Aside from the satisfaction of basking in our progress, there seems to be little point in dwelling on the past unless it guides us in the future. One lesson from dwelling on the past is that the major improvements in retinal imaging technology throughout history have sprung from advances in other fields, especially optics, microscopy, and astronomy. This repeats a pattern that has pervaded the history of vision science and ophthalmology at least as early as Kepler, an astronomer who first stated unequivocally in 1604 that the retinal image is inverted. Others from the physical sciences who have made seminal contributions to vision science and ophthalmology include Helmholtz, Herschel, Maxwell, Newton, Schiener, and Young to name just a few. It is especially interesting how long it takes for significant technical advances in other fields to migrate into the eye. Confocal imaging, OCT, and adaptive optics were all introduced in other domains decades before they were applied to the eye. Part of this delay has to do with the lack of availability or high cost of the hardware required to implement new technologies; I will never forget the $1M price tag on the only deformable mirror available when I first contemplated building an adaptive optics ophthalmoscope at the University of Rochester. It also takes time to learn a new technology and to implement the modifications required to make it work in the eye. Another delay may be the result of scientific provincialism and insularity, a delay that could be reduced by increased scrutiny of the latest developments in those fields that have historically fueled ophthalmoscopy. On the bright side, if history is any guide, the next major breakthrough in retinal imaging technology was almost certainly made decades ago and is just waiting for an enterprising scientist or engineer to translate it into the eye.
The developers of new technology for retinal imaging may be unaware that they have something in common with cave divers. Both enjoy an extreme sport that is more often than not an exercise in the management of claustrophobia. The cave diver's view of his cramped world is of almost entirely impenetrable rock. The developer of retinal imaging technology is similarly boxed in by the fundamental limits of physics and biology. Both grope along, driven by the hope of discovering a previously-missed passage that, if they can just squeeze through, will open up into whole new possibilities for exploration. The history of retinal imaging would suggest, however, that fundamental limits are rarely if ever fundamental limits of the natural world. They are almost always fundamental limits of the conceptual framework we have chosen to think in. The conventional wisdom in 1990 held that axial resolution was fundamentally constrained by geometrical optics and depth of focus. But by adding the wave properties of light to the conceptual framework, practitioners of low coherence interferometry in the eye surprised everyone with a 100-fold, and now recently a 1000-fold, improvement in axial resolution over what was thought possible before.
Though it is true that the speed of light has stood its ground as a fundamental limit for a very long time, can we really say with confidence that any limits are truly impenetrable? Progress in high-resolution retinal imaging demands that we treat these limits as invitations to overcome them. It may seem that the spectral transmittance of the cornea and lens would pose a fundamental limit on noninvasive optical imaging of the retina, forever preventing us from interrogating molecules that absorb light only in spectral regions outside the ocular transmittance window. But as shown in , Jennifer Hunter and colleagues have recently shown that two-photon imaging can excite autofluorescent molecules in the living retina that have excitation spectra in the near UV, outside the spectral pass band of the eye's optics (Hunter et al, 2011
). If this method can be made more efficient, it could open up an entirely new way to study retinal structure and function in the living eye.
Fig. 19 A. two-photon image of the cone mosaic in the living primate retina at 2.5 deg superior retina using 730 nm illumination from a Ti-Sapph laser with a pulse width of less than 70 fs. B. Reflectance image of the cone mosaic at the same location using 790 (more ...)
Another major constraint on microscopic retinal imaging is the maximum permissible light exposure that can be delivered without damaging the eye. This constraint is especially troublesome given that the typical yield in reflectance imaging is one photon back for every 10,000 that enter the pupil. The problem is compounded by the high magnification that microscopic retinal imaging demands, as well as the low efficiency of many potentially informative light-tissue interactions. But the dramatic improvements in eye tracking described earlier make it possible to maintain high spatial resolution and avoid thermal light damage by harvesting light at reduced power over longer times. Moreover, compounds have been discovered that protect the eye from photochemical light damage (eg. Maeda et al., 2006
), and the application of these prior to imaging could make it possible to observe faint retinal signals that are presently invisible. In addition, we are at the beginning of a revolution in the availability of contrast enhancing fluorophores that one can direct to particular classes of retinal neurons with increasing specificity, functional imaging capabilities, and improved quantum efficiency.
The diffraction limit is another fundamental barrier that is just now coming under scrutiny in the domain of retinal microscopy (Shroff et al. 2009
). Abbe (1837) showed nearly 140 years ago that the wave nature of light poses a fundamental barrier on the resolution of an optical system with a fixed numerical aperture. If the diffraction limit could be surpassed, then a new leap to smaller spatial scales in retinal imaging would be enabled, scales that in principle could be molecular. Most scientists and engineers long ago resigned themselves to the notion that the diffraction limit was so fundamental to the nature of light that it would never be surpassed in any practical imaging system. Remarkably, the field of microscopy has already seen the diffraction barrier fall, not once but many times, thanks to an array of new techniques including structured illumination (Gustafsson, 1999
), stimulated emission depletion (Hell and Wichmann, 1994
), and photoactivation localization microscopy (Betzig, et al., 2006
). None of these methods has yet been applied successfully to retinal imaging, and certainly the challenges of doing so are formidable. But the history of the past quarter century strongly suggests that new routes around fundamental barriers will be found, allowing us to acquire ever more information from the living retina.