The objective of this proposal is to create an Ultrafast Multiphoton Laser Activation System. This integrated laser instrument system is to be assembled to support a select set of innovative biomedical and biotechnology research programs that are implemented by multiphoton photochemistry and photophysics. Ultrafast (approximately 100 femtosecond) pulses generated by mode-locked lasers allow simultaneous absorption of two or more photons from bright pulsed illumination for molecular excitation as in the multiphoton laser scanning fluorescence microscopy used in biomedical research in living tissue. However, research on multiphoton excitation is evolving powerful photochemical tools for molecular biology and biomedical research and diagnosis. In particular, multiphoton photochemical crosslinking of the DNA of specific genes to the corresponding regulatory proteins allows assay of the molecular biology of these essential regulators of gene expression and identification of each particular regulatory enzyme-DNA pairing. The proposed laser system provides the appropriate, bright, ultrafast pulses and pulse pairs at ultraviolet (approximately 250) wavelengths to implement efficiently this crosslinking process in vitro and, it is expected, in vivo. The high-energy femtosecond pulses of the Ultrafast Laser System are provided throughout the wide range from 210 nm to 3,000 nm needed to enable or enhance the entire group of projects. Other user's applications of the Ultrafast Laser System include (1) development of caged reagents and caged neurotransmitters for localized micro-pharmacology by multiphoton photoactivation, (2) research on oxidative stress effects in disease by multiphoton oxidant activation of molecules in tissue, (3) investigations of biological photodamage mechanisms, (4) developments of several biophysical technologies, and (5) research on the photophysical mechanisms of all of the relevant multiphoton photochemical processes involved. Several biophysical technology developments are facilitated by the proposed instrument system, in particular, development of a multiphoton photochemical method to fabricate 3-dimensionally complex microfluidic systems for biotechnology devices, and photophysics for enhanced implementation of multiphoton microscopy and for biomedical endoscopic spectroscopy of intrinsic tissue fluorescence to recognize disease states. The instrument system consists of a tunable titanium:sapphire laser oscillator, regenerative amplifier, two independently tunable optical parametric oscillators, harmonic generators for frequency multiplication, delay lines and associated instrumentation in order to encompass the high pulse energy and unusually broad tuning range requirements of the select biomedical user community and to safely provide user friendly instrumentation for the biological user community.
| Fecko, Christopher J; Munson, Katherine M; Saunders, Abbie et al. (2007) Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking. Photochem Photobiol 83:1394-404 |
