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Pushing the Envelope in Biological Imaging

Research Summary

Eric Betzig develops novel optical imaging tools in an effort to open new windows into molecular, cellular, and neurobiology.

Due to its comparatively benign effect on living systems, optical microscopy has been the workhorse for dynamic studies of structure and function at the cellular level and below for more than 100 years. However, many questions at the forefront of molecular, cellular, and neurobiology remain beyond its current capabilities. At Janelia, I hope to collaborate with scientists and engineers across disciplines to extend these capabilities, to do so in ways that can be readily adopted by biologists, and to apply these methods in the service of my Janelia colleagues. There are at least five areas sorely in need of improvement.

Spatial Resolution
Transfected cells expressing fluorescent proteins contain information on the spatial organization of specific target proteins accurate at the molecular level. However, conventional optical microscopy is limited by diffraction to imaging on a scale (~250 nm) coarser by two orders of magnitude. I have an ongoing collaboration with colleagues nationwide to reach one of the "holy grails" of optical microscopy: a system capable of imaging intracellular proteins with near-molecular resolution. With multiple fluorescent labels, such a system might prove a powerful adjunct to electron microscopy, capable of determining the static structural relationship between two or more proteins of interest at the molecular level.

Temporal Resolution
Of course, a unique benefit of optical microscopy is its ability to study dynamic processes. A second holy grail is therefore to extend spatial resolution significantly beyond the diffraction limit without sacrificing temporal resolution. A key challenge here will be to compensate for the cubically decreasing number of signal-generating molecules within the probe volume as the three-dimensional spatial resolution improves. I have proposed and hope to construct an optical lattice microscope, composed of a massively parallel array of excitation foci, and further hope to apply it to new or existing superresolution methods, such as reversible fluorescence-state saturation and nonlinear structured illumination microscopy. I also intend to characterize the spatiotemporal performance of lattice microscopy, as applied to more conventional diffraction-limited imaging.

Labeling Technology
Another avenue to higher performance distinct from instrumentation is the synthesis of new optical labels having improved photophysical properties: higher saturation threshold; larger excitation cross-sections; narrower spectral linewidths; fewer and shorter intermediate dark states; improved target specificity; unambiguous response to a single environmental parameter (e.g., Ca2+ or pH); and, perhaps most importantly, reduced photobleaching and nonlinear-induced photodamage. I hope to collaborate with theoretical, physical, and biological chemists at Janelia and beyond to identify and refine these probes for application to outstanding biological questions in conjunction with new imaging modalities.

Deep-Tissue Imaging
A further level of complexity is introduced by the desire to image, at high spatiotemporal resolution, both intra- and intercellular processes in living tissues, particularly neural circuits within the brain. I intend to explore a suite of technologies such as multiphoton excitation, time-gated and/or spatial filtered detection, adaptive optics, and tomography—separately and in combination—to extend diffraction-limited imaging further in such optically heterogeneous and highly scattering environments.

Noninvasive, Data-Rich Imaging
A common thread through all this work is the desire to create a densely sampled, multidimensional measurement volume covering a broad span of space, time, and both structural and functional contrast. As more and more information is gleaned from the sample, however, the potential for photodamage, such as bleaching-induced phototoxicity, increases rapidly. It is therefore imperative that the above methods be developed with an eye toward confining the optical field to only the points of interest (such as with optical lattice and/or multiphoton excitation), minimizing parasitic interactions at those points (such as with coherent control of the excitation spectrum and/or rational design of the label's eigenstates), and maximizing the amount of information extracted from the emitted photons.

All of these efforts will be in vain if the resulting techniques cannot be refined to the point where they can be widely and routinely used by biologists having no special affinity for instrumentation. My past experiences in industry have given me a deep appreciation for the huge gulf that exists between proof-of-principle prototypes and technologies meeting this standard. I intend to target only those technologies with at least the potential to reach this level of refinement, and then lean heavily on the unique resources of Janelia, particularly the nascent innovative engineering group, to accelerate the gestation of successful concepts into turnkey instruments. To reach these goals, I also welcome collaborations with physicists, engineers, and commercial vendors having broad product development experience.