Abstract
.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.
Highlights
Multiphoton microscopy (MPM) is a well-established method for tissue structural and functional imaging.[1,2,3,4] Studies have been reported in a variety of tissues, from animal models to human samples, and the technique is used in vivo for dermatological applications.[5,6,7]MPM is of particular value for imaging extracellular matrix (ECM) architecture in label-free tissues since the combination of second-harmonic generation (SHG) and endogenous twophoton excitation fluorescence (2PEF) allows the simultaneous visualization of collagen and elastin, respectively, the main ECM proteins
The image guide (IG) used in our study is the GastroflexTM UHD confocal miniprobeTM commercialized by Mauna Kea Technologies for use with the single-photon fluorescence endomicroscope Cellvizio®
We report a nonlinear endomicroscopy apparatus employing multicore fiber IG compatible with real-time in-clinic endoscopy procedure
Summary
Multiphoton microscopy (MPM) is a well-established method for tissue structural and functional imaging.[1,2,3,4] Studies have been reported in a variety of tissues, from animal models to human samples, and the technique is used in vivo for dermatological applications.[5,6,7]MPM is of particular value for imaging extracellular matrix (ECM) architecture in label-free tissues since the combination of second-harmonic generation (SHG) and endogenous twophoton excitation fluorescence (2PEF) allows the simultaneous visualization of collagen and elastin, respectively, the main ECM proteins. Multiphoton microscopy (MPM) is a well-established method for tissue structural and functional imaging.[1,2,3,4] Studies have been reported in a variety of tissues, from animal models to human samples, and the technique is used in vivo for dermatological applications.[5,6,7]. A number of investigations on ex vivo animal[8,9,10,11] and human lung samples[12,13,14,15,16,17,18] already demonstrated the potential of the method to assess the pathological changes occurring in ECM organization. In previous studies,[19,20] the authors showed that, under the 488-nm single-photon excitation mode, the technique images the elastin network of the proximal and distal bronchial tree but does not give access to
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