Monitoring intracellular cavitation during selective targeting of the retinal pigment epithelium

Abstract

PURPOSESelective targeting of the Retinal Pigment Epithelium (RPE), by either applying trains of microsecond laser pulses or, in our approach, by repetitively scanning a tightly focused spot across the retina, achieves destruction of RPE cells while avoiding damage to the overlying photoreceptors. Both techniques have been demonstrated as attractive methods for the treatment of retinal diseases that are caused by a dysfunction of the RPE. Because the lesions are ophthalmoscopically invisible, an online control system that monitors cell death during irradiation is essential to ensure efficient and selective treatment in a clinical application. MATERIALS AND METHODSBubble formation inside the RPE cells has been shown to be the cell damage mechanism for nano-and picosecond pulses. We built an optical system to investigate whether the same mechanism extends into the microsecond regime. The system detects changes in backscattered light of the irradiating beam during exposure. Samples of young bovine eyes were exposed in vitro using single pulses ranging from 3 µs to 50 µs. Using the viability assay calcein-AM the ED 50 threshold for cell death was determined and compared to the threshold for bubble formation. We also set up a detection system on our slit lamp adapted scanning system in order to determine the feasibility of monitoring threshold RPE damage during selective laser treatment in vivo. RESULTS AND DISCUSSIONIntracellular cavitation was detected as a transient increase in backscattering signal, either of an external probe beam or of the irradiation beam itself. Monitoring with the irradiation beam is both simpler and more sensitive. We found the threshold for bubble formation to coincide with the threshold for cell damage for pulse durations up to 20 µs, suggesting that cavitation is the main mechanism of cell damage. For pulse widths longer than 20 µs, the cell damage mechanism appears to be increasingly thermal as the two thresholds diverge. We conclude that bubble detection can be used to monitor therapeutic endpoint for pulse durations up to 20 µs (or equivalent dwell time in a scanning approach). We have integrated a detection module into our slit lamp adapted laser scanner in order to determine threshold RPE damage during selective laser treatment in vivo. KEYWORDSSelective retinal photocoagulation, retinal pigment epithelium, melanosomes, cavitation, bubble formation, laser scanner, slit lamp, online detection of cell death, back scatter CORRESPONDENCE Clemens Alt Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/21/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Proc. of SPIE Vol. 4951 53 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/21/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx

Nucleation and dynamics of bubbles forming around laser heated microabsorbers

“…This is consistent with previous time resolved interferometric measurements in RPE explants [14]. Previous studies on laser induced damage of RPE cells suggested that bubble formation is the origin of cell damage when using nanosecond or microsecond laser pulses [22,23]. One probable mechanism for bubble induced cell damage among others is membrane rupture, which is caused by the volume increase of the cell due to intracellular bubble formation [7,24].…”

“…Also thermal induced cell damage by protein denaturation is not modeled, because pure thermal induced cell damage without explosive vaporization (i.e. bubble formation) is not likely to occur for laser pulses below a few tens of microseconds [22,23,26].…”

Selective Retina Therapy

“…For a safe and selective treatment, a therapeutic window with radiant exposures slightly above the MBF threshold and far below twice the MBF threshold should be considered. When investigating the cellular damage mechanisms on pulse duration close to threshold irradiation in more detail, it shows that the predominant origin is thermomechanical for pulse durations up to 20 μs and thermal for pulse durations of 50 μs and larger [36,37].…”

Laser-induced retinal damage threshold for repetitive-pulse exposure to 100-μs pulses

“…Figure 6 compares the single-pulse data of this paper to data from the literature 4,[6][7][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 data for 50-Hz exposures are poorly described by the thermal model but are well described by the PS model.…”