Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs

Evaldas Katilius; Neal W. Woodbury

doi:10.1117/12.529853

21 June 2004 Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs

Evaldas Katilius, Neal W. Woodbury

Proceedings Volume 5323, Multiphoton Microscopy in the Biomedical Sciences IV; (2004) https://doi.org/10.1117/12.529853
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

Abstract

Two- and three-photon excitation was used to investigate the properties of two fluorescent DNA base analogs: 2-aminopurine and 6-methylisoxanthopterin. 2-aminopurine is a widely used fluorescent analog of the DNA base adenine. Three-photon excitation of 2-aminopurine is achievable by using intense femtosecond laser pulses in 850-950 nm spectral region. Interestingly, the three-photon excitation spectrum is blue-shifted relative to the three-times-wavelength single-photon excitation spectrum. The maximum of the absorbance band in the UV is at 305 nm, while the three-photon excitation spectrum has a maximum at around 880 nm. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2-aminopurine for DNA-protein interaction studies. However, due to relatively small three-photon absorption cross-section, a good signal-to-noise fluorescence correlation curves take very long time to obtain. Fluorescence properties of 6-methylisoxanthopterin, the fluorescent analog of guanine, were investigated using two-photon excitation. This molecule has the lowest energy absorption band centered around 350 nm, thus, two-photon excitation is attainable using 700 to 760 nm output of Ti-sapphire laser. The excitation spectrum of this molecule in the infrared well matches the doubled-wavelength single-photon excitation spectrum in the UV. The high fluorescence quantum yield of 6-methylisoxanthopterin allows efficient fluorescence correlation measurements and makes this molecule a very good candidate for using in in vitro DNA-protein interaction studies.

Citation Download Citation

Evaldas Katilius and Neal W. Woodbury "Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); https://doi.org/10.1117/12.529853

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