Abstract
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.
Highlights
IntroductionLanthanide ions complexed with multidentate ligands (lanthanide chelates) offer interesting properties for cellular imaging and other biological and medical applications
Lanthanide ions complexed with multidentate ligands offer interesting properties for cellular imaging and other biological and medical applications
Recent work has examined methods to achieve upconverted emission from lanthanide chelates [3] either through direct excitation of lanthanide ions such as trivalent erbium or thulium [4] or by twophoton excitation of a sensitizing molecule followed by efficient energy transfer to a lanthanide ion such as europium [5,6,7,8,9,10,11,12]
Summary
Lanthanide ions complexed with multidentate ligands (lanthanide chelates) offer interesting properties for cellular imaging and other biological and medical applications. Lanthanide chelates do not self-quench, allowing for many labels to be conjugated to a molecular probe. The lanthanide chelates are small in size yet the lanthanide ions are immune to photobleaching like the much larger quantum dots [13,14] and upconverting phosphors [15,16]. Upconverted emission from lanthanide chelates tends to exhibit minimal autofluorescent background from the biological matrix. Photobleaching and autofluorescence are significant limitations for fluorescent reporters today. We report the practical application of a two-photon-excited europium chelate reporter to a cell-based assay using a microscope combining scanned excitation and CCD detection. Photobleaching, autofluorescence, and detection limits are quantified. Elevated expression of EGFR is a tumor marker for squamous carcinomas
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