Jenny Svensson

Fluorescence spectra provide information on the depth of fluorescent lesions in tissue

The fluorescence spectrum measured from a fluorophore in tissue is affected by the absorption and scattering properties of the tissue, as well as by the measurement geometry. We analyze this effect with Monte Carlo simulations and by measurements on phantoms. The spectral changes can be used to estimate the depth of a fluorescent lesion embedded in the tissue by measurement of the fluorescence signal in different wavelength bands. By taking the ratio between the signals at two wavelengths, we show that it is possible to determine the depth of the lesion. Simulations were performed and validated by measurements on a phantom in the wavelength range 815-930 nm. The depth of a fluorescing layer could be determined with 0.6-mm accuracy down to at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types of various composition. The results indicate that depth estimation of a lesion should be possible with 2-3-mm accuracy, with no assumptions made about the optical properties, for a wide range of tissues.

Tumor selectivity at short times following systemic administration of a liposomal temoporfin formulation in a murine tumor model

Meso‐tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is one of the most potent photodynamically active substances in clinical use. Treatment protocols for Temoporfin‐mediated photodynamic therapy often rely on drug‐light intervals of several days in order for the photosensitizer to accumulate within the target tissue, though tumor selectivity is limited. Here, the mTHPC localization was studied at 2–8 h following systemic administration of a liposomal Temoporfin formulation (0.15 mg kg−1 b.w.) in HT29 human colon adenocarcinoma in NMRI nu/nu mice. Photosensitizer distribution within tumor and internal organs was investigated by means of high performance liquid chromatography following chemical extraction, as well as in situ fluorescence imaging and point‐monitoring fluorescence spectroscopy. For tumor tissue, the Temoporfin concentrations at 4 h (0.16 ± 0.024 ng mg−1) and 8 h (0.18 ± 0.064 ng mg−1) were significantly higher than at 2 h (0.08 ± 0.026 ng mg−1). The average tumor‐to‐muscle and the tumor‐to‐skin selectivity were 6.6 and 2, respectively, and did not vary significantly with time after photosensitizer injection. In plasma, the Temoporfin concentration was low (0.07 ± 0.07 ng mg−1) and showed no significant variation with time. Our results indicate a rapid biodistribution and clearance from the bloodstream. Within the same type of organ, data from both fluorescence methods generally exhibited a significant correlation with the extraction results.

Spatially varying regularization based on spectrally resolved fluorescence emission in fluorescence molecular tomography

Fluorescence molecular tomography suffers from being mathematically ill-conditioned resulting in non-unique solutions to the reconstruction problem. In an attempt to reduce the number of possible solutions in the underdetermined system of equations in the reconstruction, we present a method to retrieve a spatially varying regularization map outlining the feasible inclusion position. This approach can be made very simple by including a few multispectral recordings from only one source position. The results retrieved through tissue phantom experiments imply that initial reconstructions with spatially varying priors reduces artifacts and show slightly more accurate reconstruction results compared to reconstructions using no priors.

Modeling of spectral changes for depth localization of fluorescent inclusion

We have performed modeling of fluorescence signals from inclusions inside turbid media to investigate the influence of a limited fluorescence contrast and how accurately the depth can be determined by using the spectral information. The depth was determined by forming a ratio of simulated fluorescence intensities at two wavelengths. The results show that it is important to consider the background autofluorescence in determining the depth of a fluorescent inclusion. It is also necessary to know the optical properties of the tissue to obtain the depth. A 20% error in absorption or scattering coefficients yields an error in the determined depth of approximately 2-3 mm (relative error of 10-15%) in a 20 mm thick tissue slab.

Fluorescence and absorption assessment of a lipid mTHPC formulation following topical application in a non-melanotic skin tumor model.

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [+/- standard deviation (SD)] mTHPC concentration within lesions of 6.0(+/-3.1) ngmg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.

Tissue temperature monitoring during interstitial photodynamic therapy

During δ-aminolevulinic acid (ALA) based Interstitial Photodynamic Therapy (IPDT) a high light fluence rate is present close to the source fibers. This might induce an unintentional tissue temperature increase of importance for the treatment outcome. In a previous study, we have observed, that the absorption in the tissue increases during the treatment. A system to measure the local tissue temperature at the source fibers during IPDT on tissue phantoms is presented. The temperature was measured by acquiring the fluorescence from small Cr3+-doped crystals attached to the tip of the illumination fiber used in an IPDT-system. The fluorescence of the Alexandrite crystal used is temperature dependent. A ratio of the intensity of the fluorescence was formed between two different wavelength bands in the red region. The system was calibrated by immersing the fibers in an Intralipid solution placed in a temperature controlled oven. Measurements were then performed by placing the fibers interstitially in a pork chop as a tissue phantom. Measurements were also performed superficially on skin on a volunteer. A treatment was conducted for 10 minutes, and the fluorescence was measured each minute during the illumination. The fluorescence yielded the temperature at the fiber tip through the calibration curve. The measurements indicate a temperature increase of a few degrees during the simulated treatment.

mTHPC pharmacokinetics following topical administration

Measurements of concentration of sensitizers for photodynamic therapy can provide important information in the dosimetry planning and can also give input to the optimal time for treatment. There has been skepticism towards fluorescence techniques for this purpose, as the signal depends on the fluorescence yield and optical properties of the tissue. Absorption based techniques, lack on the other hand, often the sensitivity required for many sensitizers with relative weak absorption in a wavelength region where hemoglobin absorption is dominant. A direct comparison between absorption and fluorescence techniques for measuring mTHPC concentration after topical application on hairless SKH-1 mice bearing skin carcinomas has been performed. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumor area and the concentration of mTHPC was measured at 4 and 6 hours after drug application by two methods: 1. A fluorescence imaging system capturing images at two wavelengths (500 and 650 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 650 to 500 was calculated. 2. A diffuse reflectance spectroscopy system with a fiber separation of 2 mm, providing the absorbance at 652 nm. Both systems provided consistent results related to the photosensitizer concentration. The methods show a remarkable difference in the concentration of photosensitizer in normal skin and tumor. No significant difference in mTHPC concentration in tumor could be observed between the 4 and 6h groups after drug application.

Distribution studies of m-THPC after topical application of m-THPC thermogel in a murine non-melanoma skin cancer tumor model by fluorescence spectroscopic and imaging techniques

m-THPC photodynamic therapy has been successfully studied in skin cancer, but no research effort concerning its topical application has been performed until now. Determination of the biodistribution of a special m-THPC thermogel formulation and its tumour selectivity was studied after topical application on hairless SKH-1 mice bearing non-melanoma skin carcinomas. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumour area and the concentration or demarcation of tumor by mTHPC fluorescence was measured at 2, 4 and 6 hours after drug application by three methods: 1. A fluorescence imaging system capturing images at two emission wavelengths (500 and 654 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 654 to 500 was calculated. 2. A fluorescence spectrometer with a fiber bundle for in vivo spectra recording after 420 nm excitation. 3 Each animal was euthanized and the photosensitizer was chemically extracted from liver, spleen, muscle, normal skin and tumour. The photosensitizer concentrations in the extracts and in plasma were determined by fluorescence spectroscopy. The in vivo methods showed a remarkable difference in the concentration of photosensitizer in normal skin and tumour. The highest concentration in tumour was observed 6h after drug application and the highest fluorescence intensity ratio of m-THPC in tumour to normal tissue was observed at 4 hours. Furthermore, no m-THPC was detected in normal tissues or plasma after drug topical application. In vivo and ex vivo results were consistent.

Prior information in fluorescence molecular tomography based on multispectral fluorescence emission

Fluorescence molecular tomography (FMT) suffers from inherent ill-posedness due to the vast number of possible solutions to the reconstruction problem. To increase the robustness of such a problem one need prior information. We present here a method for rendering a priori information of the position of a fluorescent inclusion inside turbid media. The method utilizes solely two spectral bands within the fluorescence spectrum emitted from the fluorophore. The method is presented and verified using experimental data from a tissue phantom. The confinement is also used to impose weights onto the voxels before the inversion of the linear set of equations describing the FMT problem.

Estimation of depth of fluorescing lesions in tissue from changes in fluorescence spectra

We present a novel method for estimating the depth of a fluorescent lesion in tissue based on measurements of the fluorescence signal in different wavelength bands. The measured fluorescence spectrum following irradiation by excitation light at the surface is a function of several parameters, because the fluorescence light has to pass through tissue with characteristic scattering and absorption properties. Thus, the intrinsic fluorescence spectrum will be altered, in a way determined by the tissue optical properties, the depth of the fluorophore, and also by the geometry of the light irradiation and the detection system. By analyzing the ratio between the signals at two wavelengths we show that it is possible to estimate the depth of the lesion. We have performed Monte Carlo simulations and measurements on an Intralipid phantom in the wavelength range 850 - 1000 nm. By taking the ratio between the signals at the wavelengths 875 and 930 nm the depth of a fluorescing layer could be determined with 0.8 mm accuracy down at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types with various composition. The results indicate that depth estimation of a lesion is possible with no assumptions made about the optical properties for a wide range of tissues.