Cellular imaging of the living human retina
Viewing the retina through the eye's cornea and crystalline lens dates back to the time of Helmholtz, inventor of the ophthalmoscope. Ophthalmoscopes have advanced substantially since then and are now an indispensable non-invasive tool for diagnosis and treatment monitoring. The three principal types are the conventional ophthalmoscope, confocal scanning laser ophthalmoscope (cSLOs), and optical coherence tomography (OCT)).123 Of these, the latter has changed the diagnosis and monitoring of retinal and optic nerve disease the most in recent years.
OCT4 raster-scans a focused beam of near-infrared light across the retina and uses a highly sensitive optical interferometer to detect very weak reflections from the largely transparent retinal layers. A computer then transforms the rastered signal into a 3D image of the ∼300μm thick retina. This clever interferometric use of the light source spectrum yields micron-scale axial resolution and much better sensitivity than other ophthalmoscopes. Axial resolution for OCT—unlike that for conventional optics—is inversely proportional to the source's spectral bandwidth and independent of the numerical aperture.
While ophthalmic OCT has excelled,5,6 it uses the same technique to probe the retina through the optics as other ophthalmoscopes. Imperfections in the eye's optics7—particularly those not correctable with glasses and contact lenses—greatly reduce opthalmoscopes' lateral resolution, or ability to resolve spatial detail at a particular depth in the retina. This blurs the retinal detail and prevents imaging at the cellular level, where vision and pathogenesis begin.
There are two general types of optical imperfections in the eye, monochromatic aberrations and chromatic aberrations. To overcome the first, we have developed a retinal instrument that combines the high axial resolution and sensitivity of OCT with the improved lateral resolution of adaptive optics (AO).8 AO is an optoelectromechanical technique originally developed to dynamically correct atmospheric turbulence that blurs images from ground-based telescopes.9 It has been applied to the conventional ophthalmoscope, cSLO, and standard OCT to correct the monochromatic aberrations of the eye.10 To overcome chromatic aberrations, we have taken advantage of the fact that the chromatic properties of the eye do not vary in time and are largely the same across eyes.11 We designed a customized achromatizing lens with a chromatic effect equal in magnitude but opposite in sign to that of the eye.
AO combined with a customized lens removes the aberrations and provides near-perfect optics for OCT imaging of the retina. Using this strategy, ultrahigh resolution (UHR) AO-OCT instruments have recently achieved an isotropic 3D resolution approaching 3×3×3μm3 in retinal tissue (see Figure 1).12–14 In addition to the roughly 3× improvement in lateral resolution, AO increases the instrument sensitivity (~7dB) and reduces the lateral size of speckle noise (3×), an unwanted byproduct of the interferometric nature of OCT.
Figure 2 shows a detailed layout of our instrument. We correct monochromatic aberrations with an AO system of a Shack-Hartmann wavefront sensor and a woofer-tweeter configuration for the wavefront corrector. The woofer-tweeter contains a high-fidelity corrector cascaded with a high-stroke corrector for near-diffraction-limited performance. For chromatic aberration compensation, the customized achromatizing lens is introduced in the beam path directly behind the fiber collimator in the sample arm.
With these UHR-AO-OCT systems, we have successfully created 3D retinal images of structures previously only visible with histology or invasive imaging, including the foveal microvasculature, bundles within the retinal nerve fiber layer, the fibers of Henle, the 3D photoreceptor mosaic, tiny drusen in age-related macular degeneration, and the tiny pores of the lamina cribosa of the optic nerve in normal and glaucoma patients12, 14. Figure 3 shows a representative UHR-AO-OCT volume acquired in one subject. For comparison, a commercial SLO image of the same retinal patch is also shown. While the commercial version is largely limited to 2D imaging of macroscopic structures, the micron-scale resolution of our device is sufficient for distinguishing—axially and laterally—the closely packed nerve fiber bundles. Bundles in the figure are about 40μm in diameter with gaps between them approaching a couple microns. These bundles transmit the visual signal to the brain and are destroyed in glaucoma, a leading cause of blindness.
The combination of OCT with AO and a customized achromatizing lens provides a powerful new imaging tool. Its 3D resolution and sensitivity in the eye substantially surpass those of other ophthalmoscopes. Future technical improvements, such as image de-warping and contrast enhancement, will add to the research and clinical utility of the device.
Financial support was provided by National Institute of Health grants 1R01 EY018339 and 5R01 EY 014743. This work was also supported, in part, by the National Science Foundation Science and Technology Center for Adaptive Optics, managed by the University of California at Santa Cruz under cooperative agreement No. AST-9876783.