Subproject #1 3D Whole-Cell PALM for Use with Genetically Expressed Proteins. The ability to merge fluorescence microscopy with appropriate labeling technologies has proven invaluable for the cell biologist, providing three dimensional views of protein distributions with high contrast and specificity, while minimizing sample perturbation. Despite these advantages, the optical diffraction limit has historically placed a lower bound of 250 nm on the smallest structures that may be resolved with optical wavelengths. A number of optical super-resolution techniques now allow spatial resolutions down to 20 nm while retaining the advantages of fluorescence microscopy. One such technique, photoactivated localization microscopy (PALM (1)), relies on the repeated stochastic photoactivation of single molecules and their subsequent localization over thousands of widefield images to provide 20-30 nm resolution in 2D and sub-100 nm resolution in 3D. PALM builds up superresolution images literally molecule-by-molecule, so maximizing the number of successful localizations is critical for resolving small structures. This procedure depends on successfully isolating the fluorescent signal emitted from a single activated molecule from the potentially much larger sea of background arising from cellular autofluorescence and extraneous activation and excitation of other molecules. For surface bound systems or thin samples existing techniques may be used to limit background, allowing even relatively dim, genetically expressed photoactivatable fluorescent proteins (PA-FPs) to be utilized. These markers are especially useful in super-resolution imaging, as they offer greater specificity and effectively higher labeling densities than brighter, but exogenously-introduced caged dyes. Imaging thicker, three-dimensional samples is problematic, however, as illumination activates and excites the entire sample, increasing background and generally precluding the use of dim PA-FPs. Furthermore, if out-of-focus molecules are not localized, they are wasted, thus decreasing the effective label density and reducing image resolution. Along with collaborators Mike Davidson (Florida State University) and Alipasha Vaziri (Janelia Farm Research Campus), we developed a technique that mitigates these problems, allowing PA-FPs to be successfully utilized for 3D superresolution in cells, exceeding a depth of 8 microns. We reduce background by confining photoactivation to the focal plane via two photon, line-scanning temporal focus activation of the sample. We have also developed model-free, 3D subdiffractive localization software that is far more tolerant of microscope aberrations than previous algorithms. The combination of these two technical innovations has enabled the imaging of a variety of cellular constructs (the nucleus, endoplasmic reticulum, vimentin, and mitochondria), with the genetically expressed protein pa-mCherry1. A paper describing this work has been published in Nature Methods, and the subdiffractive localization code we have developed is open-source, at code.google.com/p/palm3d/ We are in the process of applying the 3D PALM microscope to problems in biology. To that end, we are collaborating with Kumaran Ramamurthi (NCI) in order to perform 3D superresolution imaging of Bacillus subtilis;and Jan Liphardt(UC Berkeley) in order to perform 3D imaging of the nucleus in thymocytes. Subproject #2 Selective Plane Illumination Microscopy for worm neurobiology and embryology Selective plane illumination microscopy (SPIM (2)) is a technique whereby a sample is illuminated with a thin plane of light from the side, so that fluorescence detection occurs in a direction perpendicular to excitation. Such an experimental geometry has major advantages over conventional 3D microscopy techniques, such as confocal microscopy or 2 photon microscopy. First, acquisition speed is greatly increased relative to point-scanning methods, as the entire imaging plane is detected simultaneously. Second, excitation is confined to the focal plane, so each pixel is imaged only once during each volumetric acquisition. This drastically reduces light exposure and results in far lower photobleaching and photodamage than is possible with conventional imaging techniques. These advantages have been applied to studying whole-animal (zebrafish, drosophila) embryogenesis, and to the measurement of calcium transients in tissue slices. We have recently built a SPIM that we intend to use in constructing the first atlas of neuron positions in the developing nematode C. Elegans, in a collaboration with extramural researchers Daniel Colon-Ramos (Yale University) and Zhirong Bao (Memorial Sloan-Kettering Cancer Center). Due to the greatly reduced light dosage applied, we can already image developmental events at 30x the speed of the best available competing technology (spinning disk confocal microscopy), with equivalent signal-to-noise ratio, which should aid in embryo lineaging. Initial results from this collaboration are under review. On the technical side, we are pursuing various instrumental approaches that should allow us to image with better 3D resolution (a weakness of SPIM compared to other technologies) in living samples. We were also awarded an NIH Director's Challenge Award to use SPIM for visualizing very fast volumetric (<10 ms) dynamics in the developing zebrafish brain. Together with collaborator Harold Burgess (NICHD), we aim to induce a startle response in a live zebrafish embryo, and monitor the resulting calcium transients with SPIM. We are currently developing the tools for this experiment. Subproject 3 Structured Illumination Microscopy with a Swept Field Confocal Microscope Besides PALM, structured illumination microscopy (SIM) is another superresolution technique (3) that offers more modest resolution (2x better than the diffraction limit), but is readily compatible with live samples. SIM, while commercially available, is expensive and remains the province of relatively few labs. Together with Chris Combs (NHLBI), we are developing SIM so it can be performed on a swept field microscope. This should allow the technique to be performed by many more labs, and should offer some optical sectioning ability in addition to the resolution enhancement. (1) Betzig, E. et al. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 313, 1642-1645 (2006). (2) Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. &Stelzer, E. H. K. Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science 305, 1007-1009 (2004). (3) Gustafsson, M.G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 198, 82-7 (2000).
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