Cilia are slender, hair-like appendages that project from the surface of nearly all mammalian cells. These organelles are small, typically ranging from a few to about twenty micrometers in length, with a diameter of only 0.2 to 0.5 micrometers. They serve dual functions: acting as sensory antennae that coordinate cellular signaling pathways, and generating movement to propel cells or move fluid over tissue surfaces, such as clearing mucus from the lungs. Due to their minute size and intricate internal components, standard light microscopy lacks the necessary resolution to capture their detailed structure.
The Ideal Microscope for Cilia Surface Imaging
The classic, striking images of cilia, which show them densely covering a cell surface, are most commonly produced by the Scanning Electron Microscope (SEM). The SEM is uniquely suited for revealing the surface topography of biological samples because it visualizes the external structure rather than transmitting light through the specimen. It provides a highly detailed, three-dimensional view of the cilia’s external morphology, including their density, length, and overall organization on the cell membrane.
SEM images capture the individual cilia as distinct, thread-like projections, clearly showing their physical relationship to one another and the underlying cell surface. This method is particularly powerful for observing the dense arrangement of motile cilia, which beat in coordinated waves, or the solitary projection of a non-motile primary cilium. The resulting images show how these structures are physically organized to carry out functions like mucociliary clearance in the respiratory tract.
How Electron Microscopes Reveal Fine Details
Electron microscopes bypass the limitations of light microscopy by using an electron beam instead of light, which increases the achievable resolution. This principle relies on the significantly shorter wavelength of electrons compared to visible light, enabling the visualization of structures at the nanoscale. The Scanning Electron Microscope operates by directing a finely focused electron beam across the specimen’s surface in a raster pattern.
As the electron beam interacts with the sample, it causes the emission of various signals, most notably secondary electrons, from the surface atoms. Specialized detectors collect these secondary electrons, which are ejected from the specimen’s surface after the primary beam’s impact. The number of secondary electrons collected depends on the surface topography and the angle of the surface relative to the beam and the detector. This variation in signal intensity is used to construct the image, generating the characteristic high-magnification view with intense depth of field and an illusion of three dimensions.
Seeing Cilia’s Internal Structure
While the SEM excels at capturing the external surface, the Transmission Electron Microscope (TEM) is required to visualize the internal molecular scaffolding of the cilium. TEM reveals the internal components, providing a two-dimensional cross-section of the structure. This technique requires the sample to be fixed, embedded in a hard resin, and then sliced into extremely thin sections, typically only 50 to 70 nanometers thick, for the electron beam to pass through.
The resulting TEM image is stained with heavy metals, which scatter the transmitted electrons to create contrast, highlighting the internal microtubule arrangement known as the axoneme. This method first revealed the highly conserved “9+2” microtubule configuration characteristic of motile cilia. This structure consists of nine pairs of doublet microtubules arranged in a ring, surrounding a central pair of single microtubules.
Non-motile primary cilia display a “9+0” arrangement, lacking the central pair of microtubules and the associated motor proteins necessary for movement. TEM images allow for the precise visualization of molecular components like the dynein motor proteins, which are attached to the outer doublets and generate the force for ciliary beating. Ultimately, both SEM and TEM are necessary to provide a complete understanding of the cilium, one showing the external architecture and the other revealing the internal molecular machinery.