Abstract
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.
Highlights
Generation (SHG) microscopy[4,5,6,7,8]
Because the neural retina has a highly organized multilayered structure that is attractive as a model for self-patterning and self-driven morphogenesis[41], we thoroughly visualized rat retinal layers using SHG (2ω1), third harmonic generation (THG) (3ω1), and multiplex sum frequency generation (SFG) (ω1 +ω2), third-order sum frequency generation (TSFG), and coherent anti-Stokes Raman scattering (CARS) channels of coherent nonlinear optical microscopic (CNOM) setup shown in Fig. S2a (Fig. 1 and Fig. S3)
This conclusion was reached by observing endogenous Rootletin (Fig. 2) and by introducing exogenous truncated mutants, demonstrating that filaments assembled from the Rootletin R234 domain are the ultrastructure responsible for the SHG (Figs 3 and 4)
Summary
Generation (SHG) microscopy[4,5,6,7,8]. SHG is a frequency-doubling nonlinear optical process, which originates from coherent interaction of laser pulses with organized ensembles of non-centrosymmetric targets with significant hyperpolarizability[4,9]. Owing to recent advancements in supercontinuum (SC) generation technology[14], ultra-broadband excitation has been achieved in CNOM, which enables us to launch unique and multi-color nonlinear optical processes such as multiplex sum frequency generation (SFG)[15], third-order sum frequency generation (TSFG)[16], and CARS as well as monochromatic SHG and third harmonic generation (THG) (See Fig. S1). These methods are complementary to one another, and their utility is further increased by integrating them into a multimodal microscopic unit. The molecular mechanisms underlying these kinetics have been partially elucidated[31,32]
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