June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Time-resolved 2D-temperature monitoring on RPE explants during laser irradiation by means of a fluorescent dye
Author Affiliations & Notes
  • Ralf Brinkmann
    Medical Laser Center Luebeck, Germany
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
  • Xi Zhang
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
  • leonie Hoffmann
    Medical Laser Center Luebeck, Germany
  • Eric Seifert
    Medical Laser Center Luebeck, Germany
  • Mario Mordmüller
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
  • Dirk Theisen-Kunde
    Medical Laser Center Luebeck, Germany
  • Madoka Suzuki
    Institute for Protein Research, Osaka University, Japan
  • Yoko Miura
    Medical Laser Center Luebeck, Germany
    Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany
  • Footnotes
    Commercial Relationships   Ralf Brinkmann None; Xi Zhang None; leonie Hoffmann None; Eric Seifert None; Mario Mordmüller None; Dirk Theisen-Kunde None; Madoka Suzuki None; Yoko Miura None
  • Footnotes
    Support  BMBF FKz13N14443
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3801 – F0222. doi:
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    • Get Citation

      Ralf Brinkmann, Xi Zhang, leonie Hoffmann, Eric Seifert, Mario Mordmüller, Dirk Theisen-Kunde, Madoka Suzuki, Yoko Miura; Time-resolved 2D-temperature monitoring on RPE explants during laser irradiation by means of a fluorescent dye. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3801 – F0222.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Subvisible retinal thermal laser treatments are becoming more popular, but lack an objective dosing control. This is very demanded since the laser-induced temperature rise differs largely due to inter- and intraocular variations of light scattering and pigmentation at the fundus. Therefore we developed an optoacoustic (OA) method to determine and control the average temperature rise during laser irradiation. In order to calibrate and analyse the OA-method, the time-resolved 2D-temperature distribution was investigated in this study by means of the thermosensitive fluorescent dye, ERthermAC, being taken up into the endoplasmic reticulum of the cells.

Methods : Porcine retinal pigment epithelium (RPE)/choroid-sclera explants were incubated with the dye for 30 min and then placed in a cuvette filled with phosphate buffer saline (PBS) for laser exposure. The samples were repetitively irradiated with a slitlamp-coupled Q-switched Nd:YLF-laser (527 nm wavelength, 250 ns pulse duration) on a spot diameter of 200 µm. ERthermAC absorbs between 480 and 590 nm and fluoresces from 560 to 700 nm. The fluorescence light was decoupled from the laser path with a dichroic mirror in front of the slitlamp. The laser spot was imaged enlarged on a sensitive camera. The OA transients were measured with an ultrasonic transducer embedded in a contact lens. Calibration curves were recorded by frequently exciting the probe with 9 µJ/pulse while heating the PBS with a rate of 2 °C/min from 20 to 60 °C. For laser-induced heating a repetition rate of 1 kHz with a pulse energy of 30 µJ was applied for 200 ms.

Results : The dye was well accumulated in RPE cells. An almost linear fluorescence intensity decay of 1.87 % per 1 °C was noticed for calibration. The fluorescence emission was stable and did not bleach over the irradiation time. Laser-induced heating revealed the expected temperature-correlated pressure rise by OA and the corresponding fluorescence decrease over time, strongest at the center of the spot. Both methods are in good accordance with an average difference in temperature rise of about 9.8 % after 200 ms.

Conclusions : The thermosensitive dye ERthermAC shows high affinity to ex-vivo RPE cells and enables time-resolved 2D-temperature monitoring during laser exposure. It shows a good accordance with the OA-method and is generally a useful tool to investigate temperature-related cellular effects.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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