Two-Color Two-Photon Excitation Using Femtosecond Laser Pulses

Abstract

The use of two-color two-photon (2c2p) excitation easily extends the wavelength range of Ti:sapphire lasers to the UV, widening the scope of its applications especially in biological sciences. We report observation of 2c2p excitation fluorescence of p-terphenyl (PTP), 2-methyl-5-t-butyl-p-quaterphenyl (DMQ) and tryptophan upon excitation with 400 and 800 nm wavelengths using the second harmonic and fundamental wavelength of a mode-locked Ti:sapphire femtosecond laser. This excitation is energetically equivalent to a one-photon excitation wavelength at 266 nm. The fluorescence signal is observed only when both wavelengths are spatially and temporally overlapping. Adjustment of the relative delay of the two laser pulses renders a cross correlation curve which is in good agreement with the pulse width of our laser. The fluorescence signal is linearly dependent on the intensity of each of the two colors but quadratically on the total incident illumination power of both colors. In fluorescence microscopy, the use of a combination of intense IR and low-intensity blue light as a substitute for UV light for excitation can have numerous advantages. Additionally, the effect of differently polarized excitation photons relative to each other is demonstrated. This offers information about different transition symmetries and yields deeper insight into the two-photon excitation process.

Two-color two-photon excitation of indole using a femtosecond laser regenerative amplifier

“…Since an intense photon flux is delivered to a same focal area lasting for several minutes during our measurement, it is possible that fluorescence bleaching occurs to some extent which results in the decrease of the slope coefficient. This result is quite different from that in [22] where almost no signal is detected for irradiation of 800 nm alone from femtosecond oscillator. The discrepancy results from different single pulse energy between oscillator and amplifier.…”

“…Some reports have examined the emission intensity of indole as each of the excitation beams is rotated [21,22]. In those results, the indole intensity decreases to about one half of the maximal value for orthogonal polarized excitation beams.…”

“…In the past years, the need for higher pulse energies has triggered the rapid development of many amplifier configurations with different repetition rates from several kHz to several hundred kHz and pulse energies from microjoule to millijoule [11], which in turn have been widely used in a number of optical investigations [12][13][14][15][16][17][18]. To solve the second problem, two-color two-photon (2C2P) excitation fluorescence microscopy has been developed [19][20][21][22]. In the 2C2P technique, two excitation photons with different wavelengths k 1 and k 2 are used to excite the specimen.…”

“…Due to the development of pulse lasers since the 1990s, there has been great progress on the researches of 2C2P excitation. Gryczynski et al have observed 2C2P fluorescence with picosecond dye laser excitation of PPD in 1996 [20] and indole in 1997 [21] excitation fluorescence using a mode-locking femtosecond Ti:Sapphire laser oscillator [22]. These works involving use of pulse laser are promising steps towards the realization of a practical 2C2P fluorescence microscopy, especially the use of femtosecond laser.…”

Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy

“…The resulting decay profiles were analyzed using a single exponential decay model, yielding a fluorescence lifetime of 2.55 ns with a reduced χ 2 of 1.08 ( Figure S7a), which is consistent with literature values. 32 Fluorescence was then acquired using three photon (1c3p) excitation with an average power of 50 mW at 900 nm By increasing the 900 nm/450 nm ratio, 1c2p excitation is suppressed and the fluorescence intensity improves 28 fold with the addition of the 900 nm beam. The dashed area highlighted in Figure 3b was then imaged using the same experimental conditions and scanned with a 1 µm step size for improved resolution (Figure 3c).…”