Fluorescence molecular tomography (FMT) is quite demanding in terms of acquisition/computational times due to the huge amount of data. Different research groups have proposed compression approaches regarding both illumination (wide field structured light instead of raster point scanning) and detection (compression of the acquired images). The authors have previously proposed a fast FMT reconstruction method based on the combination of a multiple-view approach with a full compression scheme. This method had been successfully tested on a cylindrical phantom and is being generalized in this paper to samples of arbitrary shape. The devised procedure and algorithms have been tested on an ex-vivo mouse.
In recent years, an increasing effort has been devoted to the optimization of acquisition and reconstruction schemes for fluorescence molecular tomography (FMT). In particular, wide-field structured illumination and compression of the measured images have enabled significant reduction of the data set and, consequently, a decrease in both acquisition and processing times. FMT based on this concept has been recently demonstrated on a cylindrical phantom with a rotating-view scheme that significantly increases the reconstruction quality. In this work, we generalize the rotating-view scheme to arbitrary geometries and experimentally demonstrate its applicability to murine models. To the best of our knowledge this is the first time that FMT based on a rotating-view scheme with structured illumination and image compression has been applied to animals.
Since Photodynamic therapy(PDT) is able to increase the antitumor immunity, in our laboratory we examine the antitumor effect of combination of PDT,with photoactivated Aluminium disulfonated Phthalocianine(ALS2Pc),adoptive immunotherapy, with immune lymphocytes, and chemotherapy on aggressive murine tumor. Mice bearing L1210 tumor were treated at day +4 with PDT ( 5mg/Kg of AlS2Pc and 100mW/cm2 x 10’ of exposure of laser light 24hrs. later),at day +6 with Adriamycin(ADR 2mg/Kg) and at day + 7 with immune lymphocytes(IL),collected from L1210 bearing mice pretreated with PDT(2x107 cells).The results show that the combination ADR + PDT + IL demonstrates a significant synergistic antitumor effect while the chemotherapy treatment with low dose of the drug and the adotive immunotherapy treatment are slightly effective. The same positive results were obtained with the combination of PDT,Cisplatin(CDDP 2mg/Kg) and IL,while the CDDP treatment alone and the Il treatment alone are slightly effective. In conclusion these results suggest that it is possible to completely cure animals bearing advanced tumors, with a combined therapy, PDT + adoptive immunotherapy + low dose chemotherapy.
Since immune response of the host is important in the control of tumor growth and spreading, and the Photodynamic therapy (PDT) is able to increase the antitumor immunity, in our laboratory we examine the effect of PDT on immune compartment of tumor bearing mice. Lymphocytes and macrophages collected from tumor bearing mice pretreated with PDT are cytotoxic in vitro and in vivo against the parental tumor lines, in contrast the same immune cells population collected from tumor bearing mice pretreated only with laser light are unable to lyse the parental tumor cells. In adoptive immunotherapy experiments, treatment of mice bearing MS-2 tumor with adoptive transfer of immune lymphocytes collected from mice pretreated with PDT is able to significantly increase the survival time; in contrast the lymphocytes collected from mice pretreated only with laser light were not able to modify the survival time suggesting that the laser treatment alone did not increase the immune response of the host. In conclusion these results demonstrate that the PDT induce a strong immune response on the host and the stimulated lymphocytes generated could be used for an adoptive immunotherapy approach; moreover laser treatment alone (thermal effect) is unable to modulate the immune response of the host.
Photodynamic therapy (PDT) of cancer is based on the systemic administration of photosensitive drugs followed by exposure of the tumor mass to light of particular wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides for an effective therapy with efficient tumor cytotoxicity and minimal normal tissue damage. There are various studies on the effect of photoactivated photosensitizers on host immune response in tumor bearing mice. Since immunity is important in the control of tumor growth and spreading in our laboratory we examine the effect of PDT on immune compartment. Spleen hyperplasia as well as spleen and marrow hypercellularities were observed in tumor bearing mice treated with Aluminum Disulfonated Phthalocyanine (AlS2Pc) and laser light. Phytohaemagglutinin (T lymphocytes mitogen) and Lypolisaccaride (B lymphocytes mitogen), stimulation of spleen lymphocytes caused an increase in blast transformation in tumor bearing mice. Furthermore splenocytes and macrophages collected from mice treated with PDT were cytotoxic in vivo (Winn Assay) parental against tumor cells. The results observed suggest that PDT is able to modulate the immune response and oncological patients treated with PDT could become immune versus a relapse or versus the minimal residual disease.
In this paper we report on the experimental setup and the performances of a fluorescence lifetime imaging system designed by the authors for cancer detection. The experimental apparatus relies on a CCD video camera coupled to a light intensifier and on a pulsed laser. Two or more fluorescence images are acquired within suitable time windows at different delays, with respect to the excitation pulses. The images are processed using matrix algorithms implemented in the fast processor of an advanced frame grabber; then, the resulting lifetime map is displayed in pseudocolor or gray shade images. The instrumentation is fully computer controlled and the whole process takes about 1 second per lifetime image. Using a faster processor this time could be easily reduced to some milliseconds for true real time measurements. The system has been successfully applied to detect experimental tumors in mice sensitized with hematoporphyrin derivative, which is a clinically approved photosensitizing drug.
Three time-delayed fluorescence images were used to calculate the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with Hematoporphyrin Derivative (HpD). This allowed us to observe that the drug has a significantly longer fluorescence lifetime when it is incorporated in neoplastic tissues with respect to healthy surrounding ones. The imaging technique can be applied noninvasively in vivo in real time. Its diagnostic potential was tested on two murine tumor models after sensitization with low doses of HpD. In all the experimental conditions considered, the tumor could be easily identified in the lifetime images. Images were acquired also after removal of the skin overlying the tumor mass. Removal of the skin led to higher values of the lifetime contrast and allowed an even easier tumor localization.
The absorption spectrum of aluminum phthalocyanine with a mean degree of sulfonation of 2.1 (AlPcS2),
previously measured in vivo in a murine tumor model (L1210 leukemia), showed a significant (10 to 15 nm)
red shift with respect to the spectrum in solution and in vitro. To investigate whether this behavior was due
to the peculiar model considered (ascitic tumor), the absorption line shape was evaluated in vivo noninvasively in a solid tumor (murine MS-2 fibrosarcoma) by means of time-resolved reflectance. The spectrum acquired in the range of 650 to 695 nm after the injection of 2.5 or 5 mg/kg body weight AlPcS2 confirms the red shift to 680 to 685 nm. Experiments performed ex vivo suggest that the spectral shift is related to the interactions of the drug with the extracellular biological substrate and depends on the sensitizer concentration.
A system for time-gated imaging has been used to measure the spatial distribution of the fluorescence decay time in tumor-bearing mice sensitized with Hematoporphyrin Derivative. Mice were injected with five doses of the sensitizer ranging from 0.1 to 10 mg/kg body weight. Several fluorescence images of each mouse were acquired at different delays after the excitation pulses, and a matrix processing algorithm was applied to calculate the average decay time corresponding to each pixel. It was found that, for any drug dose, the decay time of the exogenous fluorescence in the tumor is always significantly longer than in normal tissues. The images created by associating a gray scale to the decay time matrix of each sample allow a reliable and precise detection of the neoplasia, even with a very low dose of the sensitizer.
The absorption spectrum of disulphonated aluminum phthalocyanine (AlS2Pc) between 650 nm and 695 nm was measured in vivo by means of time-resolved reflectance. The experiments were performed on mice bearing the L1210 leukemia 1, 4, and 7 hr after the i.p. administration of 2.5 mg/kg body weight (b.w.) of AlS2Pc. The absorption peak is centered at 685 nm, red-shifted of 10 - 15 nm with respect to the spectra obtained in solution in various environments. Measurements performed in vitro confirm the results in vivo and seem to suggest that the extracellular environment can cause the shift in the absorption line shape.
The absorption spectrum of disulphonated Aluminum Phthalocyanine (AlS2Pc) was measured in vivo in a murine tumor model by time-resolved reflectance (TRR). Mice bearing the L1210 leukemia were administered intraperitoneally 2.5 mg/kg body weight of AlS2Pc. Reflectance measurements were performed in the 650-695 nm range before and 4 h after the drug administration. The absorption coefficient at each wavelength was obtained by fitting the experimental curve using the diffusion equation. The spectrum of the photosensitizer was obtained as a difference between the absorption of treated and untreated mice. The absorption spectrum of AlS2Pc was peaked at 685 nm, showing a red-shift of approximately 15 nm with respect to the spectrum in aqueous solution.
Since immunity is important in the control of tumor growth and spreading, in our laboratory we examine the effect of photoactivated phthalocyanines on the immune compartment. Spleen hyperplasia as well as spleen and marrow hypercellularities were observed in mice bearing MS-2 fibrosarcoma treated at different times with Aluminum disulfonated Phthalocyanine and laser light. Results suggest a potential `tumor immunity' induced by PDT with photoactivated phthalocyanines and that the dyes used in PDT could play an immunopharmacological role in cancer treatment.
A time-gated fluorescence imaging technique was applied on tumor-bearing porphyrin-treated mice to study the sensitizer distribution in different organs and tissue types, and to establish whether false positives in the diagnosis of tumors (based on porphyrin fluorescence) could be generated by this localization in healthy tissues. Mice were administered 25 mg/kg body weight (b.w.) of HpD or 5 mg/kg b.w. of PII, and sacrificed 8 hr later. Time- gated fluorescence images were acquired from tumor, skin, muscle, fat, brain, heart, lung, lymph nodes, liver, bowel, spleen, and bone of both treated and untreated animals. Similar results were obtained with HpD and PII. The presence of porphyrins clearly helps the localization of the neoplastic area, which is characterized by the strongest fluorescence in delayed images. An appreciable long-living emission was observed also in bones. With the exception of the bowel, the fluorescence of other organs was weaker and, in untreated mice, short-living.
The photosensitizer of choice in photodynamic therapy (PDT) is the hematoporphyrin derivative (Hpd). However, Hpd has many characteristics which make it less than an ideal photosensitizer. The sulfonated phthalocyanines represent a new group of interesting compounds that have a strong absorption in the red part of the spectrum at 675 nm. In our laboratory we compare the efficacy of two phthalocyanines, the zincum tetrasulfonated (ZnS4Pc) and the aluminum disulfonated (AlS2Pc), on a murine tumor. Mice bearing MS-2 fibrosarcoma were treated with 5 or 25 mg/kg of ZnS4Pc or AlS2Pc and then the tumor mass was exposed to a laser light (100 mW for 10'). The results show that the treatment with AlS2Pc is significantly more therapeutically active in respect to the treatment with the same dose of ZnS4Pc. Moreover, resistance to rechallenge with the MS-2 tumor was evidenced by surviving animals. Studies are in progress with other murine tumors with different biological properties.
Time-gated fluorescence spectroscopy provides a very useful tool to evaluate the photophysical properties and the incorporation mechanisms of drugs interacting with biological substrates. In particular, taking into account that different fluorophores, even if overlapped in fluorescence spectrum, present different fluorescence lifetimes, it is possible to evidence the emission of a single molecular species by choosing a suitable observation window in the time domain. Using this technique, the effect of systemic administration on the uptake of Hematoporphyrin Derivative (HpD), its tumor localizing fraction (TLF), and disulphonated Aluminum Phthalocyanine (AlS2Pc) at the cellular level was evaluated on a murine ascitic tumor. The results obtained indicate that the TLF is the part of HpD actually retained by the cells and that AlS2Pc is incorporated more rapidly with respect to porphyrins. The observation of the gated spectra of HpD also evidenced the possibility of improving the contrast between the fluorescence of the cells and that of the drug. Thus, an imaging system has been developed which utilizes a gated, intensified, CCD camera synchronized with a subnanosecond laser- pulse excitation. The gate can be set to a minimum width of 5 ns and arbitrarily delayed with respect to the laser pulse. By optimization of the gate parameters, porphyrin fluorescence images in single cells and microscopy sections of tumor were obtained with a valuable signal- to-noise ratio.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.