Search

We have visualized living cells and tissues using an ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopic system by using a sub-nanosecond supercontinuum (SC) light source. Owing to the ultrabroadband spectral profile of the SC, we can generate multiplex CARS signals in the spectral range of 500–3800 cm−1, which covers the whole molecular fingerprint region, as well as the C-H and O-H stretching regions. Through the combination of the ultrabroadband multiplex CARS method with second harmonic generation (SHG) and third harmonic generation (THG) processes, we have successfully performed selective imaging of ciliary rootlet-composing Rootletin filaments in rat retina.

In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas.Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation.The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images.Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.

Among recent progress of optical bioimaging techniques, nonlinear optical imaging is one of the promising new-generation bioimaging methods. Nonlinear optical imaging is capable of rapid image acquisition, three-dimensional sectioning, label-free analysis, and simultaneous multimodal imaging. The multimodality of nonlinear optical processes enables us to obtain various signals simultaneously such as second harmonic generation (SHG), third harmonic generation (THG), third-order sum frequency generation (TSFG), and coherent anti-Stokes Ra-man scattering (CARS) [1]. Since images obtained by each process are based on different contrast mechanism, the combinational analysis of various nonlinear optical processes provides more detailed information on the sample. In the present study, we report recent our development of multimodal nonlinear spectral microscope and its applications to imaging of tissue samples. In particular, multimodal imaging with spectral analysis of the CARS molecular fingerprint provided molecular structural information of the tissue sample.