Single Molecule Detection of Near-Infrared Phthalocyanine Dyes

The major advantage associated with near-infrared monitoring is the fact that few compounds show intrinsic fluorescence in this region of the spectrum and hence background from other molecules is reduced. Phthalocyanine dyes provide emission at deep red and near infrared wavelengths but have excellent photostability and hence are an attractive candidate for near-infrared fluorescence bioassay applications. However, because of their small Stokes shifts, non-standard methods are needed for optimum separation of fluorescence from scattered laser light. This thesis reports the development of a custom confocal microscope that uses a lowcost laser diode operating at 667 nm for sample excitation and an angle-tuned Raman notch filter to block scattered laser radiation and provide high-throughput of fluorescence with small Stokes shift. Also, a diffraction grating is used to isolate the laser excitation wavelength from the broadband luminescence of the laser. The experimental system is used to observe photon bursts from single molecules of zinc phthalocyanine fluorophores in an ethanol solution. The autocorrelation function of the photon trace, which is measured in fluorescence correlation spectroscopy, provides a measure of the presence of photon bursts from single molecules. Experiments to characterize the limits of detection of near-infrared fluorophores in aqueous solution using the microscope are also discussed.