Characterization of a new fluorescence lifetime imaging ophthalmoscope

Abstract

Aims/Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) allows in vivo measurement of autofluorescence intensity decays of endogenous fluorophores in the ocular fundus. So far, only devices from Heidelberg Engineering based on the Spectralis system have been used in FLIO research. Here, we present and characterize a new FLIO device based on the RETImap system from Roland Consult.Methods: The device is based on a confocal scanning laser ophthalmoscope (35° field, 512 × 512 px). A ps diode laser (BDL‐SMN 473 nm, Becker & Hickl GmbH, Berlin, Germany) excites autofluorescence. The fluorescence photons are split into a short (498–560 nm, SSC) and a long (560–720 nm, LSC) spectral channel (one HPM‐100‐40 detector [Becker & Hickl GmbH] each) and are detected by time‐correlated single photon counting (SPC‐160, Becker & Hickl GmbH). We determined the maximum laser power (ILT2400, International Light Technologies, Inc. Peabody, MA, USA) and analysed the instrument's behaviour at three different laser power levels (150 μW, 200 μW and max.) in terms of laser spectrum (CAS140CT, Instrument Systems GmbH, Munich, Germany) and instrument response function (IRF). The IRF was determined using a 25 μM Eosin Y solution, mixed with a 5 M solution of potassium iodide, placed in a flat cuvette (110‐OS, Hellma GmbH & Co. KG, Müllheim, Germany) in front of the objective lens of the FLIO device. Fluorescence measurements of approximately 1‐min duration were performed three times for all three laser powers. The IRF and the full width at half maximum (FWHM) were calculated using FLIMX software (www.flimx.de).Results: The max. laser power was 280 μW. The peak wavelengths of the laser spectra were 467.6 (150 μW), 467.9 (200 μW) and 468.0 nm (280 μW). IRF FWHM in SSC were 298.6 ± 1.1 ps (150 μW), 341.0 ± 2.5 ps (200 μW) and 347.5 ± 6.0 ps (280 μW). In LSC, the IRF FWHM were 290.4 ± 3.8 ps (150 μW), 344.0 ± 3.4 ps (200 μW) and 358.8 ± 1.3 ps (280 μW). Results are mean ± standard deviation.Conclusions: A new fluorescence lifetime imaging ophthalmoscope has been characterized. The device offers a high laser power for fluorescence excitation, a large field of view, a high spatial resolution, and a sufficiently high time resolution. Thus, it is suitable for fluorescence lifetime studies.