The development of a time-resolved confocal microscope for single molecule detection of luminescent polymers

Abstract

This report details the construction and testing of a confocal microscope for use in single molecule fluorescence spectroscopy. With the prospect of making time-resolved lifetime and spectral measurements of π-conjugated polymers in the condensed phase, the system employs a pulsed 390nm, 65ps diode laser along with compact electronics for time-correlated single photon counting. Following optimisation the microscope records continuous photon arrival times with l00ps resolution, and fluorescence lifetime decays with 40ps resolution. Isolation of polymer chains has been investigated as a function of dilution by the dispersion of poly[9,9di(ethylhexyl)fluorene] (PF2/6) in a variety of host matrices. Films have been prepared by spin coating from toluene and the emission spectra, confocal lifetime and anisotropy have been investigated. Whilst the choice of host does not show any considerable influence on the spectral characteristics of the luminescent polymer, concentration and spin speed were observed to significantly influence the photophysical properties of the film. Spectral blue shifts in the emission band have been observed as the dilution of the luminescent polymer increases, reaching a maximum shift of 20nm in the most dilute samples. In line with the spectral shift, lifetime measurements show that the fluorescence decay becomes increasingly mono-exponential with dilution. In films exhibiting the maximum spectral blue shift, mono-exponential fluorescence decay was observed, for the oligomer oligo [9,9di(ethylhexyl)fluorene] (_N=20) (OF2/6) in zeonex the lifetime is found to be τ = 0.92ns. The dilution at which this occurs varies from 1x10(^-4) % w/w to 1x10(^-6) % w/w depending on spin speed and host matrix. Significantly longer that the lifetime of OF2/6 in solution, this is thought to be very close to the natural radiative lifetime of the polymer and is taken as a clear indication of chain isolation within the host matrix. Spectral blue shift and progression to mono-exponential fluorescence decay are explained in terms of the decline of low energy aggregate states and a reduction in non-radiative interchain energy transfer due to increased interchain separation. Confocal imaging using the specimen scanning technique has been developed using an electronically controlled x y piezo-stage. By optimising the optical set up the confocal image resolution is currently 0.369nm. The maximum theoretical confocal resolution at 390nm excitation is 0.268nm; the cause of this loss in resolution is thought to originate from slight non-uniformity in the excitation source.