Detection sensitivity of fluorescence lifetime imaging ophthalmoscopy for laser-induced selective damage of retinal pigment epithelium.

PURPOSE: To investigate the sensitivity of fluorescence lifetime imaging ophthalmoscopy (FLIO) to detect retinal laser spots by comparative analysis with other imaging modalities. METHODS: A diode laser with a wavelength of 514 nm was applied with pulse durations of 5.2, 12, 20, and 50 µs. The laser pulse energy was increased so that the visibility of the laser spot by slit-lamp fundus examination (SL) under the irradiator's observation covers from the subvisible to visible range immediately after irradiation. The irradiated areas were then examined by fundus color photography (FC), optical coherence tomography (OCT), fundus autofluorescence (AF), FLIO, and fluorescein angiography (FA). The visibility of a total of over 2200 laser spots was evaluated by two independent researchers, and effective dose (ED) 50 laser pulse energy values were calculated for each imaging modality and compared. RESULTS: Among examined modalities, FA showed the lowest mean of ED50 energy value and SL the highest, that is, they had the highest and lowest sensitivity to detect retinal pigment epithalium (RPE)-selective laser spots, respectively. FLIO also detected spots significantly more sensitively than SL at most laser pulse durations and was not significantly inferior to FA. AF was also often more sensitive than SL, but the difference was slightly less significant than FLIO. CONCLUSION: Considering its high sensitivity in detecting laser spots and previously reported potential of indicating local wound healing and metabolic changes around laser spots, FLIO may be useful as a non-invasive monitoring tool during and after minimally invasive retinal laser treatment.

Fractal analysis of the mandible cortical bone: correlation among fractal dimension values obtained by two processing methods from periapical radiograph and micro-computed tomography with cone-beam computed tomography.

The objectives of the present study were to assess Fractal Dimension (FD) values in the mandible cortical bone obtained from digital periapical radiographs (DPR), high-resolution microtomography (µCT), and cone-beam computed tomography (CBCT), by two processing methods: binarization (FD.b) and grayscale-based method (FD.f) and, finally, to identify the correlation among these values with other micro-architectural parameters. For this, a prospective study was conducted on 18 healthy individuals (mean age 23 ± 2.4 years old) who underwent third molar extraction. Pre-operative CBCT scans were conducted, bone fragments were removed from the retro-molar region, and DPR and µCT were performed on those bone samples. FD.b and FD.f values were calculated using three parasagittal sections for CBCT, one image for DPR, and three sections for µCT. The 3D bone microarchitecture was analyzed in µCT (voxel size: 19 µm). As a result, FD.b mean values of 1.55 ± 0.02 and 1.80 ± 0.01 were obtained for CBCT and µCT, respectively. Furthermore, FD.f mean values of 1.22 ± 0.12 for DPR, 0.99 ± 0.04 for CBCT, and 1.30 ± 0.07 for µCT were obtained. Both FD.b and FD.f values showed a good agreement. FD.f was negatively correlated with the standard deviation of the mean gray value (p = 0.003) for DPR and intra-cortical bone surface (p = 0.02) for µCT. In conclusion, image processing with or without binarization revealed different values for FD, although showing agreement. The grayscale-based method retrieved FD values correlated with the gray levels and the cortical porous network, which means that FD can be a valuable index for mandibular cortical bone evaluation. FD is associated with mineralization and microarchitecture. Nevertheless, there was no correlation between FD values obtained from low- (DPR) and high-resolution (µCT) X-ray modalities with FD obtained from the in vivo CBCT.

Temperature Increase and Damage Extent at Retinal Pigment Epithelium Compared between Continuous Wave and Micropulse Laser Application.

Continuous wave (CW) and microsecond pulse (MP) laser irradiations were compared regarding cell damage and laser-induced temperature rise at retinal pigment epithelium (RPE). The RPE of porcine RPE-choroid-sclera explants was irradiated with a 577 nm laser in CW or MP mode (5% or 15% duty cycle (DC)) for 20 ms or 200 ms at an average laser power of 20−90 mW. Cell viability was investigated with calcein-AM staining. Optoacoustic (OA) technique was employed for temperature measurement during irradiation. For 200 ms irradiation, the dead cell area (DCA) increased linearly (≈1600 µm2/mW) up to the average power of 40 mW for all modes without significant difference. From 50 mW, the increase of DCA of MP-5% significantly dropped to 610 µm2/mW (p < 0.05), likely due to the detected microbubble formation. OA temperature measurement showed a monotonic temperature increase in CW mode and a stepwise increase in MP mode, but no significant difference in the average temperature increase at the same average power, consistent with the temperature modeling. In conclusion, there is no difference in the average temperature rise between CW and MP modes at the same average power regardless of DC. At lower DC, however, more caution is required regarding mechanical damage due to microbubble formation.

Algorithms for optoacoustically controlled selective retina therapy (SRT).

OBJECTIVES: Selective Retina Therapy (SRT) uses microbubble formation (MBF) to target retinal pigment epithelium (RPE) cells selectively while sparing the neural retina and the choroid. Intra- and inter-individual variations of RPE pigmentation makes frequent radiant exposure adaption necessary. Since selective RPE cell disintegration is ophthalmoscopically non-visible, MBF detection techniques are useful to control adequate radiant exposures. It was the purpose of this study to evaluate optoacoustically based MBF detection algorithms. METHODS: Fifteen patients suffering from central serous chorioretinopathy and diabetic macula edema were treated with a SRT laser using a wavelength of 527 nm, a pulse duration of 1.7 µs and a pulse energy ramp (15 pulses, 100 Hz repetition rate). An ultrasonic transducer for MBF detection was embedded in a contact lens. RPE damage was verified with fluorescence angiography. RESULTS: An algorithm to detect MBF as an indicator for RPE cell damage was evaluated. Overall, 4646 irradiations were used for algorithm optimization and testing. The tested algorithms were superior to a baseline model. A sensitivity/specificity pair of 0.96/1 was achieved. The few false algorithmic decisions were caused by unevaluable signals. CONCLUSIONS: The algorithm can be used for guidance or automatization of microbubble related treatments like SRT or selective laser trabeculoplasty (SLT).

Fluorescence Lifetime Changes Induced by Laser Irradiation: A Preclinical Study towards the Evaluation of Retinal Metabolic States.

Fluorescence Lifetime (FLT) of intrinsic fluorophores may alter under the change in metabolic state. In this study, the FLT of rabbit retina was investigated in vivo after laser irradiation using fluorescence lifetime imaging ophthalmoscopy (FLIO). The retina of the Chinchilla bastard rabbits was irradiated with a 514 nm diode laser. FLIO, fundus photography, and optical coherence tomography (OCT) were conducted 30 min and 1 to 3 weeks after treatment. After strong coagulation, the FLT at laser spots was significantly elongated immediately after irradiation, conversely shortened after more than a week. Histological examination showed eosinophilic substance and melanin clumping in subretinal space at the coagulation spots older than one week. The FLT was also elongated right around the coagulation spots, which corresponded to the discontinuous ellipsoid zone (EZ) on OCT. This EZ change was recovered after one week, and the FLT became the same level as the surroundings. In addition, there was a region around the laser spot where the FLT was temporarily shorter than the surrounding area. When weak pulse energy was applied to selectively destroy only the RPE, a shortening of the FLT was observed immediately around the laser spot within one week after irradiation. FLIO could serve as a tool to evaluate the structural and metabolic response of the retina to laser treatments.

Investigations on Retinal Pigment Epithelial Damage at Laser Irradiation in the Lower Microsecond Time Regime.

Purpose: New lasers with a continuous wave power exceeding 15 W are currently investigated for retinal therapies, promising highly localized effects at and close to the Retinal Pigment Epithelium (RPE). The goal of this work is to evaluate mechanisms and thresholds for RPE cell damage by means of pulse durations up to 50 µs. Methods: A diode laser with a wavelength of 514 nm, a power of 15 W, and adjustable pulse durations between 2 µs and 50 µs was used. Porcine RPE-choroidal explants (ex vivo) and chinchilla bastard rabbits (in vivo) were irradiated to determine threshold radiant exposures for RPE damage \({\bar H_{Cell}}\) by calcein vitality staining and fluorescence angiography, respectively. Thresholds for microbubble formation (MBF) \({\bar H_{MBF}}\) were evaluated by time-resolved optoacoustics. Exemplary histologies support the findings. Results: \({\bar H_{{{MBF}}}}\) is significantly higher than \({\bar H_{Cell}}\) at pulse durations ≥ 5 µs (P < 0.05) ex vivo, while at 2 µs, no statistically significant difference was found. The ratios between \({\bar H_{{{MBF}}}}\) and \({\bar H_{Cell}}\) increase with pulse duration from 1.07 to 1.48 ex vivo and 1.1 to 1.6 in vivo, for 5.2 and 50 µs. Conclusions: Cellular damage with and without MBF related disintegration are both present and very likely to play a role for pulse durations ≥ 5 µs. With the lower µs pulses, selective RPE disruption might be possible, while higher values allow achieving spatially limited thermal effects without MBF. However, both modi require a very accurate real-time dosing control in order to avoid extended retinal disintegration in this power range.

Towards Automatically Controlled Dosing for Selective Laser Trabeculoplasty.

PURPOSE: Selective laser trabeculoplasty (SLT) is a treatment option for open-angle glaucoma; however, it lacks an instant evidence for successful irradiation. So far ophthalmologists use the visible appearance of permanent champagnelike bubbles (macro bubbles) as an indicator for appropriate pulse energy. We hypothesize that micro bubbles, which start energetically far below the appearance of macro bubbles, already trigger the therapeutic benefit. Here we present two methods to capture the onset of these micro bubbles. METHODS: The trabecular meshwork of freshly enucleated porcine eye globes was irradiated with a series of 15 pulses with a pulse duration of 1.7 µs and with increasing energy at a repetition rate of 100 Hz per each spot of 200 µm in diameter. An optical and an optoacoustic method have been developed and appropriate algorithms investigated towards the real-time detection of the onset of micro bubbles. RESULTS: Both observation methods are capable of detecting micro bubble nucleation. Threshold radiant exposures were found at 310 ± 137 mJ/cm2. By combination of both methods a sensitivity and specificity of 0.96 was reached. CONCLUSIONS: In case that the therapeutically demanded pressure reduction is already achieved with these micro bubbles, which needs to be proven clinically, then the methods presented here can be used in an automatic feedback loop controlling the laser irradiation. This will unburden the clinicians from any dosing during SLT. TRANSLATIONAL RELEVANCE: Automatic real-time pulse energy dosing based on the formation of micro bubbles in SLT significantly improves and facilitates the treatment for the physician.

Real-time optoacoustic temperature determination on cell cultures during heat exposure: a feasibility study.

Objective/Purpose: In order to study the effects of hyperthermia and other temperature-related effects on cells and tissues, determining the precise time/temperature course is crucial. Here we present a non-contact optoacoustic technique, which provides temperatures during heating of cultured cells with scalable temporal and spatial resolution. METHODS: A thulium laser (1.94 µm) with a maximum power of 15 W quickly and efficiently heats cells in a culture dish because of low penetration depth (1/e penetration depths of 78 µm) of the radiation in water. A repetitively Q-switched holmium laser (2.1 µm) is used simultaneously to probe temperatures at different locations in the dish by using the photoacoustic effect. Due to thermoelastic expansion of water, pressure waves are emitted and measured with an ultrasonic hydrophone at the side of the dish. The amplitudes of the waves are temperature dependent and can be used to calculate the temperature/time course at any location of probing. RESULTS: We measured temperatures of up to 55 °C with a heating power of 6 W after 10 s, and subsequent lateral temperature profiles over time. Within this profile, temperature fluctuations were found, likely owing to thermal convection and water circulation. By using cultured retinal pigment epithelial cells, it is shown that the probe laser pulses alone cause no biological damage, while immediate cell damage occurs when heating for 10 s at temperatures exceeding 45 °C. CONCLUSIONS: This method shows great potential not only as a noninvasive, non-contact method to determine temperature/time responses of cells in culture, but also for complex tissue and other materials.

Subregional areal bone mineral density (aBMD) is a better predictor of heterogeneity in trabecular microstructure of vertebrae in young and aged women than subregional trabecular bone score (TBS).

BACKGROUND: Currently, bone densitometry fails to identify nearly half of those elderly patients at immediate fracture risk. To improve clinical assessment of vertebral fracture risk, we aimed to determine how the DXA-based 2D parameter Trabecular Bone Score (TBS) relates to subregional variability in 3D trabecular microstructure in young and elderly women compared to aBMD. METHODS: T12 vertebrae from 29 women (11 young: 32 ±â€¯6 years, 18 aged: 71 ±â€¯5 years) were DXA-scanned ex vivo in anterior-posterior (AP) and lateral projection providing vertebral aBMD and TBS. Additionally, aBMD and TBS were measured for three horizontal (superior, mid-horizontal, inferior) and three vertical subregions (anterior, mid-vertical, posterior) and related to 3D microstructure indices, i.e. bone volume per tissue volume (BV/TV), trabecular number and thickness (Tb.N, Tb.Th), based on HRpQCT. RESULTS: Subregional high-resolution tomography showed significant differences in trabecular parameters for both age groups: In horizontal subregions, BV/TV was lowest superiorly, Tb.Th was highest mid-horizontally, and Tb.N was lowest mid-horizontally and highest inferiorly. Correspondingly, aBMD varied between horizontal subregions, with differences depending on projection direction. TBS varied only in lateral projections of the aged group, with lower values for the mid-horizontal subregion. In vertical subregions, BV/TV, Tb.N, and aBMD were highest posteriorly for both groups. TBS did not differ between vertical subregions. Regression analysis showed aBMD as a predictor explained more of the variance in subregional 3D microstructure compared to TBS. Stepwise multi-regression analysis revealed only three combinations of subregion, projection, and group where aBMD and TBS were both significant predictors. CONCLUSIONS: Subregional aBMD reflects variations in trabecular bone microstructure better than subregional TBS for trisected regions. Specifically, lateral aBMD identifies microstructural heterogeneities independent of age and may improve prediction of vertebral strength and susceptibility to specific fracture types.

Selective retina therapy: toward an optically controlled automatic dosing.

Selective retina therapy (SRT) targets the retinal pigment epithelium (RPE) with pulsed laser irradiation by inducing microbubble formation (MBF) at the intracellular melanin granula, which leads to selective cell disruption. The following wound healing process rejuvenates the chorio-retinal junction. Pulse energy thresholds for selective RPE effects vary intra- and interindividually. We present the evaluation of an algorithm that processes backscattered treatment light to detect MBF as an indicator of RPE cell damage since these RPE lesions are invisible during treatment. Eleven patients with central serous chorioretinopathy and four with diabetic macula edema were treated with a SRT system, which uses a wavelength of 527 nm, a repetition rate of 100 Hz, and a pulse duration of 1.7 µs. Fifteen laser pulses with stepwise increasing pulse energy were applied per treatment spot. Overall, 4626 pulses were used for algorithm parameter optimization and testing. Sensitivity and specificity were the metrics maximized through an automatic optimization process. Data were verified by fluorescein angiography. A sensitivity of 1 and a specificity of 0.93 were achieved. The method introduced in this paper can be used for guidance or automatization of microbubble-related treatments like SRT or selective laser trabeculoplasty.

Selective Retina Therapy in Patients With Chronic Central Serous Chorioretinopathy: A Pilot Study.

We evaluated visual outcomes, changes of maximum macular thickness (MMT) and subretinal fluid (SRF), and safety in patients with chronic central serous chorioretinopathy (CSC) after treatment with selective retina therapy (SRT). Retrospective cohort study of patients with chronic CSC presenting to a university-based hospital from January 2014 through January 2015 was conducted. A total of 12 eyes of 12 patients with chronic CSC lasting for at least 3 months was recruited. The follow-up period ranged from 3 to 12 months. Following evaluation of test spots at temporal arcades, SRT (Q-switched neodymium-doped yttrium lithium fluoride [Nd:YLF] laser; wavelength, 527 nm, pulse duration, 1.7 microsececond) was applied to the surrounding areas of leakage observed on fluorescein angiogram and/or pigment epithelial detachment (PED). Changes in best-correct visual acuity (BCVA), MMT, and SRF and macular sensitivity (MS) by microperimetry (MP) were evaluated. Eyes received treatment in a mean of 3.83 spots at the pulse energy of 65 to 90 µJ. Mean BCVA (logMAR) improved from 0.23 ±â€Š0.12 at baseline to 0.14 ±â€Š0.13 at 3 months. MMT decreased from 341.4 ±â€Š85.5 µm at baseline to 236.0 ±â€Š57.9 µm at 3 months. SRF completely resolved in 75% (9 eyes) at 3 months. Large PEDs (2 eyes) were flattened at 3 months. Retreatment was performed in 4 eyes. MP showed no evidence of scotoma around SRT-treated lesions. SRT treatment targeting the surrounding area of leakage point showed favorable visual and structural outcomes in chronic CSC patients without the risk of scotoma.

Safety and efficacy of selective retina therapy (SRT) for the treatment of diabetic macular edema in Korean patients.

PURPOSE: Selective retina therapy (SRT) stimulates retinal pigment epithelium (RPE) cell migration and proliferation into irradiated areas. The objective of this study was to evaluate the efficacy and safety of SRT in Korean patients with clinically significant diabetic macular edema (DME). METHODS: Prospective non-randomized interventional case series study. Twenty-three eyes of 21 patients with clinically significant DME were treated with SRT and followed for 6 months. Patients underwent an evaluation of best corrected visual acuity (BCVA) in Early Treatment Diabetic Retinopathy Study (ETDRS) letters. Microperimetry was employed to measure macular sensitivity within the central 10° field, and the central macular thickness (CMT) and maximum macular thickness (MMT) were measured. RESULTS: An improvement in BCVA of one to two ETDRS lines was observed in 41.2 % of patients and an improvement of greater than two lines in 29.4 %. Although there was no significant change in CMT (P > 0.05), MMT decreased from 465.8 ± 87.4 µm to 434.3 ± 83.9 µm (P = 0.006), and mean macular sensitivity increased from 20.8 ± 3.4dB to 22.5 ± 3.5dB (P = 0.02). CONCLUSIONS: The gains in BCVA and improvement in macular sensitivity demonstrated that SRT may be used as an effective and safe treatment modality in Korean patients with clinically significant DME.

Tissue response of selective retina therapy by means of a feedback-controlled energy ramping mode.

BACKGROUND: The purpose of the study was to evaluate the safety and selectivity of the retinal pigment epithelium lesions by using automatic energy ramping and dosimetry technique for selective retina therapy and to investigate the healing response. METHODS: Ten eyes of Chinchilla Bastard rabbits were treated with an automatic dosage controlled selective retina therapy laser (frequency doubled Q-switched Nd:YLF, wavelength: 527 nm, pulse duration: 1.7 µs, repetition rate: 100 Hz, pulse energy: linear increasing from pulse to pulse up to shut down - maximal 110 µJ, max. number of pulses in a burst: 30, retinal spot diameter: 133 µm). After treatment, fundus photography, optical coherence tomography and fluorescein angiography were performed at three time points from 1 h to 3 weeks. Histological analysis was performed. RESULTS: A total of 381 selective retina therapy laser spots were tested (range 13-104 µJ).Typical fundus photographs obtained at 1 h after irradiation showed that 379 out of 381 lesions produced by selective retina therapy were not visible ophthalmoscopically and the lesions could be detected by angiography only. Optical coherence tomography images revealed that the structure of photoreceptors was preserved, but a disrupted retinal pigment epithelium layer was observed as was expected. By 3 weeks, histology showed selective retinal pigment epithelium damage without any effect on the inner retina and focal proliferation of the retinal pigment epithelium layer. CONCLUSIONS: Automatically controlled selective retina therapy is a significant improvement in this innovative treatment. It could be demonstrated that the non-contact, reflectometric technique with a controlled pulse energy ramp is safe and selective.