Advances in Imaging Techniques for Mammalian/Human Ciliated Cell's Cilia: Insights into Structure, Function, and Dynamics

Abstract

Cilia are evolutionarily conserved, microtubule-based organelles characterized by their ultrastructures and diverse functional roles, including developmental signaling, mechanosensation, and fluid propulsion. They are widely distributed across cell surfaces and play crucial roles in cell cycle regulation and tissue homeostasis. Despite advances in studying their molecular regulation and functions, demonstrating the precise ultrastructure of cilia remains a challenge. Recent novel microscopy techniques, such as super-resolution microscopy and volume electron microscopy, are revolutionizing our understanding of their architecture and mechanochemical signaling. By integrating findings from different methodologies, this review highlights how these advances bridge basic research and clinical applications and provide a comprehensive understanding of the structural organization, functional mechanisms, and dynamic changes of cilia.

Keywords: cilia; imaging techniques; volume electron microscopy.

Figure 1

Schematic structure of the cilia. Cilia possess four primary structural elements: the basal body, axoneme, transition zone, and ciliary membrane. The transition zone is composed of Y-link structures that connect the microtubules to the ciliary membrane. Transition fibers connect the basal body to the plasma membrane. Cilia are classified according to their microtubule configuration: primary cilia have a ‘9 + 0’ microtubule arrangement (9 pairs of double microtubules), whereas motile cilia have a ‘9 + 2’ one (additional central pairs) and include ciliary movement-associated structures, such as inner dynein arms, outer dynein arms, and radial spokes (RS).

Figure 2

Initiation process of ciliogenesis. (A) Extracellular pathway: The mother centriole migrates to the cytoplasmic membrane through the mediation of its distal appendages and becomes anchored, followed by the initiation of axoneme assembly. (B) This process occurs within the cytoplasmic compartment prior to the formation of the axoneme and ciliary membrane. Preciliary vesicles (PCVs), dependent on myosin Va-mediated transport, are specifically recruited to the distal appendage of the mother centriole. These vesicles undergo coordinated membrane fusion events to generate functional ciliary vesicles (CVs). During the sheath development phase, synchronized development of the axoneme and ciliary membrane is achieved.

Figure 3

Multiciliated cell basal body production pathways: Deuterosome-dependent and PCM-dependent pathways. A deuterosome is a unique structure of multiciliated cells during the centriole amplification stage and disappears when amplification is completed. The deuterosome-dependent pathway mediates the formation of procentrioles directly, subsequently aggregates them for maturation, and ultimately releases them into the cytoplasm. The deuterosome pathway is the main mechanism of centriole generation in multiciliated cells, and about 90% of centrioles in mammalian multiciliated cells are generated through this pathway. Pericentrin, a PCM component, can recruit centromeric proteins, thereby promoting centromerogenesis. Abbreviation: PCM, pericentriolar material. Reproduced from Ref. [30]. Copyright 2020, Trends in cell biology.

Figure 4

Ciliopathies impacting human organ systems due to dysfunction of motile and non-motile cilia. The illustrations show the different ciliopathies of tissues or organ systems and highlight the key manifestations in each affected organ. Red, blue, and green to show conditions from motile cilia issues, primary cilia defects, and both types of abnormalities. Abbreviations: PKD, polycystic kidney disease; NPHP, nephronophthisis. Reproduced from Ref. [12]. Copyright 2017, Springer Nature.

Figure 5

3D OCT analysis of cilia metachronal activity in the mouse oviduct. (A) 3D OCT visualization provides a holistic view of the tubal jugular ampulla and designates specific regions for 3D+time imaging. (B) Structural OCT volumetric perspective of selected regions reveals ciliated oviductal grooves. (C) Corresponding frequency mapping. (D) 5.2 Hz phase diagram of the dominant frequency. (E) Viewing the phase distribution on the groove surface from the perspective shown in (D). (F) Plotting the change in phase over time along the line marked in plot E shows the metachronal wavefront propagation at 91 μm/s. (G) In the orthogonal orientation, no discernible waves are observed. Reproduction from Ref. [87].

Figure 6

Successful outcome of tubulin staining in a mixed population of ciliate cells, including C. inflata (1), Lacrymaria olor (2), T. thermophila (3), C. hirtus (4), and C. uncinata (5). Scale bars = 50 μm. Reproduction from Ref. [100]. Used under Creative Commons CC-BY license.

Figure 7

Principles of the three main super-resolution techniques. (A) The principle of single-molecule localization microscopy (SMLM). SMLM activates and precisely locates individual fluorescent molecules in repeated cycles. By compiling these isolated positions, it constructs a 3D super-resolution image that surpasses conventional resolution limits. (B) The principle of structured illumination microscopy (SIM). SIM uses patterned light to create low-frequency interference (Moiré pattern) with samples. By capturing shifted images, it reconstructs observable structures, but details finer than the pattern’s frequency (like the upper part of the sample) remain undetectable. (C) The principle of stimulated emission depletion (STED). STED microscopy uses a donut-shaped laser (center-aligned with the excitation spot) to suppress peripheral fluorescence via a saturation effect and narrows the emission area below the diffraction limit, enabling super-resolution imaging. Reproduced from Ref. [107], Chen, J., et al. Used under Creative Commons CC-BY license.

Figure 8

Spatiotemporal dynamics of exogenous Kif6/Kif9 in ependymal cilia. (A) 3D SIM visualization revealed spatial distributions of Kif6-GFP relative to Ift56 and Ift88, with acetylated tubulin (Ac-tub) serving as axonemal markers. (B) GI-SIM dynamic imaging captured motile trajectories of ciliary Kif6-GFP and Kif9-GFP. The mEPCs underwent adenoviral transduction to express GFP-tagged constructs, with Ift81-GFP as a positive control. Arrowheads highlight traceable GFP puncta demonstrating directional movement patterns. The trafficking trajectories of the GFP puncta projected in the first 200 frames of videos 1, 2, 3 and 4 are shown as projected images. From Ref. [111]. Used under Creative Commons CC-BY license.

Figure 9

Basic steps in structural analysis by cryo-EM. The basic process consists of several basic steps, such as sample preparation, rapid freezing of samples, TEM imaging, image processing, and structural analysis. Abbreviation: TEM, transmission EM. Reproduced from Ref. [122]. Copyright 2016, Springer Nature. 3D model from Ref. [123], Hughes et al. Used under Creative Commons CC-BY license.

Figure 10

Cryo-EM image of axonemal doublet microtubule-associated structures. (A) Left: Schematic of C. reinhardtii axoneme: nine doublet microtubule encircling central pair (gray), with RS (blue), IDA (yellow), N-DRC (green), and ODA (red). (B) Longitudinal Cryo-EM image of the 96 nm repeat of doublet microtubule structures docked into the subtomogram of the axoneme. (C) Cross-sections showing color-coded microtubule inner proteins (MIPs). The minus (-) and plus (+) tips of the microtubules are labeled in the figure, and the asterisks indicate the seams of the A microtubules. From Ref. [128]. Copyright 2019, Cell.

Figure 11

Primary cilia ultrastructure. A 3D model of primary cilia reconstructed from 33 dual-axis tomography datasets. (A) Central longitudinal slice (13.4 nm thick). (B) Amira software-based structural model. Panels C–J show microtubule changes along the cilium (view: base to tip), corresponding to the positions labeled in (B). Color-coded structures (see legend). Abbreviations: BF (basal foot), CiM (ciliary membrane), CM (cytoplasmic membrane), MtC-A/B/C (microtubule complex A/B/C-tubule), and TF (transition fiber). From Ref. [131]. Copyright 2019, Proc. Natl. Acad. Sci.

Figure 12

Overview of vEM techniques. vEM integrates TEM- and SEM-based methods for 3D ultrastructural imaging. vEM employs electron beam–sample interactions to generate serial images: TEM detects transmitted/scattered electrons through ultrathin sections (50–70 nm), while SEM captures backscattered/secondary electrons (BSE/SE) from surfaces. TEM modalities include serial-section electron tomography (ssET) reconstructing 200–300 nm slices via tilt-series imaging, serial-section TEM (ssTEM) for grid-mounted sections, and GridTape TEM automating high-throughput tape-fed imaging. SEM methods involve eFIB-SEM (enhanced ion milling with bias control), SBF-SEM (diamond knife sectioning), FIB-SEM/pFIB-SEM (ion/plasma beam milling), or sequential imaging of serial slices on a substrate such as a silicon wafer (array tomography). Ultramicrodissection is partially automated by collecting the slices on a tape. Abbreviations: FIB-SEM, focused ion beam SEM; SBF-SEM, serial block-face scanning electron microscopy; ATUM, automatic tape-collecting ultramicrotome. Reproduced from Ref. [138], Peddie, C. J., et al. Used under Creative Commons CC-BY license.

Figure 13

FIB-SEM reveals axo-ciliary synapses. From Ref. [141]. Used under Creative Commons CC-BY license.