Dominique Ettori

Laser Induced Autofluorescence Diagnosis of Bladder Tumors: Dependence on the Excitation Wavelength

PURPOSE We assessed the ability of laser induced autofluorescence spectroscopy to distinguish neoplastic urothelial bladder lesions from normal or nonspecific inflammatory mucosa. MATERIALS AND METHODS Three different pulsed laser excitation wavelengths were used successively: 308 nm. (xenium chloride excimer laser), 337 nm. (nitrogen laser) and 480 nm. (coumarin dye laser). The excitation light was delivered by a specially devised multifiber catheter connected to a 1 mm. core diameter silica monofiber introduced through the working channel of a standard cystoscope with saline irrigation. The captured fluorescence light was focused onto an optical multichannel analyzer detection system. Device performance was evaluated in 25 patients after obtaining consent and immediately before transurethral resection of a bladder tumor. Spectroscopic results were compared with histological findings. RESULTS At 337 and 480 nm. excitation wavelengths the overall fluorescence intensity of bladder tumors was clearly decreased compared to normal urothelial mucosa regardless of tumor stage and grade. At the 308 nm. excitation wavelength the shape of the tumor spectra, including carcinoma in situ, was markedly different from that of normal or nonspecific inflammatory mucosa. No absolute intensity determinations were required in this situation, since a definite diagnosis could be established based on the fluorescence intensity ratio at 360 and 440 nm. CONCLUSIONS This spectroscopic study could be particularly useful to design a simplified autofluorescence imaging device for detection of occult urothelial neoplasms.

CORRELATION BETWEEN THE KINETICS OF ANTHRACYCLINE UPTAKE AND THE RESISTANCE FACTOR IN CANCER CELLS EXPRESSING THE MULTIDRUG RESISTANCE PROTEIN OR THE P-GLYCOPROTEIN

Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins, the 170-kDa P-glycoprotein (Pgp) and the 190-kDa multidrug resistance-associated protein (MRP1). One possible pharmacological approach to overcome drug resistance is the use of specific inhibitors, which enhance the cytotoxicity of known antineoplastic agents. However, while many compounds have been proven to be very efficient in inhibiting Pgp activity only some of them are able to inhibit MRP1. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs with physicochemical properties favoring the uptake of the drug by the resistant cells. The intracellular drug retention influences its cytotoxic effect. The level of the intracellular drug content is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). In this work, the kinetics of drug uptake and the kinetics of active efflux of several anthracycline derivatives in both Pgp expressing K562/Adr cells and MRP1 expressing GLC4/Adr cells was determined. Our data have shown that in both cell lines there is no correlation between the resistance factor and the kinetics of drug efflux by these pumping systems. However, a very good correlation between the resistance factor and the kinetics of drug uptake has been established in both cell lines: the resistance factor decreases when the kinetics of drug uptake increases. This work has clearly shown that when the rate of transmembrane transport of anthracycline is high enough, the efflux mediated by the protein transporter is not able to pace with it. The protein transporter essentially operates in a futile cycle and the resistance factor is tending to one. It does not mean, however, that when the resistance factor is close to one the anthracycline is not transported by the pump.

Influence of the emission-reception geometry in laser-induced fluorescence spectra from turbid media.

Routine clinical detection of precancerous lesions by laser-inducedautofluorescence was recently demonstrated in several medicalfields. This technique is based on the analysis of complex spectrawith overlapping broad structures. However, in biological tissues, scattering and absorption are wavelength dependent, and the observedfluorescence signals are distorted when the illumination and detectiongeometry varies, making comparison of results from different groupsdifficult. We study this phenomenon experimentally in human tissuein a simple experiment: A fiber is used for the excitation and anidentical fiber is used for reception of the signal; both fibers aremaintained in contact with the tissue. We study the distortion ofthe spectra as a function of the distance between the twofibers. For correction of the spectra we show that it is possibleto use a fast and accurate ab initio Monte Carlo simulationwhen the spectral variations of the optical properties of the mediumare known. The main advantage of this simulation is itsapplicability even for complex boundary conditions or when the sampleconsists of several layers.

ULTRAVIOLET LASER-INDUCED AUTOFLUORESCENCE DISTINCTION BETWEEN MALIGNANT AND NORMAL UROTHELIAL CELLS AND TISSUES

The aim of this study was to perform a preliminary evaluation of the diagnostic potential of laser induced autofluorescence spectroscopy (LIAFS) for urothelial tumors using fluorescence intensity ratios at different wavelengths. After testing three laser excitation wavelengths (308, 337, and 480 nm) in normal and malignant bladder cell lines, 308 nm appeared to be the most promising wavelength since two fluorescence bands were observed at 360 and 440 nm; these were attributed to tryptophan (Trp) and reduced nicotinamide adenine dinucleotide (NADH) respectively. This study was then performed on freshly removed normal bladder and bladder tumor specimens exclusively using the 308-nm excitation wavelength. The tumor spectra, regardless of stage and grade, were very similar to the malignant cell spectra. However, a marked reduction of overall intensity was observed for carcinoma in situ (CIS). Normal bladder mucosa exhibited a shift of the first fluorescence band to 380 nm, indicating an overlap of Trp urothelial cell emission and collagen fluorescence derived from the lamina propria. The intensity of the NADH emission band was markedly reduced in tumor tissues compared with normal mucosa, which could indicate different redox conditions in urothelial tumors. A fluorescence intensity ratio at 360 and 440 nm can accurately discriminate normal or inflammatory mucosa from all bladder tumors, including CIS. These findings support the use of LIAFS as a new diagnostic technique for occult urothelial tumors.

Ultimate spatial resolution with Diffuse Optical Tomography

We evaluate the ultimate transverse spatial resolution that can be expected in Diffuse Optical Tomography, in the configuration of projection imaging. We show how such a performance can be approached using time-resolved measurements and reasonable assumptions, in the context of a linearized diffusion model.

Monte Carlo evaluation of laser-induced fluorescence spectra modifications due to optical properties of the medium: Application to real spectra correction

Laser induced fluorescence has often been used as a diagnostic method. Unfortunately the fluorescence signal is modified during the photons migration towards the detector. The purpose of this study is to determine the alterations of the laser induced fluorescence spectra in white matter of adult brain due to the spectral variations of the optical coefficients (mu) a((lambda) ), (mu) s((lambda) ) and of the mean cosine of the scattering angle g((lambda) ).

Time-resolved diffusing wave spectroscopy beyond 300 transport mean free paths

We presented theoretical and experimental demonstrations of the possibilities of performing time-resolved diffusing wave spectroscopy: We successfully registered field fluctuations for selected photon path lengths that can surpass 300 transport mean free paths. Such performance opens new possibilities for biomedical optics applications.

Laser-induced autofluorescence diagnosis of tumors

The feasibility of routine real-time clinical detection of precancerous lesions such as severe dysplasia and carcinoma in situ (CIS) by laser induced autofluorescence (LIAF), that is without the use of exogenous fluorescent marker, has been recently demonstrated in several medical fields. We present here an in vivo autofluorescence study we have undergone on normal, suspicious and tumoral areas of human bladder. Three different pulsed laser wavelengths were alternately used for excitation: 488 nm (Dye laser), 337 nm (Nitrogen laser), and 308 nm (XeCl excimer laser). A clinical endoscopic study was performed on 25 patients. Spectroscopic results were compared with histological analysis. For 488 nm and 337 nm excitation a single fluorescence broad band was obtained in any case, but for tumors the overall intensity was significantly reduced compared to normal mucosa. For 308 nm excitation we observed two main broad bands, centered respectively at about 360 nm and 440 nm. In case of neoplastic lesions (including carcinoma in situ), the intensity ratio [I(360nm)/I(440nm)] was always greater than 2, for normal or inflammatory areas it was less than 2. A clear diagnosis could then be achieved for 308 nm excitation with no need of absolute intensity measurements. However, in turbid media, the optical properties of the medium are wavelength dependent. As a consequence the observed fluorescence signals are different from the isolated fluorophores spectra and their shape depends also on the illumination and detection geometry. We show how this phenomenon can be taken into account for useful comparison of results from different groups.

Fourier transform acousto-optic imaging with a custom-designed CMOS smart-pixels array

We report acousto-optic imaging (AOI) into a scattering medium using a Fourier Transform (FT) analysis to achieve axial resolution. The measurement system was implemented using a CMOS smart-pixels sensor dedicated to the real-time analysis of speckle patterns. This first proof-of-principle of FT-AOI demonstrates some of its potential advantages, with a signal-to-noise ratio comparable to the one obtained without axial resolution, and with an acquisition rate compatible with a use on living biological tissue.

Synthesis and binding properties of photoactivable biotin-conjugated verapamil derivatives for the study of P-170 glycoprotein.

The design and synthesis of two photoactivable biotin-labeled analogues of verapamil (6 and 7) is reported. Preliminary evaluation of the biological profile of 6 (EDP 137) and 7 (EDP 141) shows that they have comparable affinities to that of verapamil for P-170, the protein responsible for multidrug resistance (MDR). Since both appear to bind irreversibly to the protein and the presence of biotin in their structure makes them easily detectable by avidin, they promise to be of great help in studying the protein and its mechanism of action.