Ординатура / Офтальмология / Английские материалы / Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)_John_2010

Figure 7-9: Retroiridal cysts often cause changes in iris contour. Highresolution ultrasound penetrates through the iris and readily allows differentiation of cysts, an example of which is shown here, from solid lesions.

Figure 7-12: Oversized phakic posterior chamber lens implant results in anterior displacement of iris and possible contact with crystalline lens.

Figure 7-10: Displaced posterior chamber lens implant. Lens implants are readily visualized ultrasonically due to their difference in density from surrounding media.

Figure 7-11: Anterior chamber lens implant. Haptics are indicated by arrows.

present, as is the case in this figure. Because ciliary body involvement is associated with increased risk of metastases while iris melanomas are relatively indolent, this distinction impacts upon treatment. Differentiation of solid lesion from cysts (Figure 7-9) is readily accomplished ultrasonically.

Very high frequency ultrasound allows visualization of lens implants in both the posterior (Figure 7-10) and anterior chambers (Figure 7-11) . Preoperative ultrasound biometry is of great importance for the measurement of ocular dimensions prior to phakic lens implant surgery (Figure 7-12), since white-to-white measurement is only weakly correlated with the angle and sulcus diameters.

Ultrasonic Imaging and Biometry

of the Cornea

With an approximate thickness of 0.5 mm, the need for high-resolution imaging systems is obvious. Resolution of the approximately 50 micron thick corneal epithelium requires the use of very high frequencies. A factor affecting corneal imaging and biometry is the cornea’s specularity. When imaging with a conventional sector scan probe, the corneal surface will present obliquely to the ultrasound axis, and echoes are deflected away from the probe, resulting in reduced or absent echoes outside the central cornea. This effect is exacerbated by the limited depth-of- field of the focused transducer, because only a small region of the curved cornea will be within the focal zone during a scan. The Artemis system uses an arc-scan geometry to address these issues. By moving the transducer in an arc of appropriate radius, the cornea will present approximately normal to the beam axis and remain in the focal region across its entire diameter. The Artemis also incorporates an optical subsystem that allows the patient to gaze at a fixation target while the eye position is monitored with a video camera. This is crucial in obtaining measurement reproducibility. An image of a normal cornea obtained using the Artemis is shown in Figure 7-13. Following LASIK, the flap interface is evident, as shown in Figure 7-14. Because full-width arc-scan images are so much wider than deep, it is often advantageous to display them in geometrically uncorrected format so as not to loose the axial resolution to which we are entitled. Corneal pathologies such as edema

(Figure 7-15) and scarring (Figure 7-16) can be readily visualized and their depth measured.

Biometric analysis of digitized corneal ultrasound data involves detection of peaks associated with the anterior and posterior surfaces, Bowman’s membrane and the flap, where present. The Artemis stores raw echo data rather

Figure 7-13: Artemis-2 image of normal cornea. The Artemis system, which is an immersion arc-scanner, also includes fixation lights and video monitoring of eye position during scanning.

Figure 7-14: Artemis images of post-LASIK cornea in proper geometry (top) and uncorrected rectilinear display (bottom). The corneal layers, epithelial surface (e), Bowman’s membrane (b), flap (f) and posterior surface (p) are indicated.

Figure 7-15: Example of corneal edema in high resolution arc-scan of the anterior segment.

Figure 7-16: Corneal scar superiorly (arrow). Ultrasound measurement of scar depth can be of great value in clinical management.

than the images themselves, as the image data is of lower resolution. By applying digital signal processing techniques, the exact positions of each interface are determined. The Artemis automatically detects these interfaces and displays measurements as shown in Figure

7-17 for a normal cornea. Figure 7-18 shows an unusual thickness profile in a patient with a corneal graft.

By acquiring a series of scans in a sequence of meridians, it is possible to map the thickness of each layer. Figure 7-19 shows representative B-mode images and pachymetric

maps of a post-LASIK cornea of a myope. Thickening of the cornea over the ablated central region is typical. In contrast, Figure 7-20 shows an annular thickening of the epithelium in a post-LASIK eye corrected for hyperopia. In this case as well, epithelial thickening corresponds to the ablation pattern. Images of a cornea that had received radial

keratotomy a decade previously are shown in Figure 7-21. While the incisions themselves are not evident in the B-mode images, their effect on the epithelial thickness is evident in the map. In a patient with keratoconus (Figure 7-22) , the epithelium is thinned to about 35 microns over the steepest part of the stroma with a surrounding halo of

epithelial thickening to as much as 75 microns. This remodeling of epithelium is a consequence of the bulging forward of the underlying stroma. Ultrasonic detection of this thinning pattern can be an early sign of this keratoconus that may be detectable before surface topographic changes are evident.

Conclusion

New ultrasound technologies continue to be developed for improvement in sensitivity and resolution. High-frequency arrays may become available in the near future. Prototype 35 MHz annular arrays for ophthalmic imaging have been demonstrated.16 Dynamic focusing of such arrays offers a much improved depth of field, in fact, a sixfold improvement over current single-element probes. Prototype highfrequency linear array probes have also been developed.17-18 With such arrays, it would be possible to scan at high frame rates without mechanical motion, and it might be possible to replicate an arc scan, with its advantages for corneal imaging and biometry. Transducer technology is evolving to allow higher frequency

transducers in the range of 75 MHz providing improved definition and greater sensitivity to backscatter.19 These transducers might be particularly useful in screening for conditions involving stromal changes such as keratoconus and for imaging Schlemm’s canal. While optical methods such as OCT offer superb resolution, ultrasound will continue to serve a crucial role in anterior segment imaging, diagnosis and biometry due to its ability to penetrate optic opacities and probe the layered structure of the cornea.

References

1. Mundt G, Hughes W. Ultrasonics in ocular diagnosis. Am J Ophthalmol 1956;41:488-98.

2. Purnell E. Ultrasonic interpretation of orbital disease. In: al KG, (Ed). Ophthalmic Ultrasound. St. Louis: CV Mosby Co, 1969.

3. Coleman DJ, Konig WF, Katz L. A hand-operated, ultrasound scan system for ophthalmic evaluation. American Journal of Ophthalmology 1969;68(2):256-63.

4. Baum G. Aids in ultrasonic diagnosis. Journal of the Acoustical Society of America 1970;48(6):Suppl 2,1407.

5. Coleman DJ. Reliability of ocular and orbital diagnosis with B- scan ultrasound. Ocular diagnosis. American Journal of Ophthalmology 1972;73(4):501-16.

6. Fisher YL, Bronson NR, Schutz JS, Llovera IN. Contact B-scan ultrasonography: clinicopathological correlations. Annals of Ophthalmology 1975;7(6):779-86.

7. Coleman DJ, Lizzi FL, Jack R. Ultrasonography of the Eye and Orbit. Philadelphia, PA: Lea & Febiger, 1977.

8. Coleman DJ, Silverman RH, Lizzi FL, Rondeau MJ. Ultrasonography of the Eye and Orbit. (2nd edn). Lippincott Williams & Wilkins, Philadelphia, 2005.

9. Pavlin CJ, Harasiewicz K, Sherar MD, Foster FS. Clinical use of ultrasound biomicroscopy. Ophthalmology 1991;98(3):287-95.

10.Pavlin CJ, Sherar MD, Foster FS. Subsurface ultrasound microscopic imaging of the intact eye. Ophthalmology 1990;97(2): 244-50.

11.Reinstein DZ, Silverman RH. Very high-frequency digital ultrasound: Artemis 2 scanning in LASIK. In: LASIK: Advances, Controversies, and Custom. Edited by Louis Probst, SLACK, 2004.

12.Reinstein DZ, Silverman RH, Trokel SL, Allemann N, Coleman DJ. High-frequency ultrasound digital signal processing for biometry of the cornea in planning phototherapeutic keratectomy. Arch Ophthalmol 1993;111:430-31.

13.Reinstein DZ, Silverman RH, Trokel SL, Coleman DJ. Corneal pachymetric topography. Ophthalmology 1994;101:432-8.

14.Reinstein DZ, Silverman RH, Rondeau MJ, Coleman DJ. Epithelial and corneal thickness measurements by highfrequency ultrasound digital signal processing. Ophthalmology 1994;101:140-6.

15.Reinstein DZ, Silverman RH, Raevsky T, Simoni GJ, Lloyd HO, Najafi DJ, Rondeau MJ, Coleman DJ. Arc-scanning very highfrequency ultrasound for 3-D pachymetric mapping of the corneal epithelium and stroma in laser in situ keratomileusis. J Refract Surg 2000;16:414-30.

16.Silverman RH, Ketterling JA, Coleman DJ. High-frequency ultrasonic imaging of the anterior segment using an annular array transducer. Ophthalmology In Press.

17.Lukacs M, Yin J, Pang G, Garcia RC, Cherin E, Williams R, Mehi J, Foster FS. Performance and characterization of new micromachined high-frequency linear arrays. IEEE Trans Ultrason Ferroelectr Freq Control 2006; 53(10):1719-29.

18.Ritter TA, Shrout TR, Tutwiler R, Shung KK. A 30-MHz piezocomposite ultrasound array for medical imaging applications. IEEE Trans Ultrason Ferroelectr Freq Control 2002; 49(2):21730.

19.Silverman RH, Cannata J, Shung KK, Gal O, Patel M, Lloyd HO, Feleppa EJ, Coleman DJ. 75 MHz ultrasound biomicroscopy of the anterior segment of the eye. Ultrasonic Imaging. In Press.

History of Microscopy

In the 17th century, a new invention revealed the microscopic world. The microscope marked the beginning of histology. Invented by Hooke, the Compound Microscope was used to describe pores in sections of ordinary cork.1 He coined the term “cell” to describe these pores, which was eventually applied to describe the “cells” of living systems. This early design eventually evolved into the modern microscope.

The modern Light Microscope invented by Schleiden and Schwann in 1838 has significantly advanced our understanding of living structures.1 Although numerous optical design and manufacturing enhancements have improved microscopic resolution to an impressive half-micron, the inherent limitations of this technique have significantly hampered the study of “living” tissues. For example, this technique destroys “living” tissues during the fixation and sectioning process. Consequently, many natural associations between cells and microstructures that may have existed are lost. In addition, this process creates unwanted artifacts that further obscure microstructural details.

More importantly, the optical performance of the light microscope is dreadful when used to examine thick tissue. The fundamental problem is that the image obtained not only contains light that is reflected from the focal plane, but also reflected light from structures both above and below the focal plane. Consequently, this unwanted reflected light blurs the fine structural details within the focused image; thus, decreasing contrast and both lateral and axial resolution.2

In an attempt to resolve these limitations, Maurice unveiled the Specular Microscope in 1968.3 This new microscope provided a new ability to image tissues in vivo. Maurice’s design accomplished this task by exploiting the reflectivity of tissues. In the cornea, for example, the endothelial layer is highly reflective. By being more reflective than the structures below or above, the specular microscope is able to focus this reflective light unimpeded, producing sharp images with enhanced resolution and contrast.2 Soon thereafter, Bourne and Kaufman improved upon this novel design. Their design modifications introduced a practical version into ophthalmologic practice, allowing clinically useful corneal endothelial photography at high magnification (200X).4

Unfortunately, the specular microscope generally fails to satisfactorily image the less reflective structures within the cornea, which include the stromal keratocytes and the epithelial cells.1 In addition, corneal pathologic conditions, such as scars, may induce significant light scattering.2 Although the specular microscope advanced imaging of

in vivo tissues, its limitations required a different approach and eventually this lead to the development of the confocal microscope.

Development of Confocal

Microscopy

In 1955, Marvin Minsky, when he was a Junior Fellow at Harvard University, pioneered the first Confocal Microscope.5,7,18 Unlike earlier microscopic techniques that viewed sectioned tissue in a plane perpendicular to its surface, confocal microscopy views tissue images horizontal to its surface. His invention permitted tissue examination without the light scattering artifacts prevalent in prior microscope designs. He accomplished this feat by exploiting the pinhole effect.

Minsky combined pinholes with microscope optics to produce remarkably clear images. The key was in the design. His microscope used two pinholes to produce a confocal effect. He placed the first pinhole before the condenser, which focuses the light rays onto the tissue and the second pinhole before the objective, which focuses the reflected light rays into an image. Minsky then calibrated this opticalpinhole system in a way that conjugated the tissue focal point to correspond precisely to both pinholes simultaneously. Thus, this effect permitted only light rays conjugate to the focal point within the tissue to pass, thereby blocking unwanted light rays reflected from other levels within the tissue.2,5-7 This early confocal microscopy concept is illustrated in Figure 8-1.

This blocking phenomenon eliminated the lightscattering issue prevalent in earlier light microscopes. Furthermore, Minsky’s microscope could theoretically perform a point-by-point image reconstruction by sequentially shinning a pinpoint of light across a specimen instead of flooding the entire specimen with light.

Minsky’s novel design set the focal length of the condenser and the objective lens as illustrated by the rectangle and oval respectively.18 As illustrated by the dotted line tracing in Figure 8-1, the focal point in the specimen is conjugate to the focal point at the pinhole. As one can appreciate, only the dotted light ray tracing can pass through the pinhole. If for example, reflected light from a point anterior to the focal point passes through the optical system, its light would focus before reaching the pinhole, and thus be blocked from passing through the pinhole (Figure 8-1). Likewise, reflected light from a point posterior to the focal point would focus behind the pinhole. Thus, the pinhole blocks unwanted light from passing through.

Figure 8-1: Pinhole concept. The focal lengths from the optical system to either the tissue or pinhole are identical. Therefore, only the dotted light ray tracing travels through the pinhole without being blocked. Thus, unwanted aberrant rays (red and blue) are filtered by the pinhole, creating a clear image of a single point within the tissue.

Hence, this arrangement not only illuminated, but also imaged a single point within a specimen simultaneously, blocking aberrant reflected light rays. He coined the term confocal to describe his design’s use of common focal points (Figure 8-1). His confocal technique produced sharp images with excellent contrast. Wilson and Sheppard further advanced this design that eventually lead to the modern confocal microscope.8 Modern confocal microscopy has replaced the condenser and objective eyepiece with the point illuminator and electronic detector, respectively. Thus, light from a point within the tissue can now be digitized and stored on a computer.

In order to view structures below or above the point of interest, the optical focus is changed, creating a new focal plane that can be examined. If the focal length is continually changed, then numerous optical sections can be imaged

(Figure 8-2).

Figure 8-2: In vivo confocal sectioning concept. The red and yellow planes illustrate this concept to dynamically image different planes within the block of tissue by changing the focal length of the optical system.

This ability to change focal length in real-time allowed a dynamic Z-axis scanning capability. Therefore, this technique enabled in vivo corneal scanning. In addition, digitizing the images allowed a computer to create threedimensional reconstructions. Thus, unlike earlier techniques, the natural association between cells and micro-

structures can now be examined in vivo, without using stains or dyes. However, like earlier techniques, corneal edema or opacification degraded image quality.1 This new technology improved the lateral resolution to approximately 2 μm and axial resolution to 8 - 12 μm.9 Furthermore, this design improved magnification up to 600 times.9 This significant advance allowed impressive in vivo imaging of tissues, but there was a significant trade-off, namely, the field of view is small.

To overcome this serious limitation, the concept of scanning was introduced. Just as this term implies, either the specimen must be moved past a fixed illuminated point or both the objective lens and condenser lens must be synchronously moved or scanned across the specimen within the X-Y plane.2,18 In Minsky’s device the specimen had to be moved using a precise registration system, in order to scan an entire field, which produced a multiple exposure photograph of the entire field.1 This process was tedious and time-consuming and hence never became a practical method to examine tissues.1

Tandem Scanning Confocal

Microscope

Approximately 10 years after Minsky’s original work, Petran and Hadravsky developed a novel technique that expedited the scanning process. They adapted the Nipkow Disk to improve confocal microscopy’s resolution and contrast. The Nipkow Disk was invented by Paul Nipkow in 1884 for encoding and decoding images for transmission over telegraph cables.1,5,10 This disk contains approximately 14,000 pinholes that are systematically placed 180° opposite each other, patterned in 40 identical Archimedean spirals.10,11 In other words, at any given time, there is a pinhole directly over the illuminator and detector, allowing only the light of interest to pass. The pinholes, which are 20 μm to 60 μm in diameter, are spaced at a set distance from each other to maximize the optics as explained in detail by Wilson and Sheppard.2,8 With separate light paths, or dual light path design, the illuminating light does not interfere with the light reflected from the specimen as it travels towards the detector.1 This diametric pinhole design was termed tandem and eliminated the primary cause of the decreased image contrast and the image degradation associated with white-light confocal microscopy (Figure 8-3).1

This design is critical, because, in order to scan a specimen within the X-Y plane, the disk must be rotated, while the specimen itself remains stationary. As the disk rotates, the spiraling set of pinholes act as camera shutters.

Figure 8-3: Tandem scanning confocal microscope concept. The pinhole concept is used as described in Figure 8.1. To allow for scanning, a Nipkow disk is used. When a point in the tissue is imaged, the light ray essentially travels simultaneously through two pinholes. The light ray travels through the first pinhole, through the optical system and is focused onto a point within the tissue. The reflected light is then sent back through the optical system, redirected to the second pinhole. The second pinhole filters out aberrant light rays and allows the detector to image a single point within the tissue block.

Light can only pass through the system when a set of conjugate pinholes is present before the illuminator and detector. Since the pinholes are spaced apart, consecutive, corresponding points within the X-Y plane can be systematically imaged without moving the specimen. This spiral design, in effect, acts as thousands of confocal optical systems working in parallel to scan the specimen. At any given time, only a few hundred pinholes are simultaneously present over the specimen to scan.7 However, since the disk is rotated at speeds above 40 revolutions per second, the entire specimen can be scanned several times during a single rotation.7,12 This high scan rate permitted video cameras and monitors to be used, which possess inherently high capture rates. As a result, one could observe a continuous image on a video monitor. Furthermore, since the Z-axis can be changed quickly, this system can quickly scan through tissue, allowing in vivo dynamic optical sectioning of the scanned tissue.

Advantages of Tandem Scanning Design over Prior Designs2,6

1.Subtoxic white light levels are used to create the images without using stains or dyes.

2.Noninvasive.

3.Enhanced lateral and axial resolution.

4.Enhanced contrast.

5.Dynamic, real-time images can be obtained.

6.Optical sections allow imaging from different tissue depths.

In the past, this design had a few significant disadvantages over other types of confocal microscopes.1 For a time, the calibration of the Nipkow disk itself was difficult. Since the outgoing light path and incoming light path utilize the same set of diametric pinholes in the Nipkow disk, any misalignment of the Nipkow disk resulted in severe image degradation.1 However, newer Tandem Scanning Confocal Microscopes (TSCM) contain a Nipkow disk system that is permanently aligned at the factory, eliminating this issue.1

Until recently, this microscope did not perform well during in vivo tissue examination secondary to an extremely low light-to-image capture rate. Because pinholes are used, only approximately 1% of the light sent into the system can be recovered for imaging.2 Additionally, any involuntary eye movements due to mircosaccades, pulse or respirations would result in large motion artifacts, making this novel design clinically useless. A rapid imaging capture system capable of at least 30 frames per second coupled with lowlux detectors was needed.1,9

Fortunately, technologic advances in video and electronic systems surpassed this threshold. These new capture imaging systems combined with low-lux cameras exploited the Nipkow disk’s potential. Combined with the development of a 20 X applanating cone objective lens, in vivo tandem scanning confocal microscopy of the cornea became a reality.12 Although, this design was the dominant confocal microscope used in ophthalmology, additional variations were developed.

Single-Sided Scanning Confocal

Microscope

Unlike the dual light path design of the tandem scanning confocal microscope, the single-sided confocal microscope uses a single light path design.1,2 This design utilizes the same pinhole in the rotating Nipkow disk for both the outgoing light destined for the specimen and the reflected light destined for the detector.1,2 A beam splitter mirror is required to separate the incoming and outgoing rays of light.1 The main disadvantage of this design is that the outgoing light rays, that do not pass through the Nipkow disk pinholes, reflect off the disk back toward the detector.1 These reflected light rays then interfere with specimen light rays of interest. Thus, this design potentially degrades image contrast and resolution.1

Kino and Xiao13 attempted to minimize this limitation by developing a tilted disk design. In their version, they tilted the Nipkow disk slightly.1 This tilt served to deflect