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Highly inclined thin illumination enables clear single-molecule imaging in cells

An Addendum to this article was published on 01 May 2008


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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Figure 1: HILO microscopy for molecular imaging in cells and three-dimensional imaging of the NPCs.
Figure 2: Thickness of HILO illumination beam and signal/background ratio.
Figure 3: Visualization of single GFP–importin β molecules and nuclear pores, and a kinetic analysis of the interactions.


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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.).

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Authors and Affiliations



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.

Corresponding author

Correspondence to Makio Tokunaga.

Supplementary information

Supplementary Text and Figures

Supplementary Figures (PDF 1967 kb)

Supplementary Video 1

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)

Supplementary Video 2

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)

Supplementary Video 3

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)

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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).

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