Non-linear optical microscopy proves to be an indispensable tool in natural sciences and becomes more and more attractive for clinical applications. Coherent Raman scattering, for instance, has the potential to become an in-vivo fast label-free histology tool as its chemical selectivity provides quantitative information on lipids and proteins locations and concentrations in tissues. Along with these techniques, second-harmonic generation of collagen and 2-photon excitation fluorescence broaden even more the non-linear imaging ability as collagen fibers represent an important role in human body construction. Whilst 2-photon excitation fluorescence allows to study auto-fluorescence (ex. NADH and NADHP molecules), and to excite a vast range of chromophores. However, absorption and scattering limit significantly the nonlinear imaging depth into tissues. As a solution, we offer a flexible, compact, and multimodal nonlinear endoscope (2.2 mm outer diameter, 35 mm rigid length) based on a resonantly piezo scanned hollow-core negative curvature double-clad fiber. The fiber design allows distortion-less, background-free delivery of femtosecond excitation pulses and the back-collection of nonlinear signals through the same fiber. The double-cladding of this fiber attends 10^5μm of silica collection surface which allows for a 4-fold collection improvement compared to previously used Kagomé hollow core fibers. Having a good control on the resonantly scanning fiber the endoscope can perform nonlinear imaging up to 8 frames per second over a field of view of 400μm. We demonstrate 2photon, SHG and CARS imaging in ex vivo gastric human tissue samples and in-vivo 2-photon fluorescence imaging of GFP-labeled neurons in mouse brain.
Proc. SPIE. 10859, Visualizing and Quantifying Drug Distribution in Tissue III
KEYWORDS: Second-harmonic generation, Multiphoton microscopy, 3D acquisition, Image segmentation, Skin, Collagen, Fluorescence lifetime imaging, In vivo imaging, CARS tomography, In vitro testing, 3D image processing
There is an increasing need in cosmetic research for non-invasive, high content, skin imaging techniques offering the possibility to avoid performing invasive biopsies and to supply a maximum of information on skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with <i>in vitro</i> and <i>in vivo</i> investigations of human skin, allowing its three-dimensional (3D) structure to be characterized with sub-μm resolution. Various intra-/extra-cellular constituents present specific endogenous two-photon excited fluorescence and second harmonic generation signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. In association with fluorescence lifetime imaging (FLIM) and specific 3D image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and function. Multiphoton FLIM applications in cosmetic research range from knowledge to efficacy evaluation studies. Knowledge studies aim at acquiring a better understanding of appearing skin differences, for example, with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with efficacy assessment of cosmetic ingredients in anti-aging or whitening domains. When using other nonlinear optics phenomena such as CARS (Coherent Anti-Stokes Raman Scattering), multiphoton imaging opens up the possibility of characterizing the cosmetic ingredients distribution inside the skin and founds application in other cosmetic domains such as hydration or antiperspirants. Developments in user-friendly, ultrasensitive, compact, multimodal imaging systems, on-the-fly data analysis and the synthesis of cosmetic ingredients with non-linear optical properties will certainly allow trespassing the todays frontiers of cosmetic applications.
The development of nonlinear fiber-endoscopes capable of imaging deeper in tissues and accessing internal organs represents a very attractive perspective for application of nonlinear optical microscopes to in-vivo research and diagnostics. The transmission of ultra-short laser pulses within a fiber is a critical issue in the development of such endoscopes. For instance, self-phase modulation (SPM), four-wave mixing (FWM) and Raman scattering occurring in conventional fibers severely affect transmitted pulses profiles in the time and frequency domains. Hollow-core (HC) fibers bring a solution to the problem, since propagation of the pulses in the air core limits nonlinear interactions. We employ here a novel double clad Kagomé-lattice HC fiber for the delivery of ultrafast pulses across a large spectral window (~400nm) with no pulse distortion. The epi-collection of the signal generated at the sample is efficiently performed with a specially designed outer multimode cladding. The fiber is incorporated in a prototype endoscope using a four-quartered piezo-electric tube to scan the laser beam on the sample. The low numerical aperture of the hollow-core (0.02) is efficiently increased by means of a dielectric microsphere attached to the fiber face. This results in tight focusing (~1 micron) of the beam at the HC fiber output. Resonant scanning of the fiber tip allows imaging over a field of 300 microns using low driving voltages. High-resolution images with different contrast mechanisms, such as SHG and TPEF, acquired with the prototype endoscope illustrate the potential of these fibers for nonlinear imaging in regions otherwise inaccessible to conventional optical microscopes.
Nonlinear microscopies, including two-photon excited autofluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), were used to study individual human sweat pore morphology and topically applied antiperspirant salt penetration inside sweat pore, in vivo on human palms. Sweat pore inner morphology in vivo was imaged up to the depth of 100 μm by TPEF microscopy. The 3D penetration and distribution of “in situ calcium carbonate” (isCC), an antiperspirant salt model, was investigated using CARS microscopy.