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

Abstract

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.

Keywords: fluorescence lifetime imaging ophthalmoscopy; metabolic change; retinal laser treatment.

Figure 1

Experimental set-up and procedures of laser irradiation on rabbit retina: (A) experimental set-up for laser irradiation and optoacoustic microbubble detection, (B) schematic description of the patterns for laser irradiation on the rabbit retina. Left: Four rows of 10 laser spots (blue) with increasing pulse energy and 14 marker spots (red) for orientation. Right: two rows of either 3 or 10 laser spots (blue) with the energy around the threshold for microbubble formation determined by optoacoustic measurement. Marker spots (red) for orientation were applied far from the test sites, close to the optic nerve.

Figure 2

Schematic description of the FLIO device. The fundus is excited at 473 nm with a pulsed picosecond laser. Emitted photons are detected by two hybrid photon-counting detectors either for short spectrum channel (SSC) or for long spectrum channel (LSC). TCSPC: time-correlated single-photon counting.

Figure 3

Visual streak of the rabbit eye: (A) In FLIO, there is a hyperfluorescent narrow banded area (arrow) with short FLT near the optic nerve. (B) Fundus image: the visual streak is funduscopically not recognizable. The area between the two dashed lines is the location of the visual streak estimated from previous reports, which is almost consistent with the banded area observed in FLT and FAF. FLT: fluorescence lifetime, FAF: fundus autofluorescence, ONH: optic nerve head.

Figure 4

Multimodal imaging of representative patterns, including strong irradiations (marker spots) at different points in time after laser irradiation. Two patterns were applied separately next to each other on day 0 (pattern 1) and day 9 (pattern 2). (A) 0.5 h after irradiation of pattern 1, (B) 9 days after irradiation of pattern 1, 0.5 h after irradiation of pattern 2, (C) 20 days after irradiation of pattern 1, 11 days after irradiation of pattern 2. Arrows indicate the corresponding spots in IR and OCT. Arrow head indicates the localized thinning of the outer retinal layer after coagulation. FLT: fluorescence lifetime, SSC: short spectral channel, LSC: long spectral channel, FAF: fundus autofluorescence, Color: Fundus photography, OCT: optic coherence tomography, IR: infrared reflectance image, RPE: retinal pigment epithelium, EZ: ellipsoid zone.

Figure 5

FLT at and around the marker spots: (A) A representative pseudocolor image of the τ_m in SSC at one applied pattern about 0.5 h after irradiation. (B) The FLT profiles of both channels at the lines given in (A) were analyzed with FIJI software. It is noted that one area within the Zone 2 shows shorter FLT than the surroundings (arrow). (C–H) Tukey box plots for τ_m at four different zones at different points in time in SSC and LSC. (Number of patterns analyzed: n = 19 for C and D, n = 9 for E and F, n = 4 for G and H). FLT: fluorescence lifetime, SSC: short spectral channel, LSC: long spectral channel. *** p < 0.001.

Figure 6

Histological images (hematoxylin-eosin staining) at the strong photocoagulation spots on day 0 ((A), ①) and day 9 ((D,E): ②, ③). For the image of day 0, the fundus photograph (B) and the pseudocolor FLT image of FLIO (C) were made about 0.5 h after irradiation and directly before euthanasia and eye enucleation. The arrows in B and C indicate the spot presented in the histology. For the images of day 9, the strong marker spot (②) and the coagulated site but weaker than the marker spot (③) are presented. (F,G) are the fundus photograph and the pseudocolor FLT image of FLIO (SSC) on day 0 for those spots, whereas (H,I) are those on day 9, shortly before euthanasia and eye enucleation for histology. The retinal dome-shaped morphology in histology as shown in (D) is the typical artifact seen at the medium to strong coagulation spots. Eosinophilic structureless substance and pigment clumping are observed (arrow heads in (D,E)).

Figure 7

Multimodal imaging at representative two sites irradiated with short laser pulses (5 µJ) with the energy levels slightly above microbubble formation; (A) An area irradiated with laser pulse energy at 40 µJ, (B) An area irradiated with laser pulse energy at 18 µJ. FLT and FAF in FLIO in both channels (SSC and LSC), Fundus photography (Color), and OCT images at and around laser spots on day 0 (about 0.5h after irradiation, D0) and day 7 (D7). Arrows indicate sites of laser spots, arrow heads indicate ellipsoid zone. FLT: fluorescence lifetime, FAF: fundus autofluorescence, SSC: short spectral channel, LSC: long spectral channel, OCT: optic coherence tomography.