2.1.

Subjects

The ${\mathrm{ED}}_{50}$ for multiple-pulse exposure to $100\text{-}\mu \mathrm{s}$-duration pulses at $\lambda =532\mathrm{nm}$ was measured in the macular region of the retina of Rhesus macaques (Macaca mulata). This study was conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals. All experiments involving animals used appropriate levels of anesthesia so that the subjects did not experience pain or distress.

Prior to selection for use in the study, subject animals were screened for normal retina and clear ocular media and were required to have a refractive error $<0.5$ diopters from emmetropia. In preparation for laser exposure, each animal was sedated using an intramuscular injection of Telazol. A subcutaneous injection of Atropine Sulfate was given for control of excessive salivation and bronchial secretions. Anesthesia was then induced using an intravenous administration of propofol. An intravenous line of Lactated Ringer’s solution was placed for fluid resuscitation. A retrobulbar injection of 4% lidocaine was used in the exposed eye to reduce ocular motion during the exposures. Cycloplegia and pupil dilation were induced using two drops of 0.5% proparacaine hydrochloride, 2.5% phenylephrine hydrochloride, and 1% tropicamide. A lid speculum held the eye open for exposure. The cornea was periodically irrigated with a 0.9% saline solution to maintain clarity.

2.2.

Exposure Setup

The experimental setup used to produce minimal spot size and extended diameter retinal exposures in the eye of a rhesus macaque is illustrated in Fig. 1. A Coherent Verdi G10 Diode-Pumped Solid-State $\mathrm{ND}:{\mathrm{YVO}}_4$ laser (Coherent, Santa Clara, California, USA) produced continuous-wave laser radiation at a wavelength of 532 nm. The beam was focused onto the plane of a Bentham Instruments Model 218 optical chopper (Bentham Instruments Ltd., Reading, United Kingdom) set up with two interleaved chopping blades to produce square-wave $100\text{-}\mu \mathrm{s}$-duration pulses at a PRF of either 50 or 1000 Hz. A timing shutter determined the total exposure time and, therefore, the total number of pulses.

Fig. 1

Experimental setup. ND, frequency-doubled, continuous wave $\mathrm{Nd}:{\mathrm{YVO}}_4$ laser ($\lambda =532\mathrm{nm}$). FL, focusing lens; RL, recollimating lens; OC, optical chopper; BS, brick shutter; TS, timing shutter; NDF, neutral density filters; IR, variable aperture; WBS, glass wedge beam splitter; MB, marker beam path; PC, pulse counter; OS, oscilloscope; EM, energy meter; PI, pulse integrator; FC: fundus camera; M, movable mirror.

A wedge beam splitter deflected portions of the beam into detectors to measure the pulse width and count the number of pulses. Pulse width was measured with a UDT Instruments model 247 detector (UDT Instruments, San Diego, California, USA) coupled to a Tektronix TDS3045C oscilloscope (Tektronix, Beaverton, Oregon, USA). Figure 2 shows a typical pulse temporal profile produced by the beam chopper. Pulse rate and total number of pulses were measured using a UDT Instruments model 248 detector and a Hewlett-Packard model 5316B Universal Counter (Hewlett-Packard, Palo Alto, California, USA).

Fig. 2

Typical temporal profile for the $100\text{-}\mu \mathrm{s}$ pulses for the beam having a 1000 Hz pulse repetition frequency. (a) A single pulse. (b) Average of 16 pulses.

A second wedge beam splitter deflected portions of the beam into detectors to measure the pulse energy. For single-pulse exposures, a Laser Probe RjP-765a detector (Laser Probe Inc., Utica, New York, USA) read out by a Laser Probe Rj-7620 Dual Channel Energy Meter was used to measure the pulse energy in the reference beam. Prior to exposing a retina, a Laser Probe RjP-735 detector was placed at the eye position to measure the energy incident to the eye. The ratio of the two measurements was used to calibrate the exposure system for single-pulse exposures. For multiple-pulse exposures, an International Light Technologies SED033 detector (International Light Technologies, Peabody, Massachusetts, USA) read out by an IL1700 pulse integrator was used to measure the total energy in the reference beam. This system was calibrated by placing a Scientech model 380101 detector (Scientech, Boulder, Colorado, USA) read out by a Scientech model 36-5002T2 power meter placed at the eye position prior to retinal exposures.

The central, uniform portion of the beam was selected using an adjustable aperture (labeled IR in Fig. 1). For extended retinal beam spot exposures, a 400 mm focal length lens (LNS in Fig. 1) was inserted into the setup to bring the beam to a focus some distance in front of the eye, so that the beam was diverging as it entered the eye. The diameter of the aperture IR was adjusted so that the beam divergence was 7.4 mrad, producing a $100\text{-}\mu \mathrm{m}$-diameter top-hat retinal beam spot in the macaque retina ($f_e=13.5\mathrm{mm}$). The divergence of the beam was verified using a WinCamD-UCD12 CCD camera (DataRay Inc., Bella Vista, California, USA) placed at the focal plane of a 200 mm focal length lens. Lens LNS was removed for collimated beam exposures. The beam diameter at the cornea was 3 mm for both exposure conditions.

Neutral density filters were used to attenuate the beam, determining the pulse energy for each exposure. Moveable mirrors (M) were inserted into the beam path (MB) to bypass the optical chopping system for placing marker lesions into the eye. Lens LNS was removed to provide a collimated beam of 40 mW and 100 ms pulse duration to induce marker lesions.

2.3.

Procedure

The laser-induced retinal injury threshold was measured for exposures to a collimated laser beam, leading to a minimal retinal spot size exposure, and for a beam having a 7.4 mrad divergence. Assuming an effective focal length for the macaque eye of $f_e=13.5\mathrm{mm}$, the diverging beam produced a top-hat retinal beam spot having a $1/e$ irradiance diameter of $100\mu \mathrm{m}$. The laser-induced retinal injury threshold was measured for PRFs of 50 and 1000 Hz. At the slower PRF (50 Hz), the injury threshold was measured for pulse trains of up to $N=500$ pulses (a 10-s exposure). The threshold was measured for pulse trains of up to 1000 pulses (a 1-s exposure) for the higher PRF (1000 Hz). Data from at least three eyes were used to determine the ${\mathrm{ED}}_{50}$ from each exposure condition, defined by the PRF, beam divergence, and number of pulses.

A series of marker lesions were placed around the macula to define a test exposure grid within the macula and to aid in locating the exposure sites during subsequent examination of the retina. The $\mathrm{Nd}:{\mathrm{YVO}}_4$ laser was used to produce the markers, using the beam path bypassing the beam chopper (Fig. 1).

Twenty-five sites were exposed within the macular of each eye. The pulse energy was varied from site to site, although all pulses were identical within a given exposure. Exposure sites were nominally evaluated 24 h after exposure. Retinas were photographed using a digital fundus camera and were examined visually using an ophthalmoscope. The response criterion was defined as any ophthalmoscopically visible alteration of the retina at the exposure site. Dose-response data for 35 to 70 exposures were collected for each exposure condition. Probit analysis^19,20 was used to extract an ${\mathrm{ED}}_{50}$ from each dose-response data set. The ${\mathrm{ED}}_{50}$ is defined to be the dose at which there is a 50% probability of producing a detectable retinal alteration. The dose is given as the total intraocular energy (TIE), which is the energy incident on the cornea that passes through the pupil of the eye. TIE is expressed in this paper as the energy per pulse in the pulse train.

4.1.

Threshold-Level Damage Mechanism

The ${\mathrm{ED}}_{50}$ values reported here for repetitive-pulse exposures to $100\text{-}\mu \mathrm{s}$-duration pulses are very nearly identical for the two pulse repetition frequencies, 50 and 1000 Hz (Fig. 3). In Fig. 4, PS model predictions based on the experimentally determined single-pulse ${\mathrm{ED}}_{50}$ and probit slope are compared to the experimental data.¹⁷ The data are well described by the PS model curves. Thermal models have not shown that the mechanism of thermal denaturation can lead to a condition wherein the ${\mathrm{ED}}_{50}$, expressed as energy per pulse, can be independent of the interpulse spacing (PRF). The observed result that for $100\text{-}\mu \mathrm{s}$-duration pulses the ${\mathrm{ED}}_{50}$ expressed as energy per pulse is independent of the interpulse spacing (PRF) for two widely separated frequencies supports a conclusion that the threshold-level damage mechanism for exposure to single $100\text{-}\mu \mathrm{s}$-duration pulses is microcavitation.

Fig. 4

Comparison of probability summation (PS) model calculations with the experimentally measured ${\mathrm{ED}}_{50}$ for repetitive-pulse exposures to $100\text{-}\mu \mathrm{s}$ pulses at $\lambda =532\mathrm{nm}$. The PS calculations are based on the experimentally determined single-pulse ${\mathrm{ED}}_{50}$ and probit slope. (a) Collimated beam (minimal retinal beam spot). (b) $100\mu \mathrm{m}$ retinal beam spot.

Thermal retinal injury models predict a significant difference in the ${\mathrm{ED}}_{50}$ versus number of pulses for PRFs of 50 and 1000 Hz. In Fig. 5, threshold predictions from the thermal model of Jean and Schulmeister^21,22 are compared to the experimentally measured values. The thermal model prediction for a 1000 Hz PRF conforms reasonably well to the data. However, the model provides poor prediction for the 50 Hz PRF data (Fig. 5). Differences in the model predictions for the two PRFs arise essentially from the cooling that can or cannot occur during the interpulse periods. At the lower PRF (50 Hz), the model retina has enough time to cool to its original baseline temperature through thermal diffusion to surrounding tissue. At the higher PRF (1000 Hz), there is insufficient time for thermal diffusion to cool the tissue back to the baseline temperature, and subsequent pulses hit tissue at an already elevated temperature, further increasing the temperature and, thus, the tissue damage rate.^18,23

Fig. 5

Comparison of thermal model calculations with the experimentally measured ${\mathrm{ED}}_{50}$ for repetitive-pulse exposures to $100\text{-}\mu \mathrm{s}$ pulses at $\lambda =532\mathrm{nm}$. (a) Collimated beam (minimal retinal spot size). (b) $100\mu \mathrm{m}$ retinal beam spot diameter.

It is argued that when the dominant damage mechanism for a threshold-level exposure to $N$ pulses is microcavitation, the ${\mathrm{ED}}_{50}$ expressed as energy per pulse depends only on the number of pulses and is independent of the PRF. When the dominant damage mechanism for a threshold-level exposure to $N$ pulses is thermal, the ${\mathrm{ED}}_{50}$ is dependent upon both $N$ and the PRF, and, in fact, becomes lower as the PRF is increased. It follows that in the case that the dominant mechanism for the single-pulse injury is microcavitation, the ${\mathrm{ED}}_{50}$ expressed as the energy pulse will be independent of the PRF for low frequencies, but at higher PRF, the cumulative pulse thermal damage contribution may become dominant and the ${\mathrm{ED}}_{50}$ for $N$ pulses expressed as energy per pulse will drop below the constant value predicted by the probability summation model. It further follows that, in this case, there will be a PRF wherein the ${\mathrm{ED}}_{50}$ can be equally well explained by both the microcavitation model and the thermal denaturation model.

The data of this experiment for a PRF of 1000 Hz are equally well described by both the thermal model and the PS model. The data for 50-Hz exposures are poorly described by the thermal model but are well described by the PS model. It is, therefore, reasonable to conjecture that for exposure to $100\text{-}\mu \mathrm{s}$-duration pulses, the microcavitation threshold-level damage mechanism dominates for $\mathrm{PRF}<\sim 1000\mathrm{Hz}$, while the thermal denaturation injury mechanism dominates for $\mathrm{PRF}>\sim 1000\mathrm{Hz}$. At 1000 Hz, thermal denaturation occurs at near the same level that microcavitation occurs. Data for a higher PRF are needed to clarify this conjecture. If there is a crossover between threshold-level injury mechanisms, an experiment performed at a higher pulse repetition rate, say 2000 Hz, should result in a thermal injury at a lower threshold.

Figure 6 compares the single-pulse data of this paper to data from the literature^{4,6–8,10,13,24} and shows the transition between the temporal domain of microcavitation-dominated threshold retinal injury and the temporal domain of thermal denaturation-dominated threshold retinal injury. The ${\mathrm{ED}}_{50}$ predicted by thermal models of laser-induced RPE injury is included for comparison.²⁵ The bubble formation and cell death data, obtained via ex vivo exposures in retinal explants, show that RPE cell death correlates well with the observation of microcavitation for exposure durations $<\sim 50\mu \mathrm{s}$, while for exposures $>100$ to $200\mu \mathrm{s}$, cell death occurs at radiant exposures lower than that required to produce microcavitation. The transition from microcavitation as the dominant threshold-level injury mechanism to thermal denaturation appears to occur between 50 μs and $200\mu \mathrm{s}$. The in vivo threshold data for exposures to extended sources (nominally 5 mrad visual angle) are included in this plot as the retinal image diameters are more accurately known for these data.^26–28 The in vivo data for a 24-h ophthalmoscopically visible endpoint lie consistently below the ex vivo data; however, it should be noted that the ocular transmission was set to one when calculating the retinal radiant exposure. The trend of the in vivo data follows that of the ex vivo data. This indicates that microcavitation cannot be excluded as a threshold injury mechanism for single-pulse $100\text{-}\mu \mathrm{s}$-duration exposures.

Fig. 6

The ${\mathrm{ED}}_{50}$ for laser-induced retinal injury (retinal radiant exposure) as a function of exposure duration. Open squares and closed triangles are ex vivo ${\mathrm{ED}}_{50}$ data. Microcavitation (bubble formation) correlates with cell death for exposure duration $<50\mu \mathrm{s}$ but occurs at higher radiant exposure for durations $>\sim 200\mu \mathrm{s}$. In vivo ${\mathrm{ED}}_{50}$ data are for extended source exposures. Twenty-four-hour ${\mathrm{ED}}_{50}$ data follow a trend similar to the ex vivo data. The line is the ${\mathrm{ED}}_{50}$ predicted by thermal models of laser-induced retinal pigmented epithelium injury.

4.2.

Maximum Permissible Exposure for Repetitive-Pulse Exposures

In Fig. 7, the maximum permissible exposure (MPE) levels for repetitive-pulse exposures for $100\text{-}\mu \mathrm{s}$ pulses defined in the current ANSI Z136.1-2014 (Ref. 11) and the old ANSI Z136.1-2007 (Ref. 29) are compared to the experimental data obtained in this study. For this comparison, the MPE, given in the guidelines as the corneal irradiance ($\mathrm{J}/{\mathrm{cm}}^2$), was multiplied by the area of a 7-mm pupil to give the allowable TIE. ${\mathrm{C}}_P$ is a multiplicative correction factor applied to the single-pulse MPE to determine the exposure limit for an exposure to $N$ pulses. In the ANSI Z136.1-2007, the correction factor was set as ${\mathrm{C}}_P=N^{-1/4}$ for all repetitive-pulse exposures.²⁹ As can be seen in Fig. 7(a), for collimated beam exposures to $100\text{-}\mu \mathrm{s}$ pulses, this resulted in a safety factor of $>10$ for a single-pulse exposure, which increased as the number of pulses $N$ increased. In the current ANSI Z136.1-2014, the correction factor has been set to ${\mathrm{C}}_P=1$ for all collimated beam exposures.¹¹ The data for this experiment indicate that this continues to afford an adequate safety factor for large $N$.

Fig. 7

Comparison of maximum permissible exposure (MPE) limits for repetitive-pulse exposures to $100\text{-}\mu \mathrm{s}$-duration pulses at $\lambda =532\mathrm{nm}$ with the data of this study. The solid curve is the MPE published in the 2014 edition of the ANSI Z136.1. The dashed curve is the MPE from the 2007 edition of the ANSI Z136.1. (a) Collimated beam (minimal retinal spot size). (b) $100\mu \mathrm{m}$ retinal beam spot diameter, corresponding to a source angular subtense $\alpha =5.9\mathrm{mrad}$ in the human eye.

In the human eye (effective focal length $f_e=17\mathrm{mm}$), a $100\text{-}\mu \mathrm{m}$-diameter retinal beam spot size is produced when the divergence of the incident beam is 5.9 mrad. For a single $100\text{-}\mu \mathrm{s}$-duration exposure, the MPE promulgated in the ANSI Z136.1-2014 is slightly higher than the MPE published in the 2007 version.^11,29 As seen in Fig. 7(b), both versions provide an adequate safety factor of $\sim 5$ for a single $100\text{-}\mu \mathrm{s}$-duration exposure. But setting ${\mathrm{C}}_P=1$ as is done for collimated beam exposures in the 2014 standard would result in a safety factor of only 2 for $N=1000$ pulses. For a beam having a divergence of 5.9 mrad, the ANSI Z136.1-2014 has defined ${\mathrm{C}}_P=N^{-1/4}$ for up to $N=625$ pulses, and ${\mathrm{C}}_P=0.4$ for $N>625$ pulses. This results in a safety factor that continues to be adequate for exposures to a large number of pulses.