We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals.

We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

Interstitial fibrosis is a powerful pejorative predictor of progression of nephropathies in a variety of chronic renal diseases. It is characterized by the depletion of kidney cells and their replacement by extracellular matrix, in particular, type-I fibrillar collagen, a protein scarce in normal interstitium. However, assessment of fibrosis remains a challenge in research and clinical pathology. We develop a novel methodology based on second harmonic generation (SHG) microscopy, and we image collagen fibers in human and mouse unstained kidneys. We take into account the variability in renal shape, and we develop automated image processing for quantitative scoring of thick murine tissues. This approach allows quantitative 3-D imaging of interstitial fibrosis and arterial remodeling with high accuracy. Moreover, SHG microscopy helps to raise pathophysiological questions. First, imaging of a large volume within a mouse kidney shows that progression of fibrosis is a heterogeneous process throughout the different renal compartments. Second, SHG from fibrillar collagens does not overlap with the glomerular tuft, despite patent clinical and experimental glomerulosclerosis. Since glomerulosclerosis involves SHG-silent nonfibrillar collagens, our work supports pathophysiological differences between interstitial fibrosis and glomerulosclerosis, a clearly nonfibrotic process.

Abstract. The demonstration satellite GHGSat-D, or “Claire”, launched on 21 June 2016, is the first in a planned constellation of small satellites designed and operated by GHGSat, Inc. to measure greenhouse gas emissions at the facility scale from space. Its instrument measures methane concentrations by collecting and spectrally decomposing solar backscattered radiation in the shortwave infrared using a compact fixed-cavity Fabry–Pérot imaging spectrometer. The effective spatial resolution of 50×50 m2 over targeted 12×12 km2 scenes is unprecedented for a space-based gas-sensing spectrometer. Here we report on the instrument design and forward model and retrieval procedure, and we present several examples of retrieved methane emissions observed over industrial facilities. We discuss the sources of error limiting the performance of GHGSat-D and identify improvements for our follow-on satellites. Claire's mission has proven that small satellites can be used to identify and quantify methane emissions from industrial facilities, enabling operators to take prompt corrective action.