Available online at www.sciencedirect.com ScienceDirect Functional super-resolution microscopy of the cell 1,2 1,2 1,2 Rui Yan , Bowen Wang and Ke Xu Recent advances in super-resolution fluorescence microscopy resolve the finest possible ultrastructure. The structural have led to 10 nm spatial resolution and profound impact on information, however, does not tell the full story of how cell biology and beyond. Here we discuss emerging the cell functions. The astoundingly ordered internal possibilities to encode functional information of intracellular organization achieved within the small volume of the microenvironments, for example, local pH, small-molecule cell does not stop at the level of geometrical shapes. concentrations, chemical polarity (hydrophobicity), and protein Driven far away from equilibrium, the local physicochem- activity, into diverse dimensions of the super-resolution signal, ical parameters within a cell, for example, pH, small- hence a class of approaches we collectively refer to as molecule concentrations, chemical polarity (hydrophobic- functional super-resolution microscopy. By adding remarkably ity), and protein activities, also vary greatly over short rich functional information to the already powerful super- distances. Such intracellular ‘microenvironments’ locally resolution arsenal, functional super-resolution microscopy regulate biochemical reactions and other cellular pro- transcends the structural information provided by existing cesses, and so dictate cell functions at a fundamental methods, and opens up new ways to unveil fascinating local level [10,11]. Would it be possible to unveil and visualize heterogeneities in live cells with nanometer-scale spatial such intracellular functional information with nanometer- resolution and ultimate sensitivity down to the single-molecule scale (nanoscale) spatial resolution similar to what has level. been achieved for structures with SRM? If so, what new insights can be gained toward our understanding of how Addresses the cell works? 1 Department of Chemistry, University of California, Berkeley, CA 94720, USA 2 In this review, we discuss emerging new possibilities to Chan Zuckerberg Biohub, San Francisco, CA 94158, USA answer these fascinating questions by encoding specific Corresponding author: Xu, Ke ([email protected]) functional information of intracellular microenvironments into certain, in some cases novel, dimensions of the SRM signal, a class of methodologies we collectively refer to as Current Opinion in Chemical Biology 2019, 51:92–97 functional SRM ( f-SRM; Figure 1). Although multifunc- This review comes from a themed issue on Molecular imaging tional and multiparametric fluorescence microscopy, Edited by Philipp Kukura and Sua Myong often enabled by environment-sensitive and chemical- For a complete overview see the Issue and the Editorial responsive fluorescent probes [11–16], long preceded the development of SRM, the relatively low spatial resolution Available online 15th June 2019 (300 nm) achieved with conventional, diffraction-lim- https://doi.org/10.1016/j.cbpa.2019.05.016 ited microscopy limits how localized the microenviron- 1367-5931/ã 2019 Elsevier Ltd. All rights reserved. ments can be probed. As the probed volume scales cubically with the linear dimension, a 10-fold enhance- ment in spatial resolution, which is often achieved in SRM, could reduce the probed volume by 1000 times, hence dramatic reduction of interference from the unde- sired surrounding signal when compared to the desired Introduction local signal. For SMLM-based techniques, the possibility The rapid advances in super-resolution (fluorescence) to map local properties by examining the response of each microscopy (SRM) over the past decade [1,2], including probe molecule one at a time further represent the ulti- single-molecule localization microscopy (SMLM, for mate sensitivity, effectively removing the interferences example STORM [3], (F)PALM [4,5], and PAINT [6]) between different molecules. The outstanding resolution and stimulated emission depletion (STED) microscopy and sensitivity thus offer exciting potential opportunities [7,8], have revolutionized how we perceive the world. to uncover local functional information that would other- The exquisite spatial resolution down to 10 nm, com- wise be averaged out with traditional approaches. Below bined with the inherent benefits of fluorescence micros- we summarize emerging f-SRM efforts that start to dem- copy, in particular, high molecular specificity and good onstrate such possibilities, and we group our discussion by compatibility with live samples, enables exciting discov- how the functional information is optically encoded. eries in biology and beyond [1,2,9]. Fluorescence intensity As implied by its name, the primary aim of SRM has been Fluorescence intensity is conceivably one of the most to improve the attainable spatial resolution, namely, to straightforward parameters for encoding functional Current Opinion in Chemical Biology 2019, 51:92–97 www.sciencedirect.com Functional super-resolution microscopy of the cell Yan, Wang and Xu 93 Figure 1 Figure 2 (a) Viscosity Crowding 0 min 15 min 16 min 70 Charge More + PDGF + H2O2 0 “Functional ” Chemical Super- Structural (b) concentration Resolution information 3 ns Microscopy Enzyme Polarity / activity hydrophobicity 2 ns Protein pH Confocal STED interactions Current Opinion in Chemical Biology Current Opinion in Chemical Biology f-SRM through fluorescence intensity and lifetime detections. (a) STED Functional super-resolution microscopy ( f-SRM) explores the images of EB3–HyPer2 fluorescence intensity change in an NIH 3T3 possibility to transcend the structural (shape) information offered by cell at indicated time points, after stimulation with 10 ng/mL PDGF and existing super-resolution methods and unveil multidimensional subsequent addition of 200 mM H2O2. (b) Comparison of confocal (left) information of diverse intracellular functional parameters, like those and STED (right) fluorescence lifetime images of a fixed Drosophila S2 shown in this diagram, with nanoscale spatial resolution and ultimate cell with Alexa Fluor 594-phalloidin-labeled actin. Scale bars: 2 mm. sensitivity down to single molecules. Panel (a) is adapted from [17 ]. Panel (b) is adapted from [22]. information. Indeed, many fluorescent reporters for chemical imaging are based on fluorescence turn-on photobleaching, which could be significant for the [11,13–16]. strongly illuminated SRM experiments. Mishina et al. [17 ] adopted this strategy to visualize the Fluorescence-lifetime imaging microscopy (FLIM) [19] concertation of H2O2 in live cells with STED SRM. By is a powerful, intensity-insensitive method for probing tagging HyPer2, a fluorescence turn-on fluorescent pro- local environments. The exponential decay rate of fluo- tein (FP) biosensor for H2O2, to the cytoskeleton, STED rescence emission, typically on the time scale of nano- SRM images provided 3-fold enhancement in spatial seconds, depends strongly on both the dye identity and resolution when compared to conventional confocal dye-environment interactions, thus a valuable reporter. microscopy. Treatment of the cell with platelet-derived growth factor (PDGF), as well as with H2O2, led to For SRM, although FLIM has been successfully incorpo- modest and substantial increases in the STED-measured rated with STED [20–23], the focus has been on the HyPer2 intensity (Figure 2a), respectively, attributable to unmixing of different dyes into separate color channels corresponding rises in local intracellular H2O2 concentra- for multi-target SRM [21,23]. Lesoine et al. [22] examined tion. In a more sophisticated approach, Mo et al. [18 ] Alexa Fluor 594-phalloidin-labeled actin cytoskeleton in developed biosensors based on the increased fluctuation fixed cells, and noticed varying local fluorescence lifetime in fluorescence intensity as two FPs were brought into in the STED image (Figure 2b), a result potentially con- proximity, and thus, through stochastic optical fluctuation sistent with varied quenching interactions between the imaging, showed subdiffraction features of protein kinase tagged dye and the local components of the cell. However, A (PKA) activity on the cell plasma membrane. as a rather Gaussian-like distribution was found for the measuredlifetimeatdifferent locations,itwasunclearifthe observed local variations merely reflected statistical error Fluorescence lifetime [22]. Nonetheless, given the wide usage of FLIM for Although the fluorescence intensity is easy to measure, its environment sensing [19], lifetime-resolved SRM stands limitation for reporting functional information is also as a promising direction for future f-SRM efforts. apparent. The detected intensity depends on the local concentration of the fluorescent probe, and so it is difficult Fluorescence polarization to quantify for absolute values. Interpretation of time- Fluorescence polarization and anisotropy measurements dependent signal changes is further complicated by provide information on the orientation and rotational www.sciencedirect.com Current Opinion in Chemical Biology 2019, 51:92–97 94 Molecular imaging mobility of fluorescence molecules [24]. For SRM, polar- was measured as the ratio of single-molecule emission ization-resolved SMLM has been achieved by splitting between two wavelengths centered at 580 and 640 nm. the fluorescence into polarizations parallel and perpen- Interestingly, although the mean values from single-mol- dicular to the excitation laser using