P. Taroni

Time-resolved fluorescence imaging in biology and medicine

Fluorescence lifetime imaging is a rather new and effective tool that can be used to study complex biological samples, either at microscopic or macroscopic levels. The map of the fluorescence lifetime allows one to discriminate amongst different fluorophores and to achieve valuable insights into the behaviour of emitting molecules, leading to information like local pH, oxygen concentration in cells, etc. Moreover, the distribution in space of any fluorescent marker achievable with this technique can be exploited for diagnostic purposes in medicine. After a brief introduction on the motivations for applying fluorescence lifetime imaging in biology and medicine, the basic principles of this technique will be addressed. Then, the two possible implementations of fluorescence lifetime imaging (i.e. the frequency domain and the time domain methods) will be presented. For this purpose, special attention will be devoted to practical aspects of image acquisition and processing, especially for what concerns the time domain method. Then, the analysis of the state-of-the-art systems will include a brief discussion on new concepts that have recently been introduced in this research field. Finally, two interesting applications of fluorescence lifetime imaging will be presented. The former refers to skin tumour detection and has been successfully applied in a preliminary clinical trial, the latter regards DNA chips reading and has been tested only at laboratory level, yet it has produced promising results for its future implementation in commercial systems.

Performance assessment of photon migration instruments: the MEDPHOT protocol

We propose a comprehensive protocol for the performance assessment of photon migration instruments. The protocol has been developed within the European Thematic Network MEDPHOT (optical methods for medical diagnosis and monitoring of diseases) and is based on five criteria: accuracy, linearity, noise, stability, and reproducibility. This protocol was applied to a total of 8 instruments with a set of 32 phantoms, covering a wide range of optical properties.

Spectroscopic time-resolved diffuse reflectance and transmittance measurements of the female breast at different interfiber distances

The first, to our knowledge, in-vivo broadband spectroscopic characterization of breast tissue using different interfiber distances as well as transmittance measurements is presented. Absorption and scattering properties are measured on six healthy subjects, using time-resolved diffuse spectroscopy and an inverse model based on the diffusion equation. Wavelength-tunable picosecond-pulse lasers and time-correlated single-photon counting detection are employed, enabling fully spectroscopic measurements in the range 610 to 1040 nm. Characterization of the absorption and reduced scattering coefficients of breast tissue is made with the aim of investigating individual variations, as well as variations due to different measurement geometries. Diffuse reflectance measurements at different interfiber distances (2, 3, and 4 cm) are performed, as well as measurements in transmittance mode, meaning that different sampling volumes are involved. The results show a large variation in the absorption and scattering properties depending on the subject, correlating mainly with the water versus lipid content of the breast. Intrasubject variations, due to different interfiber distances or transmittance modes, correlate with the known structures of the breast, but these variations are small compared to the subject-to-subject variation. The intrasubject variations are larger for the scattering data than the absorption data; this is consistent with different spatial localization of the measurements of these parameters, which is explained by the photon migration theory.

Antitumor efficacy of the combination of photodynamic therapy and chemotherapy in murine tumors

Photodynamic therapy (PDT) is based on the administration of tumor-localizing photosensitizers followed by light exposure of the tumor mass. The photocytotoxic effects are mainly caused by the generation of singlet oxygen. Recently, PDT has been proposed for use in combination with anticancer chemotherapy with a view to exploiting any additive antitumor effect. We investigated the effect of PDT with photoactivated aluminum disulfonated phthalocyanine (AlS2Pc) combined with the antiblastic drugs Adriamycin (ADR) and cisplatinum (CDDP) on murine tumors. Mice bearing L1210 leukemia and P388 lymphoma were treated with ADR or CDDP and subsequently treated with PDT. Low chemotherapy doses were ineffective, but the combination of antiblastic drugs + PDT had a significantly additive antitumor effect. In conclusion, with this combined therapy we were able to greatly reduce the effective doses of antiblastic drugs, thus lowering their toxic effects on normal host tissues.

Fluorescence lifetime imaging: an application to the detection of skin tumors

A portable system based on fluorescence lifetime imaging has been developed and tested for the detection of skin tumors in humans. The Heme precursor /spl delta/-aminolevulinic acid, which promotes the preferential accumulation of the endogenous Protoporphyrin IX (PpIX) in proliferative tissues, is used as an exogenous marker to target the tumor, /spl delta/-aminolevulinic acid is topically administered to the patient 1 h before the measurement. Then, using a gated intensified camera, two or more images of the sample are acquired after different delays with respect to the excitation pulses. The images are processed in real time in order to calculate the spatial map of the fluorescence decay time of the sample. The localization of the tumor is based on the longer decay time detected in neoplastic tissues as a result of the stronger emission of PpIX, which has a long decay time, and the reduction in the short living natural tissue fluorescence.

A system for time-resolved laser fluorescence spectroscopy with multiple picosecond gating

A computer‐controlled system for time‐resolved fluorescence spectroscopy is described. The system provides the simultaneous acquisition of the fluorescence decay curve, of the time‐integrated emission spectrum, and of two time‐gated spectra. Gating within the fluorescence decay is provided by setting up windows in the output of the time‐to‐amplitude converter in single photon timing instrumentation. The gate parameters (position and width) are fully programmable. Depending on the laser source used, time resolutions between 45 and 150 ps are obtained. Therefore, in most situations no deconvolution procedures are required to elaborate the spectra. The system has been characterized with the aid of suitable test solutions. The results indicate that a correct choice of the gate parameters allows complementary information (in terms of both decay times and emission spectra) of the fluorescence parameters of molecular species in molecular systems.

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL

Abstract— In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin‐type chromophores packed in highly osmiophilic subpellicular granules. Liposomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light‐induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady‐state and time‐resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L‐a‐phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.

Efficacy of photodynamic therapy against doxorubicin-resistant murine tumors

Photodynamic therapy (PDT) is a relatively new cancer treatment modality that employs light excitation of a photosensitizer to yield cytotoxic oxygen-related species. In the present study we explored whether PDT would have therapeutic effect against doxorubicin-resistant murine tumors. We compared the efficacy of PDT with aluminium disulphonated phthalocyanine (A1S2Pc) and laser light on the doxorubicin-sensitive murine tumors, B16 melanoma (B16), L1210 leukemia (L1210), P388 lymphoma (P388) and the corresponding doxorubicin-resistant lines (B16/Dx, L1210/Dx and P388/Dx). Mice bearing L1210-L1210/Dx, P388-P388/Dx and B16-B16/Dx, were treated with 5 mg/kg of A1S2Pc and laser light (100 mW/cm2 x 10 min of exposure) or with doxorubicin (10 or 12 mg/kg i.v.). The results show that PDT is active versus all tumors while doxorubicin is effective only against the three sensitive tumor lines (L1210, P388 and B16). These observations suggest that PDT might be a beneficial alternative treatment for drug-resistant tumors.

Time-Resolved Optical Spectroscopy of Wood

We have proposed and experimentally demonstrated that picosecond time-resolved optical spectroscopy in the visible/near-infrared (NIR) region (700–1040 nm) is a useful technique for noninvasive characterization of wood. This technique has been demonstrated on both softwood and hardwood samples treated in different ways simulating the aging process suffered by waterlogged woods. In all the cases, alterations of absorption and scattering spectra were observed, revealing changes of chemical and structural composition.

TIME‐GATED FLUORESCENCE OF BLEPHARISMIN, THE PHOTORECEPTOR PIGMENT FOR PHOTOMOVEMENT OF Blepharisma

Abstract— A computer‐controlled apparatus for time‐resolved laser fluorescence spectroscopy has been used to measure fluorescence lifetimes, time‐integrated and time‐gated spectra of crude extracts of blepharismin, the photoreceptor pigment of the ciliated photoresponsive protozoan Blepharisma japonicum, in ethanol, aqueous solutions and detergent micelles. The effect of hydroxyl concentration has been investigated in both alcohol and water solutions. A short‐living (0.2‐0.4 ns) molecular species, emitting at 600 nm, is predominant in aqueous solutions at pH < 11.7, whereas in pure ethanol solutions an intermediate‐living species (about 1 ns), still fluorescing at 600 nm, prevails. Upon increasing OFF concentration, a third, long‐living (about4–6 ns) molecular species, emitting at 660 nm, is formed in all the examined media. This species has been tentatively identified as the negatively charged form of the photoreceptor pigment, whereas the short‐living and the intermediate‐living fluorescence emissions have been attributed respectively to the phenolic and the quinonic neutral forms of blepharismin. The phenolic form in its ground state is suggested to be the molecular species from which proton release occurs.