Seveus L, Väisälä M, Syrjänen S, Sandberg M, Kuusisto A, Harju R, Salo J, Hemmilä I, Kojola H, Soini E
Department of Anatomy, University of Uppsala, Sweden.
Cytometry. 1992;13(4):329-38. 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 formaldehyde-fixed, wax-embedded specimens.
具有长衰变时间的荧光镧系螯合物能够抑制生物样本中快速衰变的自发荧光。这一特性使得镧系螯合物作为荧光显微镜的标记物具有吸引力。由于背景荧光的抑制，灵敏度得以提高。我们通过在窄孔径平面添加脉冲光源和斩波器，对标准落射荧光显微镜进行了改进，用于时间分辨荧光成像。使用冷却的电荷耦合器件相机进行检测，并对图像进行数字处理。将一种荧光铕螯合物与抗血清和链霉亲和素偶联。这些偶联物用于在人结肠恶性黏膜中定位肿瘤相关抗原C242，在发育中的人软骨生长板中定位II型胶原蛋白mRNA，以及在人宫颈鳞状上皮中检测人乳头瘤病毒型特异性基因序列。铕标记物特有的缓慢衰变荧光能够有效地与快速衰变的背景荧光分离。在细胞和组织水平使用铕标记物是可行的，并且在冷冻以及甲醛固定、石蜡包埋的标本的原位杂交和免疫组织化学反应中，自发荧光得到了有效抑制。
