We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.
Luminescent emission from lanthanide complexes on silver island films has been studied. Silver films with and without silica spacer layers have been prepared chemically and by thermal vacuum deposition. Enhancement of emission from lanthanide complexes on silver compared to that on bare glass depends on the optical density of the silver films (silver coverage) and sample coverage. A silica spacer layer reduces the number of molecules in quenching regions of the film and leads to better enhancement. Lanthanides with lower quantum efficiencies are expected to exhibit greater enhancement factors and our preliminary results are consistent with that prediction.
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.
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.