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

Abstract

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 I1 collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cer- vix.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. 0 1992 Wiley-Liss, Inc.

Increasing lanthanide luminescence by use of the RETEL effect

“…Time-gated measurements of long-lived luminescent lanthanide ion complexes, which previously has required expensive components, such as an intensified CCD camera (which also has the disadvantages of relatively poor resolution and low red sensitivity), rotating light choppers, and flash lamps (4,24,25), can now be performed using the relatively inexpensive combination of a UV LED with a low voltage power supply and an electronically shuttered CCD camera of the type commonly used for fluorescence microscopy. The same UV LED, although presently not as bright as a conventional mercury-xenon arc, can be used as a replacement with the additional advantage that expensive shutters to prevent photobleaching are no longer required.…”

“…This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes Ò , as molecular diagnostics for cytological and histopathological microscopic imaging. q 2006 International Society for Analytical Cytology Key terms: quantum dye; columinescence; RETEL; lanthanide; macrocycle; luminescence; time-delayed; europium; terbium Lanthanide ion complexes have found a variety of significant applications in laboratory diagnostics (1-3); however, the weak luminescence of these complexes, which is due to their comparatively low molar extinction coefficients (molar absorptivities), was a major impediment to their use in cytometry (4). The problem of increasing the emission intensity of lanthanide ion complexes has been solved by the use of the resonance energy transfer enhanced luminescence (RETEL) effect, formerly called FRE-TEL (5), combined with measurement in the dry state.…”

Time-resolved fluorescence imaging (TRFI) for direct immunofluorescence of PSA and alpha-1-antichymotrypsin in prostatic tissue sections

“…The use of¯uorescent lanthanide chelates in ISH studies, provides an attractive alternative to well established techniques. 10 Department of Clinical Chemistry, Lund University, Malmo È University Hospital, Sweden, for their expert technical assistance. Petri Aronkyto È , Wallac OY, Turku, Ari Kuusisto, Ilkka Hemmila È and Airi Toivonen, Department of Biotechnology, University of Turku, Finland are also greatly acknowledged.…”

“…Brie¯y, a Nikon Optiphot w conven-tional epi¯uorescence microscope (Nikon, Tokyo, Japan) was equipped for TRFI as originally described 10 and modi®ed according to Rulli et al 13 Using a 106objective, the tissue sections were focused on bright-®eld mode with a conventional halogen light source to avoid signal bleaching. A xenon¯ash lamp (60 W, arch length 1.5 mm, 500 Hz) (EG&G, Princeton, NJ) was used for pulsed excitation at 340 nm wavelength (coloured glass ®lter DUG 11, 320-390 nm, Schott, Mainz, Germany) and a highly sensitive stable and high dynamic range camera (model 1530-AaAUV, EG&G) was used as a cooled charge coupled photo-optical device (CCD) with dark noisè 0.1 electronsapixelas, where the size of 1 pixel is 23623mm.…”

High intensity solid‐state UV source for time‐gated luminescence microscopy

“…Lanthanide (Eu 31 or Tb 31 ) chelate fluorescent probes have exceptionally long fluorescence lifetimes reaching milliseconds for some compounds (6)(7)(8)(9)(10). The time-gated luminescence microscope (TGLM) described in this report was used in conjunction with the europium chelate fluorophore BHHST ((4,4-bis-(1,1,1,2,2,3, 3-heptafluoro-4,6-hexanedion-6-yl) sulfonyl-aminopropylester-N-succinimide-ester-o-terphenyl)) that has a fluorescence lifetime greater than 600 ls (11).…”