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The current clinical study is aimed at evaluating the clinical relevance of an innovative device (called CyPaM2 device) that for the first time provides urologists with (i) a panoramic image of the bladder inner wall within the surgery time, and with (ii) a simultaneous (bimodal) display of fluorescence and white-light video streams during the fluorescence assisted-transurethral resection of bladder cancers procedure. The clinical relevance of this CyPaM2 device was evaluated on 10 patients according to three criteria (image quality, fluorescent lesions detection relevance, and ergonomics) compared with a reference medical device. Innovative features displayed by the CyPaM2 device were evaluated without any possible comparison: (i) simultaneous bimodal display of white-light and fluorescence video streams, (ii) remote light control, and (iii) time delay for the panoramic image building. The results highlight the progress to achieve in order to obtain a fully mature device ready for commercialization and the relevance of the innovative features proposed by the CyPaM2 device confirming their interest.

Multiphoton microscopy (MPM) has the capacity to record second-harmonic generation (SHG) and endogenous two-photon excitation fluorescence (2PEF) signals emitted from biological tissues. The development of fiber-based miniaturized endomicroscopes delivering pulses in the femtosecond range will allow the transfer of MPM to clinical endoscopy. We present real-time SHG and 2PEF ex vivo images using an endomicroscope, which totally complies with clinical endoscopy regulations. This system is based on the proximal scanning of a commercial multicore image guide (IG). For understanding the inhomogeneities of the recorded images, we quantitatively characterize the IG at the single-core level during nonlinear excitation. The obtained results suggest that these inhomogeneities originate from the variable core geometries that, therefore, exhibit variable nonlinear and dispersive properties. Finally, we propose a method based on modulation of dispersion precompensation to address the image inhomogeneity issue and, as a proof of concept, we demonstrate its capability to improve the nonlinear image quality.

To approach wide-field optical properties quantification in real heterogeneous biological tissue, we developed a Dual-Step setup that couples a punctual diffuse reflectance spectroscopy (DRS) technique with multispectral imaging (MSI). The setup achieves wide-field optical properties assessment through an initial estimation of scattering with DRS, which is used to estimate absorption with MSI. The absolute quantification of optical properties is based on the ACA-Pro algorithm that has been adapted both for DRS and for MSI. This paper validates the Dual-Step system not only on homogeneous Intralipid phantoms but also on a heterogeneous gelatine phantom with different scattering and absorbing properties., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

We have developed an adaptive calibration algorithm and protocol (ACA-Pro) that corrects from the instrumental response of various spatially resolved diffuse reflectance spectroscopy (DRSsr) systems to enable the quantification of absorption and scattering properties based on a Monte Carlo-based look-up-table approach. The protocol involves the use of a calibration reference base built with measurements of a range of different diffusive intralipid phantoms. Moreover, an advanced strategy was established to take into account the experimental variations with an additional measurement of a common solid material, allowing the use of a single calibration reference base for all experiments. The ACA-Pro is validated in contact and noncontact probe-based DRSsr systems. Furthermore, the first results of a setup replacing the probe with a CCD detector are shown to confirm the robustness of the approach.

We present a two-photon microendoscope capable of in vivo label-free deep-tissue high-resolution fast imaging through a very long optical fiber. First, an advanced light-pulse spectro-temporal shaping device optimally precompensates for linear and nonlinear distortions occurring during propagation within the endoscopic fiber. This enables the delivery of sub-40-fs duration infrared excitation pulses at the output of 5 meters of fiber. Second, the endoscopic fiber is a custom-made double-clad polarization-maintaining photonic crystal fiber specifically designed to optimize the imaging resolution and the intrinsic luminescence backward collection. Third, a miniaturized fiber-scanner of 2.2 mm outer diameter allows simultaneous second harmonic generation (SHG) and two-photon excited autofluorescence (TPEF) imaging at 8 frames per second. This microendoscope's transverse and axial resolutions amount respectively to 0.8 μm and 12 μm, with a field-of-view as large as 450 μm. This microendoscope's unprecedented capabilities are validated during label-free imaging, ex vivo on various fixed human tissue samples, and in vivo on an anesthetized mouse kidney demonstrating an imaging penetration depth greater than 300 μm below the surface of the organ. The results reported in this manuscript confirm that nonlinear microendoscopy can become a valuable clinical tool for real-time in situ assessment of pathological states.

Probe-based confocal laser endomicroscopy (pCLE) allows microscopic imaging of the alveoli during bronchoscopy. The objective of the study was to assess the diagnostic accuracy of pCLE for amiodarone-related pneumonia (AMR-IP). Alveolar pCLE was performed in 36 nonsmoking patients, including 33 consecutive patients with acute or subacute interstitial lung disease (ILD), of which 17 were undergoing treatment with amiodarone, and three were amiodarone-treated patients without ILD. Nine out of 17 patients were diagnosed with high-probability AMR-IP (HP-AMR-IP) by four experts, and three separate observers. Bronchoalveolar lavage findings did not differ between HP-AMR-IP and low-probability AMR-IP (LP-AMR-IP) patients. In HP-AMR-IP patients, pCLE showed large (>20 μm) and strongly fluorescent cells in 32 out of 38 alveolar areas. In contrast, these cells were observed in only two out of 39 areas from LP-AMR-IP patients, in one out of 59 areas from ILD patients not receiving amiodarone and in none of the 10 areas from amiodarone-treated patients without ILD (p<0.001; HP-AMR-IP versus other groups). The presence of at least one alveolar area with large and fluorescent cells had a sensitivity, specificity, negative predictive value and positive predictive value for the diagnosis of AMR-IP of 100%, 88%, 100% and 90%, respectively. In conclusion, pCLE appears to be a valuable tool for the in vivo diagnosis of AMR-IP in subacute ILD patients.

Several major lung pathologies are characterized by early modifications of the extracellular matrix (ECM) fibrillar collagen and elastin network. We report here the development of a nonlinear fiber-optic spectrometer, compatible with an endoscopic use, primarily intended for the recording of second-harmonic generation (SHG) signal of collagen and two-photon excited fluorescence (2PEF) of both collagen and elastin. Fiber dispersion is accurately compensated by the use of a specific grism-pair stretcher, allowing laser pulse temporal width around 70 fs and excitation wavelength tunability from 790 to 900 nm. This spectrometer was used to investigate the excitation wavelength dependence (from 800 to 870 nm) of SHG and 2PEF spectra originating from ex vivo human lung tissue samples. The results were compared with spectral responses of collagen gel and elastin powder reference samples and also with data obtained using standard nonlinear microspectroscopy. The excitation-wavelength-tunable nonlinear fiber-optic spectrometer presented in this study allows performing nonlinear spectroscopy of human lung tissue ECM through the elastin 2PEF and the collagen SHG signals. This work opens the way to tunable excitation nonlinear endomicroscopy based on both distal scanning of a single optical fiber and proximal scanning of a fiber-optic bundle.

Hypericin is a photosensitizer expressing high affinity for cancerous cells in vivo. Diagnosis of cancer based on hypericin fluorescence imaging has been successfully assessed in several clinical trials. Our final objective will be to evaluate the potential of hypericin fluorescence imaging to improve the efficacy of cervical cancer diagnosis performed on fixed cell smears obtained from liquid-based cytology. For this purpose, the mechanism of hypericin incorporation and localization in fixed HeLa cells using different incubation media and fixation conditions was investigated. Since the duration of fixation may play an important role, the influence of fixation time on hypericin incorporation in fixed HeLa cells was studied. The uptake and distribution of hypericin in fixed HeLa cells were found to be strongly dependent on the hypericin incubation medium: for a polar organic solvent such as the alcohol-based fixative, the localization was essentially perinuclear and nuclear; for cell culture medium supplemented with serum, the localization was cytoplasmic and non-specific; the highest incorporation was observed for the serum-free culture medium but mainly as non-fluorescent aggregates. The hypericin aggregation in the incubation medium, the passive diffusion and the partitioning between the cells and hypericin carriers seemed to be the major factors accounting for these results. The localization was found to be weakly dependent on fixation time, whereas fluctuations of hypericin fluorescence at short fixation time and stabilization after two days of fixation were observed. These results suggest that the fixed cells reached a steady state after two days of fixation.

A photosensitizer is defined as a chemical entity able to induce, under light-irradiation effect, a chemical or physical alteration of another chemical entity. Thanks to their preferential retention in proliferating tissues, some photosensitizers are therapeutically used such as in photodynamic therapy (PDT). Besides, this method has already been approved for several indications. The selectivity of photosenzitizers for cells in proliferation involves both their association with low density lipoproteins (LDLs) and their ability to cross membranes under various pH conditions. The photosensitizers used are in most cases based on the porphyrin structure, but other compounds, of which far-red-light absorption properties are most compatible with biological tissues irradiation, have been developed, such as phthalocyanines. This paper presents physico-chemical studies of the interaction of a disulfonated aluminium phthalocyanine (AlPcS2) with human LDLs. The data obtained are compared with the parameters of the interaction of these lipoproteins with deuteroporphyrin (DP) and chlorin e6 (Ce6). A close attention is paid to the dynamic aspects of these phenomena. The data obtained on these simple systems then allowed us to interpret the sub-cellular localization of the photosensitizers on a human line of fibroblasts, and to evaluate the influence of LDLs on the intracellular distribution of the compounds. This last point is of major importance because the localization of such photosensitizers (in particular AlPcS2) in endocytic vesicles and their subsequent ability to induce a release of the contents of these vesicles - including externally added macromolecules - into the cytosol is the basis for a recent method for macromolecule activation, named photochemical internalization (PCI). PCI has been shown to potentiate the biological activity of a large variety of macromolecules. The comprehension of the mechanisms governing this particular sub-cellular localization could allow the design of better candidates for PCI.

The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (~ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.

Decrease in interstitial pH of the tumor stroma and over-expression of low density lipoprotein (LDL) receptors by several types of neoplastic cells have been suggested to be important determinants of selective retention of photosensitizers by proliferative tissues. The interactions of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic groups, with plasma proteins and DOPC unilamellar vesicles are investigated by fluorescence spectroscopy. The binding constant to liposomes, with reference to the DOPC concentration, is 6 x 10(3) M(-1) at pH 7.4. Binding of Ce6 to LDL involves about ten high affinity sites close to the apoprotein and some solubilization in the lipid compartment. The overall association constant is 5.7 x 10(7) M(-1) at pH 7.4. Human serum albumin (HSA) is the major carrier (association constant 1.8 x 10(8) M(-1) at pH 7.4). Whereas the affinity of Ce6 for LDL and liposomes increases at lower pH, it decreases for albumin. Between pH 7.4 and 6.5, the relative affinities of Ce6 for LDL versus HSA, and for membranes versus HSA, are multiplied by 4.6 and 3.5, respectively. These effects are likely driven by the ionization equilibria of the photosensitizer carboxylic chains. Then, the cellular uptake of chlorin e6 may be facilitated by its pH-mediated redistribution within the tumor stroma.

Cellular autofluorescence was characterized in normal human esophageal cells and in malignant esophageal epithelial cells. The study was performed under excitation at 351 nm where the cell fluorescence is mainly due to the reduced pyridine nucleotides (NAD(P)H) with a very small contribution from the oxidized flavins (FMN, FAD) or lipopigments. The autofluorescence emission of squamous cell carcinoma, adenocarcinoma on Barrett's mucosa and normal cells was characterized by microspectrofluorimetry on monolayers and by spectrofluorimetry on cell suspensions. The relative contribution of each fluorophore to the fluorescence emission of the different cell types was evaluated by a curve-fitting analysis. A statistically highly significant difference was observed between the average intensity of the raw spectra of the different cell types. Tumoral cells had a fluorescence intensity approximately twice as high as that of normal cells. The results of the NAD(P)H quantitation analyzed by microspectrofluorimetry on single living cells and spectrofluorimetry on cell suspensions were consistent with those obtained by biochemical cycling assays, showing that the amount of intracellular NAD(P)H is higher in tumoral cells than in normal cells. Bound NAD(P)H concentration was found to be quite stable whatever the cell type while the amount of free NAD(P)H showed a very important increase in tumoral cells.

The key role of gp120 in the cellular entry of HIV makes this glycoprotein an attractive target for new drugs. Various polyanions bind to the positively charged V3 loop of gp120. Here, we consider a series of anionic porphyrins bearing two sulfonate groups and two carboxylic chains with various degree of esterification. These molecules carry an overall negative charge between 4 and 2. Upon activation by light, these compounds, known as photosensitizers, produce highly reactive oxygen species able to damage amino acid chains. The interactions of these molecules with the V3 loop and a positively charged area in the C5 region were investigated in the dark by using specific antibodies and ELISA protocols. Competitive inhibition of the anti-V3 antibody was observed with an increased efficiency for the esterified compounds. No evidence for binding to the C5 region was found. In contrast, when gp120 was irradiated with light in presence of the porphyrin prior to the addition of the antibody, strong inhibition of the anti-C5 antibody was observed revealing irreversible photo-damages in this region. No effect on the V3 loop was observed. Irradiations at two wavelengths made it possible to identify porphyrin monomers as the photoactive forms despite the presence of large excess of dimers in the incubation solution. It is suggested that porphyrins bound to the V3 loop could produce photo-damages at some distance, in particular within the C5 region that contains several photosensitive amino acids.

Low-density lipoproteins (LDL) play a key role in the delivery of photosensitizers to tumor cells in photodynamic therapy. The interaction of deuteroporphyrin, an amphiphilic porphyrin, with LDL is examined at equilibrium and the kinetics of association/dissociation are determined by stopped-flow. Changes in apoprotein and porphyrin fluorescence suggest two classes of bound porphyrins. The first class, characterized by tryptophan fluorescence quenching, involves four well-defined sites. The affinity constant per site is 8.75 x 10(7) M(-1) (cumulative affinity 3.5 x 10(8) M(-1)). The second class corresponds to the incorporation of up to 50 molecules into the outer lipidic layer of LDL with an affinity constant of 2 x 10(8) M(-1). Stopped-flow experiments involving direct LDL porphyrin mixing or porphyrin transfer from preloaded LDL to albumin provide kinetic characterization of the two classes. The rate constants for dissociation of the first and second classes are 5.8 and 15 s(-1); the association rate constants are 5 x 10(8) M(-1) s(-1) per site and 3 x 10(9) M(-1) s(-1), respectively. Both fluorescence and kinetic analysis indicate that the first class involves regions at the boundary between lipids and the apoprotein. The kinetics of porphyrin-LDL interactions indicates that changes in the distribution of photosensitizers among various carriers could be very sensitive to the specific tumor microenvironment.

Dimerization of free acid and ester forms of disulfonated deuteroporphyrin is investigated in aqueous solution by absorbance and fluorescence spectroscopies. The dimerization equilibrium constant increases with the extent of esterification. In phosphate buffer saline (pH 7.4, 20 degrees C), it ranges from 1.4 x 10(6) M(-1) to 7.8 x 10(7) M(-1) for the free acid and the diethyl ester forms, respectively. The dimer formation is favored by an increase of ionic strength, as predicted by the Debye-Hückel law. The dimers display a marked shift to the blue of their Soret band. In agreement with the exciton model, a cofacial stacking of the molecules with some offset is postulated. The sulfonate groups on each molecule are likely to stand on opposite directions to reduce repulsion. Both the analysis of porphyrin self-association and careful examination of the fluorescence excitation spectra show that the dimers of disulfonated deuteroporphyrins do not fluoresce at all. The quantum yield of formation of singlet oxygen by the disulfonated deuteroporphyrins in deuterated methanol is 0.71, a value typical of monomers. In deuterated water, the yield is 0.44 for all the compounds studied though they are dimerized. The fact that nonfluorescent dimers of porphyrins can be efficient photosensitizers is emphasized.

Some guanine-rich oligonucleotides inhibit HIV infectivity through interaction with the gp120 glycoprotein. Besides, photoinactivation of viruses attracts attention for blood decontamination. The feasibility of targeting a red light-absorbing chlorin-type photosensitizer to gp120 through covalent coupling with 8-mer phosphodiester oligodeoxynucleotides is investigated. Some conjugates inhibit binding of antibodies directed to gp120. Inhibition is significantly increased upon red light activation. The activity of the conjugates correlates with their ability to self-associate, a process strongly favored by the propensity of the hydrophobic chlorin moiety to dimerize. Thus, the photosensitizer moiety both promotes structures with a higher affinity for gp120 and, upon light activation, can induce site-directed damages to the protein.

The kinetics of hematoporphyrin or deuteroporphyrin incorporation in egg phosphatidylcholine small unilamellar vesicles have been investigated by fluorescence stopped-flow measurements. The processes can be described by a fast equilibrium. The on-rate constant is nearly diffusion controlled regardless of the compound used and the pH. The affinity of these porphyrins for the vesicles is merely governed by the exit rate which depends on the structure of the porphyrin and on its charge determined by pH.

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.

Some molecular aspects underlying photochemotherapy and photodiagnosis of tumors with porphyrins are reviewed. The nature of the clinically used photosensitizer HpD is first presented along with structures of molecules found to be efficient in vitro. The possible role of pH in the preferential retention of dicarboxylic porphyrins by tumors is discussed in light of results obtained with membrane models. The uptake of dicarboxylic porphyrins by cells most likely involves passive mechanisms. Cell photoinactivation using a purified porphyrin does not depend upon the incubation time but only on the intracellular concentration of the dye. This likely reflects a poor specificity of the photoinactivation processes with regard to the cellular localization of the dye. The properties which should be presented by more efficient photosensitizers are discussed.