Fluorescence microscopy has been extensively used in biological research due to its molecular specificity, live-cell compatibility, and multicolor observation[
Laser & Optoelectronics Progress, Volume. 61, Issue 6, 0618016(2024)
Super-Resolution Fluorescence Microscopy for Cilia Investigation and Ciliopathy Diagnosis (Invited)
The last two decades have witnessed the invention and development of super-resolution microscopy (SRM) that breaks the diffraction limit of light and pushes the fluorescence microscopy resolution to several nanometers. While SRM is widely used in biological studies, such as resolving subdiffraction structures, molecular mapping, tracking single molecules, probing protein-protein interactions, and observing organelle dynamics, its direct application in translational medicine, such as disease diagnosis, is still preliminary. Despite their small size, cilia play a crucial role as organelles in cell signaling and motility, with defects in cilia leading to ciliopathy. Similar to other miniature organelles and macromolecular complexes, cilia are ideal for super-resolution imaging. In this review, we will 1) introduce cilia and ciliopathy, 2) show how SRM extends our knowledge of cilia, and 3) focus on how SRM improves the diagnosis of motile ciliopathies.
1 Introduction
Fluorescence microscopy has been extensively used in biological research due to its molecular specificity, live-cell compatibility, and multicolor observation[
Based on different principles, as shown in
Figure 1.Summary of different super-resolution imaging techniques. (a) Conventional imaging; (b) SIM; (c) SOFI; (d) STED; (e) expansion microscopy; (f) SMLM
Two other techniques—super-resolution optical fluctuation imaging(SOFI)and expansion microscopy—also achieve super-resolution imaging. 4)SOFI uses the intrinsic fluctuation properties of fluorophores and is a computational technique that relies on the post-data analysis of the fluorescence signal correlation to achieve enhanced resolution[
Recent developments in super-resolution microscopy involve the combination of different techniques to achieve sub-10-nm resolution or molecular resolution. MINFLUX[
The range of tools available for super-resolution imaging is expanding, with each offering distinct characteristics to meet various needs in biological research, such as higher spatial and/or temporal resolution, multiplexed labeling, reduced phototoxicity, and extended sample depth. In summary, these techniques have dramatically altered fluorescence microscopy and pushed it to the nanoscopy era, revolutionizing the field of biology.
2 Cilia and ciliopathy
Cilia are hair-like slender cellular organelles that arise from membrane-docked centrioles and protrude from the surface of the cell. Cilia can be motile or sensory depending on whether they beat or not.
Generally, sensory cilia are solitary and critical for signal perception and transduction. One example is the photoreceptor outer segment, which is a specialized cilium for photon reception. Sensory cilia appear in nearly all human cells and function in signaling pathways, such as Hedgehog(Hh), GPCR, RTK, and WNT signaling[
Motile cilia also possess sensory functions but are designed specifically for fluid propelling or cell motility. Motile cilia are present along the airway epithelia, brain ventricular wall, and female fallopian tube to remove mucus, circulate cerebrospinal fluids, and deliver zygotes[
Cilia defects lead to ciliopathies, a collection of rare diseases that affect nearly all human organs, including symptoms such as blindness, obesity, kidney abnormalities, and/or mental retardation[
Dysfunctions of motile cilia lead to motile ciliopathies[
Figure 2.Cilia and ciliopathy. (a) Cilia and affected organs; (b) structure of sensory and motile cilia
3 Size and architecture of cilia
For long, cilia have been regarded as futile, partly because of their small size. Despite their small size, cilia possess a delicate architecture. The centriole first docks on the plasma membrane through its distal appendage and forms the basal body of cilia, which are approximately 200-nm wide and 300‒500-nm long. Cilia then build the transition zone, a diffusion barrier that gates the ciliary contents, and the membrane-encased nine-microtubule-based axoneme grows from the transition zone. Intraflagellar anterograde and retrograde transport shuttles ciliary components into and out of cilia, building cilia and maintaining their functions. The cilia diameter is approximately 80‒200 nm, and their lengths vary from 2 to 10 μm. For motile cilia, most have two microtubule central singlets and molecular motors, such as outer dynein arm(ODA)and inner dynein arm(IDA)complexes, surrounding the microtubule doublet and power the cilia beat, as shown in
4 Super-resolution microscopy boosts research
Sensory cilia have long been ignored until the discovery of their role in Hh signaling[
4.1 Diffusion barrier
To maintain the cilia’s unique composition, a diffusion barrier gates the transport of ciliary components. The diffusion barrier sits at the base of cilia and comprises the transition zone, transition fiber, and the region in between. The transition zone is characterized by a Y link that connects the axoneme and ciliary membrane. Transition zone defects lead to ciliopathies, such as JS, Meckel-Gruber syndrome(MKS), and nephronophthisis(NPHP).
The transition zone contains at least 26 proteins[
Figure 3.Super-resolution imaging resolves ciliary architecture. (a)‒(c) Transition zone architecture revealed by STED[43], STORM[44], and expansion microscopy[48]; (d) STORM reveals an unexpected matrix zone for the transition fiber[43]; (e) organization of basal foot proteins in both sensory and motile cilia revealed by three-dimensional (3D) SIM[50]; (f) STORM shows that CatSper1 forms four linear domains along the sperm flagella[55]; (g) SIM reveals that Chlamydomonas PKD2 forms two linear domains and associates with doublets 4 and 8[57]; (h) STORM reveals the 96-nm periodicity of the RS component RSPH4A[40]; (i) ROSE-Z shows that CCDC176 binds and stabilizes doublets 1 and 9[58]
Yang et al. also resolved the organization of the transition fiber, another diffusion barrier of cilia. Under TEM micrographs, the transition fiber appears as nine-fold blades emanating from the basal body. By examining 16 transition fiber components, they found that these proteins form a cone-shaped structure with proteins, such as FBF1, located in the space between the blades, a new structure not previously recognized by TEM, as shown in
4.2 Basal foot
The basal foot is a macromolecular structure of the basal body that originates from the subdistal appendage of the centriole. Unlike the transition fiber, which exhibits a 9-fold symmetry, the number of basal feet per cilium varies among different cell types and depends on different factors[
4.3 Ciliary membrane protein
Membrane proteins have peculiar organizations, such as protein islands and functional domains. Recent research explored if ciliary membrane proteins have similar distributions. Yoon et al.[
4.4 Ciliary axoneme
The distribution of axoneme accessory proteins, such as ODAs and IDAs forms periodic structures. For instance, ODAs form 32-nm repeats, whereas IDAs, NDRC, and RS proteins form 96-nm repeats[
5 Ciliopathy investigation and diagnosis using super-resolution microscopy
5.1 Joubert syndrome study using SIM
JS is a rare ciliopathy with symptoms of brain abnormalities characterized by the molar tooth sign under nuclear magnetic resonance imaging. JS is genetically heterogeneous, and a large portion of the disease-causative genes encode transition zone components. The molecular mechanism of JS is unclear. Using STORM, Shi et al. examined the fibroblast cilia transition zone from patients with JS bearing either TCTN2 or RPGRIP1L mutations. Interestingly, they found that the MKS and NPHP complexes are absent from the patients’ cells, suggesting that transition zone absence might be a common feature[
Figure 4.Super-resolution imaging aids ciliopathy investigation and diagnostics. (a) 3D SIM reveals the disruption of the transition zone and impairment of Hh signaling in patients with JS[44]; (b) 3D SIM-based PCD molecular fingerprint for PCD diagnosis[40]; (c) PCD diagnosis based on quantitative 3D SIM[40]; (d) PCD diagnosis based on rotational polarity[40]; (e) STORM reveals the preservation of 96-nm periodicity in CCDC39 loss of function cells[40]; (f) PCD diagnosis based on MAGNIFY-SOFI[65]
5.2 PCD investigation and diagnosis using 3D SIM and STORM
Though rare, direct application of super-resolution imaging in disease diagnosis is possible, and PCD is one of the few examples that demonstrate the potential of super-resolution imaging. In the remaining sections, we focus on motile ciliopathy PCD. We first introduce the traditional methods of PCD diagnosis and then illustrate how super-resolution imaging improves the diagnosis. Finally, we discuss how the diagnosis can be further optimized by integrating multiplexed labeling and high-speed video microscopy(HSVM).
5.3 Different diagnostic approaches for PCD
Early diagnosis is essential for initiating therapies to slow PCD progression because a delay in diagnosis may lead to lung collapse or transplantation, threatening patients’ lives. However, for a long time, PCD has been underdiagnosed because of its genetic heterogeneity, technical limitations, and lack of awareness. It may take years for patients with PCD to receive a confirmed diagnosis. Currently, two different methods are used to conclude the diagnosis:TEM and/or DNA sequencing. Even when the two techniques are combined, ~30% of patients with PCD clinical symptoms do not receive a confirmed diagnosis[
TEM was once regarded as the gold standard for PCD confirmation, specifically for ciliary ultrastructure defects. However, TEM mainly examines the absence of ODAs and/or IDAs, microtubule disorganization, and central pair defects and may introduce false-negative results. Generally, next-generation sequencing platforms enable the simultaneous querying of multiple exons, and compared with other methods, molecular genetic testing is an easy and quick option. However, unexpected mutations may occur in new PCD genes and untranslated regions, which can only be detected using the more expensive and time-consuming whole genome sequencing. Additionally, for known PCD causative genes, the mutations could be variants of unknown significance(VUS), which are common and increase the complexity of the diagnosis.
Two other techniques have recently emerged to aid PCD diagnosis:HSVM and fluorescence microscopy. Ciliary beat information can be extracted from the HSVM of multiciliated cells;however, it is insufficient to confirm PCD. Most PCD diagnostics only examine the cilia beat frequency but do not adequately differentiate PCD because the cilia beat amplitude, waveform, and coordination are vital to evaluating the ciliary beat. Additionally, chronic infections can disable ciliary motility, leading to false-positive diagnosis.
5.4 PCD diagnosis using fluorescence microscopy and 3D SIM
For most motile ciliopathies, molecular defects impair the assembly of the sophisticated axoneme of cilia. Immunofluorescence can help illustrate the absence of these components and contribute to the diagnosis. Shoemark etal.[
To further validate the fluorescence microscopy-based diagnosis and increase the resolution, Liu et al. first generated a molecular fingerprint of ~25 PCD proteins by staining control multiciliated cells and using 3D SIM, as shown in
To test whether 3D SIM and antibody panel staining combination can be developed as an independent diagnostic tool, the authors further tested two patients with different antibody panels. The first patient with DNAH5 and DNAH11 mutations was diagnosed with 15 PCD protein antibodies, and the result suggested the loss of all ODA components:DNAH5, DNAH11, and DNAI1. The second patient, with mutations in DNAH11 and TEM inconclusive, was diagnosed with 10 PCD protein antibody panels, and the result suggested the absence of DNAH11.
When cilia beat coordinately, the basal feet point toward the direction of the cilia beat, a phenomenon called rotational polarity[
Antibody panel and quantitative 3D SIM-based diagnosis is straightforward and is likely to be implemented in clinical settings. Restricted by the availability of patients’ cells, this study only examined 31 patients with the most common PCD mutations. The sensitivity and robustness of this method require more testing of more patients and PCD mutations in the future. Note that the more antibodies in the panel, the more patient cells and labor required. To address this issue, an antibody multiplexed labeling strategy is required to optimize diagnostics in the future.
5.5 PCD investigation using 3D SIM and STORM
To further investigate whether super-resolution imaging can detect structural defects in patients with PCD, this study[
5.6 PCD diagnosis using MAGNIFY-SOFI
3D SIM-based diagnosis relies on sophisticated microscopes, which may not be accessible in many hospitals/institutes. Contrarily, expansion microscopy and SOFI can be performed using a regular fluorescence/confocal microscope.
To explore the possibility of applying expansion microscopy and SOFI to PCD diagnosis, Klimas et al.[
This study provides an economical and speedy method for assessing the motile cilia architecture using super-resolution microscopy instead of TEM. TEM requires cutting samples into thin cross-sections and examining different cross-sections to locate motile cilia. MAGNIFY-SOFI is fluorescence imaging-based and does not involve sample cutting. However, this study was limited to pan staining of the entire motile cilia proteome. As molecular specificity is one major advantage of fluorescence microscopy, it would be encouraging to observe different components of the cilia axoneme stained by different antibodies and to compare the differences between healthy controls and patients with PCD in the future.
6 Future perspectives on ciliopathy diagnosis
Despite the exciting progress made by the two groups, disease diagnosis using super-resolution microscopy is still at a preliminary stage.
One major challenge is the heterogeneity of the disease and the scarcity of patients. With the application of CRISPR KO in human airway cells, it would be helpful to test new and compare different diagnostics using CRISPR KO multiciliated cells[
Although the structure of cilia is well studied, their motility is only well characterized in a few model organisms and sperm cells. HSVM records ciliary motility with a high temporal resolution, and additional cilia beat parameters can be extracted to aid diagnosis. HSVM is also restricted by light microscopy resolution and sensitive cilia beat analysis which requires isolating a single cilium. Therefore, it is worthwhile to label motile cilia with fluorescent markers and apply high-speed super-resolution imaging techniques to distinguish single cilia for high-quality data analysis. Integrating cilia beat and antibody panel data will provide a more comprehensive characterization of cilia.
7 Summary
Super-resolution imaging has demonstrated its effectiveness in revealing the ciliary structure and diagnosing motile ciliopathy. The new generation of super-resolution imaging techniques that achieve nano- or angstrom spatial resolution and unprecedented time resolution are expected to revolutionize this field. High-speed super-resolution tracking techniques, such as MINFLUX[
Regarding ciliopathy diagnosis, the sensitivity and accuracy of super-resolution imaging will be further evaluated by integrating patient data and checking CRISPR loss of function cells. The protocols can be further developed and optimized to facilitate diagnosis. Given that motile cilia are such sophisticated structures that they may reflect any cilia defects and multiciliated cells are easily accessible, other ciliopathies, such as sensory ciliopathies, can be possibly diagnosed by examining airway multiciliated cells.
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Zhen Liu, Yang Wu. Super-Resolution Fluorescence Microscopy for Cilia Investigation and Ciliopathy Diagnosis (Invited)[J]. Laser & Optoelectronics Progress, 2024, 61(6): 0618016
Category: Microscopy
Received: Dec. 1, 2023
Accepted: Dec. 23, 2023
Published Online: Mar. 22, 2024
The Author Email: Liu Zhen (zhenliu@ust.hk)