A number of new technologies have been developed from our work over the last few years that are now at the core of our discovery efforts in the areas of HIV/AIDS and cancer biology. Two areas where progress that been especially noteworthy are summarized below. The development of the technological infrastructure for 3D imaging in our laboratory has enabled the analysis, over the last year, of numerous HIV-1/SIV spike and spike complex structures. This is unprecedented, given that in 2008, we achieved a breakthrough by determining the first three HIV-1 spike complex structures. Our streamlined experimental and computational procedures allow us to operate round the clock, remotely, and with average instrument uptimes of 85% for data collection. Over the coming years, we will continue to refine this pipeline by adding tools for separating distinct conformations using in-line, unsupervised classification procedures. One advance that is proving to be useful is the development of data processing strategies that allow clear separation of the closed and open states, as well as unliganded and antibody liganded states of trimeric Env when they are present in mixtures. We have shown that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual spikes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity of Env in heterogeneous SIV and HIV-1 mixtures. We have validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded HIV-1 Env complexes simultaneously present in a mixture. Knowledge of the spatial distributions of proteins, metabolites and elements within the cell is potentially important for understanding function in health and disease. Submicrometer resolution is required for localizing subcellular regions of interest, and techniques based on optical technology, such as fluorescence microscopy, have proven to be very useful for monitoring discrete chemical changes within and around cells. However, fluorescence imaging is restricted to imaging natively fluorescent molecules or those that have been specifically attached to analytes of interest. In contrast, mass spectrometry-based techniques provide unique opportunities for achieving simultaneous detection of multiple, unlabeled cellular components. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used extensively for tissue imaging, although the typical spatial resolutions achieved (around 20 to 50 microns) make it unsuitable for subcellular imaging of most mammalian cells. Secondary ion mass spectrometry (SIMS)-based approaches, with SIMS primary ion beams as small as 50 nm to 500 nm, are capable of much higher resolution, and are therefore useful for single cell chemical imaging mass spectrometry. Despite these advances, SIMS imaging continues to yield few biological discoveries and only modest success with subcellular molecular imaging because of several shortcomings such as smaller mass ranges compared to MALDI-MS (typically less than 500 m/z for SIMS), higher primary beam-induced molecular damage levels, and generally low molecular ionization probabilities of target molecules. Further, there have also been challenges in developing sample preparation protocols that maintain the chemical integrity of the cells, in minimizing molecular damage from the incident beam that limits the ability to generate 3D data sets, and achieving the theoretical limits of better spatial resolution. We have made advances both in specimen preparation and in strategies to extend the imaging into the third dimension by combining SIMS with focused ion beam (FIB) milling. Using this approach we have imaged chemical species within intact mammalian cells using secondary ion mass spectrometry, with simultaneous mapping of subcellular elemental and molecular species along with intrinsic membrane-specific cellular markers. Results from imaging of both the cell surface and cell interior exposed by site-specific focused ion beam milling demonstrate that in-plane resolutions of approximately 400 nm can be achieved. The results from mapping cell surface phosphatidylcholine and several other molecular ions present in the cells establish that spatially-resolved chemical signatures of individual cells can be derived from novel multivariate analysis and classification of the molecular images obtained at different m/z ratios. The methods we have established for specimen preparation and chemical imaging of cell interiors provide the foundation for obtaining 3D molecular maps of unstained mammalian cells, with particular relevance for probing the subcellular distribution of small molecules, such as drugs and metabolites.