Abstract

The data situation of laser-induced damage measurements after multiple-pulse irradiation in the ns-time regime is limited. Since the laser safety standard is based on damage experiments, it is crucial to determine damage thresholds. For a better understanding of the underlying damage mechanism after repetitive irradiation, we generate damage thresholds for pulse sequences up to N = 20 000 with 1.8 ns-pulses using a square-core fiber and a pulsed Nd:YAG laser. Porcine retinal pigment epithelial layers were used as tissue samples, irradiated with six pulse sequences and evaluated for damage by fluorescence microscopy. The damage thresholds decreased from 31.16 µJ for N = 1 to 11.56 µJ for N = 20 000. The reduction indicates photo-chemical damage mechanisms after reaching a critical energy dose.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

1. Introduction

Lasers are classified for safety reasons based on their potential to cause injury to the human eye and skin. For this purpose, the accessible emission limits (AELs) per laser safety class are defined for different pulse durations, wavelengths and specified by application-related correction factors [1]. These AELs are correlated to the maximum permissible exposures (MPEs), which indicate the human exposure limits to prevent injury to the human eye and skin.

For the determination of the AELs for multiple irradiation of pulsed exposures, a correction factor was introduced in the laser safety standard IEC 60825-1, which was empirically derived from damage experiments on non-human primates (NHPs) and on explants [2]. The damage experiments showed that the energy of a single pulse of a pulse train to induce damage, decreases due to the underlying mechanism of cell damage. In the thermal damage regime (sub-microsecond range until a few seconds), a pulse additivity could be observed [3] and showed a good agreement with the empiric derived correction factor for multiple-pulses. This kind of damage mechanism could be explained and modeled using the Arrhenius integral based on the thermo-kinetic relationship [4,5]. Since a damage-causing energy decrease was also observed in the thermo-mechanical regime [69] (nanoseconds until the sub-microsecond range), this correction factor is also used for these pulse durations, although the damage processes regarding the additive behavior in the thermo-mechanical regime [8,9] are not yet fully understood. Since the data set for short pulse laser irradiation is incomplete, further damage thresholds are generated and discussed for repeated irradiation and briefly possible explanations of additive behavior are presented in this paper. The aim of this work is to generate data of high pulse numbers that can later be used to describe the trend of damage thresholds and deduce reduction trends based on the underlying cell death mechanism.

2. Interactions of multiple-pulse irradiation in the thermo-mechanical damage regime

Thermo-mechanical damage is caused by the appearance of small microbubbles on the melanosome surface of retinal pigment epithelium (RPE) cells, which inevitably lead to cell death [10,11]. The question as to how the microbubbles induce cell death has not yet been fully clarified. It has been demonstrated that the cell membrane was destroyed but the damage procedure remains still unclear, although this mechanism is absolutely crucial for understanding the intercellular processes of repetitive sub-threshold irradiation (compared to single pulse thresholds). For a better understanding of the occurring effects, the process or combination of processes causing the lethal response of the cell need to be recognized. For this reason, possible interactions [12] were identified that can be induced by repeated sub-threshold irradiation. These interactions relate to cumulative processes and the description of the occurrence of a statistically independent event and are explained in more detail in the following sections.

2.1 Accumulation effects

This hypothesis is based on the assumption that the tissue is sustainably sensitized by irradiation. The irradiation thus affects the tissue in terms of an accumulation, since the influence is increased by the frequency of occurrence (number of pulses of a pulse sequence). In this section, accumulations are presented, which can be (a) reversible as well as (b) non-reversible. With (a) reversible accumulation, it is possible for the tissue to return to its previous condition between the pulses. An example could be background heating in the tissue at high repetition rates. An (b) irreversible accumulation effect, would indicate a permanent change in environmental conditions. This would mean that the tissue would progressively change and the pulse pause would not contribute to regeneration. Studies by Qiang et al. [13] have shown this fatigue in other cell types.

The main difference between the two theses is the assumption of an ability to "reset" the cell conditions in the reversible case. Assuming a non-reversible effect, it can be concluded that even time pauses between the pulses are not able to restore the cell to its initial position.

2.2 Probability summation model

The probability summation model (PSM) is a statistical method for describing the occurrence of an event by increasing probability. Here, the PSM serves as a methodical approach for the prediction of a damage after multiple laser irradiation with low-dose energy. This model is based on the probit analysis which was introduced by Finney [14] for dose-response curves in other fields of application back in the 1970s, and later on, the PSM was used by Menendez [15] for laser irradiation damage prediction. This approach is partly still used today to predict damage and compared with other established methods such as the correction factor from the laser safety standard [16,17] .

The PSM is based on the assumption that the response to any exposure of a pulse train can be considered as an independent event to previous pulses. All previous exposures of a pulse sequence have no effect on the retina in kind of a sensitization or a desensitization. Assuming that the probability $P$ of a retinal response to each single pulse $p_{\mathrm {Single}}$ of a pulse train is identical, the probability $P(N)$ of inducing a retinal response after $N$ pulses can be calculated according Eq. (1) [15,16]:

$$P(N)=1-(1-p_{\mathrm{Single}})^{N}$$
Consequently, the exposure that represents a 50% probability of injury ($\mathrm {ED_{50}}$) concludes a value of $P(N)$ = 0.5. The answer probability for each pulse can then be determined by solving Eq. (1) for $p_{\mathrm {Single}}$:
$$p_{\mathrm{Single}}=1-(0.5)^{1/N}$$
In order to determine $p_{\mathrm {Single}}$, the ProbitFit developed by Lund [16] was used. This analysis yields a dose-response curve (based on the assumption of a log-normal distribution) which is characterized by the $\mathrm {ED_{50}}$ as inflection point. The resulting curve indicates the probability of occurrence of an event (damage) with increasing energy doses. This means that the individual probability sought from Eq. (2) in the ProbitFit indicates an energy dose at which a damage occurs in the respective repetitive irradiation.

For a prediction of the $\mathrm {ED_{50}}$ for $N$ = 10, the single pulse $\mathrm {ED_{50}}$ data from previous reports [12] is used: For the calculation of the $\mathrm {ED_{50}}$ according to Eq. (1), Eq. (2) needs to be solved for 10 pulses which results in a $p_{\mathrm {Single}}$ = 0.067. In Fig. 1, this probability point is marked in green colour and thus indicates which dose is necessary after a probabilistic summation to produce damage at a tenfold irradiation.

 

Fig. 1. Dose-response curve for the single exposure evaluation. The probability $p_{\mathrm {Single}}$ of 0.067 is marked schematically in green and indicates the probabilistic summation to produce damage after 10 pulses. The PSM predicts an according pulse energy of 30.63 µJ to be necessary to provoke damage at a tenfold irradiation.

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3. Methods and materials

3.1 Use of porcine explants

The experiments were carried out on explants, as the dosimetry control of diameter, beam profile and real interaction energy during irradiation is easier compared to NHP. The ex vivo samples were obtained from fresh porcine eyes purchased from a local slaughterhouse. During transport, the unopened eyeballs were stored in a dark and insulated cool box. For sample preparation (see Fig. 2) the fresh eyes were then rinsed with Hanks balanced saline solution (HBSS). The surrounding tissue was removed and the bulbus was opened with a needle (see Fig. 2(a)). The bulbus was cut concentrically at the equator (see Fig. 2(b)). The anterior part of the eye and the vitreous body were removed. The sensory retina was removed by gentle withdrawal or rinsing with HBSS to expose the underlying RPE in a trefoil shape (see Fig. 2(c)). The sclera and the underlying choroid were not removed. The black pigmented parts of the ocular fundus were cut into rectangular pieces and clamped in holding devices (see Fig. 2(d)) and inserted into the HBSS and into a nutrient medium.

 

Fig. 2. Sample preparation of porcine eyes: (a)  Opening (b) Cut (c) Trefoil shape (d) Holding constructions to flatten the sample.

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After irradiation, RPE cell vitality was checked using the fluorescent dyes Calcein-Acetoxy-methylester (Calcein-AM) (stock 1 µg/µl) and Propidium iodide (PI) (stock 1 mg/ml). HBSS was mixed (a) with Calcein-AM (1:200 to 5 µM) and independently (b) with PI (1:100 to 15 µM) and incubated for 20 minutes. Calcein-AM is transformed to Calcein in living cells by esterases which can be excited by blue light (excitation maximum at 494 nm, emission at 517 nm) and appears bright under the microscope. Non-vital cells appear dark. PI is used as a dye which penetrates only damaged cellular membranes. Intercalation complexes are formed by PI with double-stranded deoxyribonucleic acid (DNA), which causes an amplification of fluorescence (excitation maximum at 488 nm, emission at 590 nm - 617 nm). Thus, damaged cells appear bright, and vital cells dark [18].

3.2 Procedure

In Fig. 3 the setup for the determination of the laser-induced damage thresholds is shown schematically. A Q-Switched frequency-doubled Nd:YAG laser (Crylas, FDSS 532-1000, Germany) produced temporal Gaussian pulses with a full width at half maximum (FWHM) of 1.8 ns at a wavelength of 532 nm. The spatial mode was $\mathrm {TEM_{00}}$. Furthermore, the long term pulse energy stability (regarding 6 h) was less than $\pm \,5\%$ and the pulse-to-pulse-stability was less than 5% root mean square (rms). Since the maximum exposure time in our experiments for a spot remained 800 s, we have neglected the deviation (less than 3% in our application) for the calculation of systematic uncertainties.

 

Fig. 3. Optical setup for laser-induced measurements with top hat profile. The pulsed Nd:YAG is coupled into a multimode fiber to excite mode mixing. The squared beam profile at the distal tip of the fiber is imaged onto the samples. By using a beam splitter, the applied energy can be measured on the samples during irradiation. A camera is used to secure the top hat on the sample by recognition of the squared shape of the image. (a) Schematic illustration of the setup (b) Spatial beam profile at the sample position.

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The beam was coupled into a square-core-fiber (Thorlabs, FP150QMT, Germany) with a numerical aperture (NA) of 0.39 and a length of 20 m to excite mode mixing for the generation of a top hat beam profile at the distal fiber tip. The square-core-shape has the advantage that the imaging of a square shaped beam profile (see Fig. 3(b)) projected onto the plane of the sample position can be controlled with high precision with the attached camera (IDS, UI-1540SE-M-GL, 1280 x 1024 pixel, Germany). The camera as well as the sample position were readjusted by means of an adjustment laser (Thorlabs, CPS532, Germany). In the imaging position, the top hat beam profile was thus ensured at all times by the squared shape recognition. The image position of the adjustment laser had a deviation $\leq$ 2 % and was neglected at this point. Subsequently, the fiber tip was imaged by an asphere (Thorlabs, ACL3026U-A, Germany) and an objective (Thorlabs, LMH-5X-532, Germany) onto the plane of the sample position. A beam splitter (Thorlabs, BS013, Germany) was used to deflect portions of the beam into an energy meter (Coherent, LabMax-TOP, Model No. 1104622, USA) to measure pulse energy and to count the number of pulses. Before and after the experiments the pulse length was measured by a high-speed free-space detector (Thorlabs, DET025AL/M, Germany) at the position of the sample. The detector was coupled to an oscilloscope (Teledyne LeCroy, waverunner 6100A, 1 GHz, 10 GS/s). Prior to the experiments, a BeamViewer (Coherent, LaserCam-HR II, USA) was used to examine the spatial beam profile (see Fig. 3(b)).

The setup was used to apply a square beam profile with an edge length of 319 µm $\pm \,3\,$µm to the samples. In the experiment, the samples were irradiated with pulse trains of N = 1, 10, 100, 1 000, 10 000 and 20 000 to determine the damage threshold in terms of the $\mathrm {ED_{50}}$. The pulse repetition frequency (PRF) was set to 25 Hz for each experiment to prevent background heating [19]. The room temperature was set to 21$^{\circ }$C. In a previous study [12], the samples were immersed and moistened in a medium, but not completely, so that the irradiated area was also in contact with air. To minimise oxidative stress on the cells, we fully immersed the samples this time.

In order to take into account the biological variability between and within individuals, we have indicated the number of eyes studied in Table 1. In addition, each series of experiments (related to the pulse sequence) was carried out on several dates to take environmental influences into consideration.

Tables Icon

Table 1. $\mathrm {ED_{50}}$ measured for exposure to a 319 µm squared top hat of 1.8-ns-duration pulses at $\mathrm {\lambda }$ = 532 nm. 95% confidence intervals on the $\mathrm {ED_{50}}$ are given in parentheses. A $"-"$ indicates data insufficient to obtain confidence intervals. The slope is defined by the ratio of $\mathrm {ED_{84}}$ to $\mathrm {ED_{50}}$. Intensity modulation factor was 1.15 $\pm$ 0.1.

4. Results

The thresholds obtained for the explants are listed in Table 1 for the 319 µm squared top hat exposures. The $\mathrm {ED_{50}}$ is expressed as the energy per pulse. Table 1 includes 95% confidence intervals on the $\mathrm {ED_{50}}$, the slope $b$ of the ProbitFit, the number of total exposures as well as the exposures corrected by the intensity modulation factor (IMF) [20]. The slope of the ProbitFit and the standard deviation $\mathrm {\sigma }$ of the log-normal dose-response probability distribution are related through $b$ = $\frac {1}{\mathrm {\sigma }}$ [14,16].

The results from Fig. 4 show that the damage-induced energy of a single pulse of a pulse sequence decreases with increasing number of pulses. The slope defines the ratio between $\mathrm {ED_{84}}$ to $\mathrm {ED_{50}}$ and is generally used as a quality feature, thus a step function is ideal for describing the transition from "no damage" to "damage" in dependence of pulse energy [2]. The slope $b$ in this study was never higher than 1.14 indicating an obvious transition for damage definition.

 

Fig. 4. Damage thresholds of multiple pulse irradiation with spot edge length d = 319 µm and pulse duration of $\mathrm {\tau }$ = 1.8 ns with PRF = 25 Hz. Error bars indicate 95% confidence limits. A decrease of the individual pulse energy of a pulse sequence can be observed with increasing number of pulses.

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At pulse sequences between N = 10, N = 100, and N = 20 000 the overlap between "damage" and "no damage" was too low, which meant that no confidence limits could be defined. In addition, the calculated exposures (incident energy of the top hat beam profile on the sample) were corrected by the IMF, assuming that the peaks in this profile caused damage to the sample. The IMF was calculated by evaluating the beam profile in advance. For this, the profil was investigated for 30 transversal cross-sections of several recordings concerning peak occurrence. The IMF is defined by the ratio of maximum occurred peaks to the averaged measured level using a top hat fit. The detailed evaluation of the IMF can be found in a previous report [20]. In order to quantify the uncertainties precisely, we have shown the 95% confidence intervals ($1.96\mathrm {\sigma }-$interval) in brackets in Table 1. The exposure columns, which can be calculated by $\mathrm {ED_{50}}$, diameter and IMF correction, were examined in more detail by means of error propagation to indicate the uncertainty in the $1\mathrm {\sigma }-$interval. The uncertainty of the $\mathrm {ED_{50}}$ can be indicated by the above mentioned relation to the slope. The diameter was determined with a measurement uncertainty of 319 $\pm$ 3 microns. The IMF was determined with a value of 1.15 $\pm$ 0.1. These initial parameters were used to calculate the exposure in the last two columns of Table 1.

As expected, the damage threshold of the pulse sequences for the individual pulse decreases indicating an interaction between the pulses (see Sec. 2). Above all, it is very noticeable that especially in the range of higher pulse numbers the decrease is particularly strong.

5. Discussion

5.1 Application of the probability summation model

The PSM is an established method to predict the probability of laser-induced damages based on an increasing pulse number. Therefore, we compared the measured $\mathrm {ED_{50}}$s with the PSM (cf. Figure 5). Based on the slope of a measurement series, it can be deduced how the $\mathrm {ED_{50}}$s changes and decreases for multiple pulses. This model can thus be generated not only from the single pulse value (N = 1) but also from higher pulse numbers and predict how the damage threshold $\mathrm {ED_{50}}$ will decrease. [17]

 

Fig. 5. Damage thresholds of multiple pulse irradiation with spot edge length d = 319 µm and pulse duration of $\mathrm {\tau }$ = 1.8 ns with PRF = 25 Hz. Error bars indicate 95% confidence limits. A decrease of the individual pulse energy of a pulse sequence can be observed with increasing number of pulses. PSM models (dashed) of the individual multiple-pulse thresholds were generated, but are independent of the starting point and in any case able to describe sufficiently restrictive the reduction.

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It can be concluded from Fig. 5 that the models do not appear adequate for describing the respective damage $\mathrm {ED_{50}}$s. The predictions of the PSM underestimate in any case the risk of damage. This result indicates that the probability summation cannot be used for low pulse numbers (N < 1 000) due to underestimation of the hazard. The applicability and thus qualification of the PSM for higher number of pulses (N > 1 000) can be examined by further investigations in the higher pulse number range. Since the course of the PSM is based on the slope of the underlying measurement, this is also the reason for the deviation from the real measured values: Although a low slope indicates a clear transition between the binary response (damage or no damage), it also influences the trend of probability summation predictions. Applying the PSM to our data leads to the conclusion that this description of multiple-pulse data is not appropriate.

Another issue about the PSM is that for unknown input parameters such as pulse duration and damage range, no prediction can be made for individual pulses, since PSM is based on the injury thresholds of individual pulse exposure. This assumption is also one of the major weaknesses of the model approach, since the course and prediction are based on the quality and biological variability of a single pulse exposure and its slope [17,21].

5.2 Investigation of accumulation effects

From the previous section we concluded that the statistical PSM is unsuitable for describing our data. Therefore, in this section we deal with accumulating effects from Sec. 2. Assuming a stimulating effect, this would suggest that the tissue is sensitized after each pulse.

The use of the explants had the advantage that the fluorescence images (see Fig. 6) provide indirect information on the metabolic activity of the irradiated cells: By using the fluorescent dye Calcein-AM (see Fig. 6(a)), the increased metabolic activity in the irradiated cells was detected. The cells were particularly stimulated shortly before the damage (especially bright spots). This behaviour indicates biochemical processes that support the theory of resulting free radicals with cytotoxic effects. Below the injury threshold the irradiated cells light up brightly upon stimulation, suggesting that these cells have converted more Calcein-AM into Calcein, which can be excited after conversion.

 

Fig. 6. Fluorescence microscope images (a) Activation and excitation of the converted calcein after sub-threshold irradiation: (1) Regular excited, vital hexagonal RPE structures can be recognized. (2) Destroyed cell membranes by intensity modulation peaks in the beam profile. (3) Cells with a higher brightness level, which indicates an increased metabolic activity. (b) Fluorescence microscope image of exposures with an edge length of 319 µm and a pulse duration of 1.8 ns. Hexagonal structure of single RPE cells are visible. Green bright cells represent living cells. Red bright cell nuclei are excited by the PI through destroyed cell membrane.

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In Fig. 6(b), we show an exemplary photograph on the samples with the irradiated square area. Bright green cells indicate vitality due to calcein. The red dots on the other hand are assigned to cell nuclei that could only be excited by PI after the cell membrane had been destroyed.

Another investigation concerns the temperature increase in the tissue through multiple-pulse irradiation. This background heating can hypothetically lead to thermal damage or promote photo-chemical processes within the cell. In the following sections we will discuss the evidence for photo-chemical processes. At this point, however, it cannot be excluded that a combination of interactions can also occur.

Apart from these considerations regarding thermal or photo-chemical damage, photo-mechanical aspects still have to be considered. The stress fatigue of the cells can also contribute to the lethal response after irradiation. The most recent work by Qiang et al. [13], describes the stress and failure tendencies of red blood cells. These results clearly demonstrate the important role of mechanical fatigue in influencing the physical properties of biological cells. They provide further insights into accumulated membrane damage, which cannot be excluded in our experiments.

5.3 Indication of a photo-chemical damage

The strong reduction of the damage thresholds (between N = 1 000 and N = 10 000) indicates a further damage mechanism in the retinal tissue. Figure 7 shows that from the high number of pulses (longer operating beam durations) a dose is reached which leads to damage. This damage can be described by photo-chemical effects, since critical limit doses are characteristic for this type of damage [22,23]. Photo-chemical effects typically occur at longer exposure times and shorter wavelengths of the visible spectrum [24,25]. Nevertheless, it is known that the subcellular reactions as a consequence of the photo-chemical effect also occur during shorter exposures, but are not the dominant damage mechanism [26].

 

Fig. 7. Photo-chemical damage occurs from a dose of about $200\,\mathrm {J/cm^{2}}$ (green line) or lower (considering the decreased slope from N = 1 000). Previous doses that caused the damage based on thermo-mechanical mechanism and have not been of photo-chemical origin. The further slight increase in the effective dose for damage may be due to repair mechanisms.

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The results from Fig. 4 indicate that at the lower pulse numbers in our studies, the photo-chemical effect is probably not decisive for the damage. Thus, for the shorter emission durations the thermo-mechanical damage dominates. This assumption can also be observed in a transition region (between N = 1 000 and N = 10 000), where the damage thresholds drop more strongly than before. This transition could be interpreted as a photo-chemical effect with regard to the higher dose. According to the laser safety standard, this total exposure time (400 s) and longer, might lead to both thermal and photo-chemical damage. In the case of single pulse exposures or low pulse numbers, the thermo-mechanical or thermal damage threshold is expected to be significantly lower than photo-chemical damage. Higher pulse numbers therefore require less single pulse energy as they have to apply the same dose (neglecting internal cell repair mechanisms [27]) to trigger oxidative stress (photo-chemical damage [25]).

The studies of Ham et al. [24] have shown that photo-chemical damage was observed: The damage experiments were performed in vivo NHP. A wavelength of 488 nm was applied with cw-radiation. The results were evaluated after 48 h. Ham et al. concluded that for exposure times longer than 1 000 s (488 nm), the damage seems to be photo-chemical.

The observation of photo-chemical effects (see Fig. 8) in terms of RPE disruption has already been observed in vivo in NHP for different wavelengths: In a study of Zhang et al. [28] in Rochester, the photo-chemical effects were demonstrated as primary damage. They determined the $\mathrm {ED_{50}}$ to be 82 µ$\mathrm {J/cm^{2}}$, the lethal dose $\mathrm {ED_{100}}$ was 140 µ$\mathrm {J/cm^{2}}$ for the cw-exposure using 594 nm. Despite their use of a cw-laser and a different spot size on the samples (which should be negligible under the assumption of photo-chemical effects), the data show a similar behaviour. Their threshold data fit well to the data of this work. An even greater difference would also have been expected since the samples in this study were irradiated at controlled room temperature. However, it is also possible that the photo-chemical processes are only triggered once the body temperature of the NHP is exceeded.

 

Fig. 8. Comparison of integrated damage thresholds with other studies on photo-chemical effects (a) For different emission durations. Experimentally determined damage thresholds of this study (squares) with data from Ham et al. [24] (circles) and NHP experiments from Zhang et al. [28] (triangles). The studies of Ham et al. [24] were examined for different wavelengths of different cw-irradiation durations. Saturation can be identified for the shorter wavelength (b) Wavelength dependence: The data of this work (squares) fit well with the study of Zhang et al. [28] (triangles), for different wavelengths. For longer pulse durations, the dose necessary to cause damage is only slightly higher (probably due to repair mechanisms).

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Figure 8 shows the comparison of the measured damaging exposure (for the entire pulse train) of this work with the two published comparable studies [24,28]: In each publication a kind of a "saturation" can be noticed, starting from a certain emission duration. The data from Ham et al. indicate this transition range only for the shorter wavelengths from emission durations of $<$ 10 s. In the experiment of this study, the "transition" can be observed from about 400 s (corresponding to N $=$ 10 000). Longer emission durations required more energy to cause damage to the samples, but this increase was low. The slight increase can possibly be explained by cell-internal repair processes. The work of Ham et al. shows a similar course of damaging exposure. In his study of different wavelengths, he examined the necessary damaging dose for several irradiation times. The curves from Ham et al. for wavelengths of 488 nm and 514.5 nm show a similar bending behaviour as observed in this study. The longer wavelengths, on the other hand, do not indicate this "saturation" trend. [24]

The Zhang et al. [28] and Ham et al. [24] studies show a similar wavelength dependence for the necessary damaging exposure: A time frame was investigated for wavelengths from 476 nm to 594 nm indicating a wavelength dependence to the damage threshold similar to the values in the literature. Furthermore, Zhang underlines an underestimated photo-chemical RPE-disruption for longer wavelengths.

At this point, it cannot be definitively concluded that photo-chemical damage occurs for the longer exposures. However, there is a lot of evidence for this, besides the good agreement with the data of Zhang et al. [28] and Ham et al. [24].

6. Outlook

The strongly decreasing damage thresholds at the high number of pulses suggest that either an accumulating effect occurs or photo-chemical processes are induced. In order to identify the dominant damage mechanism, the experiments could be repeated with a higher PRF (approx. 1 kHz). Since the time intervals are significantly shorter than in our experiments, it can be evaluated to what extent these "pauses" have an influence on the damage threshold.

For further evaluation of photo-chemical processes, several approaches can be applied: Backscattering [29] or interferometric [30] measurements can be used to determine whether microbubbles have occurred at all in the pulse sequence. Furthermore, photo-chemical processes are very temperature-dependent. A study of the damage thresholds at higher temperatures (e.g. body temperature) seem to be useful.

Prospective studies should investigate long-term damage. RPE cell cultures (primary cells) could be used to approach the model of an in vivo organism. Intercellular processes can be observed and understood over a longer period of time. The underlying damage mechanism could thus possibly be better understood.

7. Conclusion

In this work, the laser damage thresholds of explants were determined for multiple pulses up to N = 20 000 in the ns-time damage range using a Q-switched, frequency-doubled Nd:YAG laser (532 nm wavelength, 1.8 ns pulse, 25 Hz, 319 µm edge length, squared top hat, porcine RPE). We observed how the damaging individual pulse energy of a pulse train decreases with increasing pulse train, contrary to the predictions of probability summation model. The degree of reduction is especially strong at pulse numbers above 1 000, which indicates further damage mechanisms such as photo-chemical effects which seem to dominate above an energy dose of almost 200 $\mathrm {J/cm^{2}}$.

Acknowledgments

The authors would like to thank Sven Schnichels, Heidi Mühl and Agnes Fietz of the Eye Clinic in Tübingen, Germany for supporting sample preparation. Furthermore, we would like to thank Brian Lund for providing the ProbitFit software.

Disclosures

The authors declare that there are no conflicts of interest related to this article.

References

1. IEC 60825-1, Safety of Laser Products - Part 1: Equipment Classification and Requirements (International Electrotechnical Commission, Geneva, 2014), 3rd ed.

2. D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002). [CrossRef]  

3. K. Schulmeister and M. Jean, “Manifestation of the strong non-linearity of thermal injury,” in International Laser Safety Conference, vol. 2011 (LIA, 2011), pp. 201–204.

4. R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).

5. S. L. Jacques, “Ratio of entropy to enthalpy in thermal transitions in biological tissues,” J. Biomed. Opt. 11(4), 041108 (2006). [CrossRef]  

6. R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000). [CrossRef]  

7. G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005). [CrossRef]  

8. B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014). [CrossRef]  

9. B. J. Lund, D. J. Lund, and P. R. Edsall, “Damage threshold from large retinal spot size repetetive-pulse laser exposures,” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 84–87.

10. B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996). [CrossRef]  

11. J. Neumann and R. Brinkmann, “Boiling nucleation on melanosomes and microbeads transiently heated by nanosecond and microsecond laser pulses,” J. Biomed. Opt. 10(2), 024001 (2005). [CrossRef]  

12. S. Ramos, W. Stork, and N. Heussner, “Multiple-pulse damage thresholds on the retinal pigment epithelium layer using top hat profiles,” in Optical Interactions with Tissue and Cells XXXI, vol. 11238 (International Society for Optics and Photonics, 2020), p. 112380D.

13. Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019). [CrossRef]  

14. D. J. Finney, Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve (Cambridge University Press, 1952).

15. A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993). [CrossRef]  

16. B. J. Lund, “The probitfit program to analyze data from laser damage threshold studies,” Tech. rep., Northrop Grumman Corp., San Antonio, TX, infomation technology (2006).

17. C. D. Clark and G. D. Buffington, “On the probability summation model for laser-damage thresholds,” J. Biomed. Opt. 21(1), 015006 (2016). [CrossRef]  

18. W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995). [CrossRef]  

19. M. A. Mainster, “Decreasing retinal photocoagulation damage: principles and techniques,” in Seminars in Ophthalmology, vol. 14 (Taylor & Francis, 1999), pp. 200–209.

20. S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.

21. D. H. Sliney and D. J. Lund, “Do we over-state the risk of multiple pulsed exposures?” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 93–98.

22. D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

23. International Commission on Non-Ionizing Radiation Protection, ICNIRP guidelines on limits of exposure to laser radiation of wavelengths between 180 nm and 1,000 µm,” Heal. Phys. 105(3), 271–295 (2013).

24. W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979). [CrossRef]  

25. M. B. Rozanowska, “Light-induced damage to the retina: current understanding of the mechanisms and unresolved questions: a symposium-in-print,” Photochem. Photobiol. 88(6), 1303–1308 (2012). [CrossRef]  

26. D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

27. G. Griess and M. Blankenstein, “Additivity and repair of actinic retinal lesions,” Investig. Ophthalmol. & Vis. Sci. 20, 803–807 (1981).

28. J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

29. J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004). [CrossRef]  

30. J. Neumann, “Mikroskopische Untersuchungen zur laserinduzierten Blasenbildung und - dynamik an absorbierenden Mikropartikeln,” Ph.D. thesis (2005).

References

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  • |

  1. IEC 60825-1, Safety of Laser Products - Part 1: Equipment Classification and Requirements (International Electrotechnical Commission, Geneva, 2014), 3rd ed.
  2. D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
    [Crossref]
  3. K. Schulmeister and M. Jean, “Manifestation of the strong non-linearity of thermal injury,” in International Laser Safety Conference, vol. 2011 (LIA, 2011), pp. 201–204.
  4. R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).
  5. S. L. Jacques, “Ratio of entropy to enthalpy in thermal transitions in biological tissues,” J. Biomed. Opt. 11(4), 041108 (2006).
    [Crossref]
  6. R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
    [Crossref]
  7. G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
    [Crossref]
  8. B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
    [Crossref]
  9. B. J. Lund, D. J. Lund, and P. R. Edsall, “Damage threshold from large retinal spot size repetetive-pulse laser exposures,” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 84–87.
  10. B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
    [Crossref]
  11. J. Neumann and R. Brinkmann, “Boiling nucleation on melanosomes and microbeads transiently heated by nanosecond and microsecond laser pulses,” J. Biomed. Opt. 10(2), 024001 (2005).
    [Crossref]
  12. S. Ramos, W. Stork, and N. Heussner, “Multiple-pulse damage thresholds on the retinal pigment epithelium layer using top hat profiles,” in Optical Interactions with Tissue and Cells XXXI, vol. 11238 (International Society for Optics and Photonics, 2020), p. 112380D.
  13. Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
    [Crossref]
  14. D. J. Finney, Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve (Cambridge University Press, 1952).
  15. A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
    [Crossref]
  16. B. J. Lund, “The probitfit program to analyze data from laser damage threshold studies,” Tech. rep., Northrop Grumman Corp., San Antonio, TX, infomation technology (2006).
  17. C. D. Clark and G. D. Buffington, “On the probability summation model for laser-damage thresholds,” J. Biomed. Opt. 21(1), 015006 (2016).
    [Crossref]
  18. W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
    [Crossref]
  19. M. A. Mainster, “Decreasing retinal photocoagulation damage: principles and techniques,” in Seminars in Ophthalmology, vol. 14 (Taylor & Francis, 1999), pp. 200–209.
  20. S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.
  21. D. H. Sliney and D. J. Lund, “Do we over-state the risk of multiple pulsed exposures?” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 93–98.
  22. D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.
  23. International Commission on Non-Ionizing Radiation Protection, ICNIRP guidelines on limits of exposure to laser radiation of wavelengths between 180 nm and 1,000 µm,” Heal. Phys. 105(3), 271–295 (2013).
  24. W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
    [Crossref]
  25. M. B. Rozanowska, “Light-induced damage to the retina: current understanding of the mechanisms and unresolved questions: a symposium-in-print,” Photochem. Photobiol. 88(6), 1303–1308 (2012).
    [Crossref]
  26. D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.
  27. G. Griess and M. Blankenstein, “Additivity and repair of actinic retinal lesions,” Investig. Ophthalmol. & Vis. Sci. 20, 803–807 (1981).
  28. J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).
  29. J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004).
    [Crossref]
  30. J. Neumann, “Mikroskopische Untersuchungen zur laserinduzierten Blasenbildung und - dynamik an absorbierenden Mikropartikeln,” Ph.D. thesis (2005).

2019 (1)

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

2016 (2)

C. D. Clark and G. D. Buffington, “On the probability summation model for laser-damage thresholds,” J. Biomed. Opt. 21(1), 015006 (2016).
[Crossref]

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

2014 (1)

B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
[Crossref]

2013 (1)

International Commission on Non-Ionizing Radiation Protection, ICNIRP guidelines on limits of exposure to laser radiation of wavelengths between 180 nm and 1,000 µm,” Heal. Phys. 105(3), 271–295 (2013).

2012 (1)

M. B. Rozanowska, “Light-induced damage to the retina: current understanding of the mechanisms and unresolved questions: a symposium-in-print,” Photochem. Photobiol. 88(6), 1303–1308 (2012).
[Crossref]

2006 (1)

S. L. Jacques, “Ratio of entropy to enthalpy in thermal transitions in biological tissues,” J. Biomed. Opt. 11(4), 041108 (2006).
[Crossref]

2005 (2)

G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
[Crossref]

J. Neumann and R. Brinkmann, “Boiling nucleation on melanosomes and microbeads transiently heated by nanosecond and microsecond laser pulses,” J. Biomed. Opt. 10(2), 024001 (2005).
[Crossref]

2004 (1)

J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004).
[Crossref]

2002 (1)

D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
[Crossref]

2000 (1)

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

1996 (1)

B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
[Crossref]

1995 (1)

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

1993 (1)

A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
[Crossref]

1985 (1)

R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).

1981 (1)

G. Griess and M. Blankenstein, “Additivity and repair of actinic retinal lesions,” Investig. Ophthalmol. & Vis. Sci. 20, 803–807 (1981).

1979 (1)

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[Crossref]

Berger, D. P.

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

Birngruber, R.

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).

Blankenstein, M.

G. Griess and M. Blankenstein, “Additivity and repair of actinic retinal lesions,” Investig. Ophthalmol. & Vis. Sci. 20, 803–807 (1981).

Brinkmann, R.

G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
[Crossref]

J. Neumann and R. Brinkmann, “Boiling nucleation on melanosomes and microbeads transiently heated by nanosecond and microsecond laser pulses,” J. Biomed. Opt. 10(2), 024001 (2005).
[Crossref]

J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004).
[Crossref]

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

Bubel, T.

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

Buffington, G. D.

C. D. Clark and G. D. Buffington, “On the probability summation model for laser-damage thresholds,” J. Biomed. Opt. 21(1), 015006 (2016).
[Crossref]

Clark, C. D.

C. D. Clark and G. D. Buffington, “On the probability summation model for laser-damage thresholds,” J. Biomed. Opt. 21(1), 015006 (2016).
[Crossref]

Clarke, A.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[Crossref]

Dao, M.

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

Dengler, W. A.

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

Du, E.

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

Edsall, P. R.

B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
[Crossref]

B. J. Lund, D. J. Lund, and P. R. Edsall, “Damage threshold from large retinal spot size repetetive-pulse laser exposures,” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 84–87.

Elmlinger, P.

S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.

Fiebig, H. H.

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

Finney, D. J.

D. J. Finney, Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve (Cambridge University Press, 1952).

Gabel, V.

R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).

Gabel, V.-P.

D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
[Crossref]

Gaines, V. D.

B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
[Crossref]

Gerstman, B. S.

B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
[Crossref]

Griess, G.

G. Griess and M. Blankenstein, “Additivity and repair of actinic retinal lesions,” Investig. Ophthalmol. & Vis. Sci. 20, 803–807 (1981).

Ham, W. T.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[Crossref]

Heussner, N.

S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.

S. Ramos, W. Stork, and N. Heussner, “Multiple-pulse damage thresholds on the retinal pigment epithelium layer using top hat profiles,” in Optical Interactions with Tissue and Cells XXXI, vol. 11238 (International Society for Optics and Photonics, 2020), p. 112380D.

Hillenkamp, F.

R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).

Huettmann, G.

G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
[Crossref]

Hunter, J. J.

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

Hüttmann, G.

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

Jacques, S. L.

S. L. Jacques, “Ratio of entropy to enthalpy in thermal transitions in biological tissues,” J. Biomed. Opt. 11(4), 041108 (2006).
[Crossref]

B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
[Crossref]

Jean, M.

K. Schulmeister and M. Jean, “Manifestation of the strong non-linearity of thermal injury,” in International Laser Safety Conference, vol. 2011 (LIA, 2011), pp. 201–204.

Lin, C. P.

J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004).
[Crossref]

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

Liu, J.

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

Lund, B. J.

B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
[Crossref]

B. J. Lund, D. J. Lund, and P. R. Edsall, “Damage threshold from large retinal spot size repetetive-pulse laser exposures,” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 84–87.

B. J. Lund, “The probitfit program to analyze data from laser damage threshold studies,” Tech. rep., Northrop Grumman Corp., San Antonio, TX, infomation technology (2006).

Lund, D. J.

B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
[Crossref]

B. J. Lund, D. J. Lund, and P. R. Edsall, “Damage threshold from large retinal spot size repetetive-pulse laser exposures,” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 84–87.

D. H. Sliney and D. J. Lund, “Do we over-state the risk of multiple pulsed exposures?” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 93–98.

D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

Mainster, M. A.

M. A. Mainster, “Decreasing retinal photocoagulation damage: principles and techniques,” in Seminars in Ophthalmology, vol. 14 (Taylor & Francis, 1999), pp. 200–209.

MCheney, F. E.

A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
[Crossref]

MCrump, P.

A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
[Crossref]

Mellerio, J.

D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
[Crossref]

Menendez, A. R.

A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
[Crossref]

Mertelsmann, R.

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

Mueller, H. A.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[Crossref]

MZuclich, J. A.

A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
[Crossref]

Neumann, J.

J. Neumann and R. Brinkmann, “Boiling nucleation on melanosomes and microbeads transiently heated by nanosecond and microsecond laser pulses,” J. Biomed. Opt. 10(2), 024001 (2005).
[Crossref]

J. Neumann, “Mikroskopische Untersuchungen zur laserinduzierten Blasenbildung und - dynamik an absorbierenden Mikropartikeln,” Ph.D. thesis (2005).

Qiang, Y.

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

Ramos, S.

S. Ramos, W. Stork, and N. Heussner, “Multiple-pulse damage thresholds on the retinal pigment epithelium layer using top hat profiles,” in Optical Interactions with Tissue and Cells XXXI, vol. 11238 (International Society for Optics and Photonics, 2020), p. 112380D.

S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.

Rögener, J.

J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004).
[Crossref]

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

Rogers, M. E.

B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
[Crossref]

Roider, J.

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

Rozanowska, M. B.

M. B. Rozanowska, “Light-induced damage to the retina: current understanding of the mechanisms and unresolved questions: a symposium-in-print,” Photochem. Photobiol. 88(6), 1303–1308 (2012).
[Crossref]

Ruffolo, J. J.

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[Crossref]

Rumohr, M.

G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
[Crossref]

Sabarinathan, R.

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

Schüle, G.

G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
[Crossref]

Schulmeister, K.

D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
[Crossref]

K. Schulmeister and M. Jean, “Manifestation of the strong non-linearity of thermal injury,” in International Laser Safety Conference, vol. 2011 (LIA, 2011), pp. 201–204.

Schulte, J.

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

Sliney, D. H.

D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
[Crossref]

D. H. Sliney and D. J. Lund, “Do we over-state the risk of multiple pulsed exposures?” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 93–98.

Stork, W.

S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.

S. Ramos, W. Stork, and N. Heussner, “Multiple-pulse damage thresholds on the retinal pigment epithelium layer using top hat profiles,” in Optical Interactions with Tissue and Cells XXXI, vol. 11238 (International Society for Optics and Photonics, 2020), p. 112380D.

Stuck, B. E.

D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

Suresh, S.

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

Thompson, C. R.

B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
[Crossref]

Williams, D. R.

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

Zhang, J.

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

Anti-Cancer Drugs (1)

W. A. Dengler, J. Schulte, D. P. Berger, R. Mertelsmann, and H. H. Fiebig, “Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays,” Anti-Cancer Drugs 6(4), 522–532 (1995).
[Crossref]

Heal. Phys. (1)

International Commission on Non-Ionizing Radiation Protection, ICNIRP guidelines on limits of exposure to laser radiation of wavelengths between 180 nm and 1,000 µm,” Heal. Phys. 105(3), 271–295 (2013).

Health Phys. (3)

A. R. Menendez, F. E. MCheney, J. A. MZuclich, and P. MCrump, “Probability-summation model of multiple laser-exposure effects,” Health Phys. 65(5), 523–528 (1993).
[Crossref]

D. H. Sliney, J. Mellerio, V.-P. Gabel, and K. Schulmeister, “What is the meaning of threshold in laser injury experiments? implications for human exposure limits,” Health Phys. 82(3), 335–347 (2002).
[Crossref]

R. Birngruber, F. Hillenkamp, and V. Gabel, “Theoretical investigations of laser thermal retinal injury,” Health Phys. 48(6), 781–796 (1985).

Investig. Ophthalmol. & Vis. Sci. (2)

G. Griess and M. Blankenstein, “Additivity and repair of actinic retinal lesions,” Investig. Ophthalmol. & Vis. Sci. 20, 803–807 (1981).

J. Zhang, R. Sabarinathan, T. Bubel, D. R. Williams, and J. J. Hunter, “Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina,” Investig. Ophthalmol. & Vis. Sci. 57, 2220 (2016).

Investigative ophthalmology & visual science (1)

G. Schüle, M. Rumohr, G. Huettmann, and R. Brinkmann, “RPE damage thresholds and mechanisms for laser exposure in the microsecond-to-millisecond time regimen,” Investigative ophthalmology & visual science 46(2), 714–719 (2005).
[Crossref]

J. Biomed. Opt. (5)

B. J. Lund, D. J. Lund, P. R. Edsall, and V. D. Gaines, “Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-µs pulses,” J. Biomed. Opt. 19(10), 105006 (2014).
[Crossref]

S. L. Jacques, “Ratio of entropy to enthalpy in thermal transitions in biological tissues,” J. Biomed. Opt. 11(4), 041108 (2006).
[Crossref]

J. Neumann and R. Brinkmann, “Boiling nucleation on melanosomes and microbeads transiently heated by nanosecond and microsecond laser pulses,” J. Biomed. Opt. 10(2), 024001 (2005).
[Crossref]

J. Rögener, R. Brinkmann, and C. P. Lin, “Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells,” J. Biomed. Opt. 9(2), 367–372 (2004).
[Crossref]

C. D. Clark and G. D. Buffington, “On the probability summation model for laser-damage thresholds,” J. Biomed. Opt. 21(1), 015006 (2016).
[Crossref]

Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery (2)

R. Brinkmann, G. Hüttmann, J. Rögener, J. Roider, R. Birngruber, and C. P. Lin, “Origin of retinal pigment epithelium cell damage by pulsed laser irradiance in the nanosecond to microsecond time regimen,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 27(5), 451–464 (2000).
[Crossref]

B. S. Gerstman, C. R. Thompson, S. L. Jacques, and M. E. Rogers, “Laser induced bubble formation in the retina,” Lasers in Surgery and Medicine: The Official Journal of the American Society for Laser Medicine and Surgery 18(1), 10–21 (1996).
[Crossref]

Photochem. Photobiol. (2)

W. T. Ham, H. A. Mueller, J. J. Ruffolo, and A. Clarke, “Sensitivity of the retina to radiation damage as a function of wavelength,” Photochem. Photobiol. 29(4), 735–743 (1979).
[Crossref]

M. B. Rozanowska, “Light-induced damage to the retina: current understanding of the mechanisms and unresolved questions: a symposium-in-print,” Photochem. Photobiol. 88(6), 1303–1308 (2012).
[Crossref]

Proc. Natl. Acad. Sci. (1)

Y. Qiang, J. Liu, M. Dao, S. Suresh, and E. Du, “Mechanical fatigue of human red blood cells,” Proc. Natl. Acad. Sci. 116(40), 19828–19834 (2019).
[Crossref]

Other (12)

D. J. Finney, Probit Analysis: A Statistical Treatment of the Sigmoid Response Curve (Cambridge University Press, 1952).

S. Ramos, W. Stork, and N. Heussner, “Multiple-pulse damage thresholds on the retinal pigment epithelium layer using top hat profiles,” in Optical Interactions with Tissue and Cells XXXI, vol. 11238 (International Society for Optics and Photonics, 2020), p. 112380D.

M. A. Mainster, “Decreasing retinal photocoagulation damage: principles and techniques,” in Seminars in Ophthalmology, vol. 14 (Taylor & Francis, 1999), pp. 200–209.

S. Ramos, P. Elmlinger, W. Stork, and N. Heussner, “Influence of the beam profile on laser-induced thresholds using explants,” in Tissue Optics and Photonics, vol. 11363 (International Society for Optics and Photonics, 2020), p. 113631D.

D. H. Sliney and D. J. Lund, “Do we over-state the risk of multiple pulsed exposures?” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 93–98.

D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

IEC 60825-1, Safety of Laser Products - Part 1: Equipment Classification and Requirements (International Electrotechnical Commission, Geneva, 2014), 3rd ed.

B. J. Lund, D. J. Lund, and P. R. Edsall, “Damage threshold from large retinal spot size repetetive-pulse laser exposures,” in International Laser Safety Conference, vol. 2009 (LIA, 2009), pp. 84–87.

K. Schulmeister and M. Jean, “Manifestation of the strong non-linearity of thermal injury,” in International Laser Safety Conference, vol. 2011 (LIA, 2011), pp. 201–204.

D. J. Lund and B. E. Stuck, “Retinal injury thresholds for blue wavelength lasers,” in International Laser Safety Conference, vol. 2003 (Laser Institute of America, 2003), pp. 50–56.

J. Neumann, “Mikroskopische Untersuchungen zur laserinduzierten Blasenbildung und - dynamik an absorbierenden Mikropartikeln,” Ph.D. thesis (2005).

B. J. Lund, “The probitfit program to analyze data from laser damage threshold studies,” Tech. rep., Northrop Grumman Corp., San Antonio, TX, infomation technology (2006).

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Figures (8)

Fig. 1.
Fig. 1. Dose-response curve for the single exposure evaluation. The probability $p_{\mathrm {Single}}$ of 0.067 is marked schematically in green and indicates the probabilistic summation to produce damage after 10 pulses. The PSM predicts an according pulse energy of 30.63 µJ to be necessary to provoke damage at a tenfold irradiation.
Fig. 2.
Fig. 2. Sample preparation of porcine eyes: (a)  Opening (b) Cut (c) Trefoil shape (d) Holding constructions to flatten the sample.
Fig. 3.
Fig. 3. Optical setup for laser-induced measurements with top hat profile. The pulsed Nd:YAG is coupled into a multimode fiber to excite mode mixing. The squared beam profile at the distal tip of the fiber is imaged onto the samples. By using a beam splitter, the applied energy can be measured on the samples during irradiation. A camera is used to secure the top hat on the sample by recognition of the squared shape of the image. (a) Schematic illustration of the setup (b) Spatial beam profile at the sample position.
Fig. 4.
Fig. 4. Damage thresholds of multiple pulse irradiation with spot edge length d = 319 µm and pulse duration of $\mathrm {\tau }$ = 1.8 ns with PRF = 25 Hz. Error bars indicate 95% confidence limits. A decrease of the individual pulse energy of a pulse sequence can be observed with increasing number of pulses.
Fig. 5.
Fig. 5. Damage thresholds of multiple pulse irradiation with spot edge length d = 319 µm and pulse duration of $\mathrm {\tau }$ = 1.8 ns with PRF = 25 Hz. Error bars indicate 95% confidence limits. A decrease of the individual pulse energy of a pulse sequence can be observed with increasing number of pulses. PSM models (dashed) of the individual multiple-pulse thresholds were generated, but are independent of the starting point and in any case able to describe sufficiently restrictive the reduction.
Fig. 6.
Fig. 6. Fluorescence microscope images (a) Activation and excitation of the converted calcein after sub-threshold irradiation: (1) Regular excited, vital hexagonal RPE structures can be recognized. (2) Destroyed cell membranes by intensity modulation peaks in the beam profile. (3) Cells with a higher brightness level, which indicates an increased metabolic activity. (b) Fluorescence microscope image of exposures with an edge length of 319 µm and a pulse duration of 1.8 ns. Hexagonal structure of single RPE cells are visible. Green bright cells represent living cells. Red bright cell nuclei are excited by the PI through destroyed cell membrane.
Fig. 7.
Fig. 7. Photo-chemical damage occurs from a dose of about $200\,\mathrm {J/cm^{2}}$ (green line) or lower (considering the decreased slope from N = 1 000). Previous doses that caused the damage based on thermo-mechanical mechanism and have not been of photo-chemical origin. The further slight increase in the effective dose for damage may be due to repair mechanisms.
Fig. 8.
Fig. 8. Comparison of integrated damage thresholds with other studies on photo-chemical effects (a) For different emission durations. Experimentally determined damage thresholds of this study (squares) with data from Ham et al. [24] (circles) and NHP experiments from Zhang et al. [28] (triangles). The studies of Ham et al. [24] were examined for different wavelengths of different cw-irradiation durations. Saturation can be identified for the shorter wavelength (b) Wavelength dependence: The data of this work (squares) fit well with the study of Zhang et al. [28] (triangles), for different wavelengths. For longer pulse durations, the dose necessary to cause damage is only slightly higher (probably due to repair mechanisms).

Tables (1)

Tables Icon

Table 1. E D 50 measured for exposure to a 319 µm squared top hat of 1.8-ns-duration pulses at λ  = 532 nm. 95% confidence intervals on the E D 50 are given in parentheses. A " " indicates data insufficient to obtain confidence intervals. The slope is defined by the ratio of E D 84 to E D 50 . Intensity modulation factor was 1.15 ± 0.1.

Equations (2)

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P ( N ) = 1 ( 1 p S i n g l e ) N
p S i n g l e = 1 ( 0.5 ) 1 / N

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