In vivo endoscopic autofluorescence microspectro-imaging of bronchi and alveoli

Fibered confocal fluorescence microscopy (FCFM) is a new technique that can be used during a bronchoscopy to analyze the nature of the human bronchial and alveolar mucosa fluorescence microstructure. An endoscopic fibered confocal fluorescence microscopy system with spectroscopic analysis capability was developed allowing real-time, simultaneous images and emission spectra acquisition at 488 nm excitation using a flexible miniprobe that could be introduced into small airways. This flexible 1.4 mm miniprobe can be introduced into the working channel of a flexible endoscope and gently advanced through the bronchial tree to the alveoli. FCFM in conjunction with bronchoscopy is able to image the in vivo autofluorescence structure of the bronchial mucosae but also the alveolar respiratory network outside of the usual field of view. Microscopic and spectral analysis showed that the signal mainly originates from the elastin component of the bronchial subepithelial layer. In non smokers, the system images the elastin backbone of the aveoli. In active smokers, a strong autofluorescence signal appears from alveolar macrophages. The FCFM technique appears promising for in vivo exploration of the bronchial and alveolar extracellular matrix

Dynamics of pH-dependent self-association and membrane binding of a dicarboxylic porphyrin: a study with small unilamellar vesicles

AbstractSteady-state and stopped-flow measurements of the absorbance and fluorescence of aqueous solutions were performed to characterize the pH-dependent ionization and aggregation states of deuteroporphyrin. Porphyrin self-association promoted by neutralization of the carboxylic groups takes place within a few milliseconds impeding characterization of the monomer ionization states. Extrapolation at infinite dilution of the values obtained from steady-state measurements yielded the pKs of the carboxylic groups (6.6, 5.3) and inner nitrogens (4.1, 2.3). The kinetics of interactions of the porphyrin with unilamellar fluid state dioleoylphosphatidylcholine vesicles was examined in a large pH range, with focus on the entry step. From alkaline pH to a value of 6.5, the entrance rate is maximal (1.69×106 M−1 s−1 versus phospholipid concentration). It decreases to 2.07×105 M−1 s−1 at lower pH with an apparent pK of 5.39. This effect appears to be related to the formation of porphyrin dimer rather than to the protonation of inner nitrogen. In keeping with previous data, these results support the concept of a pH-mediated selectivity of carboxylic porphyrins for tumor. They also indicate that the propensity of these molecules to self-associate at low pH could yield to some retention in acidic intracellular vesicles of the endosome/lysosome compartment

Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins

AbstractThe 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

Tetrapyrrole-photosensitizers vectorization and plasma LDL: A physico-chemical approach

International audienc

in vivo imaging of the bronchial and alveolar walls microstructure using fibered confocal autofluorescence microscopy

International audienc

Microspectro-imagerie confocale endoscopique des alvéoles pulmonaires : analyse spectrale comparative de l'autofluorescence des sujets sains et des patients présentant une pneumopathie induite par l'amiodarone

National audienc

Endoscopic autofluorescence micro-spectroimaging of alveoli: comparative spectral analysis of amiodarone-induced pneumonitis patients and healthy smokers

International audienc

Autofluorescence spectro-endomicroscopy of alveoli: comparative spectral analysis of healthy smocker volonteers and amiodarone-induced pneumonitis patients

International audienceFluorescence endomicroscopy with spectroscopic analysis capability was used during bronchoscopy, at 488nm excitation, to record, simultaneously, autofluorescence (AF) images and associated emission spectra of the alveoli of healthy smoking volunteers (HS) and amiodarone treated non-smoker patients (ATNS). Alveolar fluorescent cellular infiltration was observed in both groups. Our objective was to assess the potential of spectroscopy in differentiating these two groups. Each normalized spectrum was modeled as a linear combination of several components: cellular flavins (FAD) and another cellular component, namely lipopigments, modelled by a Voigt profile, extracellular matrix (ECM) elastin and tobacco tar; HbO2 absorption was also taken into account. Besides the FAD and elastin contributions, the addition of a tobacco tar component is necessary to account for HS group spectral shape. The ATNS patients autofluorescence spectra result from the contribution of elastin, FAD and lipopigments, the tobacco tar component being found to zero. In conclusion, spectral analysis is able to differentiate between similar cellular infiltrated images from healthy smokers and ATNS patients

Equilibrium and kinetic studies of the interactions of a porphyrin with low-density lipoproteins.

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