feature Voices in methods development To mark the 15th anniversary of Nature Methods, we asked scientists from across diverse felds of basic biology research for their views on the most exciting and essential methodological challenges that their communities are poised to tackle in the near future. Polina Anikeeva: Clifford Oliver Fiehn: Neural engineering Brangwynne: We Metabolomics has benefitted have a detailed has become an from decades of understanding of integral cornerstone innovation in micro- the conditions under of biological and nano-electronics, which distinct states research. Biological photonics, materials of non-living matter interpretations science, chemistry form, codified in rely on accurate and synthetic biology. phase diagrams that identification Our current ability to reflect underlying of metabolites. Credit: Andrew integrate these fields thermodynamic driving forces. Can we Yet, currently, compound annotations LaNoue with each other and achieve a similar quantitative understanding lack confidence scoring; this needs to with neuroscience, of liquid–liquid phase separation within change! Data reports should become however, pales in comparison with the living cells? To truly understand intracellular more harmonized, with cloud processing scale and complexity of neuronal signaling. self-assembly, and its functional and for large data sets and kits of internal Understanding the nervous system in the pathological dysregulation in devastating standards to assess metabolite levels. Even context of health and disease will demand diseases, the answer needs to be yes. New in-depth untargeted discovery assays should a paradigm shift, from refinement of technologies are needed to probe and become cheaper and use fast-turnaround individual device components to integration engineer intracellular phase behavior, and standardized protocols. Data needs to of multiple signaling capabilities, to address should interface with deep proteomics, become findable, accessible, interoperable the richness of communication within metabolomics and genomics readouts of and re-usable for large-scale analyses. neural circuits. Such a paradigm shift biological function. These technologies Metabolome atlases of compound levels highlights the need for fluid exchange will also elucidate non-equilibrium driving in organs and cells are needed to compare of ideas between the fields and demands forces within the complex intracellular individual studies against animal models and understanding of fundamental physical milieu, and provide the foundation for a human population health data. Eventually, principles at the core of each technology. rigorous understanding of living matter. the community should tackle the biggest bottleneck: interpreting metabolomics data Edward Boyden: Ibrahim I. Cissé: sets by extending database queries towards Over the last few To detect a single automatic literature text mining. decades, we have fluorescent molecule, seen the invention it must either be Petra Fromme: of new technologies dilute or one must Biological for imaging brain turn off any other processes are highly activity, controlling nearby fluorescent dynamic, but most brain activity, molecule. Although biomolecular and mapping the ability to structure Credit: Matt Staley, the molecular localize individual determination Credit: Justin Knight HHMI/Janelia composition and fluorophores is approaches only wiring of the brain. advantageous and show a static picture. An important methodological challenge has led to development of super-resolution X-ray-free electron Credit: Mary Zhu will be to optimize these technologies and fluorescence microscopy, an implication of lasers (XFELs) incorporate them into a single workflow, so needing sparse fluorescent molecules is the have revolutionized that scientists can systematically investigate concentration limit of a few nano-molar or structural biology with femtosecond how the molecular composition and wiring less that it imposes. Practically, this means pulses: structures can be determined of the brain yields its emergent dynamics, that, at molecular resolution, live-cell before destruction takes place, enabling the which in turn generates behavior and fluorescent microscopes only capture discovery of the dynamics of biomolecular pathology. For example, experimental the more strongly interacting biomolecules, reactions ‘on the fly’. However, access to workflows that enable imaging activity and are blind to most assemblies of XFELs is limited, with only five facilities throughout a brain circuit, then perturbing weaker affinities. However, the growing in the world. Compact XFELs, which its dynamics, and finally mapping the appreciation for biomolecular condensates aim to shrink XFELs from 1 mile to 30 molecules and wiring throughout, may and in vivo phase transitions will likely feet, could bring XFEL technology to the yield new insights into the mechanisms force us to come up with clever ways to laboratory scale, opening the field to the underlying complex brain functions and unveil the blind spots of in vivo single- broad scientific community. Combined with dysfunctions. molecule microscopy. ultrafast spectroscopy, this will enable the NATURE METHODS | VOL 16 | OCTOBER 2019 | 945–951 | www.nature.com/naturemethods 945 feature determination of the dynamics of molecular or duplicate the interdisciplinary training of our next and electronic structural transitions genome have been generation of life scientists will also simultaneously, in real time, in the future. characterized at the be essential. atomic level, and Anne-Claude the CRIPSR–Cas9 Grant Jay Jensen: Gingras: Proteomics genetic engineering The history of cell research is currently revolution has biology has been undergoing a burst helped dissect their punctuated by of exciting technical functions. These major advances in developments, in Credit: Institut Curie technologies provide imaging technology. terms of improving profound insights Cryo-EM imaging throughput, into how genome methods have quantification and structure relates to genome regulation and recently enjoyed an the ability to analyze gene expression. The main technological amazing ‘resolution Credit: Annie Tong, very small samples by challenge today is to follow the dynamics revolution’. In the future, the range of Sinai Health System mass spectrometry. of genome folding and function over time samples that can be imaged will expand to These improvements in living cells, integrating imaging and both much smaller and much larger targets. are already being applied to profile protein genomic data. We also have to address the For imaging macromolecules, electrons abundance, but they can also be employed behavior and role of the repetitive portion have profound advantages over X-rays in in functional proteomics. Coupled with, for of the genome, which may dictate many that they can be focused to high resolution, example, CRISPR technologies and advances of its architectural and regulatory features. revealing phases as well as amplitudes. in protein labeling and crosslinking The repeat fraction of the genome has been Because of this, and because imaging in 3D techniques, advanced proteomics methods somewhat of a blind spot for the analysis of is better than 2D, the way of the future will will provide fine details of cellular genome architecture, yet it may contain key be to image macromolecules using cryo- organization, as well as of changes in the architectural and regulatory features. electron tomography. Eventually, this will be association, localization and functions of true across scales and context from crystals proteins following perturbations. This will Stefan W. Hell: Now of small purified proteins to enormous require the acquisition of multi-faceted that the ultimate macromolecular complexes inside tissues, datasets, and one of the next challenges resolution limit but there are formidable technical challenges will be to develop tools to facilitate their in fluorescence to be overcome in sample preparation, visualization and re-use by the broader microscopy—that instrumentation and analysis. scientific community. is, 3D resolution of the size scale of Rachel Karchin: Casey S. Greene: We a molecule—has Cancer researchers are generating data been reached with are working with at an unprecedented MINFLUX, we high-dimensional scale and at levels of should seriously think beyond fluorescence. data: genomic, resolution ranging Coming up with molecular signals that are transcriptomic, from environmental as specific as fluorescence but do not require proteomic and sensors to molecular labeling with reporter molecules; that would epigenomic, from profiling of be something. bulk sequencing individual cells. It can to single-cell be tempting to search Elizabeth sequencing of tens of thousands of cells. Credit: Anna Greene through large-scale Hillman: The latest New imaging technologies will provide 2D datasets to identify microscopes are and 3D views of the cancer cells and their results that support existing notions. A near- revealing the inner environments. Longitudinal studies will term challenge is to develop techniques that workings of living make it possible to model the dynamics integrate data to illuminate under-studied organisms like never of these changes in many dimensions. We processes or reveal relationships that are at before: dynamics imagine it will be feasible to associate the odds with our expectations. Uniting machine of motion, high- dynamics of omics measurements and learning methods