Fluorescence Lifetime Imaging with Near-Field Scanning Optical Microscopy

Abstract

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.

Phase separation in polyfluorene – polymethylmethacrylate blends studied using UV near‐field microscopy

“…The polymer poly (9,9‐dioctylfluorene) (PFO) has recently received particular attention in NSOM studies (Teetsov, 2000, 2002; Kwak et al ., 2001), as it is a state‐of‐the‐art material used in organic light emitting diodes (OLEDs) (Grice et al ., 1998). PFO emits blue fluorescence with high quantum efficiency (in excess of 50% in a thin film; Virgili et al ., 2000), and is characterized by a high hole carrier mobility (Redecker et al ., 1999).…”

“…Because of this, a number of studies have shown that fluorescence NSOM can be used to determine the composition of phase‐separated thin polymer films (DeAro, 1997; Hsu, 1998; Webster, 1998; Stevenson et al ., 2001a; Stevenson et al ., 2001b), even those containing two or more fluorescent polymers in a common transparent matrix (Stevenson et al ., 1999). Furthermore, NSOM has been combined with fluorescence decay‐lifetime measurements to study the relative importance of non‐radiative channels in thin‐films of conjugated polymers (Kwak et al ., 2001; Nabetani et al ., 2001). Other work has used near‐field photoconductivity measurements to study the structure of stretched aligned conjugated‐polymer films (DeAro & Buratto, 1999).…”

Fluorescent Metal Nanoshells: Lifetime-Tunable Molecular Probes in Fluorescent Cell Imaging

“…However, the imaging measurements are often influenced by intrinsic weaknesses of organic fluorophores, including low emission intensity, fast photobleaching, and strong photoblinking [4]. With an advance in fluorescence lifetime imaging microscopy (FLIM), lifetime-resolved fluorescence imaging is now widely used as a valuable tool for cell imaging [5,6]. The use of FLIM may provide certain advantages.…”

Enhancement of the weak scattered signal in apertureless near-field scanning infrared microscopy

“…10 This work has taken place along with notable developments in apertured near-field methods and other new optical and infrared microscopies. [11][12][13][14][15][16][17][18][19][20] In apertureless near-field IR microscopy, the development of several imaging techniques, including constant height imaging, detection of the scattered signal at higher harmonics of the tip motion, heterodyne detection, and homodyne detection, [21][22][23][24] in addition to the availability of new IR, tunable sources, have set the stage for significant advances. One of the primary challenges in apertureless nearfield microscopy is the detection of the weak scattered field.…”