Graefes Arch Clin Exp Ophthalmol (2014) 252:2025–2026 DOI 10.1007/s00417-014-2813-1
LETTER TO THE EDITOR
Fluorescence lifetime imaging ophthalmoscopy in glaucoma L. Ramm & S. Jentsch & R. Augsten & M. Hammer
Received: 3 June 2014 / Revised: 19 August 2014 / Accepted: 22 September 2014 / Published online: 22 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Dear Editor: With the aim to detect retinal changes at cellular level, fluorescence lifetime imaging ophthalmoscopy (FLIO) was conducted in primary open-angle glaucoma. In addition to the loss of retinal ganglion cells, metabolic alterations and tissue remodeling are conceivable [1, 2]. Using a modified laserscanning ophthalmoscope, the autofluorescence of different fundus regions was investigated in 43 glaucoma patients (64.9 ±11.4 years) and 54 healthy controls (65.3±11.8 years, p= 0.85). Twenty-five patients and 39 controls were phacic, and 18 patients and 15 controls were pseudophacic. Subjects without serious systemic diseases, diabetes mellitus, or ocular pathologies (except previous cataract surgery) were included. Patients under anti-coagulant therapy with fluorescent coumarin-derivatives were excluded. All investigations were approved by a local institutional review board, and written informed consent was obtained. As the anti-glaucomatous
A
treatment was not interrupted, the intraocular pressure was lower than 22 mmHg in all cases. The FLIO method was described by Schweitzer et al. [3]. Exciting the fundus with light of short wavelengths leads to fluorescence of different substances. Therefore, the measured signal is composed of the sum of several fluorophores. Investigating the fluorescence decay time allows, at least partly, a separation of single fluorophores [3, 4]. Using the fluorescence lifetime imaging ophthalmoscope, the fluorescence decay can be allocated to three components, and thereby described through the lifetime parameters τ1–3 and amplitudes α1–3. For a global characterization of the fluorescence, the amplitude-weighted mean of all decay times, τm, is calculated. Fluorescence was excited with a wavelength of 448 nm. The emission was captured in two spectral channels (Ch1: 490– 560 nm, Ch2: 560–700 nm) and the autofluorescence properties in different fundus regions were analyzed (Fig. 1). Fur-
C
B 100 ps
330 ps
Fig. 1 Mean fluorescence lifetime in the short wavelength channel. The squares represent the fundus areas for evaluating the fluorescence lifetimes. The area of the macula and its surroundings (a), the arcade (b) and the optic nerve head (c) were analyzed separately L. Ramm : S. Jentsch : R. Augsten : M. Hammer (*) Department of Ophthalmology, University Hospital Jena, Bachstr. 18, 07743 Jena, Germany e-mail: [email protected]
2026
Graefes Arch Clin Exp Ophthalmol (2014) 252:2025–2026
Table 1 FLIO - Results in phacic subjects. Mean values and standard deviations of the parameters showing differences in one or more of the regions are presented. The t-test (shaded in blue) or the Mann–Whitney U test (white/grey) was employed. *significant N α3 (Ch1) [%] α2 (Ch2) [%] τ3 (Ch2) [ps]
glaucoma paents controls glaucoma paents controls p-value glaucoma paents controls p-value glaucoma paents controls p-value
macula
inferior arcade
opc nerve head
25 39 3.01 ± 1.12 3.49 ± 0.97 0.023* 23.62 ± 2.3 22.7 ± 2.8 0.041* 2278.9 ± 198.64 2396.99 ± 196.46 0.023*
24 39 2.92 ± 1.29 3.23 ± 0.88 0.06 23.88 ± 2.2 23.03 ± 2.62 0.028* 2259.39 ± 220.63 2346.32 ± 184.27 0.055
25 25 10.06 ± 2.36 10.75 ± 3.4 0.405 23.01 ± 2.46 22.31 ± 2.92 0.147 3788.71 ± 753.5 4231.05 ± 1048.17 0.093
thermore, a classification of the participants according to their eye lens was necessary [3]. Depending on whether the data was normally distributed or not, the significance of differences in mean fluorescence parameters between patients and controls was analyzed using ttests or the Mann–Whitney U test. In phacic participants, differences between patients and healthy controls were found for the parameters α3 (Ch1), α2 (Ch2), and τ3 (Ch2), as shown in Table 1. There were no significant differences between the groups of pseudophacic patients. The significant differences of the parameters in phacic subjects might be caused by the fluorescence of the crystalline lens itself, since no differences were found by investigating subjects with a non-fluorescent, implanted intra-ocular lens. The fluorescence of the crystalline lens is characterized by relatively long lifetimes, and it mainly influences the third fluorescence decay component (α3, τ3) [3]. A possible explanation for the high impact of the lens fluorescence in the macular region could be the lower fluorescence intensity in the macula as well as the high amount of short-lifetime fluorophores located in this area. The investigation of time-resolved autofluorescence has been successfully used to examine other diseases, for example age-related macular degeneration [5, 6]. Here, augmented lipofuscin sedimentation might lead to a change of the fluorescence signal [7]. The alteration of the autofluorescence in diabetic retinopathy [8] could result from the accumulation of advanced glycation endproducts [7]. Likewise, Tetzel et al. reported a higher amount of advanced glycation end products (AGEs) in the retina and optic nerve head in glaucoma patients [9]. However, a smaller magnitude than in diabetic retinopathy is probable. Using two-photon excited fluorescence microscopy, Peters et al. investigated the fluorescence properties in different retinal layers of porcine eyes. In the ganglion cell layer, the major contribution to the fluorescence intensity was descended from Müller cells [10]. Therefore, a glaucomatous degeneration of retina ganglion cells, is not seen by fluorescence lifetime
imaging. To what extent the neuronal retina can be observed by this method needs to be further investigated. Conflict of interest L. Ramm: none S. Jentsch: none R. Augsten: none M. Hammer: none
References 1. Chang EE, Goldberg JL (2012) Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement. Ophthalmology 119:979– 986. doi:10.1016/j.ophtha.2011.11.003 2. Flammer J, Mozaffarieh M (2007) What is the present pathogenetic concept of glaucomatous optic neuropathy? Surv Ophthalmol 52(6): S162–S173. doi:10.1016/j.survophthal.2007.08.012 3. Schweitzer D (2010) Metabolic mapping. In: Holz F (ed) Medical retina: focus on retinal imaging. Springer, Berlin, Heidelberg, pp 107–122 4. Schweitzer D, Hammer M, Schweitzer F, Anders R, Doebbecke T, Schenke S, Gaillard ER (2004) In vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Opt 9:1214–1222 5. Schweitzer D, Quick S, Schenke S, Klemm M, Gehlert S, Hammer M, Jentsch S, Fischer J (2009) Vergleich von Parametern der zeitaufgelösten Autofluoreszenz bei Gesunden und Patienten mit früher AMD. Ophthalmologe 106:714–722. doi:10.1007/s00347-009-1975-4 6. Wildner K, Jentsch S, Klemm M, Schweitzer D, Augsten R, Hammer M (2012) Zeitaufgelöste Autofluoreszenz: Vergleich zwischen Gesunden und Patienten mit altersbezogener Makuladegeneration (AMD) bei Pseudophakie [Abstract]. Ophthalmologe 109:67. doi: 10.1007/s00347-012-2684-y 7. Jentsch S, Hammer M, Wildner K, Dawczynski J, Deutsch L, Klemm M, Schweitzer D (2012) In vivo Untersuchungen zur zeitaufgelösten Autofluoreszenz bei retinalen Erkrankungen des menschlichen Auges [Abstract]. Klin Monatsbl Augenheilkd 229:V38. doi:10. 1055/s-0032-1327164 8. Deutsch L (2012) Evaluierung des Fluorescence Lifetime Imaging vom Augenhintergrund bei Patienten mit Diabetes mellitus [Dissertation]. Friedrich-Schiller-Universität, Jena 9. Tezel G, Luo C, Yang X (2007) Accelerated aging in glaucoma: immunohistochemical assessment of advanced glycation end products in the human retina and optic nerve head. Invest Ophthalmol Vis Sci 48:1201–1211. doi:10.1167/iovs.06-0737 10. Peters S, Hammer M, Schweitzer D (2011) Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples. In: So PTC, Beaurepaire E (eds) Advanced Microscopy Techniques II. SPIE Proceedings Vol. 8086:1–10
LETTER TO THE EDITOR
Fluorescence lifetime imaging ophthalmoscopy in glaucoma L. Ramm & S. Jentsch & R. Augsten & M. Hammer
Received: 3 June 2014 / Revised: 19 August 2014 / Accepted: 22 September 2014 / Published online: 22 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Dear Editor: With the aim to detect retinal changes at cellular level, fluorescence lifetime imaging ophthalmoscopy (FLIO) was conducted in primary open-angle glaucoma. In addition to the loss of retinal ganglion cells, metabolic alterations and tissue remodeling are conceivable [1, 2]. Using a modified laserscanning ophthalmoscope, the autofluorescence of different fundus regions was investigated in 43 glaucoma patients (64.9 ±11.4 years) and 54 healthy controls (65.3±11.8 years, p= 0.85). Twenty-five patients and 39 controls were phacic, and 18 patients and 15 controls were pseudophacic. Subjects without serious systemic diseases, diabetes mellitus, or ocular pathologies (except previous cataract surgery) were included. Patients under anti-coagulant therapy with fluorescent coumarin-derivatives were excluded. All investigations were approved by a local institutional review board, and written informed consent was obtained. As the anti-glaucomatous
A
treatment was not interrupted, the intraocular pressure was lower than 22 mmHg in all cases. The FLIO method was described by Schweitzer et al. [3]. Exciting the fundus with light of short wavelengths leads to fluorescence of different substances. Therefore, the measured signal is composed of the sum of several fluorophores. Investigating the fluorescence decay time allows, at least partly, a separation of single fluorophores [3, 4]. Using the fluorescence lifetime imaging ophthalmoscope, the fluorescence decay can be allocated to three components, and thereby described through the lifetime parameters τ1–3 and amplitudes α1–3. For a global characterization of the fluorescence, the amplitude-weighted mean of all decay times, τm, is calculated. Fluorescence was excited with a wavelength of 448 nm. The emission was captured in two spectral channels (Ch1: 490– 560 nm, Ch2: 560–700 nm) and the autofluorescence properties in different fundus regions were analyzed (Fig. 1). Fur-
C
B 100 ps
330 ps
Fig. 1 Mean fluorescence lifetime in the short wavelength channel. The squares represent the fundus areas for evaluating the fluorescence lifetimes. The area of the macula and its surroundings (a), the arcade (b) and the optic nerve head (c) were analyzed separately L. Ramm : S. Jentsch : R. Augsten : M. Hammer (*) Department of Ophthalmology, University Hospital Jena, Bachstr. 18, 07743 Jena, Germany e-mail: [email protected]
2026
Graefes Arch Clin Exp Ophthalmol (2014) 252:2025–2026
Table 1 FLIO - Results in phacic subjects. Mean values and standard deviations of the parameters showing differences in one or more of the regions are presented. The t-test (shaded in blue) or the Mann–Whitney U test (white/grey) was employed. *significant N α3 (Ch1) [%] α2 (Ch2) [%] τ3 (Ch2) [ps]
glaucoma paents controls glaucoma paents controls p-value glaucoma paents controls p-value glaucoma paents controls p-value
macula
inferior arcade
opc nerve head
25 39 3.01 ± 1.12 3.49 ± 0.97 0.023* 23.62 ± 2.3 22.7 ± 2.8 0.041* 2278.9 ± 198.64 2396.99 ± 196.46 0.023*
24 39 2.92 ± 1.29 3.23 ± 0.88 0.06 23.88 ± 2.2 23.03 ± 2.62 0.028* 2259.39 ± 220.63 2346.32 ± 184.27 0.055
25 25 10.06 ± 2.36 10.75 ± 3.4 0.405 23.01 ± 2.46 22.31 ± 2.92 0.147 3788.71 ± 753.5 4231.05 ± 1048.17 0.093
thermore, a classification of the participants according to their eye lens was necessary [3]. Depending on whether the data was normally distributed or not, the significance of differences in mean fluorescence parameters between patients and controls was analyzed using ttests or the Mann–Whitney U test. In phacic participants, differences between patients and healthy controls were found for the parameters α3 (Ch1), α2 (Ch2), and τ3 (Ch2), as shown in Table 1. There were no significant differences between the groups of pseudophacic patients. The significant differences of the parameters in phacic subjects might be caused by the fluorescence of the crystalline lens itself, since no differences were found by investigating subjects with a non-fluorescent, implanted intra-ocular lens. The fluorescence of the crystalline lens is characterized by relatively long lifetimes, and it mainly influences the third fluorescence decay component (α3, τ3) [3]. A possible explanation for the high impact of the lens fluorescence in the macular region could be the lower fluorescence intensity in the macula as well as the high amount of short-lifetime fluorophores located in this area. The investigation of time-resolved autofluorescence has been successfully used to examine other diseases, for example age-related macular degeneration [5, 6]. Here, augmented lipofuscin sedimentation might lead to a change of the fluorescence signal [7]. The alteration of the autofluorescence in diabetic retinopathy [8] could result from the accumulation of advanced glycation endproducts [7]. Likewise, Tetzel et al. reported a higher amount of advanced glycation end products (AGEs) in the retina and optic nerve head in glaucoma patients [9]. However, a smaller magnitude than in diabetic retinopathy is probable. Using two-photon excited fluorescence microscopy, Peters et al. investigated the fluorescence properties in different retinal layers of porcine eyes. In the ganglion cell layer, the major contribution to the fluorescence intensity was descended from Müller cells [10]. Therefore, a glaucomatous degeneration of retina ganglion cells, is not seen by fluorescence lifetime
imaging. To what extent the neuronal retina can be observed by this method needs to be further investigated. Conflict of interest L. Ramm: none S. Jentsch: none R. Augsten: none M. Hammer: none
References 1. Chang EE, Goldberg JL (2012) Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement. Ophthalmology 119:979– 986. doi:10.1016/j.ophtha.2011.11.003 2. Flammer J, Mozaffarieh M (2007) What is the present pathogenetic concept of glaucomatous optic neuropathy? Surv Ophthalmol 52(6): S162–S173. doi:10.1016/j.survophthal.2007.08.012 3. Schweitzer D (2010) Metabolic mapping. In: Holz F (ed) Medical retina: focus on retinal imaging. Springer, Berlin, Heidelberg, pp 107–122 4. Schweitzer D, Hammer M, Schweitzer F, Anders R, Doebbecke T, Schenke S, Gaillard ER (2004) In vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Opt 9:1214–1222 5. Schweitzer D, Quick S, Schenke S, Klemm M, Gehlert S, Hammer M, Jentsch S, Fischer J (2009) Vergleich von Parametern der zeitaufgelösten Autofluoreszenz bei Gesunden und Patienten mit früher AMD. Ophthalmologe 106:714–722. doi:10.1007/s00347-009-1975-4 6. Wildner K, Jentsch S, Klemm M, Schweitzer D, Augsten R, Hammer M (2012) Zeitaufgelöste Autofluoreszenz: Vergleich zwischen Gesunden und Patienten mit altersbezogener Makuladegeneration (AMD) bei Pseudophakie [Abstract]. Ophthalmologe 109:67. doi: 10.1007/s00347-012-2684-y 7. Jentsch S, Hammer M, Wildner K, Dawczynski J, Deutsch L, Klemm M, Schweitzer D (2012) In vivo Untersuchungen zur zeitaufgelösten Autofluoreszenz bei retinalen Erkrankungen des menschlichen Auges [Abstract]. Klin Monatsbl Augenheilkd 229:V38. doi:10. 1055/s-0032-1327164 8. Deutsch L (2012) Evaluierung des Fluorescence Lifetime Imaging vom Augenhintergrund bei Patienten mit Diabetes mellitus [Dissertation]. Friedrich-Schiller-Universität, Jena 9. Tezel G, Luo C, Yang X (2007) Accelerated aging in glaucoma: immunohistochemical assessment of advanced glycation end products in the human retina and optic nerve head. Invest Ophthalmol Vis Sci 48:1201–1211. doi:10.1167/iovs.06-0737 10. Peters S, Hammer M, Schweitzer D (2011) Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples. In: So PTC, Beaurepaire E (eds) Advanced Microscopy Techniques II. SPIE Proceedings Vol. 8086:1–10
