Dr. Evaldas Katilius Profile

Dr. Evaldas Katilius

Faculty Research Associate at Arizona State Univ

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SPIE Journal Paper | 1 July 2006 Open Access

Multiphoton excitation of fluorescent DNA base analogs

Evaldas Katilius, Neal Woodbury

JBO, Vol. 11, Issue 04, 044004, (July 2006) https://doi.org/10.1117/12.10.1117/1.2337521

KEYWORDS: Luminescence, Analog electronics, Fluorescence correlation spectroscopy, Molecules, Absorption, Ultraviolet radiation, Diffusion, Photons, Sapphire lasers, Absorbance

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Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5×10–50 cm4s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

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Proceedings Article | 21 June 2004 Paper

Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs

Evaldas Katilius, Neal Woodbury

Proceedings Volume 5323, (2004) https://doi.org/10.1117/12.529853

KEYWORDS: Luminescence, Molecules, Fluorescence correlation spectroscopy, Absorption, Diffusion, Analog electronics, Ultraviolet radiation, Absorbance, Sapphire lasers, Molecular lasers

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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.

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