Clinical Techniques in Ophthalmology
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In this technique illumination system is placed on the side of cornea or lens i. By this way a parallelepiped illuminated area is created. Angle can be changed between degrees. Magnification may vary between 10xx Figure 4.
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By focusing on the anterior side of the parallelepiped light, corneal epithelial alterations and protein deposits and debris in a contact lens wearer can be viewed. Endetolial pigment changes, keratic precipitates, other deposits and striations on the endotelium can be viewed by using parallelepiped technique [ 2 ]. Crystalline lens transparency and opacities can be evaluated by using with a 0.
Different from the corneal examination illumination, system should be set to a smaller angle, 10 to 45 degree. Detailed examination can be performed by directing the light towards the pupil area and focusing different layers of the lens [ 2 ]. Wide beam illumination is useful for the inspection of contact lens surface.
By this method, protein deposits, mucus secretion, corneal nerve fibers, infiltrative keratitis, corneal opacities, iris and lens surface can be viewed. Setup of the method is similar to parallelepiped method except slit width is wider then corneal depth. Light intensity should get reduced and angle of the illumination adjusted depending on the surface of under inspection, generally more than 45 degrees [ 2 ]. Conical beam illumination is useful in viewing the inflammatory cells and proteins in the anterior chamber.
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In this method width and height of the slit light beam is reduced until obtaining a point light. Height is reduced to mm. Light intensity is set to maximum. Examination should be done in dark conditions Figure 5 [ 2 ]. The number of cells in the anterior chamber is assessed from side to side of the anterior chamber in medium magnification and high-angle illumination.
In this technique light beam is projected to a different area other than the focal point of the observation system. Illumination system is get moved off from its click position by releasing a knob Figure 6. Therefore two systems do not coincide at same point. Procedure is similar to parallelepiped illumination technique exceptionally, in this method, observed area is not directly the illuminated area but just beside the anterior border of parallelepiped area. With this method, transparency loss or loose contrast differences in transparent structures can be viewed such as subtle corneal opacities, bulbar conjunctival vessels and lens opacities [ 2 ].
Scleral scatter is used for especially small changes in large transparent areas. Central corneal clouding, bullous keratopathy, corneal scars, contact lens edges can be examined with this method. In this method, parallelepiped beam focused on central cornea then after releasing the knob illumination system moved out its click position and beam can be projected to nasal or temporal limbus Figure 7.
With correct positioning of the illumination system, a halo of scatter light around the cornea can be viewed [ 2 ].
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Any condition which alters corneal transparency obstructs the internal reflecting light and becomes visible as a whitish color over pupil background. Retro illumination can be used in visualization of central corneal opacities, lens and vitreous opacities, iris defects and pigment liberation.
Procedure of the retro illumination is started with releasing of the knob. Angle between illumination and observing system should be set about zero. Then illumination system is focused on the object which is intended to be observed. When the beam is projected through the edge of the pupil, the bright light reflex returning from the retina can be seen Figure 8A.
If the beam is projected to the iris, corneal opacities and any vascularisation in the cornea or iris can be viewed by the reflected light Figure 8 B [ 2 ]. Confocal microscopy is a non-invasive histological imaging technique. It uses reflected light from the living tissue. Therefore, it is an in vivo imaging method of the living cornea. It uses focused light or laser beam. A bright light beam is projected and focused through an objective lens to the cornea.
Then reflected light spot is collected from the illuminated tissue area by objective lens.
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With the help of beam splitter, reflected light is separated from the light mixture and directed to the detection unit. Reflected light reaches to the detection apparatus by passing a pin-hole. Detector transcodes the reflected light into electrical signal and records to the storage media Figure 9. The pin-hole at the entrance of detector apparatus filters the light coming from outside the intended focal point. This filtration helps grabbing sharper and clearer images than conventional light microscopy [ 3 ]. For two-dimensional imaging, the sample is scanned sequentially in different focal planes.
There are three types of confocal imaging techniques. Tandemn scanning confocal microscopy was developed by Petran and Hadravsky [ 4 ]. Basic part of the system is the real time point illumination and point detection. It was developed by Nipkow in [ 5 ]. System provides real-time images at true color and marginal image quality based on the low intensity of reflected light [ 6 ]. Future development in tandemn scanning was done by Xiao et al [ 7 ]. They used set of pin-holes on the same side of Nipkow disk for illumination and detection.
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The pinholes need to be as small as possible to eliminate the scattered light [ 4 ]. Design was simple but it has disadvantage of low intensity of illumination for image formation.
Clinical version was produced but no longer in production [ 3 ]. Scanning slit confocal microscopy is an alternative scanning method to point scanning which uses a slit illumination. It scans over the back focal plane of the microscope. Advantage of slit scanning is that many points on the axis of the slit are scanned at the same time and therefore scanning time is markedly decreased. Scanning slit systems also have superior light brightness compared to point scanning. In comparison to pin-hole systems, slit scanning systems have lower axial and transverse resolution.
Slit height, width and amount of light can be adjusted in slit scanning systems. By this way, visualization quality and optic section area can be modified based on the specimen i. Laser scanning confocal microscopy was developed by Webb in [ 8 - 10 ]. A coherent laser beam is used as a light source and specimen scanned by laser beam with the help of galvanometer scanning mirrors which provides fast scanning. Reflected light is refocused and projected to the photomultiplier through the pin-hole aperture. Heidelberg Retinal Tomograph is an example of in vivo confocal imaging systems.
It uses nm diode laser as a light source to acquire and analyze the optic nerve for glaucomatous damage.
With the help of detachable optical attachment, HRT II was modified to high resolution confocal laser scanning microscope by Stave et. Clinical applications of confocal microscopy: Qualitative properties of cornea can be documented such as corneal thickness measurement, depth of surgical interfaces, densities of stromal and endothelial cells, density or nerves and reflectance and scatter at various depths [ 12 ]. Reliable measurements highly depend on experienced technician and grabbing good quality images.