Imaging has been enhanced so much over the past ten years that researchers now have the ability to view living cells, and even whole organisms, from the micro- to nano-scale in real time. This quantum leap in visualization possibilities is leading to major advances in our understanding of how cells function and dramatically impacting experimental programs. The improvements provide not just a clearer image, but the ability to access when and where genetically or biochemically defined molecules, signals or processes are formed, transformed and dissipated in space and time. Keeping cell biologists up to date with these cutting-edge developments in molecular imaging is the goal of the Journal of Cell Science's Imaging Article Series. The aim is not only to help researchers navigate among the bewildering array of imaging techniques, but also to provide guidance for selection of optimum approaches for particular research needs. What follows is a brief overview of some of the more exciting developments that will be tracked in this Article Series over time.
The molecule-imaging revolution has grown out of parallel developments in optical probes, imaging strategies, microscope instrumentation and analysis tools. Synergistic relationships in these areas have led to ever-increasing new ways of non-disruptively probing cells and tissue.
The availability of new optical probes, for example, has permitted tagging and localization of an enormous variety of proteins, providing essential information about protein geography and organization. Most prominently, the genetically encoded green fluorescent protein (GFP) has evolved from just a few to more than 100 different varieties, including variants with different spectra, and the ability to sense pH or metabolites, and to photoactivate or reversibly photoswitch. At the same time, the depth penetration of red fluorescent proteins has opened the possibility of greatly enhanced deep-tissue imaging. Moreover, rapid progress in the synthesis and generation of photolabile caged compounds and quantum dots has created additional possibilities, including their use as biosensors or indicators to measure biochemical parameters. Finally, detection of intrinsic signals (autofluorescence and higher harmonic generation) has provided still further ways to interrogate specimens without the introduction of exogenous probes.
Creative imaging techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS) and chromophore-assisted light inactivation (CALI) have been developed to make optimum use of these new optical probes. Together, they provide a way to measure protein diffusion, protein–protein interactions, and protein transport, function and turnover inside cells. The resulting information has revolutionized our understanding of the dynamic spatial organization and function of molecules, molecular assemblies and compartments within cells.
The rapid technological innovations in microscope systems have similarly led to unprecedented imaging possibilities at both the micro- and nano-scale. Nearly all elements of the conventional fluorescent microscope have been modified to allow fast, multispectral imaging at high resolution. This has included modifications that increase the speed of image acquisition (~120 images/second), minimize spectral emission overlap for multispectral imaging or modify the light path. Microscope systems incorporating these modifications include commercial light scanning confocals, spinning disk confocals and wide-field microscopes with total internal reflection. These systems make it relatively straightforward to determine the kinetic properties of proteins and their molecular transactions in different physical locations within single cells by FRAP, FRET or FCS.
Other technological innovations in instrumentation have made it possible to have higher resolution in the study of whole, live organisms. For example, in selective plane illumination microscopy (SPIM), illumination comes from a sheet of laser light 2–8 micrometers thick. This sheet is used to optically section a sample, turned in various directions to illuminate successive planes. In this way, the cells in a whole, intact embryo can be completely imaged, enabling gene and protein expression patterns to be tracked over days. In situ imaging of whole organisms is now also possible with fiber-optic microscopes, the equivalent of having a confocal microscope on the tip of a flexible fiber-optic cable. This makes it possible to explore the properties of cell populations, such as those in the cavities of internal organs or in the pathways of blood capillaries, in the context of the whole organism.
At the opposite end of the scale, unprecedented magnification has been achieved with innovative imaging strategies and microscope systems. Until recently, optical resolution below ~200 nm has been impossible owing to the diffraction limit of light. New super-resolution microscopy techniques have pushed the limits of temporal and spatial detection to ~20 nm. One approach has been to use nonlinear optical strategies to reduce the focal spot size. These illumination-based techniques include stimulated emission depletion (STED) microscopy and saturated structured illumination microscopy (SSIM), with the former typically achieving tenfold higher resolution than conventional fluorescence imaging. Still higher resolution has been achieved with the introduction of probe-based super-resolution techniques that exploit the stochastic activation of fluorescence. These include photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), which rely on photoconvertible fluorescent proteins or dyes, respectively. Structures labeled by an ensemble of photoconvertible molecules too dense to be imaged simultaneously can be resolved with these techniques with nanometric precision through successive imaging of the activated molecules, providing finer spatial resolutions of cellular structures.
Finally, numerous nano-based analysis tools are now available for studying the behavior of molecular components in the complex environment of the living cell. These include microinjection instruments that allow chemicals to be locally applied, laser tweezers that allow cells to be physically manipulated, new transfection reagents and fabricated surfaces on which to grow cells. With such nano-based tools, together with the optical probes, microscope innovations and innovative imaging strategies now available, cell biologists are making inroads into obtaining a fundamental inventory of cell function at many levels of organization. By integrating information from multiple subcellular mechanisms, it is now possible to build a comprehensive, hierarchical framework for integrating the rapidly expanding insights of cell biology.
