plant with information that is used to guide specific genetic and physiological processes. Changes in gene expression underlie adaptation to, or avoidance of, the light-compromised environment. These changes have been well described and are mostly attributed to a decrease in the red light to far-red light ratio and/or a reduction in blue light fluence rate. In most cases, these changes rely on the integration of red/far-red/blue light signals, leading to changes in phytohormone levels. Studies over the last decade have described distinct responses to green light and/or a shift of the blue-green, or red-green ratio. Responses to green light are typically low-light responses, suggesting that they may contribute to the adaptation to growth under foliage or within close proximity to other plants. This review summarizes the growth responses in artificially manipulated light environments with an emphasis on the roles of green wavebands. The information may be extended to understanding the influence of green light in shade avoidance responses as well as other plant developmental and physiological processes.

Self-calibrated algorithms for diffuse optical tomography and bioluminescence tomography using relative transmission images

Reconstruction algorithms for diffuse optical tomography (DOT) and bioluminescence tomography (BLT) have been developed based on diffusion theory. The algorithms numerically solve the diffusion equation using the finite element method. The direct measurements of the uncalibrated light fluence rates by a camera are used for the reconstructions. The DOT is self-calibrated by using all possible pairs of transmission images obtained with external sources along with the relative values of the simulated data and the calculated Jacobian. The reconstruction is done in the relative domain with the cancelation of any geometrical or optical factors. The transmission measurements for the DOT are used for calibrating the bioluminescence measurements at each wavelength and then a normalized system of equations is built up which is self-calibrated for the BLT. The algorithms have been applied to a three dimensional model of the mouse (MOBY) segmented into tissue regions which are assumed to have uniform optical properties. The DOT uses the direct method for calculating the Jacobian. The BLT uses a reduced space of eigenvectors of the Green's function with iterative shrinking of the permissible source region. The reconstruction results of the DOT and BLT algorithms show good agreement with the actual values when using either absolute or relative data. Even a small calibration error causes significant degradation of the reconstructions based on absolute data.