Fluorescence Quenching by Colloidal Heavy Metals: Implications for Correlative Fluorescence and Electron Microscopy Studies

Abstract

Colloidal heavy metals, such as gold, can be produced in a variety of sizes in the nanometer range. For labeling purposes the metal particles can be directly conjugated to various ligands or to antibodies via nonspecific hydrophobic interactions [1,2]. The heavy metal colloids are electron dense and have a distinct spherical shape and thus are very useful for both TEM and SEM applications [1,2,3]. They have a high extinction coefficient and produce a visible, inflated diffraction image and thus can be identified in several forms of light microscopy [1]. However, the use of the heavy metal colloids as light microscopic labels, particularly in correlative studies, is often problematic. Either the particle size or specimen geometry limits information attainable by interference based light systems. Enlarging smaller particles via silver enhancement may provide visibility in light microscopy, but the spatial resolution necessary for EM is lost. Thus, for correlative studies both in living and fixed systems, probes which combine fluorescence and colloidal heavy metal particles are useful. If colloidal particles of differing shapes or elemental compositions are used for high resolution co-localization studies, it would be advantageous to link a particular probe with a specific fluor such that co-localization at the light microscopic level (as for living cells) via fluorescence can be directly correlated with co-localization at the molecular level via EM [4]. Unfortunately, studies using UV-excited fluoresceinated identifier proteins such as FITC or rhodamine conjugated to colloidal metal particles result in quenching of the fluorescence. The degree of quenching seen with indirect labeling via fluorescent labels has not been well understood and is a possible alternative to direct conjugates. The use of mixtures of fluoresceinated identifier proteins and colloidal metal labeled proteins is also an alternative but competition between probes reduces the overall efficiency of labeling and hence detectability and spatial resolution for either probe.

FluoroNanogold: an important probe for correlative microscopy

“…Thus, separate attachment of the NG to a hinge thiol, and the fluorescent labels to amino groups elsewhere in the Fab', provides sufficient separation to allow bright fluorescence. However, for larger gold particles, the Förster distance increases significantly and conjugation of gold and fluorescent labels to the same probe results in drastic loss of fluorescence [45,76]. For example, with 6-nm gold, useful fluorescence intensity requires that the gold and fluorophore be delivered to the target conjugated to separate antibodies [46], thus not achieving a true dual label probe.…”

“…Attempts to prepare combined fluorescent and gold labeled probes by the adsorption of fluorescently labeled antibodies to colloidal gold particles have produced limited success [12,13,27], due primarily to quenching of the fluorescence by the gold particle absorption [45]. Gold nanoparticles efficiently absorb fluorescence, and this becomes a severe problem in larger gold particles.…”

Multiple Correlative Immunolabeling for Light and Electron Microscopy Using Fluorophores and Colloidal Metal Particles

“…In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. With the fluorophore conjugated to the secondary antibody and colloidal particles to the primary antibody, we found quenching to be reduced by 50% in the case of 5-nm particles and by 20% in the case of 18-nm particles (Kandela et al 2003; Kandela and Albrecht in press). For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies.…”

“…This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric miscroscopy (TEM). However, direct conjugation of both colloidal metal particle and fluorescent dye to the same antibody molecule results in nearly total quenching of the fluorescent signal (Kandela et al 2003). In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths.…”

Correlative Labeling Studies in Light and Electron Microscopy

“…For correlative labeling purposes, an ideal situation is one in which a single label contains a fluorescent dye and a colloidal metal particle. However, the combination of both a fluorescent dye and a colloidal metal particle on the same molecule leads to quenching of the fluorescent signal [3]. The percentage of quenching varies depending on the size of the colloidal metal particle.…”