Adaptive optics provides us with the ability to take microscopic pictures of the living human retina. We now have the luxury of being able to view high resolution images of fine structures in the eye, such as individual photoreceptors, blood vessels, and nerve fibers. One of the first scientific imaging experiments our lab undertook with this technology was to determine the arrangement and relative numbers of the three cone classes in the living eye. For hundreds of years, scientists, such as Thomas Young (1801), have proposed that the retina contains three different types of cones whose signals are combined to provide us with normal color vision. These three types of cones are typically designated as L-, M-, or S-cones and represent classes of photoreceptors that are primarily sensitive to long wavelength light (L), medium wavelength light (M), and short wavelength light (S) within the visible spectrum. However, it was not possible to determine the arrangement of these cone types in a human eye before the development of a high-resolution imaging modality such as adaptive optics. Our laboratory has successfully mapped the distribution of all three cone classes in living human eyes, as well as in a living macaque monkey.

Segregating the three cone classes

We classify individual cones by comparing images of the photoreceptor mosaic when the photopigment is fully bleached to those when the photopigment is dark-adapted or selectively bleached with different wavelengths of light. When segregating the three cone types, we take advantage of the slight differences in the spectral sensitivities of the S, M, and L cones, as shown in the top left panel of the figure below. For example, the absorptance spectrum for the S cones is nicely separated from the similar M and L cone spectra. In order to determine the location of the S cones, we take images of the photoreceptor mosaic using 550 nm light after bleaching the mosaic with 550 nm light. The S cones are primarily unaffected by a bleaching light of this wavelength whereas the M and L cones absorb strongly at this wavelength and will have little absorptance after the bleach (top right panel). Therefore, the retinal absorptance images after a 550 nm exposure will show a relatively sparse array of dark cones, representing the S cones, amongst a sea of bright cones, representing the M and L cones. To segregate the M and L cones, we use a 650 nm and 470 nm light to selectively bleach the L and M cones, respectively, and then image the retina with 550 nm light. The absorptance images for the 650 nm bleach contain dark, low absorptance L cones that have been heavily bleached and bright, high absorptance M cones that were spared from bleaching (lower left panel). For the 470 nm bleach (lower right panel), the absorptance image contains M cones that are now selectively bleached, but the difference in the residual L and M cone absorptance levels is less dramatic due to their similar spectra. Using this technique, we are able to correctly identify nearly all of the photoreceptors within a given patch of retina for a given observer.

Organization of the three cone classes in the human retina

With adaptive optics, Austin Roorda and David Williams (1999) were able to determine the packing arrangement of all three cone classes for the first time in the human retina. Fig. 12 shows the scatter plot of the absorptances of single cones following a 470 and 650 nm bleach for two male subjects. The left panel is obtained from a color normal subject, and the right panel is obtained from a protanope, or an observer who lacks L cone pigment and does not possess normal color vision. For the color normal subject, the cloud of points in the left frame produces a bimodal distribution. The lower distribution represents the L cones as it shows higher absorptance after the 470 nm bleach, while the top distribution represents the M cones. The results obtained from the protanope produces only a single distribution, representing this subject's M cones, and helps to confirm our method of segregating L and M cones. This single distribution observed in the figure is again due to the fact that protanopes lack and L cone pigment. Fig. 13 shows the cone arrangements for two color normal subjects. These are pseudocolor images in which we have falsely colored the cones to be blue, green, or red in order to represent which photoreceptors are S, M, and L cones, respectively. All three cone classes were randomly distributed in both subjects. In addition, the relative number of L- and M-cones differs greatly between these two subjects. The subject on the left has a L to M cone ratio of 3.79 while the subject on the right possesses a L to M ratio of 1.15, yet both subjects possess normal and similar color vision.

Organization of the three cone classes in the primate retina

We also succeeded in classifying cones in a macaque monkey (Roorda et al., 2001). The L to M cone ratio was about 1.4 for the macaque and these cones were randomly distributed. Unlike the human cone mosaics, however, the Scones were regularly distributed, confirming previous reports regarding the arrangements of the Scones in the monkey (Bumstead & Hendrickson, 1999; Shapiro et al., 1985).

Young T., (1801). On the mechanism of the eye. Philosophical Transactions of the Royal Society of London, 91, 23-88.

Shapiro M.B., Schein S.J., & Monasterio F.M., (1985). Distribution and development of short-wavelength cones differ between macaque monkey and human fovea. Journal of Comparative Neurology, 403, 501-516.

Bumstead K. & Hendrickson A., (1999). Distribution and development of short-wavelength cones differ between macaque monkey and human fovea. Journal of Comparative Neurology, 403, 501-516.

Roorda A., & Williams D.R., (1999). The arrangement of the three cone classes in the living human eye. Nature, 397, 520-522.

Roorda A., Metha A.B., Lennie P., & Williams DR, (2001). Packing arrangement of the three cone classes in primate retina. Vision Research, 41,(10-11) 1291-1306.

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