3.2.1.

Biomicroscopy

The effective use of slit-lamp biomicroscopy to overcome vitreous transparency necessitates maximizing the Tyndall effect. Although this can be achieved in vitro, as described earlier, there are limitations to the illumination-observation angle that can be achieved clinically. This is even more troublesome in the presence of meiosis, corneal and/or lenticular opacities, and limited patient cooperation. Essential to the success of achieving an adequate Tyndall effect are maximizing pupil dilation in the patient, since the Tyndall effect increases with an increasingly subtended angle between the plane of illumination and the line of observation (up to a maximum of 90 deg, as in dark-field slit-lamp microscopy in vitro), and dark adaptation in the examiner. Some observers claim that green light enhances the Tyndall effect, although this has never been explained or tested scientifically.

Preset lens biomicroscopy slightly increases the available illumination-observation angle, offers dynamic inspection of the vitreous in vivo, and provides the capability of recording the findings in real time.¹⁸ Initially introduced on a wide scale for use with a Hruby lens and currently practiced by using a hand-held 90-diopter lens at the slit lamp, this technique is purportedly best performed with a fundus camera and the El Bayadi-Kajiura lens promoted by Schepens, Trempe, and associates. This approach has been used in many seminal studies of the role of vitreous in various disease states.¹⁸ However, there has not been widespread use of this approach, probably because it is heavily dependent upon subjective interpretation of the findings and there is thus unreliable reproducibility from facility to facility. It would be desirable to have a method of determining the presence or absence of posterior vitreous detachment (PVD) that is more objective. Furthermore, there have been no studies correlating preset lens biomicroscopy findings in patients suspected of having a posterior vitreous detachment with some type of corroborating evaluation, such as histology or intraoperative findings.

3.2.2.

Ophthalmoscopy

Of all the parts of the eye that are routinely evaluated by clinical inspection, the vitreous is perhaps the least amenable to standard examination techniques. This is because, as previously mentioned, examining thr vitreous is an attempt to visualize a structure designed to be virtually invisible.¹⁹ With the direct ophthalmoscope, light rays emanating from a point in the patient’s fundus emerge as a parallel beam that is focused on the observer’s retina and an image is formed. However, incident light reaches only the part of the fundus onto which the image of the light source falls and only light from the fundus area onto which the observer’s pupil is imaged reaches that pupil. Thus the fundus can be seen only where the observed and the illuminated areas overlap and where the light source and the observer’s pupil are aligned optically. This restricts the extent of the examined area. Also, because of a limited depth of field, this method is rarely used to assess the vitreous structure.

Indirect ophthalmoscopy extends the field of view by using an intermediate lens to gather rays of light from a wider area of the fundus. Although binocularity provides stereopsis, the image size is considerably smaller than with direct ophthalmoscopy and only significant alterations in vitreous structure, such as the hole in the prepapillary posterior vitreous cortex (Fig. 7) that is visible after posterior vitreous detachment, vitreous hemorrhage, or asteroid hyalosis, are reliably diagnosed by indirect ophthalmoscopy.

Figure 7

Preset lens biomicroscopy of posterior vitreous detachment. The optic disk and retinal vessels are to the left; the slit beam of illumination is in the center; and the posterior vitreous cortex (linear structure to the right) can be seen detached anteriorly.

The scanning laser ophthalmoscope (SLO), also developed at the Schepens Eye Research Institute in Boston, enables dynamic inspection of the vitreous in vivo, features tremendous depth of field, and offers real-time recording of findings. Monochromatic green, as well as other wavelengths of light are also available for illumination.²⁰ To date, however, the SLO has really only improved our ability to visualize details in the prepapillary posterior vitreous, such as Weiss’s ring (Fig. 8). In spite of the dramatic depth of field possible with this technique, the SLO does not adequately image the vitreous body and an attached posterior vitreous cortex, probably because of limitations in the level of resolution. Thus, posterior vitreous detachment, which is by far the most common diagnosis to be found when imaging the vitreous clinically, is not reliably determined by the SLO. Indeed, there is an increasing awareness among vitreous surgeons that the reliability of the clinical diagnosis of total posterior vitreous detachment by any existing technique is woefully inadequate. This awareness arises from the fact that vitreous surgery following clinical examination often reveals findings that are contradictory to preoperative assessments.

Figure 8

Scanning laser ophthalmoscopy (SLO) of posterior vitreous. The SLO image of a detached posterior vitreous cortex demonstrates the appearance of Weiss’s ring surrounding the prepapillary hole. (Reprinted with permission from Ref. 18.)

3.2.3.

Ultrasonography

Ultrasound is an inaudible acoustic wave that has a frequency of more than 20 kHz. The frequencies used in ophthalmology are generally in the range of 8 to 10 MHz. Although these very high frequencies produce wavelengths as short as 0.2 mm, these are not short enough to adequately assess a normal internal vitreous structure such as the fibers described earlier. Even the posterior vitreous cortex, about 100 μ m at its thickest point in the normal state, is below the level of resolution of conventional ultrasonography. The utility of this technique results from the fact that strong echoes are produced at “acoustic” interfaces found at the junctions of media with different densities and sound velocities, and the greater the difference in density between the two media that create the acoustic interface, the more prominent the echo. Thus, age-related or pathological phase alterations within the vitreous body are detectable by ultrasonography.

Oksala, who in the late 1950s and early 1960s was among the first to employ B-scan ultrasonography to image the vitreous, summarized his findings of aging changes in 1978.²¹ In that report of 444 “normal” subjects, Oksala defined the presence of acoustic interfaces within the vitreous body as evidence of vitreous aging and determined that the incidence of such interfaces was 5 between the ages of 21 and 40 years, and fully 80 for individuals over 60 years of age. In clinical practice, however, only relatively profound vitreous abnormalities such as asteroid hyalosis, vitreous hemorrhage, and intravitreal foreign bodies (if sufficiently large) are imaged by ultrasonography. At the vitreo–retinal interface, the presence of a posterior vitreous detachment is often suspected on the basis of B-scan ultrasonography but can never be definitively established, since the level of resolution is not sufficient to reliably image the posterior vitreous cortex, which is only a little more than 100 μ m thick at its thickest point. However, recent studies have successfully used this technique to determine the presence of a split in the posterior vitreous cortex, called vitreoschisis (Fig. 9), of patients with proliferative diabetic vitreoretinopathy.²² The success achieved in using ultrasound to identify this important pathological entity probably results from the fact that this tissue is abnormally thickened by nonenzymatic glycation of vitreous collagen and other proteins.²² In the absence of such advanced disease, however, the diagnosis of PVD cannot be definitively established by ultrasonography.

Figure 9

Ultrasonographic imaging of vitreoschisis. Splitting of the vitreous cortex (arrow) can occur and mimic posterior vitreous detachment. In diabetic patients, blood can be present in the vitreoschisis cavity. I, inner wall; P, posterior wall of vitreoschisis cavity within the posterior vitreous cortex. (Photograph courtesy of Dr. Ronald Green. Reprinted with permission from R. L. Green and S. F. Byrne, “Diagnostic ophthalmic ultrasound,” in Retina, S. J. Ryan, Ed., The C. V. Mosby Company, St. Louis, 1989.)

3.2.4.

Optical coherence tomography

Introduced in 1991, optical coherence tomography (OCT) is a new technique for high-resolution cross-sectional imaging of ocular structure.²³ It is based on the principle of low coherence interferometry, where the distances between and sizes of structures in the eye are determined by measuring the “echo” time it takes for light to be backscattered from the different structures at various axial distances. The resolution of all echo-based instrumentation (such as ultrasound and OCT) is based upon the ratio of the speed of the incident wave to that of the reflected wave. As described earlier, clinical ultrasonography is performed with a frequency of approximately 10 Hz and has a 150-mm resolution. Although recently introduced ultrasound biomicroscopy has increased the frequency (up to 100 Hz), and thus has a spatial resolution of 20 μm, penetration into the eye is no more than 4 to 5 mm. Light-based devices, such as the OCT, use an incident wavelength of 800 nm and have increased axial resolution to 10 μm, providing excellent imaging of retinal architecture, although vitreous applications are less useful. The limitations of OCT include the inability to obtain high-quality images through opaque media, such as dense cataract or vitreous hemorrhage. The use of OCT is also limited to cooperative patients who are able to maintain fixation for the full acquisition time of 2.5 s per section. Thus, to date OCT has primarily been used to image, and to some extent quantitate, retinal laminar structure. Some vitreous applications have been useful, especially those that involve imaging the vitreo–macular interface in patients with macular holes²⁴ (Fig. 10). However, the exact nature and molecular composition of these preretinal tissue planes cannot be definitively deduced using OCT, and little information can be garnered about structures within the vitreous body.

Figure 10

Optical coherence tomography (OCT) of a macular hole. Evolution of an impending macular hole in a fellow eye to a stage II hole. (Top left) OCT shows a cystic formation in the inner part of the foveal center. The elevation of the foveal floor and the adherence of the partially detached posterior vitreous cortex suggest vitreo-retinal traction. (Top right) Two months later the roof of the cyst has opened on the nasal side of the macula, constituting an incompletely detached operculum to which the posterior vitreous cortex adheres. (Bottom) A composite optical coherence tomogram shows the convex elevation of the partially detached posterior vitreous cortex, suggesting anteroposterior vitreous traction. (Reprinted with permission from Ref. 24.)

3.2.5.

Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy. The NMR technique is based upon the fact that when placed in a magnetic field, magnetic nuclei, such as water protons, tend to orient their magnetic vectors along the direction of the magnetic field. The time constant for this orientation, known as the longitudinal relaxation time, T₁, reflects the thermal interactions of protons with their molecular environment. Magnetic vectors that have previously been induced to be in phase with each other undergo a “dephasing” relaxation process that is measured by the transverse relaxation time, T₂. It is the transverse relaxation time T₂ that reflects inhomogeneities within the population of protons. Protons oriented by a magnetic field absorb radio waves of a frequency appropriate to induce transactions between their two orientations. Such absorption is the basis of the NMR signal used to index relaxation times. Relaxation times in biological tissues vary with the concentration and mobility of water within the tissue. Since the latter is influenced by the interaction of water molecules with macromolecules in the tissue, this noninvasive measure can assess the gel to liquid transformation that occurs in the vitreous during aging¹⁴ and disease states, such as diabetic vitreopathy.^{15 16} These considerations led Aguayo et al.;²⁵ to use NMR to study the effects of pharmacological vitreolysis^{26 27} on bovine and human vitreous specimens and intact bovine eyes in vitro. Collagenase induced measurable vitreous liquefaction, more so than hyaluronidase (Fig. 11). Thus, this noninvasive method could be used to evaluate age and disease-induced synchisis (liquefaction) of the vitreous body, although it is not clear that this technique would adequately evaluate the vitreo–retinal interface. There have, curiously, been few recent studies that have employed NMR spectroscopy in research or clinical applications on the vitreous.

Figure 11

NMR spectroscopy of pharmacologic vitreolysis. Progressive saturation. (a) 300 ms, (b) 1200 ms, (c) 2000 ms, (d) 3920 ms, and (e) 7864 ms images of a bovine eye 12 h after injection with 0.2 ml of 100 U/ml collagenase solution. Note the bright area (x) in the posterior pole. This area of enhanced relaxation (shorter T₁) was absent immediately after injection. In this view the optic nerve is in the edge of the plane of the image. (Reprinted with permission from Ref. 25.)

Raman spectroscopy. This form of spectroscopy was first described in 1928 by C. V. Raman in India. Raman spectroscopy is an inelastic light-scattering technique in which molecules in the vibrational mode in the study specimen absorb energy from incident photons, causing a downward frequency shift, which is called the Raman shift. Because the signal is relatively quite weak, current techniques employ laser-induced stimulation with gradual increases in the wavelength of the stimulating laser so as to be able to detect the points at which the Raman signal becomes apparent as peaks superimposed on the broad background fluorescence. The wavelengths at which these peaks are elicited are characteristic of the chemical bonds, such as aliphatic C―H (2939 cm ⁻¹), water O―H (3350 cm ⁻¹), C=C and C―H stretching vibrations in π-conjugated and aromatic molecules (1604 cm ⁻¹ and 3057 cm ⁻¹) , etc. To date, most applications of this technique in the eye have been for analysis of lens structure and pathology.²⁸ The use of near-infrared (IR) excitation wavelengths is particularly effective in the lens, since these wavelengths have better penetration in opacified cataractous lenses.

Vitreous studies²⁹ employed excised samples of human vitreous obtained during surgery. The near-IR excitation at 1064 nm was provided by a diode-pumped neodymium-doped: yttrium aluminum garnet (Nd:YAG) continuous wave (cw) laser with a diameter of 0.1 mm and a power setting of 300 mW. Backscattering geometry with an optical lens collected scattered light, which was passed through a Rayleigh light rejection filter into a spectrophotometer. The results (Fig. 12) showed that this technique was able to detect peaks at 1604 and 3057 cm ⁻¹ in the vitreous of diabetic patients that were not present in controls. Further research and development is needed to reliably interpret such results and refine the methodology for noninvasive use in situ. While this has already been achieved in the lens, it is not clear that this will be possible for vitreous applications.

Figure 12

Raman spectroscopy of diabetic vitreopathy. A Fourier transform of Raman spectra (FT-RS) (using a laser power of 300 mW, a laser spot diameter of 0.1 mm, and 250 co-added scans) of control and diabetic human vitreous collagen. Samples were pooled into one specimen for the patients with diabetes (n=7) and one for the control group (n=10). The graphs represent the actual FT-RS spectra of these two specimens. Quantitative analysis of the peak area at 1604 cm ⁻¹ showed a threefold increase in samples from patients with diabetes compared with controls. The broad background was corrected for when drawing the baseline of the peak at 1455 cm ⁻¹. (Reprinted with permission from Ref. 29.)

3.2.6.

Dynamic light scattering

Dynamic light scattering (DLS) is an established laboratory technique for measuring the average size (or size distribution) of microscopic particles as small as 3 nm in diameter that are suspended in a fluid medium where they undergo random Brownian motion. Light scattered by a laser beam passing through such a dispersion will have intensity fluctuations in proportion to the Brownian motion of the particles. Since the size of the particles influences their Brownian motion, analysis of the scattered light intensity yields a distribution of the size(s) of the suspended particles. Visible light from a laser diode (power 50 μW) is focused into a small scattering volume inside the specimen (excised lens or vitreous, autopsy or living eye). The detected signal is processed via a digital correlator to yield a time autocorrelation function (TCF). For dilute dispersions of spherical particles, the slope of the TCF provides a quick and accurate determination of the particle’s translational diffusion coefficient, which can be related to its size via a Stokes-Einstein equation, provided the viscosity of the suspending fluid, its temperature, and its refractive index are known. For the lens and vitreous, a viscosity of η=0.8904 centipoise, a refractive index of n=1.333, and a temperature of 25°C for in vitro studies and 37°C for in vivo studies were used to determine macromolecule sizes.

Studies^{30 31 32} of the lens and vitreous have employed DLS instrumentation that was developed by the National Aeronautics and Space Administration to conduct fluid physics experiments on-board the space shuttle and space station orbiters. The beam input from a semiconductor laser (670-nm wavelength) at 50 μW power was projected into the specimens and the scattered signal was collected by the DLS probe for 10 s. The signal was then detected by an avalanche photodiode detector system. A TCF was constructed using a digital correlator card. The slope of the TCF provides a measure of particle sizes in the selected measurement sites (volume=50 μm ³). Studies³¹ in the lens found that DLS was significantly more sensitive than Scheimpflug photography in detecting early changes in the lens.

When the DLS probe was used to obtain measurements from the entire vitreous body, scanning was performed in conjunction with a micropositioning assembly, which controlled detector position in the x-, y-, and z -planes. This enabled semiautomated measurements from a sufficient number of sites within the bovine vitreous body to create a three-dimensional map of the distribution of the average particle sizes of vitreous macromolecules (Fig. 13). Furthermore, in studies³² of autopsy human eyes, DLS was able to detect the structural changes¹⁶ resulting from diabetic vitreopathy²⁵ (Fig. 14). It is interesting that these DLS findings appear to corroborate the findings of dark-field slit microscopy (Fig. 6) where glycation of vitreous proteins resulted in cross-linking of collagen fibrils and aggregation into large bundles of fibrils. It is plausible that these are detected by DLS as particles of larger size with more variability than that seen in the nondiabetic control in this preliminary study. Future studies with more advanced instrumentation will determine if this phenomenon can be detected in a clinical setting, confirming these preliminary in vitro results.

Figure 13

Dynamic light scattering (DLS) of bovine vitreous. A three-dimensional map of DLS measurements in the bovine vitreous body in situ. The x -axis is the horizontal distance from a central (near-optical) axis of the eye. The y -axis is the distance along the sagittal (front-to-back) plane from the central starting point of measurements on the optical axis of the eye. From this starting point, the location of which is represented as the z coordinate and is defined as millimeter posterior to the lens along the optical axis in the central vitreous, measurements were obtained at 0.5-mm steps in both the x and y directions. Along the z -axis (in the sagittal plane), measurements were obtained every 2 mm, beginning at 14 mm behind the back surface of the lens to 24 mm posterior to this point. The heterogeneous distribution of macromolecules throughout the vitreous body can be appreciated in these plots, with the height of the lines representing the average particle size for all molecular constituents (cumulant fit of both fast hyaluronan and slow collagen) at the measurement site. (Reprinted with permission from Ref. 30.)

Figure 14

Dynamic light scattering of diabetic vitreopathy. Eyes were obtained at autopsy and prepared by removing the cornea, iris, and lens from the globe, leaving the posterior capsule of the lens intact. DLS measurements were made along the anteroposterior axis at steps 0.5 mm apart, beginning behind the posterior capsule of the lens. The abscissa represents the distance from the lens capsule. The ordinate shows the particle sizes determined from the “slow component” of the time relaxation curves. In the 72-year-old patient with diabetes (solid triangles) there were larger and more varied particle sizes than in the 70-year-old nondiabetic (open triangles).