George T. Timberlake

Scanning Laser Ophthalmoscopy: Clinical Applications

The scanning laser ophthalmoscope (SLO) provides a high-quality television image of the retina using less than 1/1000 of the light required for conventional indirect ophthalmoscopy. The SLO employs a new ophthalmoscopic principle in which a dim laser beam scans across the fundus, and light is collected only from one retinal point at a time. Since the instrument is highly light efficient, illumination levels are comfortable for the patient, and fluorescein angiography can be performed with one tenth of the usual fluorescein dose. Since a continuous, large depth of field view is displayed on the SLO screen and stored on video tape, repeated dynamic inspection of the vitreous, retina and vitreoretinal interface is afforded. In addition, any graphical material that can be displayed on a microcomputer monitor (such as text of video games) can also be impressed on the retinal pattern formed by the sweeping laser beam. The graphical material is thus observed directly by the patient and on the patients retina by the clinician. Since the exact retinal locus of each point in the graphical material is viewed directly, it is possible to perform perimetry directly on the retina, to measure acuity at arbitrary retinal loci, to study how patients with macular disease use residual functional retina for reading, and to perform distortometry with a retinal (Amsler-type) grid.

Corneal Light Scattering and Visual Performance in Myopic Individuals With Spectacles, Contact Lenses, or Excimer Laser Photorefractive Keratectomy

Corneal transparency and visual performance are currently receiving much attention after excimer laser surgery. To date, emphasis has been on comparison of eyes on which laser surgery has been performed to eyes with emmetropia. A more appropriate comparison should be between eyes on which laser surgery has been performed and eyes with other forms of correction for myopia. Forward-scattered light, back-scattered light, and visual acuity were investigated and data were collected from 35 myopic individuals with various types of correction for myopia (spectacles, hard and soft contact lenses, and excimer laser surgery). Forward-scattered light was measured by using a new computerized technique, back-scattered light was measured with a charge coupled device-camera system, and visual acuity was measured with a computerized system at various levels of contrast. Spectacles, hard contact lenses, and excimer laser surgery are all superior to soft contact lenses in terms of light scatter and low-contrast visual acuity and excimer laser photorefractive keratectomy produces comparable results to spectacles one year postoperatively. At low-contrast visual acuity, mean visual acuity was 2.45 minutes of arc for the spectacle wearers, 3.21 minutes of arc for the hard contact-lens wearers, and 5.04 minutes of arc for the soft contact-lens wearers. Excimer laser patients had a mean visual acuity of 9.04 minutes of arc three months postoperatively, and 2.53 minutes of arc after one year. A mean value of 2.4% contrast for forward light scatter was obtained for spectacle wearers compared with a level of 3.84% contrast for hard contact-lens wearers and 16.1% contrast for soft contact-lens wearers. The mean value for excimer laser patients was 20% contrast three months postoperatively and 2.1% contrast one year postoperatively.

Corneal haze after excimer laser refractive surgery: objective measurements and functional implications.

Postoperative changes in corneal transparency is a major concern after PRK. Data were obtained from 69 human eyes treated with excimer laser photorefractive keratectomy in order to evaluate a relationship between objective measurements of corneal transparency and visual performance. A CCD-camera device was employed and by using polarizing filters the system could discriminate between reflected and scattered light. We observed two groups of postoperative behaviour in corneal transparency. Each group showed an increase in both scattered and reflected light signals with a maximum at around two months postoperatively. In the majority group (70%) this was followed by a subsequent decline of the scattered light signal, whereas the combined signal generated by reflected and scattered light showed a biphasic curve with a second peak at around 4 to 5 months postoperatively. In the minority patient group (30%) the timebase variations in pattern of both signals were indistinguishable throughout the period of observation. In all patients we observed a good correlation between the signal generated by scattered light alone and the reduction in the 5% contrast visual acuity performance, whereas correlation was poor when the combined signal of reflected and scattered light was considered. These disturbances in low contrast visual performance were only significant during the first three to four months postoperatively and thereafter most patients returned to their preoperative value. Eighteen percent of our patients discontinued topical steroids postoperatively. No differences in either corneal transparency or final refraction was observed. We consider that PRK is an effective form of refractive surgery and the marginal loss of corneal transparency should only be a problem during the first three or four months postoperatively.

Corneal light scattering after excimer laser photorefractive keratectomy: the objective measurements of haze.

BACKGROUND After photorefractive keratectomy using excimer lasers (193 nm), most corneas show a marginal loss of transparency (haze) and the assessment of its magnitude in clinical studies has been subjective. To address this problem, we have developed a new device for the objective measurement of haze by measuring corneal light scattering. METHODS A CCD-camera was fixed at 40 degrees to a slit-lamp microscope and connected via frame-grabber to a computer. By incorporating polarizing filters, the system could discriminate between reflected and scattered light. The intensity of light coming from the cornea was measured in gray scale levels using in-house image analysis software. The system was calibrated against three different sizes of microspheres (0.25, 0.50, and 5.00 microns) which corresponded to the size of cellular and extracellular elements known to occur at sites of corneal surgery. Data were obtained from three treated human eyes with measurements before treatment and at five different postoperative intervals with a maximum follow up of 4 months. RESULTS All three sizes of microspheres caused disturbances in gray scale levels (36 to 255 units) in the same range of those observed in corneal measurements. Disturbances in corneal light scattering were noted from 1 week postoperatively and persisted throughout the period of observation. We observed an increase in reflected and scattered light until the 2nd postoperative month followed by a subsequent decline. CONCLUSIONS It appears that this device is very useful to defect and measure objectively disturbances in corneal transparency after excimer laser photorefractive keratectomy.

A Novel Method for Measuring Saccade Profiles Using the Scanning Laser Ophthalmoscope

Most existing techniques for accurately measuring angular eye position vs time during a saccade (the saccade profile) need either contact to the eye or are restricted in time resolution. In this paper we introduce a new noninvasive method, with high spatial and temporal resolution, for determining saccade profiles using a scanning laser ophthalmoscope (SLO). This method uses the fact that images of a moving object taken with the SLO are not blurred (as are images from video cameras) but show a tilt with respect to images of the same stationary object. A mathematical framework is given that allows determination of a saccade profile from a restricted number of consecutive SLO video fields of the fundus during a saccade. The angular resolution obtained by this method is below 0.1 deg, and the maximum time resolution near 1 msec. Measured saccade profiles could be well approximated by a gamma function, the first derivative of which yields the saccade velocity profile. Measurements of peak saccade velocity as a function of saccade amplitudes (main sequence) using our method show good agreement with results obtained by other authors.

A Photoactivated Diazopyruvoyl Cross-linking Agent for Bonding Tissue Containing Type-I Collagen¶

On the basis of the earlier examples of diazopyruvoyl (DAP) groups reported by Lawton for covalent binding and cross‐linking of proteins and oligopeptides and our recent demonstration that a coumaryl diazopyruvamide was used to label Type‐I collagen, we have extended our investigations to the synthesis and cross‐linking capabilities of a bis‐DAP polyethylene glycol to cross‐link Type‐I collagen. The new photoactivated cross‐linking agent, N,N′‐bis(3‐diazopyruvoyl)‐2,2′‐(ethylenedioxy)bis(ethylamine) (DPD, 2), has been designed and synthesized specifically to “weld” collagenous tissues by cross‐linking Type‐I collagen. A working model for the photochemical welding studies of collagenous tissues was developed using gelatin strips (gel strips) composed of denatured Type‐I collagen. Gel strips are transparent to near‐UV and visible light, uniform in thickness, and have reproducible composition. Furthermore, the availability of nucleophilic amine sites in gel strips was demonstrated by reaction with o‐phthalaldehyde, producing a fluorescent derivative of the protein. Gel strips were coated with a solution of DPD in chloroform 7 irradiated at 320–390 nm, and the resulting bonded gel strips were tested for the strength of the weld. The welds were generally brittle and had average tensile strengths that exceeded 100 N/cm2. Welds were not formed in the absence of light or DPD. Scanning electron microscopy studies revealed a pockmarked surface from severed welds. Welds of rabbit Achilles tendon were also obtained using the tethered diazopyruvamide. These welds were much weaker, having an average tensile strength of 11.95 N/cm2 for DPD–2,2′‐ethylenedioxy(bis)ethylamine comonomers in the cross‐linking reaction. In both studies the welds obtained by this method were significantly stronger than the controls.

Automated Clinical Visual Acuity Testing

We developed a method for testing visual acuity using a general-purpose microcomputer which displays visual acuity targets on a television monitor, controls a staircase psychophysical testing procedure, and provides a printed record of mean visual acuity and standard deviation. This automated procedure can be used to increase the precision of clinical visual acuity testing and to determine if changes in a patients visual acuity are statistically significant.

Retinal image location of hand, fingers, and objects during manual tasks.

Purpose. A new method was developed using the scanning laser ophthalmoscope (SLO) to investigate the effects of central visual loss on eye-hand coordination in manual tasks. Using the SLO, the retinal positions of the hand, fingers, and objects are imaged and recorded while a subject performs a manual task. Method. A video camera images the subject’s hand and objects to be manipulated in the SLO laser-beam raster, producing a video image of a subject’s hand, fingers, and objects on the subject’s retina while the objects are manipulated. A subject with bilateral central scotomas and an age-matched control subject with normal vision traced an ellipse with the index finger, tapped four disks in sequence, and carried out a pattern duplication task with pegs. Retinal positions of the fovea or preferred retinal locus (PRL), fingers, and objects were measured from digitized SLO images. Results. In all tasks, the fovea or PRL was directed to an object or position before the fingers arrived. This lead time was much greater for the scotoma subject than the control subject (∼1400 vs. ∼400 ms, respectively). The scotoma subject was much less accurate in placing the PRL and fingers on objects and required substantially more time for task completion than the control subject. Conclusions. The coordination of foveal fixation and finger placement found with the SLO method was similar to that found by others using eyetracking techniques with visually normal subjects. The presence of a central scotoma and use of a PRL caused marked deterioration in the quality of this coordination. Unlike eyetracking methods, the SLO technique does not require calibration because the positions of the fingers and objects are directly observable on the retina. This method could be useful in studying eye-hand coordination of individuals with scotomas that affect foveal vision.

Photoactivated coumaryl-diazopyruvate fluorescent label for amine functional groups of tissues containing type-I collagen

Abstract The design, synthesis and application of a new fluorescent-labeling reagent for collagen has been developed as a prerequisite for the design of a photoactivated collagen-crosslinking compound for surgical wound closure. The amine groups in collagen are the targets of a rational design for a new fluorophore because natural collagen crosslinks are formed between primary (1°) amine groups of lysine and hydroxylysine. The availability of 1° amines for crosslinking in native collagenous tissues was evaluated by reacting tendon and corneal samples with o-phthalaldehyde and dansyl chloride, fluorophores commonly used for the detection of 1° and 2° amines. The resulting fluorescent collagen fibrils indicated the presence of amines in native tissue. Subsequently, a photoactivated fluorescent label for 1° and 2° amines, coumaryl gamma-amino-butyric acid diazopyruvate (CGDP), was designed and synthesized. CGDP was first used to photolabel poly-l-lysine, forming a fluorescent, covalent bond to the 1° amine. CGDP was then photoreacted with corneal and tendon tissue samples to produce CGDP fluorescent–labeled samples that were statistically significantly more fluorescent than were the controls. These experiments support the postulate that 1° or 2° (or both) amines in native collagenous tissues are available to serve as targets for photoactivated collagen crosslinkers for wound closure.

Collagen Cross Linking Agents: Design and Development of a Multifunctional Cross Linker

A new cross linking reagent based on the first‐generation polyamidoamine dendrimer (G.1 PAMAM) has been synthesized by reaction of the PAMAM with eight equivalents of p‐nitrophenyl diazopyruvate. The resulting water‐soluble octadiazopyruvoyl PAMAM (8G.1 DAP, 1.3) was shown to undergo Wolff rearrangements upon photolysis in methanol at λ > 300 nm to yield the methyl esters of the ketenes formed from the loss of nitrogen. 8G.1 DAP also forms strong bonds with dehydrated collagen with glass as high as 36 N cm−2. Collagen to collagen bonds with tensile strengths as high as 92 N cm−2 were observed with fully dehydrated tissues. The bonding decreased rapidly with increasingly hydrated tissue possibly due to the increased distance between the collagen fibrils and the competition of H2O for the free ketene functions.