Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study.

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS4) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C6 symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane. The R ratio changes for the 2D crossing fibers observed in cornea tissue. The 3D crossing of fibers also affects R ratio in cartilage tissue. The distinctly different dependence of R on crossing and tilting of fibers is demonstrated in collagen and TPPS4 aggregates, due to the achiral molecular susceptibility ratio having values below and above 3, respectively. The polarimetric microscopy results correspond well with the analytical expressions of amplitude and R and C ratios dependence on the crossing angle of the fibers. The experimentally measured SHG intensity and R and C ratio maps are consistent with the computational modeling of various fiber configurations presented in the preceding article. The demonstrated SHG intensity and R and C ratio dependencies on fibril configurations provide the basis for interpreting polarimetric SHG microscopy images in terms of 3D ultrastructural organization of fibers in each voxel of the samples.

Interactions of semiconductor Cd-based quantum dots and Cd2+ with gut bacteria isolated from wild Salmo trutta fry.

Background: With the rapid development of nanotechnology, more and more nanoproducts are being released into the environment where they may both pose ecological risks and be toxic to living organisms. The ecotoxicological impact of quantum dots (QDs), a class of nanoparticles (NPs), on aquatic organisms is becoming an emerging issue, this due to their nano-specific properties, to the physico-chemical transformation in the environment and to the possible release of toxic metals from their structure such as Cd. Methods: In this work, (i) spectroscopic measurements of commercially available Cd-based QDs (CdSe/ZnS-COOH) were made at various pH values (5.0 and 7.0) to study their interactions (at a concentration of 4 nm) with various strains of Gram-positive and Gram-negative gut bacteria after short-term exposure and (ii) the antibacterial efficacy of QDs and Cd2+ (at a concentration 0.09-3.56 mM) against gut bacteria isolated from wild freshwater Salmo trutta fry was studied at different temperatures (15 °C and 25 °C) and pH values (5.0 and 7.0) by applying a well-established disc diffusion assay. Results: Twenty-six gut bacterial isolates from wild Salmo trutta fry were identified as Aeromonas spp., A. popoffii, A. salmonicida, A. sobria, Carnobacterium maltaromaticum, Buttiauxella sp., Listeria sp., Microbacterium sp., Shewanella putrefaciens and Serratia sp. Cd-based (CdSe/ZnS-COOH) QDs at a concentration of 4 nm were found to be stable in aqueous media (with pH 7.0) or starting to form aggregates (at pH 5.0), thus, apparently, did not release heavy metals (HMs) into the media over 48 h in conditions of light or dark and did not show antibacterial efficacy on the gut bacteria isolated from wild Salmo trutta fry after short-term (9 h and 48 h) incubations. Cd2+ was found to produce significant dose-dependent toxic effects on bacterial growth, and the size of the inhibition zones on some of the tested strains significantly correlated with temperature. The most sensitive and the most resistant to Cd2+ were the Gram-positive bacteria, for which the minimum inhibitory concentration (MIC) values of Cd2+ were 0.09-0.27 mM and 3.11-3.29 mM respectively and varied significantly between the tested temperatures (15 °C and 25 °C). The MIC values of Cd2+ for the Gram-negative bacteria (18 out of 22 strains) ranged from 0.44 to 0.71 mM and did not differ significantly between the tested temperatures. Among the selected Gram-positive and Gram-negative strains, those with the higher sensitivity towards Cd2+ also revealed relatively stronger signals of QDs photoluminescence (PL) when transferred after incubation into fresh medium without QDs. In addition, the formation of endogenous metalloporphyrins observed spectroscopically in some bacterial strains indicates certain differences in metabolic activity that may play a protective role against potential oxidative damage.

Photoinactivation of Salmonella enterica exposed to 5-aminolevulinic acid: Impact of sensitization conditions and irradiation time.

The photodynamic inactivation (PDI) represents the potential alternative to traditional antibiotic therapy, and can be applied to treat various bacterial infections, including those caused by Gram-negative bacterial strains. One of the treatment modalities is based on the capacity of bacterial cells to synthesize the excess amounts of porphyrins after exposure to an externally applied 5-aminolevulinic acid (5-ALA), which makes them photosensitive and leads to reduced survival after irradiation with an appropriately selected light source. This study focuses on the sensitization and the photoinduced inactivation of Salmonella enterica cells in PBS containing 0.5 mM 5-ALA, incubated at 37 °C for 4 h or for 20 h and afterwards irradiated with violet LED light (11.1 mW/cm2, a peak at 400 nm). It has been found that both amounts and composition of endogenous porphyrins not only depended on the incubation duration, but also were affected by externally induced photo- and chemo-oxidation reactions. The application of different sensitization conditions has revealed that the increasing amounts of endogenously produced porphyrins do not ensure the proportional reduction of bacterial cell survival numbers. The comparative investigations also demonstrated that the presence of endogenously produced porphyrins in the medium results in secondary sensitization of bacterial cells and causes a notably stronger photoinactivation effect in comparison to their externally applied standards.

Inactivation of Opportunistic Pathogens Acinetobacter baumannii and Stenotrophomonas maltophilia by Antimicrobial Photodynamic Therapy.

Acinetobacter baumannii and Stenotrophomonas maltophilia are opportunistic pathogens causing hospital infections with limited treatment options due to bacterial multidrug resistance. Here, we report that antimicrobial photodynamic therapy (aPDT) based on the natural photosensitizers riboflavin and chlorophyllin inactivates A. baumannii and S. maltophilia. The riboflavin and chlorophyllin photostability experiments assessed the photomodifications of photosensitizers under the conditions subsequently used to inactivate A. baumannii and S. maltophilia. A. baumannii planktonic cells were more sensitive to riboflavin-aPDT, while biofilm bacteria were more efficiently inactivated by chlorophyllin-aPDT. S. maltophilia planktonic and biofilm cells were more susceptible to chlorophyllin-aPDT compared to riboflavin-aPDT. The results suggest that riboflavin- and chlorophyllin-aPDT can be considered as a potential antimicrobial treatment for A. baumannii and S. maltophilia inactivation.

Application of Antimicrobial Photodynamic Therapy for Inactivation of Acinetobacter baumannii Biofilms.

Acinetobacter baumannii is a dangerous hospital pathogen primarily due to its ability to form biofilms on different abiotic and biotic surfaces. The present study investigated the effect of riboflavin- and chlorophyllin-based antimicrobial photodynamic therapy, performed with near-ultraviolet or blue light on the viability of bacterial cells in biofilms and their structural stability, also determining the extent of photoinduced generation of intracellular reactive oxygen species as well as the ability of A. baumannii to form biofilms after the treatment. The efficacy of antimicrobial photodynamic therapy was compared with that of light alone and the role of the photosensitizer type on the photosensitization mechanism was demonstrated. We found that the antibacterial effect of riboflavin-based antimicrobial photodynamic therapy depends on the ability of photoactivated riboflavin to generate intracellular reactive oxygen species but does not depend on the concentration of riboflavin and pre-incubation time before irradiation. Moreover, our results suggest a clear interconnection between the inactivation efficiency of chlorophyllin-based antimicrobial photodynamic therapy and the sensitivity of A. baumannii biofilms to used light. In summary, all the analyzed results suggest that riboflavin-based antimicrobial photodynamic therapy and chlorophyllin-based antimicrobial photodynamic therapy have the potential to be applied as an antibacterial treatment against A. baumannii biofilms or as a preventive measure against biofilm formation.

Protein-stabilized gold nanoclusters for PDT: ROS and singlet oxygen generation.

Suitable properties as well as eco-friendly synthesis of photoluminescent Au nanoclusters (NCs) make them promising compounds for biomedical diagnostics and visualization applications. However, the potential photochemical activity of such agents on cancerous cells is largely unknown. The nanoclusters (BSA-Au NCs) were synthetized in the presence of BSA (an average hydrodynamic diameter was about 9.4 nm, while the size of the metal cluster was <1.3 nm according to atomic force microscopy measurements) and possessed a broad photoluminescence band at 680 nm in buffered (pH 7.2) aqueous medium. The photochemical activity was studied by adding two fluorescent probes (dihydrorhodamine or Singlet Oxygen Sensor Green) for detection of reactive oxygen species in samples irradiated at 405 nm to minimize direct excitation of the probes. The photoluminescence measurements evidenced the capability of BSA-Au NCs to generate reactive oxygen species upon light exposure, while the observed sensitivity of the photoluminescence properties might be used to indicate photooxidative processes in the medium. The viability test performed on breast cancer cells after incubation with BSA-Au NCs and subsequent irradiation revealed notable difference in induced phototoxicity between two cell lines, which was not the case after the corresponding treatment using the photosensitizer chlorin e6.

Light-mediated effects of CdTe-MSA quantum dots on the autofluorescence of freshwater green microalgae: Spectroscopic studies.

The water-soluble semiconductor quantum dots (QDs) serve as optically detectable models of nanoparticles and are commonly applied as photoluminescent markers in biological systems. The unicellular algae represent a popular model system suitable to evaluate pollution-induced effects. There is growing experimental evidence that release of metal ions cannot account for potential toxicity of metal containing nanoparticles, however, the underlying mechanisms are not clearly understood. Surrounding environment and illumination conditions are among the most important factors affecting the stability of QDs as well as the interaction between nanoparticles and cells such as microalgae. The measurements of changes in photoluminescence (PL) of QDs and autofluorescence (AF) of microalgae can thus be used as a non-invasive screening method for detecting mutual effects of nanoparticles and algae cells on each other under natural conditions. In this study, CdTe quantum dots (a peak of PL at 550 nm) capped with a mercaptosuccinic acid (MSA) were introduced into aqueous ionic medium containing wild type green freshwater microalgae Scenedesmus and Chlorella sp. cells under artificial and natural ambient illumination. The spectroscopy and microscopy techniques were applied to observe both the influence of the microalgae on the spectral properties of negatively charged CdTe-MSA quantum dots and the effects of nanoparticles on the microalgae. The presence of algae cells revealed a protecting effect on both medium-dependent and radiation-induced changes in photoluminescence properties of QDs, which could be related with the increased stability of the capping layer. The effects on cellular AF intensity and the interaction of QDs with cellular surface depended on type of microalgae. The observed changes in AF spectral properties and AF induction signals can't be explained only by the photodegradation of QDs and have revealed the ability of nanoparticles to retard the photoadaptation of wild type microalgae under naturally varying illumination conditions.

Sensitization of Salmonella enterica with 5-aminolevulinic acid-induced endogenous porphyrins: a spectroscopic study.

Photodynamic therapy (PDT) of bacterial strains presents an attractive potential alternative to antibiotic therapies in search of the solution for the chemoresistance problem. The efficacy of the treatment is dependent on the interaction of photochemically active substances called photosensitizers (PSs) with the bacterial cell wall or their intracellular accumulation. In addition to exogenous PSs, other molecules such as 5-aminolevulinic acid (5-ALA), a natural precursor of heme, are gaining interest. When provided exogenously to cells, 5-ALA uptake results in the overproduction of various photoactive porphyrins. The pattern of their intracellular accumulation and release to the surroundings depends on incubation conditions such as the applied 5-ALA concentration, cell density and incubation duration. The detection of endogenously synthesized porphyrins in samples of Salmonella enterica cells and supernatants was accomplished after 4 h and 20 h incubation periods by means of fluorescence spectroscopy. The relative proportions of different types of porphyrins were assessed by modeling the registered spectra with the fluorescence spectra of standard porphyrins. After the shorter incubation period, the dominant porphyrins in the supernatant medium were coproporphyrins. The longer incubation period shifted the relative proportion of intracellular porphyrins from protoporphyrin IX towards water-soluble porphyrins such as uroporphyrin I, which interfered with additional by-products. The time-dependent changes in compositions of both intracellular and extracellular porphyrins imply that 5-ALA-induced sensitization might have triggered a complex protective mechanism of bacterial cells. Thus, identification and evaluation of the relative amounts of porphyrins, which accumulate in bacterial cells and are extruded outside after different time periods, could provide access to valuable information, working towards more efficient applications of 5-ALA-based antibacterial PDT.

Cell surface markers and exogenously induced PpIX in synovial mesenchymal stem cells.

The aim of present study was to assess the expression of surface markers and the accumulation of protoporphyrin IX in synovial mesenchymal stem cells (SMSCs). SMSC from patients with rheumatoid arthritis (RA, n = 5) and osteoarthritis (OA, n = 5-6) were characterized and their PpIX accumulation rates were evaluated by flow cytometry. The expression of the 21 out of 24 tested surface markers, related to stem-like features and aggressiveness of cells showed no statistically significant differences between RA and OA groups. However, the cells from RA group had the significantly lower levels of expression for the integrin-associated protein CD47 and the grow factor receptor CD271 (P = 0.018), while the higher levels of cell membrane zinc-dependent metalloproteinase CD10 (P = 0.006), as compared to the cells from OA group. Comparison of the mean intensities of PpIX fluorescence revealed no statistically significant differences between the RA and OA groups, as well as no relation to proliferation rates or cell size, although some conspicuous distinction in PpIX accumulation was observed in certain specimens within these groups, suggesting possibilities of this method application for characterization of individual SMSC populations. CD10, CD47, and CD271 were differently expressed in RA and OA SMSC, while had no direct association with the PpIX fluorescence intensity.

Suppression of a Specific Intracellular Uptake Pathway by a Saturating Accumulation of Quantum Dots.

Quantum dots (QDs) play an active role in triggering biological effects and should not be viewed as ordinary carriers for biomedical applications; therefore, the aim of this study was to investigate the molecular mechanisms involved in the saturating accumulation of non-targeted, carboxylated QDs, the related specific internalization pathways and the induced changes in the endocytotic cycle in NIH3T3 cells. We determined that the saturating accumulation of QDs suppressed the internalization of subsequently introduced QDs that had an identical chemical composition. However, the reinitiation of uptake was detected in the NIH3T3 cells after 8 h of incubation in medium without QDs. A very small suppressive effect of accumulated QDs was observed on uptake via the clathrin-mediated endocytosis pathway and macropinocytosis. In contrast, uptake via the caveolin-mediated pathway was almost completely prevented. Deeper insight into the suppression mechanism was obtained by transiently transfecting NIH3T3 cells with the plasmid pEGFP-C1-Caveolin-1. In these transfected cells, the usual intracellular presence of Caveolin-1 near the plasma membrane was not observed after long-term incubation with QDs. The putative application of QDs for diagnostic visualization in combination with certain anticancer substances was also evaluated. The QDs did not affect the intracellular photosensitization pattern of the amphiphilic molecule chlorin e6 in the case of photodynamic therapy. However, the saturating accumulation of QDs increased the resistance of NIH3T3 cells to the widely used anticancer drug cisplatin.

Multidimensional visualization of healthy and sensitized rabbit knee tissues by means of confocal microscopy.

This study combines several fluorescence detection methods to distinguish structural features of the synovium and cartilage tissues and to visualize the localization of endogenous porphyrins in the sensitized tissues. Specimens of synovium and cartilage tissues obtained from rabbits with antigen-induced monoarthritis after intra-articular 5-aminolevulinic acid methyl ester injection and those from healthy rabbits were investigated ex vivo by means of fluorescence spectroscopy, fluorescence intensity, and lifetime microscopy. The presence of endogenous porphyrins was confirmed with the fluorescence spectra measured on sliced sensitized specimens. Application of the lifetime-gating method on fast fluorescence lifetime imaging microscopy images, allowed separate visualization of tissue structures possessing different average lifetimes. The presence of the structures has been validated by histopathological imaging based on conventional rapid hematoxylin­eosin staining of the specimens. The fluorescence lifetime of endogenous protoporphyrin IX has been assessed and employed for visualization of sensitized tissues.

Induction of protoporphyrin IX in patient-derived synoviocytes, cartilage explants and chondrons after application of 5-aminolevulinic acid or its methyl ester.

OBJECTIVE: To compare the accumulation of protoporphyrin IX between synoviocytes of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and cartilage explants (CE) as well as chondrons of patients with OA after the application of 5-aminolevulinic acid (ALA) or its methyl ester (ALA-Me). MATERIALS AND METHODS: Samples of synovial and cartilage tissues were obtained from joint replacement surgeries. The accumulation of PpIX was determined by measuring fluorescence spectra from 2 × 10(5) synoviocytes or chondrons suspended in a glass tube or directly from CE surface after 2, 4, 8 and 24h of incubation with ALA or ALA-Me. RESULTS: No differences were found between the average fluorescence intensity values of PpIX in synoviocytes of patients with RA and OA. These values were non-significantly higher after incubation with ALA in comparison with ALA-Me at almost all time points. The average fluorescence intensity of PpIX in CE and chondrons was about ten times lower than in synoviocytes. The presence of preparation of hyaluronic acid (HA) significantly enhanced PpIX induction in chondrons versus treatment only with ALA. CONCLUSIONS: A potential for the selective synovial sensitization with endogenous PpIX in comparison with cartilage tissue has been demonstrated in vitro after application of ALA or ALA-Me.

Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection.

BACKGROUND: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. METHODS: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker(®). RESULTS: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4 °C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37 °C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5-8 µm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. CONCLUSION: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison with incubation as well as the limited transfer of quantum dots from vesicles into the cytosol and vice versa support the endocytotic origin of the natural uptake of quantum dots. Quantum dots with proteins adsorbed from the culture medium had a different fate in the final stage of accumulation from that of the protein-free quantum dots, implying different internalization pathways.

Visualization of human heart conduction system by means of fluorescence spectroscopy.

The conduction system of the heart is a specific muscular tissue, where a heartbeat signal originates and initiates the depolarization of the ventricles. The muscular origin makes it complicated to distinguish the conduction system from the surrounding tissues. A surgical intervention can lead to the accidental harm of the conduction system, which may eventually result in a dangerous obstruction of the heart functionality. Therefore, there is an immense necessity for developing a helpful method to visualize the conduction system during the operation time. The specimens for the spectroscopic studies were taken from nine diverse human hearts. The localization of distinct types of the tissue was preliminary marked by the pathologist and approved histologically after the spectral measurements. Variations in intensity, as well as in shape, were detected in autofluorescence spectra of different heart tissues. The most distinct differences were observed between the heart conduction system and the surrounding tissues under 330 and 380 nm excitation. The spectral region around 460 nm appeared to be the most suitable for an unambiguous differentiation of the human conduction system avoiding the absorption peak of blood. The visualization method, based on the intensity ratios calculated for two excitation wavelengths, was also demonstrated.

Two-photon photodynamic therapy of C6 cells by means of 5-aminolevulinic acid induced protoporphyrin IX.

Photodynamic therapy (PDT) has received increased attention as a treatment modality for malignant tumors as well as non-oncologic diseases such as age-related macular degeneration (AMD). An alternative to excite the photosensitizer by the common one-photon absorption is the method of two-photon excitation (TPE). This two-photon photodynamic therapy has the potential of improving the therapeutic outcome due to a highly localized photodynamic effect. The present study investigated the two-photon excited PDT performing in vitro experiments where C6 rat glioma cells were irradiated with a pulsed and focused fs Ti:sapphire laser emitting light at 800 nm. The irradiance distribution of the laser beam was carefully analyzed before the experiment and the applied irradiance was known for each position within the irradiated cell layer. Cells were divided into four groups and one group was incubated with 5-ALA and irradiated 4-5h later. The survival of this group was tested after irradiation by means of ethidium bromide and acridine orange staining and compared to a control group, which was irradiated under the same conditions, but not incubated with 5-ALA before. Both groups showed necrotic areas depending on the applied irradiance, the value of which at the margin of the necrotic area could be deduced from its size. 5-ALA incubated cells became necrotic after irradiation with a mean irradiance above 6.1 x 10(10) W/cm(2), while non-incubated cells remained viable. Cells of both groups became necrotic when treated with an irradiance above 10.9 x 10(10) W/cm(2). The observed affected area of the cell layers was between 0.13 mm(2) and 1.10 mm(2). Since the irradiation of non-incubated cells below the mean power density of 10.9 x 10(10) W/cm(2) induced no necrosis, apparently no thermal damage was induced in the cells and necrosis of the 5-ALA incubated cells can be ascribed to the photodynamic effect induced by two-photon excitation. The successful photodynamic treatment of a large area of a monolayer cell culture induced by two-photon excitation offers new perspectives for photodynamic treatment modalities.

Spectroscopic study of TPPS4 nanostructures in the presence of bovine serum albumin.

The influence of bovine serum albumin (BSA) on the formation of J-aggregates of meso-tetra(4-sulfonatophenyl)porphine (TPPS4) in aqueous acid solution (pH 1.3) has been investigated by means of absorption and fluorescence spectroscopy. TPPS4 concentration was kept constant at 2 microM while BSA concentration was varied to get TPPS4 : BSA molar ratios from 1 : 0.005 to 1 : 20. In the presence of protein at all used concentrations the intensity of J-aggregates absorption band was higher than that in the pure solution. Spectral changes indicated that the dynamic equilibrium of the aggregated TPPS4 species was highly dependent on the molar ratio between TPPS4 and BSA. Small relative concentrations of BSA (TPPS4 : BSA, 1 : 0.005-1 : 0.1) had a stimulating effect on formation of J-aggregates. Several fractions of J-aggregates located in protein and aqueous moieties were detected in mixed solutions at intermediate BSA concentrations (TPPS4 : BSA, 1 : 0.5-1 : 8), when the absorbance intensity of the J-aggregates was the highest. At the highest used BSA concentrations (TPPS4 : BSA, 1 : 10-1 : 20) the spectral properties of the remaining J-aggregates were similar to those typical for pure porphyrin solution. Additionally, the split of the Soret band into two with peaks at 440 nm and 423 nm was followed by the simultaneous appearance of Q bands and reflected the formation of TPPS4-BSA complexes including both protonated and deprotonated TPPS4 forms.

Spectroscopic study of ALA-induced endogenous porphyrins in arthritic knee tissues: targeting rheumatoid arthritis PDT.

The inflamed synovium of rheumatoid arthritis exhibits many features typical for neoplastic tissue implying that the photodynamic therapy might be an efficient modality for chronic poliarthritis. The accumulation of endogenously produced porphyrins after administration of exogenous 5-aminolevulinic acid (ALA) in a rabbit model of rheumatoid arthritis was evaluated by fluorescence spectroscopy. Independent of the way, intravenously or intra-articularly, in which ALA was administered to the experimental animals, the highest fluorescence intensity of endogenously produced porphyrins was detected in the tissues of the inflamed joints. Besides, the application of ALA had a systemic sensitising effect on the whole organism of rabbits. The highest amount of endogenously produced porphyrins in the inflamed joints measured from the surface of the skin above the synovium tissues was detected 1-3 h after the administration of ALA. Fluorescence measurements performed on the tissue specimens ex vivo showed the predominant accumulation of porphyrins in the synovium of the inflamed joints. The fluorescence of porphyrins was also observed in the cartilage tissues taken from knee joints. However, the fluorescence spectra features indicated that the composition of porphyrins detected in the cartilage tissues was different than that in the synovial tissues. The selective accumulation of porphyrins in the inflamed synovial tissues stands up for the application of photodynamic therapy in the treatment of rheumatoid arthritis and implies the possibility to use optical non-invasive methods based on fluorescence detection of endogenously produced porphyrins for diagnostics of inflamed tissues.

Autofluorescence of transplantable hepatoma A22 (MH-A22): prospects of tumor tissue optical biopsy.

AIM: Autofluorescence of experimental tumor (hepatoma A22 (MH-A22)) was employed to discriminate the optical differences between necrotic and non-necrotic tumor, hemorrhagic tumor and healthy tissue. METHODS: The experiment was performed ex vivo using the transplantable tumor from the right haunch of hybrid mice (C57Bl/CBA). Blue LED light (lambda em=405 nm) was applied for autofluorescence excitation and fibre optics based spectrofluorimeter was used for spectra detection. RESULTS: We observed that necrotic tumor tissue is characterized by the absence of endogenous porphyrins fluorescence, and registered spectra do not possess differences in the red spectral region (600-710 nm) in comparison with normalized autofluorescence spectra of muscle. Moreover, only certain segments of non-necrotic tumor bear the fluorescence of endogenous porphyrins. CONCLUSIONS: Based on the experimental results, we suggest that the absence of long-waved fluorescence differences between necrotic tumor tissue and healthy tissue, e.g. muscle can impede the demarcation between healthy and tumor tissue. The uneven distribution of endogenous porphyrins in non-necrotic tumor tissue as well as the absence of endogenous porphyrins fluorescence in the small experimental tumors complicates the localization of cancerous tissue based on the autofluorescence registration.