Imaging the distribution of gold nanoparticles attached to target cells is of critical importance for numerous studies, including plasmonic cell transfection, drug delivery, and plasmonic photothermal and photoionization therapy. Thanks to their unique optical properties, gold nanoparticles can be characterized by a variety of optical spectroscopy techniques; the characterization of individual nanoparticles, however, requires high-resolution microscopy techniques such as dark-field microscopy, two-photon excitation fluorescence microscopy, or, alternatively, transmission electron microscopy for resolving the particles’ structure and shape. When attached to living cells, individual gold nanoparticles are much more difficult to resolve because their scattering properties are still low, even under plasmonic resonance illumination, compared to the characteristic scattering of cellular organelles. To overcome this problem, some researchers tend to increase nanoparticle concentration considerably; such practice often results in high cytotoxicity and considerable particle aggregation. The talk will outline the difficulties associated with gold nanoparticle detection in cells and propose solutions that integrate two-photon excitation fluorescence microscopy, absorption spectroscopy and scattering electron microscopy, for mapping particle distribution within the cells and estimating the level of particle aggregation. We will discuss our approach for estimating nanoparticle distribution within three-dimensional cell cultures, where light penetration is limited and electron microscopy is ineffective. Two-photon microscopy of these cultures after conjugation to gold nanoparticle revealed subsurface aggregates that were widely dispersed across the culture. These findings assist our current efforts to better control particle delivery into tissue phantoms for effective targeting of cancer cells by plasmonic phototherapy.