Time‐resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization

• Published in: Cytometry (New York, N.Y.) Vol. 13; no. 4; pp. 329 - 338
• Main Authors: Seveus, Lahja, Väisälä, Mikko, Syrjänen, Stina, Sandberg, Minna, Kuusisto, Ari, Harju, Raimo, Salo, Juha, Hemmilä, Ilkka, Kojola, Hannu, Soini, Erkki
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 1992; Wiley-Liss
• Subjects: Alkaline Phosphatase; Antigens, Neoplasm - analysis; Autofluorescence; Biological and medical sciences; Biomarkers, Tumor - analysis; Brain Chemistry; CCD‐camera; Collagen - genetics; Colonic Neoplasms - chemistry; Colonic Neoplasms - immunology; digital imaging; DNA Probes, HPV; Europium; Female; Humans; image processing; Immunoenzyme Techniques; Immunohistochemistry - methods; lanthanide chelates; Medical sciences; Microscopy, Fluorescence - instrumentation; Nucleic Acid Hybridization; Organometallic Compounds; Papillomaviridae - isolation & purification; Pyridines; RNA, Messenger - analysis; Tumors; Uterine Cervical Neoplasms - diagnosis; Uterine Cervical Neoplasms - microbiology; Human; Immunohistochemistry; Molecular hybridization; Tumor associated antigen; In situ; Localization; Fluorescence microscopy
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/cyto.990130402

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time‐resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD‐camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehydefixed, wax‐embedded specimens.