Principle of scanning laser tomography

The scanning laser tomograph (Zinser et al, 1989; 1990) is a type of confocal microscope that images structures in the fundus of the eye in optical sections in vivo and reconstructs their anatomy three-dimensionally using software. The imaging has two crucial features: sequential point-by-point laser imaging and a confocal optical system.

PoinUby-point laser imaging. A HeNe laser with a wavelength in the red spectrum, usually 632.8 nm or 670 nm, is focused at a given point in a particular plane of an object o f interest. The light reflected from this point is separated from the incident beam o f light and deflected by a beam splitter to a light detection unit where it is registered. This registered information is digitised and stored in computer memory. Adjacent point locations are imaged in sequence so that a total of, say, 256 points are measured in a horizontal and vertical grid to give a resolution o f 65,536 pixels per image. More points imaged per unit distance give a higher resolution. In point- by-point imaging, filters and polarisers can be placed in the path o f the light reflected from each point. This allows the properties of the imaging system to be easily and specifically manipulated to improve the quality of the system and extract desired information. For example, the combination of a polarising beam splitter and quarter-wave retarder ensures that it is mainly specularly reflected light which is detected and registered (Bartsch and Freeman, 1994). In commercial systems, this principle is used to detect the internal limiting membrane, the principal specular reflector in the normal human retina (Knighton, 1995).

Confocal optics. In the confocal system, a pinhole aperture is positioned like a diaphragm in front of the light detector at a location that is optically conjugate with the focal point and focal plane of the laser beam, as shown in Figure 4.1. Light reflected at the focal plane is focused in the plane o f the aperture and passes to the detector without being diminished in intensity. Light reflected from outside the focal plane is not focused at the aperture so that only a fraction o f its light reaches the detector where it is registered at a lower intensity. The intensity of the registered signal is maximal precisely where the focal plane is confocal with the pinhole aperture as illustrated in Figure 4.2. In tomography, point-by-point scanning is conducted in sequential optical planes through the depths of a structure. The confocal arrangement ensures that spatial resolution remains high both perpendicular to and along the optical axis.

Axial intensity distribution. Each point in a topography image has its own axial intensity distribution based on the registered intensity at the same point in each of

Beam splitter Laser source Computer Beam scanner Detector Monitor Image buffer Laser Confocal pinholes Point source Beam splitter Detector Objective Object in focal plane not in focal plane

FIG U R E 4.1. Set up for scanning laser tomography. Top: standard wiring;

bottom: confocal optics.

Optical

axis

Focus position

Laser

beam

focus

Intensity

the 32 tomographic sections. The peak of the axial intensity distribution profile represents registered light o f greatest intensity, corresponding to the principal reflecting surface; in Figures 4.2, 4.3 and 4.4 the y-axis value at the peak represents light intensity at the principal reflecting surface, and the x-axis value indicates its location on the optical axis (z-axis). The distribution of axial intensity is gaussian at the foveola but tends to vary with retinal location (Bartsch and Freeman, 1993); knowing this helps in choosing a method to locate the distribution’s peak. Reflections from deeper layers of the retina at different fundus locations contribute differently to the axial intensity distribution (Bartsch and Freeman, 1994). The HRT assumes that the peak o f the axial intensity distribution corresponds to the inner limiting membrane and locates this peak by calculating the centre of gravity o f the distribution profile (Zinser et al, 1990).

Commercial systems. The Heidelberg Retina Tomograph (HRT; Heidelberg Engineering, Germany) images in 32 evenly spaced serial sections through an object of interest with 256x256 points imaged per section, as shown in Figure 4.5 (Zinser et al, 1989). A tomographic image series is thus generated. A newer version of the Heidelberg Retina Tomograph has been introduced, called the FCRT II, which uses the same principles of imaging as its predecessor. Main differences relate to the HRT II’s fixed imaging distance and fixation target, higher image resolution, and better user-friendliness in the acquiring and processing of images. The TopSS (Laser Diagnostics Technologies, San Diego, CA) has a similar optical and imaging design to the HRT but different image analysis software. The Zeiss scanning laser ophthalmoscope scans a matrix of 500 x 600 points in eight optical planes (Cioffi et al, 1993). Reference to scanning laser tomography in the rest of this thesis relates to the Heidelberg Retina Tomograph (‘HRT T) unless otherwise stated.

Topographic image series and single topography images, Cartesian coordinates representing horizontal, vertical and optical axes (x, y, z) identify the location of each image point in each optical plane. Sequential images at each optical plane within tomographic image series are stacked in register by the software and errors of shifting, tilting and rotation corrected to reconstruct a new image called a single topography image and is described more in Section 4.1.5.

X = 2 . 0 3 9 M M

im

F IG U R E 4.3. Gaussian axial intensity distribution. Left cohmn=\nXQns\Xy {top)

and topography (bottom) images o f the same optic nerve head. Right colimn=

axial intensity {top) and topographical height {bottom) have gaussian

distributions at the optic nerve head location indicated by the cross-hair.

FIGURE 4.4. Non-gaussian axial intensity distribution. Left column: Intensity

{top) and topography {bottom) images o f the same optic nerve head. Right

co/i/WA7=Topographical height {top) and axial intensity {bottom) at the cross-hair

have distributions that are skewed to the right.

% % % ^ I ^ 1

^ *5

FIGURE 4.5. Tomography o f the optic nerve head to produce a tomographic

image series. Top /ç/M series o f images through the optic nerve head in 32

sequential planes lying 50pm apart along the visual axis. Other panels: images at

different levels starting vitread. The tomographic level o f each image is indicated by the green box in its accompanying image series (inset) and white horizontal line

in the profile plot adjacent to each image; middle /c//=section 9, bottom

/ç//=section 13, top right=SQcX\on 20, middle rzg/z/=section 23, bottom

right=^QC\\on 31.