122 10  Metabolic Mapping

ocular lens was implanted into his eye. As shown in Fig. 10.13a and c, both the amplitude a1 in the shortwavelength channel and the fluorescence intensity as the sum of all detected photons are reduced at the coagula-

10  tion spots. That is, the fluorescence of RPE is eliminated, emitting with a short lifetime. In contrast, the long lifetimes of connective tissue are detectable at the coagulation spots (Fig. 10.13b). The force of each laser spot can be evaluated according to the detectable lifetime.

Measurements of dynamic fluorescence open the way for objective estimation of laser therapy in diabetes. As the lifetime t2 in the range of 1,000–1,500 ps in the shortwavelength channel is characteristic of unsupplied retinal tissue, its contribution should be reduced after laser coagulation.

Summary for Clinicians

■Time-resolved measurement of endogenous fluorophores in different spectral ranges in combination with selected spectral excitation is a new method for the investigation of cellular metabolism.

■The origination of pathological changes can be determined in a reversible state.

■Individual adapted therapy might be possible on the basis of lifetime measurements.

■Measurement of lifetime is suited to therapy control.

References

 1. Schweitzer D, Leistritz L, Hammer M et al (1995) Calibration-free measurement of the oxygen saturation in retinal vessels of men. Ophthalmic Technol V, SPIE 2393: 210–218

 2. Feke GT (2006) Laser Doppler instrumentation for the measurement of retinal blood flow: theory and practice. Bull Soc Belge Ophtalmol 302:171–184

 3. Leitgeb RA, Schmetterer L, Hitzenberger CK et al (2004) Real-time measurement of in vitro flow by Fourier-domain color Doppler optical coherence tomography. Opt Lett 29(2):171–173

 4. Beach JM, Schwenzer KJ, Srinivas S et al (1999) Oximetry of retinal vessels by dual-wavelength imaging: calibration and influence of pigmentation. J Appl Physiol 86(2): 748–758

 5. Schweitzer D, Hammer M, Kraft J et al (1999) In vivo measurement of the oxygen saturation of retinal vessels in healthy volunteers. IEEE Trans Biomed Eng 46 (12): 1454–1465

 6. Schweitzer D, Lasch A, van der Vorst S et al (2007) Änderung der retinalen Sauerstoffsättigung bei Gesunden

und in frühen Stadien der diabetische Retinopathie nach Atmung von 100% Sauerstoff. Klin Monatsbl Augenheilkd 224:402–410

 7. Stryer L (1991) Biochemie. Spectrum Akademischer, Verlag GmbH Heidelberg

 8. Boettner EA, Wolter JR (1962) Transmission of the ocular media. Invest Ophthalmol 1:776–783

 9. La Schiazza O, Bille JF (2008) High-speed two-photon excited autofluorescence imaging of ex vivo human retinal pigment epithelial cells toward age-related macular degen-

eration diagnostic. J Biomed Opt 13(6):064008

10.American National Standard for the safe use of laser ANSI Z 136.1–2000 (2000) Laser Institute of America, Orlando, FL

11.Schweitzer D, Schenke S, Hammer M et al (2007) Towards metabolic mapping of the human retina. Microsc Res Tech 70:410–419

12.Sionkowska A, Skopinska J, Wisniewski M et al (2007) Spectroscopic studies into the influence of UV radiation on elastin in the presence of collagen. J Photochem Photobiol B Biol 86:186–191

13.Skala MC, Riching KM, Gendron-Fitzpatrick A et al (2007) In vivo multiphoton microscopy of metabolic oxidationreduction states and fluorescence lifetimes in normal and pre-cancerous epithelia. Proc Natl Acad Sci U S A 104(49): 19494–19499

14.Yagi K, Tanaka F, Nakashima N et al (1983) Picosecond laser fluorometry of FAD of D-amino acid oxidasebenzoate complex. J Biol Chem 258(6):3799–3802

15.Nakashima N, Yoshihara K, Tanaka F et al (1980) Picosecond fluorescence lifetime of the coenzyme of D-amino acid oxidase. J Biol Chem 255 (11):5261–5263

16.König K, Riemann I (2003) High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 8 (3): 432–439

17.Schneckenburger H, Wagner M, Weber P et al (2004) Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode. J Fluoresc 14 (5): 649–654

18.Wu Y, Zheng W, Qu J (2006) Sensing cell metabolism by time-resolved autofluorescence. Opt Lett 31(21): 3122–3124

19.Schweitzer D, Jentsch S, Schenke S et al (2007) Spectral and time-resolved studies on ocular structures. In: Schweitzer D, Fitzmaurice M (eds) Diagnostic optical spectroscopy in biomedicine IV. Proceedings of the SPIE, 6628,pp 662807- 1–662807-12

20.Schweitzer D, Kolb A, Hammer M et al (2000) Tau mapping of the autofluorescence of the human ocular fundus. SPIE 4164:79–89

21.Goldenberg-Cohen N, Dadon S, Avraham BCR et al (2008) Molecular and histological changes following central retinal artery occlusion in a mouse model. Exp Eye Res 87: 327–333

22.Lakowicz JR (2007) Principles of fluorescence spectroscopy, 3rd edn. Kluwer, Academic, Plenum, New York

23.Becker W (2005) Advanced time-correlated single photon counting techniques. Springer Series in Chemical Physics 81. Springer, Berlin

24.Schweitzer D, Hammer M, Schweitzer F et al (2004) In vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Opt 9(6)1214–1222

25.Schweitzer D, Quick S, Schenke S et al (2009) Vergleich von Parametern der zeitaufgelösten Autofluoreszenz bei Gesunden und Patienten mit früher AMD. Ophthalmologe 9(6):714–722

26.AREDS Research Group (2001) A randomized, placebocontrolled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report No 8. Arch Ophthalmol 119(10):1417–1436

27.Eldred GE, Katz ML (1988) Fluorophores of the human pigment epithelium: separation and spectral characterization. Exp Eye Res 47:71–86

28.Schweitzer D (2010) Quantifying fundus autofluorescence. In: Lois N, Forrester JV (eds) Fundus autofluorescence. Lippincott Williams & Wilkins, Philadelphia

References 123

29.Bindewald A, Bird AC, Dandekar SS et al (2005) Classification of fundus autofluorescence patterns in early age-related macular diseases. Invest Ophthalmol Vis Sci 46:3309–3314

30.Birol G, Budzynski E, Wangsa-Wirawan ND et al (2005) Retinal arterial occlusion leads to acidosis in the cat. Exp Eye Res 80:527–533

31.Wakita M, Nishimura G, Tamura M (1995) Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ. J Biochem 118:1151–1160

32.WangHW,GukassyanV,ChenCTetal(2008)Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells. J Biomed Opt 13(5):054011