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Keratocyte Density and Size in Conscious Humans by Digital Image

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Keratocyte Density and Size in Conscious Humans by Digital Image Keratocyte density and J.I. PRYDAL, F, FRANC, P,N, DILLY, .. M,G, KERR MUIR, M,e. CORBETT, size In conscIous J. MARSHALL humans by digital image analysis of confocal images Abstract stroma of conscious humans. These gave independent automated measurements of the Purpose Confocal microscopy can give images density and size of keratocytes. The technique of high magnification and resolution in and results in normal subjects are presented in undisturbed living tissue. It provides new this paper. Changes during wound healing will information about the cellular structure of the be presented in a later publication. cornea. Our aim was to measure the density, Klyce and Beuerman2 examined keratocyte size and distribution of keratocytes. structure using electron microscopy. Long Methods Healthy cornea in four subjects was cytoplasmic processes tapering to about 1 J,Lmin examined using tandem scanning confocal diameter were described. In our work such microscopy. Methods for digital analysis of processes were not seen. They were probably images were developed. too fine to be resolved by the optical system. Results Keratocyte density in confocal cross­ JJ Prydal Thus confocal images of keratocytes show only sections was greatest immediately under M,G, Kerr Muir a portion of the cell, perhaps just the nucleus or Bowman's membrane (maximum 800 cells/ M,e. Corbett the nucleus and part of the cell body. In this J, Marshall mm2) and decreased sharply towards posterior paper, keratocytes imaged by confocal St Thomas' Hospital and cornea (minimum 65 cells/mm2). Cross­ microscopy are referred to as 'cells', as the exact UMDS sectional cell size ranged from 78 to 211 I-lm2, London SE1 7EH, UK portion seen in images is unknown. but did not correlate with depth in the tissue. Keratocytes function to maintain the collagen JI. Prydal � Conclusions Results are consistent with those fibres and extracellular matrix of the corneal Department of of earlier work using histological and stroma.2 Earlier work concerning their density Ophthalmology (Box 41) biochemical techniques in isolated tissue. The Addenbrooke's Hospital and distribution was done using conventional methods we have developed enable studies of Hills Road microscopy or biochemical methods in excised ongoing processes in conscious humans and Cambridge CB2 2QQ, UK tissue?-6 Petroll et al? used a laser scanning can be used to examine diseased tissue as well Tel: +44 (0)1223 464969 confocal microscope to examine isolated rabbit as the response to injury. Fax: +44 (0)1223 778282 cornea. Images were digitised for computer e-mail:prydal@ analysis, but each cell was marked manually to elmc.demon,CQ,uk Key words Confocal microscopy, Cornea, enable measurement of their density. Corneal stroma, Image analysis, Keratocytes F, Franc Institute of Physics Department of Applied Confocal microscopy can give images of high Materials and methods Optics Academy of Sciences of magnification, resolution and contrast Equipment Czech Republic throughoutthe cornea in conscious co-operative A tandem scanning confocal microscope Prague human subjects.1 Conventional histological (Tandem Scanning Corporation, Reston, Czech Republic techniques of sectioning, fixation and staining Virginia, USA) was used with 100 W mercury are not required to identify structures. It P,N, Dilly arc lamp and X24 objective (numerical aperture St George's Hospital provides the opportunity to examine ongoing 0.6). The microscope has been described by Medical School physiological and pathological processes in Cavanagh et al.l The position of the camera and Department of Anatomy undisturbed living tissue. final condensing lens was adjusted so that as London SW17 ORE, UK Video recordings of confocal microscopy can much as possible of the image fell on the We would like to thank the give several thousand different images in each photocathode of the camera, thus increasing the Iris Fund and Special subject. Imaging of such large amounts of field of view (630 X 420 I-lm). Images were Trustees of St Thomas' information has not been possible in the past recorded using a DACE VT100O silicon Hospital London for their generous support. M,e.e. and new techniques are required to extract intensified tube camera (SIT) and Panasonic holds the William's usable data. We developed computerised S-VHS recorder (AC-7355). Optical sections are Fellowship for Medical and methods for analysing cellular structure in the curved with the apex away from the eye under Scientific Research Eye (1998) 12, 337-342 © 1998 Royal College of Ophthalmologists 337 examination. The radius of curvature is similar to that of the cornea O. Hill, Tandem Scanning Corporation, personal communication). Petroll et al.B determined axial resolution of optical sections to be 9 JLm. A special feature of this confocal microscope is that the plane of focus can be advanced through the cornea without movement of the objective or object. A separate unit (Oriel controller 180011) controls motor-driven lenses within the objective. It can be set to move the focal plane in or out of the tissue, either continuously or in specified steps. A readout gives the distance moved by lenses in the objective. Using computer models of the optics, the manufacturer has provided a polynomial equation converting this reading to the distance moved Fig. 1. Grey-scale confocal image of keratocytes in superficial stroma. by the optical section through the stroma. Single video field digitised to 8 bits, resolution 768 X 512. Field of view 630 J1-m X 420 J1-m. The section marked is that used in image processing, 256 X 256 pixels, 210 X 210 p.m. Procedure However, images were brighter in the centre than at Four subjects (2 men, 2 women; age 23-39 years) were the edge and thus a suitable threshold level in the centre fully informed of the nature of the procedure and gave was not appropriate for the entire image, and could their consent to be examined. The cornea was incorrectly portray the size and shape of cells. Variations anaesthetised with 0.4% oxybuprocaine hydrochloride in brightness were due in part to the camera. Also and the subject's head supported by chin and forehead illumination was brighter in the centre. It was possible to rests. Subjects were instructed to fixate a small light. adjust a condenser lens to produce more even Immersion fluid was 2% methykellulose. After contact illumination, but this reduced image contrast. between objective and eye, the focal plane was advanced Even within small sections of images where and the position of the microscope adjusted until images background intensity was nearly constant, careful of superficial epithelium were circular. Optical sections manual setting of an appropriate threshold level was were then tangential to the cell surface. The plane of necessary to select cell shapes. Several other small bright focus was advanced into the most superficial layer of areas were included in the resultant binary image. It was keratocytes and then in steps of 50 JLm (25 JLm in one of presumed that these bright spots were not from cells and the subjects). Pauses for about 5 s were recorded at each were considered to be 'noise'. level. Depth readout was recorded verbally on the audio track. When the endothelial surface was reached fine adjustments were made to image the layer of keratocytes Procedure for image analysis just superficial to Descemet's membrane. Several approaches were used to overcome these problems. An outline of the procedure is given below, Digitising of images followed by a detailed description. Single fields of video recordings were digitised to a resolution of 768 X 512 pixels and 256 grey levels Outline (Overlay Frame Grabber, Imaging Technology, Woburn, 1. Contrast was enhanced. This made the range of Massachusetts, USA). Ten fields at each level were brightness levels the same in all images: from 0 to 255 digitised for all subjects, giving a total of 100-200 images (arbitrary units). per subject. The only criteria used in selecting images 2. There was less variation in background intensity were that they should not be blurred or show stromal across small areas of the image. Small sections from nerves. The presence of nerves complicated computer the centre of images were processed. Images of the analysis and made measurements unreliable. background were computed and then subtracted from the recorded image. This resulted in images with more uniform background. They contained cell shapes, but Problems in analysis of images also noise. A sample image is shown in Fig. 1. Keratocytes appeared 3. A threshold brightness level was set at a point as bright objects against a dimmer background. In our between 0 and 255. Pixels less than the threshold were early attempts at processing, binary images (two set to zero, those greater to a value 1. This gave a brightness levels) were produced from grey-scale images binary image. (256 brightness levels). Pixels with values greater than a 4. Binary images contained cell shapes but also 'noise' threshold level were set to 1 and those with smaller (not cell structure). 'Noise' was removed from images. values to O. Groups of pixels that were too small to be cells, or 338 40 were of a different shape from cells, were removed. This gave images in which the back,ground was at value zero and cell structures at value 1. 30 5. Such images could then be processed to give Q information about cell structure. .... 0 :z: rJ) 20 w II: Details :z: I- 1. Contrast enhancement Contrast was enhanced by the , 10 process of 'normalisation .9 A new value for each pixel of an image was determined using the equation ] 0 [ (P-mi�) P' = 255 X (max-mill) 0 10 20 30 40 50 60 70 MEAN INTENSITY where P' is the new pixel value, P is the original pixel Fig.
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