Raman microscopy is a powerful method to provide spatially resolved information about the chemical composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles, either resting on a surface or optically trapped at a laser focus, where the confocal collection volume optimizes the signal from the particle. In this work, the sensitivity and spatial selectivity of detecting Raman scattering from single particles was determined as a function of particle size. An inverted confocal Raman microscope was used to acquire spectra of individual surface-bound and optically trapped polystyrene particles with sizes ranging between 200 nm and 10 microm. The particles are in contact with aqueous solution containing perchlorate ion that served as a solution-phase Raman-active probe to detect interferences from the surrounding medium. The collection volume is scanned through single particles that are attached to the surface of the coverslip, and the sensitivity and selectivity of detection are measured versus particle size. The results compare favorably with a theoretical analysis of the excitation profile and confocal collection efficiency integrated over the volumes of the spherical particles and the surrounding solution. This analysis was also applied to the detection of particles that are optically trapped and levitated above the surface of the coverslip. The results are consistent with the optical trapping of particles at or near the excitation beam focus, which optimizes excitation and selective collection of Raman scattering from the particle.

Optical trapping of small structures is a powerful tool for the manipulation and investigation of colloidal and particulate materials. The tight focus excitation requirements of optical trapping are well suited to confocal Raman microscopy. In this work, an inverted confocal Raman microscope is developed for studies of chemical reactions on single, optically trapped particles and applied to reactions used in solid-phase peptide synthesis. Optical trapping and levitation allow a particle to be moved away from the coverslip and into solution, avoiding fluorescence interference from the coverslip. More importantly, diffusion of reagents into the particle is not inhibited by a surface, so that reaction conditions mimic those of particles dispersed in solution. Optical trapping and levitation also maintain optical alignment, since the particle is centered laterally along the optical axis and within the focal plane of the objective, where both optical forces and light collection are maximized. Hour-long observations of chemical reactions on individual, trapped silica particles are reported. Using two-dimensional least-squares analysis methods, the Raman spectra collected during the course of a reaction can be resolved into component contributions. The resolved spectra of the time-varying species can be observed, as they bind to or cleave from the particle surface.

In-vivo and in-vitro investigations indicate that a newly developed polyazamacrocyclic chelate of Tb(III) has superior properties for use as an abnormal tissue marker. In addition to tissue selectivity, this molecule is unique because of its low toxicity, attractive fluorescent properties, rapid pharmokinetics, and relatively high water solubility. The complex Tb-3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13 -triene (Tb-PCTMB) has also been shown to exhibit strongly shifted emission (delta lambda--280 nm), moving the detection frequency away from autofluorescence backgrounds, and good quantum efficiencies (phi = 0.51), providing high brightness. Fluorescence imaging was used to quantify Tb-PCTMB at the picomolar level in tissues and to show the significant difference in affinity for the chelate by adenocarcinoma cells HT-29 versus normal epithelial cells (IEC-6). Topical application, or lavage introduction, under endoscopy was used to instill a millimolar aqueous solution of Tb-PCTMB into a dimethylhydrizene-treated Sprague Dawley rat large intestine containing a suspect growth. Subsequent in vitro fluorescence detection and standard histological evaluation confirmed enhanced uptake by adenocarcinoma tissue. Semiquantitative signal interrogation was employed to show the potential for using Tb-PCTMB as a contrast enhancement marker for disease detection.

Polyazamacrocyclic chelates of terbium are shown to be useful in diagnostic medical imaging as tissue site-selective markers. Spectroscopic properties and biodistribution are studied for three terbium(III) species: 3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(15),11,13-triene (abbreviated as PCTMB); 3,6,9-tris(methylene phosphonic acid)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(13),11,13-triene (abbreviated as PCTMP); and N,N'-bis(methylene phosphonic acid)-2,11-diaza [3.3]-(2,6)pyridinophane (abbreviated as BP2P). The respective aqueous molar absorptivities are found to be 3424, 2513, and 3281/2210 L mole−1 cm−1. Fluorescence quantum efficiency is determined against rhodamine 19 in basic ethanol and rhodamine 6G in ethanol. These values are 0.48, 0.21, and 0.40 for Tb-PCTMB, Tb-PCTMP, and Tb-BP2P, respectively. Biodistribution studies performed in Sprague–Dawley rats indicate tissue site-selectivity. Fluorescence images of bone tissues are presented and demonstrate the potential for using the lanthanide chelates to perform site-directed in vivo imaging for the early identification of abnormal tissue.