Electron Microscopy

The examination of inner ear hair cells is essential for the accurate diagnosis of trauma to the inner ear ultrastructure, as a result of exposure to high intensity anthropogenic sounds.  Previous studies of the hair cells have been conducted using a light microscope (Jensen, 1994) to reveal hair cell orientations in the upside-down catfish (Synodontis nigriventris), whilst Lu and Popper (1998) examined the polarisation of ciliary bundles in the end organs of the sleeper goby (Dormitator latifrons) using immunocytochemicals and a confocal imaging technique.  However, the Scanning and Transmission Electron Microscopes (SEM and TEM) are able to produce high quality images at considerably higher magnifications than light microscopes, and has been used to considerable effect when examining inner ear ultrastructure (see Lovell, et al., 2005a, and b).

Figure 1. The Jeol Scanning Electron Microscope (insert: hair cells from D. labrax)

Click on image for expanded view of hair cells.

Fresh inner ear samples are placed in chilled fixative (2.5% glutaraldehyde in 0.1 M cacodylate buffer with 3.5% sodium chloride) for 48 hours, prior to dehydration through a graded ethanol series ranging from 35% through 50%, 70% and 90% to absolute ethanol, prior to critical point drying.  Fully desiccated samples are mounted on a specimen stub, and coated with c. 8 nm of gold in an Emitech K 550 sputter coater.

For Transmission Electron Microscopy (TEM) Freshly excised tissue from the inner ear is fixed by placing it into a 2.5% Glutaraldehyde in Sodium Cacodylate buffer (0.1 M pH7.2) for at least one hour, then rinsed twice in Cacodylate buffer for 15 minutes each rinse.  The inner ear tissue is then secondary fixed in a 1% Osmium Tetroxide in Sodium Cacodylate buffer (0.1M pH7.2) for one hour.  The tissue is then rinsed twice in buffer, then Dehydrated in an ethanol series. Once in 100% ethanol, the tissue is placed in increasing concentrations of Spurr’s Resin until it is fully infiltrated to 100%.   The sample is then placed in Beem capsules ready for polymerisation of the resin, which was achieved by placing  it in a 70 ºC oven overnight in accordance with Glauert  (1975).  The resulting resin blocks are sectioned using a Reichert-Jung Ultracut and a Micro Star diamond knife (insert of Figure 3).  The sections are usually placed on thin bar copper grids, and stained firstly with a saturated ethanol solution of Uranyl Acetate and then a second stain of Reynolds Lead Citrate (15 minutes each stain) (Lewis and Knight 1977). The fully processed images are taken using the Jeol 1200 EX II TEM and the images captured with an SIS Mega view III (Figure 2).

Figure 2.  The Jeol 1200 EX II Transmission Electron Microscope

The Transmission Electron Micrograph (TEM) section in Figure 3.b shows the complete hair cell from the sea bass D. labrax, sectioned lengthwise from the ciliary bundle at the top of the Figure, to the innervating nerves at the bottom, and the receptor cell from the prawn presented in the same way.  In Figure 3.b, the longest of the cilia with the dark core is the kinocilia, and the shorter hairs are the stereocilia.  The peripheral saccular nerve fibres can be seen in an area where the membrane of the axon terminal is in close proximity to the membrane of the neuron.  Information travels across the synapse by way of neurotransmitters, which diffuses across the synaptic cleft to the postsynaptic membrane.  If sufficient neurotransmitter is secreted, an action potential is generated in the neuron.  The crosswise section through the top of the cell (Figure 3.b), shows that the cuticular plate is almost void of obvious structures, except for a number of fine tracts created by actin filaments extending from the cilia base into the cuticular plate (Lovell et al., in press).   The sides of the receptor cell are buttressed by supporting cellular structures, and the base of the receptor cell sits above two further supporting cells with large nuclei.  A high powered micrograph of the section taken through the cuticular plate, 3.c shows the cilia base and actin filaments (af) that “root” the stereocilia to the hair cell.

Figure 3.  Transmission and scanning electron micrographs of the hair cells from the ear of the bass (D. labrax), and the prawn (P. serratus) (from Lovell et al., 2005a).Click on image for expanded view of hair cells.