Multiphoton microscopy enables sub-micron resolution, label-free structural and functional imaging of living tissues with contrast from multiple modalities, including second harmonic generation and two-photon excited fluorescence. We developed a fast, large area multiphoton exoscope (FLAME) portable system with enhanced label-free molecular contrast for macroscopic imaging of human skin with microscopic resolution. It combines optical and mechanical scanning mechanisms with deep learning image restoration to produce 3D sub-cellular resolution images that encompass sub-mm2 to cm2 scale areas of tissue within minutes. We demonstrate the performance and utility of the instrument by fast ex vivo and in vivo imaging of human skin.
KEYWORDS: Skin, In vivo imaging, Associative arrays, Tumors, Melanoma, Image resolution, Diagnostics, Denoising, Convolutional neural networks, Chemical elements
Multiphoton microscopy can provide sub-micron resolution images of living tissues in their
native environment with chemical contrast. We recently reported on a fast large area multiphoton
exoscope (FLAME) for rapidly mapping out macroscopic tissue areas (cm-scale) with microscopic
resolution. In this presentation we demonstrate the imaging capability and the clinical utility of this
system by performing a pilot study on ex vivo imaging of benign and malignant pigmented lesions of
human skin. We identify morphological features such as cytological atypia, lentiginous hyperplasia,
migration of melanocytes and demonstrate the value of sampling large tissue volumes for capturing the
lesion heterogeneity.
Multiphoton microscopy (MPM) can provide sub-micron resolution images of living tissues in their native environment with contrast from multiple modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). Recent advances of MPM in clinical skin imaging demonstrated the unique potential of this technology as a label-free research and clinical tool for a broad area of applications such as melanoma and non-melanoma skin cancer detection, monitoring pigmentary skin disorders, characterizing keratinocyte metabolism, etc. In this contribution we demonstrate the ability of this microscope to provide sub-micrometer resolution ex-vivo images of large areas of skin tissue (up to 5x5 mm2) in <1 minute. We demonstrate the importance of high-speed, high-resolution mesoscopic imaging on cancerous skin tissues that present heterogeneous morphology to show the ability of the instrument to capture both benign and malignant areas of the lesion.
Our group has recently developed a method for characterizing distribution of a topical drug within skin using two-photon fluorescence lifetime imaging (FLIM) and phasor analysis. Here, we expand on this work by describing a multimodal approach for rapidly visualizing multiple components in tissue using FLIM and coherent Raman imaging (CRI). By employing a non-Euclidian FLIM phasor analysis for a three-component system informed with the vibrational signature of one of the components retrieved with CRI, we were able to semi-quantitatively describe the spatial distribution of drugs in tissue with molecular specificity and cellular resolution.
We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean phasor analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.
KEYWORDS: Excitons, Semiconductors, Nanowires, Microscopy, Femtosecond phenomena, Near field scanning optical microscopy, Near field optics, Visualization, Spatial resolution, Temporal resolution
Excitonic excitations play an important role in the optical response of low-dimension nanoscale semi-conducting materials. During its lifetime, excitons may diffuse or migrate in particular directions, thus constituting a form of excitation information transfer on the nanoscale. The details of the spatio-temporal evolution of excitons remain unclear, because it has been challenging to directly visualize this process with nanometer spatial resolution and femtosecond temporal resolution. Here we describe pump-probe measurements at the nanoscale, using the photo-induced force microscopy (PiFM) and near-field scanning optical microscopy (NSOM) at ambient conditions. We analyze the spatial and temporal characteristics of the excitons in quasi-1D semiconductor nanowires, and provide unprecedented views of their evolution.
We present pump-probe and Raman measurements on individual plasmonic nano-junctions. The time-resolved measurements reveal differences between capacitive and conductive junctions, and paint a detailed picture of the ultrafast electrodynamics of the nano-junction. The insights gleaned from these measurements help interpret the ultrafast response of single molecules placed in the junction.
We have previously demonstrated a total internal reflection, wide-field CARS microscope, where the signal is enhanced with the aid of a thin gold layer that supports surface plasmon polariton resonances. This surface-enhanced CARS microscope is capable of generating images of lipid structures in close proximity (<100 nm) to the glass substrate at excitation densities that are 4 orders of magnitude lower than in point-scanning CARS microscopy. In this contribution, we demonstrate its application to visualizing lipids in aqueous media, including imaging of cells, with a unique surface-sensitive contrast that cannot be obtained with conventional CARS microscopy.
We present combined surface-enhanced stimulated Raman scattering (SE-SRS) and surface-enhanced coherent anti-Stokes Raman scattering (SE-CARS) measurements on individual plasmonic antennas dressed with bipyridyl-ethylene molecules. By carefully optimizing the conditions for performing SE-SRS experiments, we have obtained stable and reproducible molecular surface-enhanced SRS spectra from single nano-antennas. Using surface-enhanced Raman scattering (SERS) and transmission electron microscopy of the same antennas, we confirm that the observed SE-SRS signals originate from only one or a few molecules. We highlight the physics of surface enhancement in the context of coherent Raman scattering and derive sensitivity parameters under the relevant conditions. The implications of single molecule SRS measurements are discussed.
Surface enhanced Raman scattering (SERS) is a popular technique for detecting and analyzing molecules at very low concentrations. The sensitivity of SERS is high enough to detect single molecules. It has proven difficult, however, to perform similar measurements in the so-called nonlinear optical regime, a regime in which the molecule is responding to multiple light pulses. Nonetheless, recent experiments indicate that after careful optimization, it is possible to generate signals derived from nonlinear analogs of SERS. Such measurements make it possible to view molecular vibrations in real time, which amounts to the femto- to pico-second range. In this contribution, we discuss in detail under which conditions detectable surface-enhanced coherent Raman signals can be expected, provide experimental evidence of coherent Raman scattering of single molecules, and highlight the unique information that can be attained from such measurements.
We have developed a coherent Raman scattering microscope that combines total internal reflection illumination with surface plasmon resonance. The excitation geometry is based on an objective-type Kretschmann configuration, which allows widefield excitation of surface plasmon polariton modes in a thin gold film on a glass substrate. The surface plasmon fields enhance the excitation efficiency, enabling image acquisition at 10 frames/s. Since the evanescent field extends only over a length scale of ~100 nm, structures close the substrate surface are observed while bulk contributions are suppressed. We discuss the operational principles of this microscope in detail and point out its applications in cell biology.
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