Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window

Abstract

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm2), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.

Molecular identification of second harmonic generation (SHG) sources in mouse brain by multimodal imaging with ultra-broadband multiplex coherent anti Stokes Raman scattering (CARS)

“…3(a). In this region, no CARS image gave the same contrast with the SHG image, and no distinct features were found in the Im[χ (3) ] spectra at the SHG spots. Morphologically, this region is composed of layers of neurons.…”

“…Multiplex CARS imaging at the Raman shift of 1248(Amide III), 934(the C-C stretch), and 860 cm -1 (C-C stretch from the pyrrolidine ring of proline) exhibits similar contrasts with the SHG image. Notably, the spatially averaged Im[χ (3) ] spectrum over the SHG active region surrounded by yellow dashed line in SHG image shows a prominent peak attributed to collagen [6] as shown in Fig. 2(b).…”

“…Since the spectral profile of the raw CARS signal was distorted due to interference of the vibrationally resonant CARS signal with the non-resonant background, we used the maximum entropy method (MEM) [5] to retrieve Im[χ (3) ] spectra. The SHG and CARS signals were extracted by fitting each spectral profile using a Gaussian function, and their respective amplitudes for each spatial position were mapped out.…”

“…Microtubules, myosin, collagen [1], and Rootletin [2] have been reported as SHG-active molecules in living organisms. In our previous study, we reported the multiple SHG signals in the mouse brain using a 1.55-m femtosecond fiber laser source [3]. However, due to the lack of molecular specificity, it was difficult to assign the molecular origins based solely on the SHG signal.…”

Deep tissue imaging with multiphoton microscopy in the short-wavelength infrared windows

“…To date, most of the THG imaging has been performed using excitation wavelengths 17 ,18, 19 between 1000 and 1350 nm, also known as NIR II optical window 21 . However the range (1550 -1850 nm) which is known 21,22 as NIR III optical window has significant advantages regarding deep tissue imaging, such as longer attenuation length 20,21,22 and none or very low THG signal absorption from tissue as the latter appears at longer wavelengths 21 . However, biomedical imaging is very little explored in this spectral range due to a sparsity of laser light sources at these wavelengths 23 .…”