Jeanne P. Haushalter

Upconversion from aqueous phase lanthanide chelates

We have prepared and characterized several lanthanide ion complexes of multidentate ligands or chelates in an effort to develop new upconverting luminescent labels that can be immune to autofluorescence and photobleaching. This study has involved the characterization of various chelates of Nd, Er, and Tm with respect to relative luminescent efficiency and excited-state lifetimes and explored various two-photon stepwise excitation mechanisms. Using peak laser powers near 100 kW, the upconversion emissions of Nd in Nd(EDTA)2(5-) at 386 nm, Er in Er(DPA)3(3-) at 550 nm, and Tm in Tm(DPA)3(3-) at 480 nm, at levels of approximately 10(-12) moles can be detected.

Cell assay using a two-photon-excited europium chelate

We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu3+ emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles.

Strategy for photostable proximity bioassays using lanthanides

We report initial findings for research aimed at creating photostable lanthanide chelate reporters for proximity assays. These reporters take advantage of the nanometer-scale distance dependence of fluorescence enhancement for molecules in the vicinity of noble metal nanoparticles and also capitalize on some unique properties of lanthanide chelates. This approach promises to lead to proximity assays that do not suffer from photobleaching and offer very high on/off enhancement ratios. Results for lanthanide chelates on silver island films and in colloidal suspensions are reported. Enhancement factors range from 1 to 2 orders of magnitude, with larger enhancements for strongly quenched lanthanides.

Near-infrared fluorescent labeling of tissue transglutaminase substrates for imaging tumor boundaries

A novel strategy is developed to optically image tumor boundaries by cross-linking near-infrared fluorescent-labeled tissue trans glutaminase substrates into tumor boundary tissue.

Optical response measurements of a new class of upconverting luminescent reporters

We have prepared and characterized several lanthanide ion complexes of multidentate ligands or chelates in an effort to develop new luminescent reporters that will be immune to autofluorescence and photobleaching. Our study has involved the characterization of various chelates of Eu, Er, and Tm with respect to relative luminescent efficiency and excited state lifetimes. Included in the list of chelates studied are TTFA, EDTA, DPA, DOTA and DTPA as well as mixed and double chelates. In addition to determining the relative efficiencies and luminescence lifetimes of the lanthanide chelates, we have explored various excitation mechanisms and determined optimum excitation wavelengths. This paper will address the various hurdles encountered in the development of this new class of reporters.

Optical property measurements of a novel type of upconverting reporter

We have recently developed a new type of reporter (upconverting chelate) for biomedical diagnostics. For this reporter, the light is absorbed and emitted by a lanthanide ion, rather than an organic molecule, as is the case for a typical fluorescent dye. These materials do not photobleach and have no autofluorescent background. We focus in this paper on neodymium ions complexed with the familiar chelating agents, EDTA, DPA, DTPA and DOTA. We have performed experimental measurements with one- and two-color laser light excitation for different chelate compounds. The samples are excited using two Nd:YAG-pumped dye laser systems that provide laser light near 587 nm and 800 nm. For one-color excitation, the emitted light depends quadratically on the incident laser power, as expected. Three strongly emitting lines are observed, located near 360 nm, 387 nm, and 417 nm. We observed more efficient upconversion in EDTA although the DPA chelates show comparable ground state absorbance. We have studied the influence of temporal delay between the two laser pulses and obtained the decay lifetime of the first intermediate state in the various chelated compounds.

Enzyme-based Labeling of Tumor Boundaries

We are developing a method for labeling tumor boundaries. An enzyme involved in wound healing covalently links a fluorescent-labeled substrate into the growing tumor boundary. We have performed in vitro assays to study this method.

Fluorescent Tissue Transglutaminase Substrates for Tumor Boundary Imaging

A novel strategy is developed to image tumor boundaries optically by crosslinking fluorescent tissue transglutaminase substrates with tumor tissues.

Development of photostable proximity bioassays

We are developing a novel reporter for proximity assays based on enhanced lanthanide luminescence promoted by metal nanoparticles. This method should provide high on/off ratios using photostable labels.

Fluorescent labeling of tumor boundaries

We are developing a new method for labeling tumor boundaries using an enzyme involved in tumor growth.