Second-generation instrument for second harmonic detection of two-photon excited fluorescence
Continued progress is reported in the development of two-photon absorption instrumentation. Noise considerations in second harmonic detection fluorometry are discussed and improvements in two-photon excited fluorescence detection are reported. The use of short focal length microscope objectives for excitation and collection optics allows spatial discrimination of the two-photon induced fluorescence from linear background interferences. Combined with second harmonic detection, this arrangement provides detection limits as low as 200 pM. Suggestions are made for a third generation instrument.^ To test the analytical utility of this instrument, the feasibility of identifying and quantitating bacteria obtained from soil samples was examined. The inherent selectivity of two-photon absorption combined with the excellent detection limits offered by second harmonic detection result in the possibility of reducing assay blank limitations associated with one-photon techniques. The development and spectroscopic characterization of some mono- and di-substituted naphthalenes for monitoring bacteria has resulted in the discovery of a new class of fluorescent probes for enzymatic analyses. ^
Major Professor: Fred E. Lytle, Purdue University.