Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.

Electron microscopy (EM) has been instrumental in our understanding of complex biological systems. Although EM reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An EM-based bioimaging technology capable of localizing individual proteins and resolving protein-protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. Here, we synthesized small lanthanide-doped nanoparticles, and measured the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results suggest that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nano-labels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colors, so that the imaging of biomolecules in a multicolor EM modality may be possible.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Molecular trafficking within engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of 15 mm. Here we present a new 3D tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 mm. The system has a response time of 1 ms with a temporal resolution down to 50 ms in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built upon spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for 3D particle tracking and allows for two-photon, multi-color imaging. 3D tracking of epidermal growth factor receptor (EGFR) complexes at a depth of 100 mm in tumor spheroids is demonstrated. Our 3D tracking microscope is built upon spatiotemporally multiplexed two-photon excitation and uses time-gated analysis via a photon counting histogram to discern the molecular 3D position. Feedback control then steers the excitation to lock-on to the single molecule as it travels at a high speed. The molecular trajectories are reconstructed from the recorded actuator positions from the feedback control loop operating at 1-5 ms. Dynamics down to 50 ms can be inferred from analysis of the photon counting histogram. In our method, the first PMT channel is used for particle tracking while the second and the third PMT channels can be used for two-photon scanning microscopy, colocalization analysis, and energy transfer studies. We have coined this technique TSUNAMI (Tracking Single particles Using Nonlinear And Multiplexed Illumination). E. Perillo et al., ''Deep and high-resolution three-dimensional tracking of single particles using nonlinear and multiplexed illumination,'' Nature Communications, 2015.