GAIDA Perspectives and Limits of 3D Fundus Microscopy Chapter25

8/8/2019 GAIDA Perspectives and Limits of 3D Fundus Microscopy Chapter25

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25Perspectives andLimits of Three-Dimensional FundusMicroscopyGerhard Gaida

SummaryConfocal scanning microscopy is a newmethod in microscopy which opens the wayto precise three-dimensional measurementof objects. Its application to ophthalmoscopy is discussed centered around the concept of a three-dimensional elementary resolution element. On the fundus this elementis a cylinder with radius 10 11m (lateral resolution) and length 300 11m (depth resolution, FWHM). The local fundus topography,e.g.3 of the papilla, can be determined combining resolution-limited measurement withlow patient stress. Broad application of thismethod is promising for early diagnosis ofglaucoma.

25.1 IntroductionConfocal scanning microscopy is a newform of microscopy which has become feasible by recent progress of optomechanicaland electrooptical scanning technologies. Itextends the range of conventional microscopy for dimensional measurement intothree dimensions. Applied to ophthalmoscopy, it enables new types of measurementof fundus structures.The optical principle of scanning microscopy is illustrated in Fig 25- 1.A parallel beam of laser light is focussedby the microscope objective onto the specimen where it forms a single illum ination

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+"-

4- "- "-

radius R *canningunitFig 25-1 The optical principle of scanning microscopy.

spot. The light scattered by the specimenwhich enters the objective forms a beamparallel to the illuminating laser light. Asemireflecting beamsplitter directs it to anauxiliary objective. This objective forms animage of the illuminated spot in a plane optically conjugate to the focus plane. A smallpinhole called a confocal stop transmits thelight to the detector, where its intensity isconverted to an electrical signal.An image is formed by ~ o v i n g the illumination spot over the specimen sequentially in time. This is usually accomplished by an optomechanical or electrooptical scanning unit located between thebeam splitter and the objective. The varyingelectrical signal then modulates the brightnees of a display device, e.g. a video display. The point by point correspondence ofthe displayed spot to the illuminated spoton the fundus is guaranteed by a video electronics unit.

The principle of confocal microscopy can be applied to ophthalmoscopy byregarding the refracting media of the eyeas the objective and the fundus as the object.This method is then called confocal laserscanning ophthalmoscopy. As the eye playsthe role of the microscope objective in thiscase, the optical properties of the refracting254

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Fig 25-2 Lateral resolution of a confocal microscopeas a function of stop radius.

media of the eye are the limiting factor forthe resolution of a well-built instrument.

25.2 Resolution of confocalmicroscopy

In addition to the focal length f and the numerical aperture NA, the radius R of theconfocal stop is an important parameterdetermining the resolution of a confocalmicroscope1.2 . In the following, the lateralresolution (in the image plane) and depthresolution (along the line of sight) will bediscussed separately.

25.3 Lateral resolutionThe lateral resolution of a scanning microscope without confocal stop is determinedby the size of the laser spot formed in theimage plane. It can be calculated using theAiry formula and appropriate values of thelaser wavelength and the numerical apertureof the objective. The Laser spot is then imaged in the plane of the confocal stop usingthe objective a second time. I f the confocalstop is larger than the image of the laserspot, all the light will enter the detector.


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obje c tou t of focus

in fo cu s

Fig 25-3 The optical principle of depth resolution ofa confocal microscope.

However, if the confocal stop is smaller,only the central part of the laser spot willcontribute to the image, thereby providingsome potential to improve the resolutionover conventional microscopy. Fig 25-2shows the dependency of the resulting resolution (normalized to the resolution without stop) on the confocal stop radius, including all the effects of diffraction. Forlarge stops there is no improvement. Forstops of the order of the Airy spot (markedby the line rA on the abscissa) a slight improvement results. For very small stops theimprovement reaches 30 % in the limit ofvanishingly small stops. For practical purposes, however, this improvement cannot beused for ophthalmoscopy because the stopthen transmits only a small fraction of thelight scattered by the object. The resultingdegradation in image quality (signal-tonoise ratio) cannot be tolerated in the caseof the eye fundus where one works at thephoton noise limit.

25.4 Depth resolutionThe origin of confocal depth resolution isillustrated in Fig 25-3. In a defocus sed object plane not only the small Airy spot il-

10

10

Fig 25-4 Depth resolution of a confocal microscopeas a function of stop radius.

luminates the object but a much larger blurspot. This blur spot is imaged onto the confocal spot plane which in turn is not in focuswith respect to the object plane. This resultsin an even larger blur spot. The confocalstop transmits only a small fraction of thescattered light , resulting in a dark image.Confocal microscopy therefore has the unusual ability to suppress light coming from

.planes above or below the focal plane.Analogous to the convention

used for the lateral resolution, the depth resolution of a confocal system can be definedas the range of depth values around thefocus where the confocal stop transmitsmore than half of the scattered light. Aslarge stops transmit even strongly defocussed light, the stop radius strongly influences the depth resolution. Fig 25-4shows this dependency for the case of a isotropically scattering object. At stop sizesgreater than the Airy spot rA he contributionof geometrical optics (lower line) dominates, while for small stops diffraction effects become more important. A stop comparable to the Airy spot is therefore optimalfor depth measurement. I t transmits mostof the light but provides the maximum depthresolution possible.

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lateral

rA = 0.61 Mdepth

dz = 0.68 }. ( nfarf eye lengtha pupil radius}. wavelengthn refractive indexof vitreous

Eye

0. 3 mm

0.01 mm

Fig 25-5 The elementary resolution element at thefundus using the optical configuration of the eye.

25.5 Three-dimensionalresolution elementLateral resolution and depth resolution canbe combined to form a three-dimensionalresolution element in object space. I t hasthe shape of a cylinder. Its radius and heightare given by the formulae in Fig 25-5 asa function of laser wavelength, objectivefocal length and aperture radius. In scanning ophthalmoscopy the human eye servesas the objective. Using the parameters ofthe Gullstrand eye at 3 mm pupil diameterand a wavelength of 633 nm, the resolutionelement on the fundus is pencil-shaped withabout 10 !-lm width by 300 !-lm length. Withthe thickness of the retinal nerve fiber layercomparable to the depth resolution, the internal structure of the retina cannot be resolved. Additional assumptions which arehighly questionable are necessary to obtainsub-pixel resolution.

Fig 25-6 Reconstruction of the object geometry from a sequence of images at different focal planes.

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/(%,y)

-I- - 2r--r-- I{%,y)

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confocal depth measurement Z1 z. Z, z, z.z{%,y)256

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25.6 Measurement oftopography

The depth resolution of confocal microscopy can be used to measure the geometryof the object by analysis of a sequence ofimages taken at equidistant focal planes asillustrated in Fig 25-6. For each picture element (x,y) the intensity as a function ofdefocus (z) forms a bell-shaped curve. Theposition of the maximal intensity is the position of the object at this particular pictureelement (zo). Taken together, the independent measurements of all picture elements form a topographic map of the object.

Because several independentimages at different focal planes contributeto the topographic map, its depth valuesshow an accuracy better than the depth resolution. In clinical use, a standard deviation of 50 11m can be reached for measurement of the papillary topography. However,the data reduction procedure makes the as-

References1. Wilson, T. , Carlini , A.R. Theory and practice of

scanning optical microscopy. Academic Press, London 1984.

sumption that the object is a purely scattering surface which is not the case for thepapilla. The reflecting and scattering constituents of the retina and the papilla (pigment epithelium, nerve fiber layer, innerlimiting membrane etc.) have a thicknessvarying between 200 11m and 500 11m. Dueto the limited depth resolution (300 11m)this inner structure or changes in it cannotbe resolved. Therefore only the mean depthof these layers can be determined with better accuracy.

25.7 ConclusionThe application of confocal scanning microscopy to fundus imaging has brought a newdimension to ophthalmoscopy, the objectivemeasurement of papilla topography. Broadapplication of this new diagnostic techniquepromises to open new horizons in the diagnostics of early phases of glaucoma.

2. Hellmuth, T. , Seidel, P., Siegel , A. Spherical aberration in tonfocal microscopy, Proc SPIE 1028 : 28(1989).

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GAIDA Perspectives and Limits of 3D Fundus Microscopy Chapter25