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.
We present the Dual-Step system and method we developed to achieve 2D quantitative maps of optical properties. It is non-contact, quantitative for both absorption and scattering, large field, and spectrally resolved. The present study shows the results obtained on rats and figures the interest of the approach to address complex in-vivo samples.
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.
KEYWORDS: Optical properties, Algorithm development, Monte Carlo methods, Imaging systems, Signal detection, Optical transfer functions, Spectroscopy, Absorption, Scattering, Electroluminescent displays
We validate a non-contact Diffuse Reflectance Spectroscopy (DRS) system as a first stage to approach quantitative multi-spectral imaging technique. The non-contact DRS system with separated illumination and detection paths was developed with different progressive set-ups which were all compared to a well-founded contact DRS system. While quantitation of the absorption coefficient is well achieved with the existing method, the calculation of the scattering coefficient is deteriorated by the non-contact architecture measurements. We have therefore developed an adaptive reference-based algorithm to compensate for this effect.
This work deals with label free multiphoton imaging of the human lung tissue extra-cellular matrix (ECM) through
optical fibers. Two devices were developed, the first one using distal scanning associated to a double clad large mode
area (LMA) air-silica microstructured fiber, the second one using proximal scanning of a miniature multicore image
guide (30000 cores inside a 0.8 mm diameter). In both cases, the main issue has been efficient linear and nonlinear
distortion pre-compensation of excitation pulses. By inserting before the delivery fiber a compact (10 cm × 10 cm
footprint) grisms-based stretcher (a grating in close contact with a prism) made of readily available commercial
components, we achieved as short as 35-femtosecond-duration pulses that were temporally compressed at the direct exit
of a 2-meter-long fiber. Interestingly, this femtosecond pulse fiber delivery device is also wavelength tunable over more
than 100 nm inside the Ti: Sapphire emission band. With the help of distal scan system, those unique features allowed us
to record elastin (through two-photon fluorescence) and collagen (through second harmonic generation) fibered network
images. These images were obtained ex-vivo with only 15 mW @ 80 MHz of IR radiation delivered to the alveoli or
bronchus tissues. 3D imaging with 400-μm-penetration depth inside the tissue was possible working with a 2-meter-long
LMA fiber. With the help of proximal scanning, the miniature image guide allowed us to perform endoscopic real time
microimaging of the ECM ex vivo.
During glioblastoma surgery, delineation of the brain tumour margins remains difficult especially since
infiltrated and normal tissues have the same visual appearance. This problematic constitutes our research
interest. We developed a fibre-optical fluorescence probe for spectroscopic and time domain measurements.
First measurements of endogenous tissue fluorescence were performed on fresh and fixed rat tumour brain
slices. Spectral characteristics, fluorescence redox ratios and fluorescence lifetime measurements were analysed.
Fluorescence information collected from both, lifetime and spectroscopic experiments, appeared promising for
tumour tissue discrimination. Two photon measurements were performed on the same fixed tissue. Different
wavelengths are used to acquire two-photon excitation-fluorescence of tumorous and healthy sites.
Fibered confocal fluorescence microscopy (FCFM) with spectroscopic analysis capability was used
during bronchoscopy, at 488nm excitation, to record autofluorescence images and associated emission spectra of the
alveoli of 5 healthy smoking volunteers and 7 non-smoking amiodarone-induced pneumonitis (AIP) patients.
Alveolar fluorescent cellular infiltration was observed in both groups. Our objective was to assess the potential of
spectroscopy in differentiating these two groups.
Methods: We previously demonstrated that in healthy smokers alveolar elastin backbone and tobacco tar contained in
macrophages contribute to the observed signal. Each normalized spectrum was modeled as a linear combination of 3
components: Sexp(λ) = Ce.Se(λ)+Ct.St(λ)+CG.SG(λ), Ce, Ct and CG are amplitude coefficients. Se(λ) and St(λ) are
respectively the normalized elastin and tobacco tar emission spectra measured experimentally and SG(λ) a gaussian
spectrum with tunable width and central wavelength. Levenbergt-Marquardt algorithm determined the optimal set of
coefficients.
Results: AIP patient autofluorescence spectra can be uniquely modelized by the linear combination of the elastin
spectrum (Ce = 0.61) and of a gaussian spectrum (center wavelength 550nm, width 40nm); the tobacco tar spectrum
coefficient Ct is found to be zero. For healthy smoking volunteers, only two spectral components were considered:
the tobacco tar component (Ct = 1,03) and the elastin component (Ce = 0).
Conclusion: Spectral analysis is able to distinguish cellular infiltrated images from AIP patients and healthy smoking
volunteers. It appears as a powerful complementary tool for FCFM.
Fibered confocal fluorescence microscopy (FCFM) is an emerging technique that can be used during bronchoscopy to analyze the nature of the human bronchial mucosa and alveolar network fluorescence microstructure. An endoscopic fibered confocal fluorescence microscopy system with spectroscopic analysis capability was developed allowing realtime, simultaneous images and emission spectra acquisition, at 488 nm excitation, using a flexible miniprobe. This flexible 1.4 mm miniprobe can be introduced into the working channel of a flexible endoscope and gently advanced through the bronchial tree up to the alveoli. FCFM in conjunction with bronchoscopy is able to image the in vivo autofluorescence microstructure of the bronchial mucosa but also the alveolar respiratory network outside of the usual field of view. In the normal bronchi, reproducible images were obtained, characterized by a highly organized fibered network. Precancerous lesions exhibited alterations of this fibered network. 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.
The objectives of this study are to characterize the autofluorescence spectra of normal and tumoral esophageal epithelial cells and to link the cellular spectra with a data basis of in vivo tissular spectra.
Our preliminary results show that no difference in spectral distribution can be observed between squamous cell carcinoma, adenocarcinoma and normal cells. A statistical significant difference is observed between the average intensity of the raw spectra of the different cell types. Nucleus autofluorescence presents the same spectral shape as cytoplasm, but with lower intensity.
The reaction of singlet oxygen with vinyl porphyrins, which was first described in the case of protoporphyrin, is reinvestigated as a route to chlorin type molecules. Various dicarboxylic chlorins were thus prepared and characterized for their chemical and photosensitizing properties. All of them efficiently photosensitize the production of singlet oxygen. Some of these compounds might be formed during photodynamic therapy with hematoporphyrin derivative or Photofrin. Their biological relevance is discussed.
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