LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular analysis of Her-2/neu oncogene amplification

Abstract

Light emitting diodes (LED), which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2) gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH) and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER (Amplified Fluorescence by Transmitted Excitation of Radiation) LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green) were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue) LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own experiences emphasize the high potential of this technology to provide a serious alternative to conventional fluorescence microscopy in routine pathology; representing a sustainable technological progress, this low-cost technology will clearly give direction also to the growing field of molecular pathology.

Figure 1

Detection of Her-2/neu gene expression in human breast cancer tissue by FISH analysis. Two exemplary tissues of breast cancer exhibiting no amplification (A, B) or strong amplification of Her-2/neu gene (C, D) are shown (400× magnification). ZyGreen labeled Her-2/neu gene signals captured by advanced Nikon fluorescent microscope in combination with conventional 100W mercury vapor lamp (A, C) are directly compared with those captured by standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER LED Fluorescence Microscope Kit (B, D). Cerb-B2 gene expression was detected by a 480 nm Fluorescence light cassette combined with a 510 nm long pass emission filter mounted to the Axiostar microscope. Equal quality of the captured pictures was achieved by integration times between 1 and 3 seconds (A, C, 100W mercury vapor) and 6 and 10 seconds (B, D, LED).