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Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5, 159–161 (2008). https://doi.org/10.1038/nmeth1171

Three-dimensional image of the nuclear pore complexes (NPCs) in a cell reconstructed from z-scanned serial images (see Fig. 1c and Supplementary Fig. 2). A 3D image of the NPCs on the nuclear envelope was reconstructed from z-scanned serial images. Although deconvolution was not used, clear point-like images of NPCs were obtained. The distribution of NPCs is not uniform and they are often in line. The larger the depth z, the darker the image. This is because of the spherical aberration of the objective. Photobleaching was linearly corrected using to-and-fro scanned images. The depth z was corrected for refraction at the coverslip-specimen surface (see Supplementary Methods). Digitonin-permeabilized cells were incubated with 100 nM GFP-importin β, 1 μM RanGDP and energy sources. Scale bar, 5.0 μm. (MOV 2473 kb)

We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.

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It is our intention to lead with transparency and accountability, to build relationships and trust with members of the community who have been left out of the historical narrative. Our goal is for the Springfield History Museum to offer a more inclusive representation of all the people who have both shaped and been shaped by Springfield and rural east Lane County. For these people to be empowered as stewards of their owns stories, and to entrust the Springfield History Museum with the preservation, documentation, interpretation and display of their stories through exhibits, archives and digital collections.

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The culminating works from this project will all become part of the Springfield History Museum archive, available to the public. Selected images and narratives will also become part of the permanent heritage exhibit on display at the museum.

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M.T. devised and performed microscopy, analysis, kinetics studies and all experiments except biological specimen preparation, and wrote the paper; N.I. contributed biological materials and designed transport experiments; K.S.-S. contributed to image analysis, 3D experiments and 3D microscopy.

Department of Genetics, School of Life Science, The Graduate University for Advanced Studies, Mishima, 411-8540, Shizuoka, Japan

The land we call Springfield today is home to more than 60,000 people, all of whom have unique personal and ancestral stories that contribute to our collective history, and our rich, multicultural present.

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We use interviews and photographs to share stories that illuminate the intersectionality of our Springfield identity. This year our skilled team includes Kunu Bearchum (Northern Cheyenne, Ho-Chunk), Princess Mason (Klamath, Western Shoshone) and Megan England (Siletz) who are working with the community to explore the American Indian and Alaska Native experience in the land we now call Springfield and rural east Lane County, Oregon. The team is supported by exhibit advisor Dr. David Lewis (Grand Ronde).This exhibit will open in December, 2024.

We thank K. Shinkura and K. Takada for assistance, M. Hiroshima, S. Kose, Y. Ue and K. Ebe for technical help, S. Goto and K. Kinosita, Jr. for discussions, and R. Triendl and C. Rowthorn for critical reading of the manuscript. This work was supported by Dynamic Biology Project of New Energy and Industrial Technology Development Organization (M.T.), the Toray Science Foundation (M.T.), Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (M.T., N.I., K.S.-S.), and the Advanced and Innovational Research Program of MEXT (M.T.).

ILLUMINATION invites us to discover more about our neighbors, our forebears, and perhaps a little more about ourselves along the way…

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Single GFP-importin β molecules mediating the cargo transport at the bottom of a nucleus (see Fig. 3a). Single molecules were visualized on the bottom surface of a nucleus at video rate. The fluorescent spots are the images of single molecules of GFP-importin β, which are observed only when GFP-importin β molecules are bound to and interacting with NPCs, because, when dissociated, they are under vigorous Brownian motion and are also away from the focus plane. Occasionally, lateral rapid movement of single molecules from one NPC to an adjacent NPC was observed. Images were averaged over 16 frames (0.5 s). Digitonin-permeabilized cells were incubated with 0.5 nM GFP-importin β, 10 nM importin β, 1 μM MBP-IBB (cargo), 700 nM RanGDP and energy sources. Scale bar, 5.0 μm. (MOV 1558 kb)

Single molecule imaging of microinjected GFP-importin β mediating the cargo transport in a living cell. First 0.33 s) Brightfield images of the tip of a glass microneedle (upper left) and a nucleus at which microinjection was aimed. The black stain at the lower center is a blot of brightfield optics. Next 2.33 s) Fluorescence images at the moment of microinjection a few μm above the bottom of the nucleus. 3 μM GFP-importin β in PBS containing 0.7% BSA was microinjected into the cytoplasm at the upper left. Next 4.60 s) Single molecule images of GFP-importin β 1 min 40 s to 48 s after microinjection at the bottom of the nucleus. Single molecules were visualized as bright spots. Last 0.23 s) Fluorescence images at 7 min and 29 s after microinjection at 2 μm above the bottom surface of the nucleus. GFP-importin β did not accumulate in the nucleus, whereas GST-NLS-GFP did. Images were averaged over 8 frames (0.27 s). Scale bar, 5.0 μm. (MOV 2513 kb)

The Springfield History Museum is actively working to address representation in our collection. Museum institutions have historically used gatekeeping and erasure in collection practices. We are working to address this harm with projects and special exhibits like ILLUMINATION, that intentionally center the stories and experiences of those who have been historically marginalized and who are currently underrepresented in our collection.