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In summary, we have developed a method that permits concurrent and selective visualization by EELS and EFTEM of two cellular components that can be labeled by photosensitizing fluorescent tracers or by peroxidases that can oxidize Ln-DAB2. Many fluorescent dyes are known to generate sufficient singlet oxygen via triplet sensitization of molecular oxygen so the method should be of wide scope. The use of genetically encoded singlet oxygen generators, such as miniSOG when fused to proteins of interest, also permits the selective visualization by fluorescence and correlated EFTEM of their cellular localization in samples ranging from tissue culture cells to complex tissues such as the mammalian brain. Ln-DAB2 is efficiently precipitated by HRP for immunoperoxidase labeling but with some limitations. Unexpectedly, we found that HRP or its genetically encoded versions and the more recently developed APEX (), a mutated ascorbate peroxidase, act as photosensitizers and precipitate Ln-DAB2 (or DAB) when illuminated between 400 and 650 nm (data not shown). This property limits their use with multicolor EM unless they are introduced after the first round of Ln-DAB2 photooxidation, as described above ( Figures 2 and 3 ). Both peroxidases contain a heme prosthetic group that has not been reported to photosensitize molecular oxygen but, if insufficient heme is available in the cell, protoporphyrin IX () is known to bind both HRP and APEX (). We speculate that trace-bound amounts of this efficient photosensitizer () are responsible for the photooxidation of Ln-DAB2 by these enzymes over the range of illuminating wavelengths that match that of protoporphyrin IX absorbance. APEX2 was engineered to improve heme binding but still deposited DAB upon illumination. Chemical inactivation () of APEX2 to permit a second Ln-DAB2 to be photooxidized by another photosensitizer was ineffective. Further mutation of APEX or methods for increasing cellular heme availability during APEX expression will probably be required for their use in multicolor EM. Despite these limitations, multicolor EM has demonstrated the feasibility of selective deposition of metals by DAB oxidizers that are organelle stains, genetically targetable or encodable, or immunoreactive. EFTEM, although predominately used today for chemical analysis of materials, has been applied to biological samples using the generally weak signals from endogenous elements (), but is sufficiently sensitive to detect and distinguish many of the 14 stable lanthanides when precipitated as Ln-DAB2. The sensitivity of the method is probably not limited by Ln-DAB oxidation as it photooxidizes at a comparable rate to DAB which can yield close to single-molecule detectability after extended illumination, staining with osmium, and conventional TEM (our unpublished results). There is negligible Ln background signal in EELS spectra of non-photooxidized cells, so similar sensitivity might be expected, but EELS is an inherently insensitive technique. The major current limitation is probably the noise introduced by sample drift during the long energy-filtered exposures required for the images.