In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra (hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dualwavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack

Enhanced resonance energy transfer in gold nanoparticles bifunctionalized by tryptophan and riboflavin and its application in fluorescence bioimaging

Gold nanoparticles were functionalized by amino acid tryptophan and vitamin riboflavin - a resonance energy transfer (RET) pair of biomolecules. The presence of the gold nanoparticles resulted in 65% increase in RET efficiency. Because of enhanced RET efficiency, the photobleaching dynamics of the fluorescent molecules at the surface of the nanoparticles is different from that of molecules in solution. The observed effect was used for detection of the functionalized nanoparticles within biological material rich with autofluorescent species. Synchrotron radiation deep-ultraviolet fluorescence microscopy is used to study the photobleaching dynamics of the fluorescence centers within human hepatocellular carcinoma Huh7.5.1 cells incubated with the nanoparticles. The fluorescent centers were classified according to their photobleaching dynamics, which enabled the discrimination of the cell areas where the accumulation of the nanoparticles takes place, even though the particles were smaller than the spatial resolution of the images

X-ray-induced radiophotodynamic therapy (RPDT) using lanthanide micelles: Beyond depth limitations

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DUV Autofluorescence Microscopy for Cell Biology and Tissue Histology Biology of the Cell Biology of the Cell

International audienceAutofluorescence spectroscopy is a powerful tool for molecular histology and for following metabolic processes in biological samples as it does not require labelling. However, at the microscopic scale, it is mostly limited to visible and near infrared excitation of the samples. Several interesting and naturally occurring fluorophores can be excited in the UV and deep UV (DUV), but cannot be monitored in cellulo nor in vivo due to a lack of available microscopic instruments working in this wavelength range.To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system.In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology

Fleroxacin uptake by <i>Enterobacter aerogenes</i> population. A

<p>. Fluorescence emission spectra of Fle (λ_exc = 283 nm) detected from Glycin-HCl-induced lysis of <i>Enterobacter aerogenes</i> strain EA289. EA289 were incubated with Fle (2 µg/ml) for 30 min in follow conditions: (− ·− ·) Fle; (−−−) Fle + Glu (0.4%); (–) Fle + CCCP (10 µM). <b>B</b>. Comparison of Fle concentration uptake determined from lysated bacteria. <i>Enterobacter aerogenes</i> strains EA289 and EA298 were incubated with Fle at different concentrations (1, 2 or 8 µg/ml) alone or with Glu (0.4%) or CCCP (10 µM).</p

Individual bacteria microspectro-fluorescence measurement. A

<p>. Transmission image of <i>Enterobacter aerogenes</i> EA289 bacteria. White arrow indicates on bacterium from which one of the fluorescence spectra of <b>fig. 3B</b> was taken. Scale bar corresponds to 3 µm. <b>B</b>. <b> </b>Fluorescence emission spectra (recorded by UV-VIS microspectrofluorimetry at λ_exc = 290 nm) from two individual Fle-untreated bacteria EA289. Fluorescence emission spectrum (− ·− ·) corresponds to bacterium marked on <b>fig. </b><b>3A</b>; (–) spectrum corresponds to bacterium not in the field of view.</p

Comparison of fluorescence intensity and concentration of Fleroxacin in individual bacteria and lysated bacteria. A

<p>. <b> </b>Fluorescence intensity of Fle measured by UV-VIS microspectrofluorimetry from individual EA289 bacteria. EA289 were incubated with Fle (64 µg/ml) only or with Glu (0.4%) or CCCP (25 µM). <b>B</b>. <b> </b>Comparison of Fle concentration uptake determined from lysated bacteria. <i>Enterobacter aerogenes</i> EA289 were incubated with Fle (64 µg/ml) alone or with Glu (0.4%) or CCCP (25 µM).</p

Fleroxacin uptake by individual <i>Enterobacter aerogenes</i>. A

<p>. Fluorescence spectra of Fle (750 ng/ml) in PBS pH = 7: (−−−) excitation spectrum at λ_emis = 420 nm; (–) emission spectrum at λ_exc = 313 nm. <b>B</b>. Transmission (left), fluorescence (middle) and merge (right) images of Fle (64 µg/ml)-treated EA289 bacteria. Scale bar corresponds to 3 µm. <b>C</b>. Transmission (left), fluorescence (middle) and merge (right) images of Fle (64 µg/ml)- and CCCP- (25 µM) treated EA289 bacteria. Scale bar corresponds to 3 µm. <b>D</b>. Percentage of maximum fluorescence intensity of Fle within single bacteria from <b>fig. 2C. E</b>. Fluorescence intensity detected from Fle channel (λ_exc = 290 nm; DM 300 nm; BP filter 420≤ λ_emis ≤480 nm) from individual EA289 bacteria as a function of treatment conditions: (a) EA289 with no additions; (b) EA289 incubated with Fle (64 µg/ml); (c) EA289 incubated with Glu; (d) EA289 co-incubated with Glu and Fle (64 µg/ml); (e) EA289 incubated with CCCP (25 µM); (f) EA289 co-incubated with CCCP (25 µM) and Fle (64 µg/ml).</p