This is a summary of some of the research I did as a graduate student with Melanie Campbell and Bill Bobier at the University of Waterloo, School of Optometry.

    Scanning laser ophthalmoscopy (Webb, 1979) is a retinal imaging technique that is based on the standard scanning laser microscope. The important difference is that in scanning laser ophthalmoscopy, the optics of the eye serve as the objective lens. Scanning laser ophthalmoscopes and microscopes, when equipped with a confocal aperture, offer fundamentally better performance than conventional imaging instruments (Wilson, 1984). The confocal SLO generates high contrast images and can do optical slicing through weakly scattering media making it ideal for imaging the multilayered retina. My aim was to explore the high-resolution capabilities of the instrument. Resolution of retinal images is limited by blur caused by the aberrations in the optical system of the eye. However, I found that imaging could be improved if I carefully aligned the beam to image through the least aberrated areas of the optical system.
    To facilitate high resolution imaging, the Waterloo CSLO was adapted to image a 1.5 degree field on the retina with 512 X 512 pixels at 30 frames per second. To ensure a high cut-off frequency, the entrance beam size was 5 mm. To collect as much light as possible, the entire dilated pupil was used in the return path. Precise control of the entrance beam position was done with an infra-red pupil position monitor (see figure 1).
 

Figure 1: Schematic of the Waterloo CSLO. The red shows the path of the scanning laser beam. Raster scanning is done with a combination of a rotating polygon and a galvanometer scanning mirror. A series of telescopes adjust the diameter and scanning angle of the beam.

Series of images were taken with different entrance beam positions for three subjects.  Single frame images were noisy so the best series were registered and summed to show the real structure. The images are best appreciated by looking at video-taped recording of the real-time images.
 

Figure 2: Registered sum of a sequence of CSLO frames for three subjects. While not all photoreceptors are resolved, the registered sum shows features as small as 1 minute of arc, which is the expected size of a cone photoreceptor at that eccentricity.
 

The photoreceptor array was studied by taking the average power spectra from a sequence of images (see figure 3). The power spectrum is useful because it is useful to detect periodic structure and is translation invariant.
 

Figure 3. Average power spectra of a series of frames at different retinal locations for three subjects. In each spectra, a ring or a plateau can be seen, commonly referred to as Yellot's ring (Yellot, 1982). This ring lies at the dominant spatial frequency of the photoreceptor array.

We measured the average location of the ring in each of the above figures to estimate the photoreceptor spatial frequency as a function of retinal eccentricity (see figure 4).

Figure 4. We plotted our estimates of photoreceptor spacing along with values from Williams (1988) and Curcio et al. (1990). Our estimates fall neatly between the two lines.

The confocal scanning laser ophthalmoscope has been used to provide the first real-time images of photoreceptors in the living human eye. While the instrument provided good enough image quality to measure photoreceptor spacing, it did not have enough contrast to image all photoreceptors. Further improvement in image quality can be obtained by using adaptive optics to compensate the aberrations.

Interesting References

Atkinson, M.R., Roorda, A. & Campbell, M.C.W. (1995) Imaging of individual photoreceptors: Optical possibilities beyond the incoherent resolution limit. Investigative Ophthalmology and Visual Science Supplem. 36, 188
Campbell, M.C.W., Glasser, A. & Roorda, A. (1997) Clinical Implications of Changes in Lens and Ocular Imaging Properties. In: Lakshminarayanan, V. (Ed) Basic and Clinical Applications of Vision Science, (pp. 83-91). Dordtrecht, Netherlands: Kluwer.
Curcio, C.A., Sloan, K.R., Kalina, R.E. & Hendrickson, A.E. (1990) Human photoreceptor topography. J. Comp. Neurol. 292, 497-523.
Dreher, A.W., Bille, J.F. & Weinreb, R.N. (1989) Active optical depth resolution improvement of the laser tomographic scanner. Appl. Opt. 28, 804-808.
Elsner, A.E., Burns, S.A., Hughes, G.W. & Webb, R.H. (1992) Reflectometry with a scanning laser ophthalmoscope. Appl. Opt. 31, 3697-3710.
Liang, J., Williams, D.R. & Miller, D. (1997) Supernormal vision and high-resolution retinal imaging through adaptive optics. J. Opt. Soc. Am. A 14, 2884-2892.
Miller, D., Williams, D.R., Morris, G.M. & Liang, J. (1996) Images of cone photoreceptors in the living human eye. Vision Res. 36, 1067-1079.
Roorda, A. & Campbell, M.C.W. (1997) Confocal scanning laser ophthalmoscope for real-time photoreceptor imaging in the human eye. Vision Science and its Applications: Technical Digest (OSA, Washington, D. C. ) 1, 90-93.
Roorda, A., Campbell, M.C.W. & Cui, C. (1997) Optimal entrance beam location improves high resolution retinal imaging in the CSLO. Investigative Ophthalmology and Visual Science Supplem. 38, 1012
Webb, R.H. (1990) Scanning Laser Ophthalmoscope. In: Masters, B.R. (Ed) Noninvasive Techniques in Ophthalmology, (pp. 438-450). New York: Springer-Verlag.
Webb, R.H., Hughes, G.W. & Delori, F.C. (1987) Confocal scanning laser ophthalmoscope. Appl. Opt. 26, 1492-1499.
Webb, R.H., Hughes, G.W. & Pomerantzeff, O. (1979) Flying spot TV ophthalmoscope. Appl. Opt. 19, 2991-2997.
Williams, D.R. (1988) Topography of the foveal cone mosaic in the living human eye. Vision Res. 28, 433-454.
Wilson, T. (1990) The role of the pinhole in confocal imaging systems. In: Pawley, J.B. (Ed) The Handbook of Biological Confocal Microscopy, (pp. 99-113). New York: Plenum Press.
Wilson, T. & Carlini, A.R. (1989) The effect of aberrations on the axial response of confocal imaging systems. J. Microsc. 154, 243-256.
Wilson, T. & Sheppard, C.J.R. (1984) Theory And Practice of Scanning Optical Microscopy, London: Academic Press.
Yellot Jr, J.I. (1982) Spectral analysis of spatial sampling by photoreceptors: Topological disorder prevents aliasing. Vision Res. 22, 1205-1210.