Although autofluorescent proteins starting from the Green Fluorescent Protein of Aequorea Victoria have revolutionized molecular and cell biology, they have major limitations, such as irreducible size (>200 residues) and optical spatial resolution (conventionally >200 nm). To circumvent these limitations, we are developing genetically encoded tags for electron microscopy. Controlled local photogeneration of singlet oxygen (1O2, the metastable excited state of O2) is useful for generating electron-microscopic contrast, rapidly inactivating proteins of interest, reporting protein proximities over tens of nanometers, and ablating cells by photodynamic damage. Arabidopsis phototropin, a blue light photoreceptor containing flavin mononucleotide (FMN) as its chromophore, can be engineered into a small (106-residue) Singlet Oxygen Generator (“miniSOG”), which absorbs maximally at 448 and 473 nm. Quantum yields for fluorescence and SO generation are 0.30 and 0.47. MiniSOG binds endogenous FMN very tightly (dissociation constant ~ 0.1 nM), so cells upregulate their total FMN to keep miniSOG saturated, without any obvious toxicity in the absence of illumination. Although the green fluorescence of miniSOG is weak and bleachable, it shows that fusions of miniSOG to a variety of proteins in mammalian cells appear to localize correctly, even inside organelles when appropriate. After fixation, illumination of miniSOG to generate 1O2 efficiently polymerizes 3,3’-diaminobenzidine into an osmiophilic deposit, enabling correlative electron microscopy with spatial resolution on the order of nanometers. In an initial biological application, electron microscopy shows that the closely related cell-adhesion molecules SynCAM1 and SynCAM2, separately fused to miniSOG, predominantly localize respectively to the pre-synaptic and post-synaptic sides of mammalian CNS synapses. MiniSOG may do for electron microscopy what GFP did for optical microscopy. Autofluorescent proteins are also of very limited direct utility in human patients, because humans are too thick and opaque for most applications of fluorescence, and introduction of foreign genes into patients faces formidable technical and ethical barriers. We therefore remain interested in synthetic molecules with novel amplifying mechanisms for homing on diseased tissues. Activatable cell penetrating peptides (ACPPs) are polycationic cell penetrating peptides (CPPs) whose cellular uptake is minimized by a polyanionic inhibitory domain and then restored upon proteolysis of the peptide linker connecting the polyanionic and polycationic domains. Local activity of proteases able to cut the linker causes amplified retention in tissues and uptake into cells. Tumor uptake of ACPPs is up to 4 fold higher with a matrix metalloproteinase substrate (PLGLAG) as the linker than with a negative control composed of D-amino acids. Conjugation of ACPPs to macromolecular carriers such as dendrimers prolongs pharmacokinetics and increases delivery of payload (Cy5 or Gd-DOTA or both in the same molecule) to tumor for far-red or MR imaging (Olson et al, PNAS 2010). The dual labeled probe with Cy5 and Gd-DOTA enables whole body magnetic resonance imaging followed by fluorescence-guided surgery. Such fluorescence guidance improves tumor-free survival in two animal models (Nguyen et al, PNAS 2010). Chemotherapeutic drugs also gain efficacy when targeted to the tumor by ACPPs. Thrombin-cleavable ACPPs accumulate in atherosclerotic plaques and experimental stroke models, so vascular pathologies can also be imaged. Thus the ability of ACPPs to deliver imaging and therapeutic payloads with enzymatic amplification to protease-expressing tissues in vivo offers clinical potential. Furthermore, we have used phage display to discover peptides that home to peripheral nerves. When these peptides are fluorescently labeled and injected intravenously, all the peripheral nerves become fluorescent for a few hours without apparent physiological effects. A custom-built multispectral fluorescence imager then permits the surgeon to overlay the fluorescence images of the tumor or atherosclerotic plaque vs. the nerves on the traditional reflected-light color image continuously in real time. We believe such molecular fluorescence guidance will greatly improve surgical accuracy.