Transmission and confocal fluorescence microscopy and time-resolved fluorescence spectroscopy combined with a laser trap: investigation of optically trapped block copolymer micelles

Optical trapping was combined with transmission microscopy (TM), confocal and nonconfocal fluorescence scanning microscopy (CFSM and FSM, respectively), and confocal and nonconfocal time-resolved fluorescence spectroscopy (CTRFS and TRFS, respectively) to study latex particles and block copolymer micelles. Dye-labeled latex particles of various size, in polymer composite films as well as optically trapped in solution, were studied with CFSM to characterize the limits of the setup. CFSM revealed that the resolution in the x- and y-directions was near the theoretical limit, i.e., 200−250 nm. CTRFS on the labeled latex particles revealed that the decay time of the label was not influenced by the polymer matrix nor the optical trap. Poly(tert-butylstyrene-block-sodium methacrylate) micelles (diameter approximately 30−40 nm) in deuterated aqueous solutions could be optically trapped, this region of high copolymer micelle concentration being referred to as a trapped cluster. In the transmission images, trapped clusters of 1.5−2 μm diameter were detected. Fluorescence images were obtained using perylene as a fluorophore that is specifically dissolved within the block copolymer micelles. The size of the trapped cluster, estimated from TM and FSM images, increases with increasing irradiation time and power, respectively. In the TM images, the trapped cluster appears as a dark spot (low transmission) with a bright (high transmission) corona-like ring around it. The appearance of the corona is explained as a light deflection phenomenon; i.e., the trapped cluster acts as lens due to a lateral refractive index gradient. When the corona is taken into account when the diameter of the trapped clusters is calculated, a very good agreement is found between TM and FSM. Long irradiation times lead to the formation of large trapped clusters, which are stable for about 10 s, with diameters of several hundred nanometers, while, for short irradiation times, the trapped cluster is smaller and disappears within a time less than 1 s. With CFSM it could be shown that the trapped particle has a spot size of approximately 1.7 μm in the region of the IR laser focus, while the diameter extends up to 5 μm without using the confocal imaging capability. The reason for this is that the conditions for optical trapping are fulfilled not only in but also above and below the focal region. Due to the high numerical aperture, a dumbbell-like shape of the trapped cluster results.