High resolution reflectance images of nerve fiber bundles in the RNFL exhibit a speckled texture at long wavelengths that becomes less apparent at short wavelengths (). This appearance may be understood by considering the size of a coherence volume relative to the RNFL thickness. shows schematic drawings of the approximately cylindrical coherence volumes produced by the IMR and calculated with Equations 2
. With quasi-monochromatic illumination from an incandescent source, reflectance speckle arises mostly from temporal interference, that is, interference between scattered components of a wave and itself delayed in depth. At 660 nm, the coherence length is comparable to the thickness of a typical nerve fiber bundle (~15 μm) and rays scattered from throughout a bundle contribute to the speckled texture. At 440 nm, however, the coherence length is only a fraction of the bundle thickness and the resulting speckles from different depths are superimposed on the average reflectance. Short-wavelength speckles, therefore, have lower contrast, as seen in C. In this view of the origin of RNFL reflectance speckle, the speckle pattern is a property of the RNFL, associated with its internal structure. To demonstrate this concept and to rule out a possible contribution of tissue movement to the change of speckled texture, we used fixed retinas and purposely shifted the retina laterally and in depth. These movements caused little change in the speckled texture of the RNFL, confirming that the speckles were a property of the tissue itself and not of its position in the optical system.
Figure 6. Schematic of coherence volumes in the RNFL. Rays scattered at points within a coherence volume (gray cylinder) can produce interference effects. The size of a coherence volume increases with increasing wavelength. At 440 nm, L = 6 μm and d = 1.4 (more ...)
The speckled texture of RNFL reflectance images was studied at 660 nm. A time-lapse image series showed that the speckle pattern changed over a period of a few minutes. The speckles appeared and faded but did not move along bundles. We quantified the change in a series of subimages with CCs between the initial and subsequent speckle patterns, which provided a measure of their similarity. For normally prepared retinas in conditions designed to maintain physiological activity, the CC was high at the beginning of an image series, indicating similarity between the speckle patterns. With the lapse of time, the CC decreased gradually until after a few minutes it varied around a plateau determined by features common to all subimages. The time constant of an exponential fit to the decrease was used to quantify the rate of change of CCs. Fixation of tissue structure with paraformaldehyde did not affect the qualitative appearance of the RNFL speckle, but eliminated the temporal change, suggesting that speckle dynamics resulted from temporal change of reflecting structures.
Compared with the gradual decrease of CCs in bundle areas, the CC of gap areas decreased abruptly to a plateau. The average τ = 0.09 minute was approximately the interval used for collecting an image series. The result suggests that the reflectance of gap areas was not correlated between two consecutive images and reflectance at each pixel changed randomly. The time courses of CCs in gaps were similar for unfixed and fixed retinas, suggesting that the random change of gap reflectance was due to the noise of the optical measurements.
Consideration of structural change within axons immediately suggests a role for axonal transport. Axonal transport is achieved by the binding of molecules, vesicles, and organelles (so-called cargo) to molecular motors that generate movement along the axons.20
A direct role for axonal transport as the source of the temporal change in speckle is problematic, however, because the structures that scatter light in the RNFL are known to be cylinders oriented along the axon bundles,28
with approximately half the reflectance coming from MTs.25,26
We hypothesize that movement in depth of scattering structures occurs as a secondary consequence of axonal transport. depicts a possible biophysical model for this concept. The model in shows the component of transport that uses MTs as tracks. In this model, the movement of the motor-cargo complexes along MTs changes the spacing between MTs and hence the relative phase of the reflected light. Within a coherence volume as depicted in , these phase changes sum coherently to produce a spatial change in the speckle pattern without movement along bundles. Although explicitly shows MTs, other cylindrical scattering structures could be similarly displaced by axonal transport and contribute to speckle dynamics. Further, other displacement mechanisms could be involved, and a more general hypothesis would link speckle change to unspecified axonal dynamic activity.
Figure 7. Biophysical model of RNFL reflectance speckle. Axonal MTs, an important contributor to RNFL reflectance, act as tracks to guide motor-cargo complexes along axons. In this model, longitudinal movement of the complexes changes the spacing between MTs and (more ...)
To test the hypothesis that temporal change of RNFL reflectance speckle is associated with axonal dynamic activity, we performed three experiments expected to alter this activity. The first, mentioned above, was tissue fixation, which resulted in the speckle pattern becoming highly correlated over time (D). The second experiment used normally prepared retinas perfused with a physiologic solution at a lower temperature. Because low temperature slows axonal transport30
and other physiological processes, the slower decay of the CC measured at 24°C () demonstrates a link between change of speckle and axonal dynamic activity. The third experiment attempted to relate temporal change of RNFL speckle to axonal transport by depolymerizing MTs with colchicine. MTs are a key cytostructure for transferring proteins and cellular components along the axons,21–23
and MT depolymerization was expected to disrupt axonal transport.32
The CC time courses () obtained with different durations of MT depolymerization confirmed this expectation; the decay of CC became slower with the colchicine treatment and the time constant increased with the duration of treatment.
Change of axonal transport is often an early sign in many optic neuropathic diseases, such as glaucoma.33–40
Optical assessment of RNFL is currently limited to evaluating RNFL structure. This study demonstrated that change of RNFL reflectance speckle reveals axonal dynamic activity, perhaps axonal transport. This new concept may allow a novel approach for assessing the RNFL, namely, detecting physiological activity of axons, which may precede loss of axonal structure.
This study used an in vitro preparation of retina to eliminate the confounding effects of other ocular tissues on measurements. Recently reported RNFL images obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) reveal a similar speckled texture of RNFL reflectance in human eyes (Scoles DH, et al. IOVS
2012;53:ARVO E-Abstract 6954).41
If it can be demonstrated that the apparent AOSLO speckle also arises from interference, it may be possible to adapt AOSLO technology for noninvasive assessment of axonal dynamic activity in clinical practice.