CELL STRUCTURE AND FUNCTION 27: 333–334 (2002)
© 2002 by Japan Society for Cell Biology
PREFACE
Live Cell Imaging: Approaches for Studying Protein Dynamics in Living Cells
Tokuko Haraguchi
CREST Research Project of the Japan Science and Technology Corporation, Kansai Advanced Research
Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan
ABSTRACT. In the last decade, the long-standing biologist’s dream of seeing the molecular events within the
living cell came true. This technological achievement is largely due to the development of fluorescence microscopy
technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed
us to determine the subcellular localization, mobility and transport pathways of specific proteins and even
visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular
dynamics can provide important information about cellular events that cannot be obtained by other methods.
Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and
cellular functions. In this special issue of review articles, we review various imaging technologies of microscope
hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.
molecules can be toxic to the cells. These difficulties have
been solved to make live cell imaging possible (Spector
et al., 1997; Haraguchi et al., 1999). In early days, fluorescence staining of living cells was accomplished by microinjection of purified protein labeled with a fluorescent dye.
This method successfully visualized several fluorescentlytagged proteins such as histone, tubulin and actin in living
embryos of Drosophila (Minden et al., 1989; Hiraoka et al.,
1991; Sullivan et al., 1990). However, applications of this
method were limited by its need for purification of the protein. For this limitation, it was difficult to apply this method
to insoluble proteins such as integral membrane proteins.
The advent of the jellyfish green fluorescent protein (GFP)
made it possible to fluorescently label proteins in living
cells without protein purification. Using GFP, it is now possible to generate fluorescent proteins that could be hardly
purified by biochemical methods. Since then, a wide variety
of GFP variants have been developed (Chalfie et al., 1994;
Heim et al., 1994; Miyawaki et al., 1997; Tsien, 1997;
Miyawaki, this issue), and have been widely used in many
fields of cell biology.
Microscope hardware technology and fluorescence staining technology have mutually stimulated their technological development. A computerized, fluorescence microscope
system capable of recording multiple-color images of living
cells has been developed based on a wide-field microscope
(Hiraoka et al., 1991; Haraguchi et al., 1999; Swedlow and
Platani, this issue). This microscope system is also capable of obtaining high-resolution three-dimensional images
by computational image processing of deconvolution
Physicochemical properties of proteins are mostly examined in a test tube under defined experimental conditions.
These conditions can be well controlled in a uniform
solution, but may not necessarily reflect the situation in
the living cell. In living cells or organisms, proteins are
working in a much more complex environment that involves
many other intracellular molecular components within
structured compartments, and these structures are not solid
ones, but instead are repeating continuous reorganization
during cellular activities. For proper cellular functions, the
right molecules must come to the right place at the right time.
Thus, to understand the functions of the protein in living
cells, it is important to know the properties of the protein
in the temporal and spatial context of the cell. Fluorescence
microscopy is one of the most widely used tools for
localizing proteins in intracellular compartments, with its
advantages for molecular selectivity in imaging and capability
of live observation, allowing us to visualize specific
molecules in living cells.
In the 1980s, the fluorescence imaging of specific proteins in living cells became a practical laboratory approach.
Observation of fluorescently-stained living cells on a microscope stage required special considerations for experimental
conditions such as the control of temperature, pH, nutrition
and other growth conditions. In addition, exposure to the
excitation light used to visualize these fluorescently-stained
Kansai Advanced Research Center, Communications Research Laboratory,
588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan.
Tel: +81–78–969–2241, Fax: +81–78–969–2249
E-mail: tokuko@crl.go.jp
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T. Haraguchi
Haraguchi, T., Shimi, T., Koujin, T., Hashiguchi, N., and Hiraoka, Y.
2002. Spectral imaging for fluorescence microscopy. Genes Cells, 7:
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Heim, R., Prasher, D.C., and Tsien, R.Y. 1994. Wavelength mutations and
posttranslational autoxidation of green fluorescent protein. Proc. Natl.
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Hiraoka, Y., Swedlow, J.R., Paddy, M.R., Agard, D.A., and Sedat, J.W.
1991. Three-dimensional multiple-wavelength fluorescence microscopy
for the structural analysis of biological phenomena. Seminars in Cell
Biology, 2: 153–165.
Hiraoka, Y., Shimi, T., and Haraguchi, T. 2002. Multispectral imaging
fluorescence microscopy for living cells. Cell Struct. Funct., 27:
367–374 (this issue).
Ichihara, A., Tanaami, T., Isozaki, K., Sugiyama, Y., Kosugi, Y.,
Mikuriya, K., Abe, M., and Uemura, I. 1996. High-speed confocal
fluorescence microscopy using a Nipkow scanner with microlenses – For
3-D imaging of fluorescent molecule in real-time. Bioimages, 4: 57–62.
Lansford, R., Bearman, G., and Fraser, S.E. 2001. Resolution of multiple
green fluorescent protein color variants and dyes using two-photon
microscopy and imaging spectroscopy. J. Biomedical Optics, 6: 311–
318.
Minden, J.S., Agard, D.A., Sedat, J.W., and Alberts, B.M. 1989. Direct
cell lineage analysis in Drosophila melanogaster by time-lapse, threedimensional optical microscopy of living embryos. J. Cell Biol., 109:
505–516.
Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura,
M., and Tsien, R.Y. 1997. Fluorescent indicators for Ca2+ based on
green fluorescent proteins and calmodulin. Nature, 388: 882–887.
Miyawaki, A., Griesbeck, O., Heim, R., and Tsien, R.Y. 1999. Dynamic
and quantitative Ca2+ measurements using improved cameleons. Proc.
Natl. Acad. Sci. USA, 96: 2135–2140.
Miyawaki, A. 2002. Green fluorescent protein-like proteins in reef
Anthozoa animals. Cell Struct. Funct., 27: 343–327 (this issue).
Nakano, A. 2002. Spinning-disk confocal microscopy – a cutting-edge
tool for imaging of membrane. Cell Struct. Funct., 27: 349–355 (this
issue).
Paddock, S.W. 2000. Principles and practices of laser scanning confocal
microscopy. Mol. Biotechnol., 16: 127–149.
Sako, Y. and Uyemura, T. 2002. Total internal reflection fluorescence
microscopy for single-molecule imaging in living cells. Cell Struct.
Funct., 27: 357–365 (this issue).
Spector, D.L., Goldman, R.D., and Leinwand, L.A. 1997. Cells: a laboratory manual. Volume 2: Light microscopy and cell structure (Cold
Spring Harbor Laboratory Press, New York) pp. 75.1–75.13.
Sullivan, W., Minden, J.S., and Alberts, B.M. 1990. daughterless-abo-like,
a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development, 110:
311–323.
Swedlow, J.R. and Platani, M. 2002. Live cell imaging using wide-field
microscopy and deconvolution. Cell Struct. Funct., 27: 335–341 (this
issue).
Tokunaga, M., Kitamura, K., Saito, K., Iwane, A.H., and Yanagida T.
1997. Single molecule imaging of fluorophores and enzymatic reactions
achieved by objective-type total internal reflection fluorescence microscopy. Biochem. Biophys. Res. Commun., 235: 47–53.
Tsien, R.Y. 1998. The green fluorescent protein. Annu. Rev. Biochem., 67:
509–544.
(Agard et al., 1989; Fay et al., 1989; Swedlow and Platani,
this issue). Several types of confocal microscope systems
were also developed to obtain high-resolution images in
living cells (reviewed in Paddock, 2000). Compared with
scanning confocal microscopes, spinning-disk confocal
microscopes provide the capability of high-speed image
acquisition, and thus are appropriate for obtaining images
of living cells (Ichihara et al., 1996; Nakano, this issue).
Newly-developed technology of multispectral imaging
using a tubable filter, grating or prism provided a unique
capability of spectral separation of fluorescence images, and
has extended the versatility of fluorescent dyes that can
be used for live cell imaging (Lansford, 2001; Haraguchi
et al., 2002; Hiraoka et al., this issue). Total internal reflection fluorescence microscopy (TIRFM) has made it possible
to detect single fluorescent molecules by significantly
decreasing background fluorescence (Tokunaga et al, 1997;
Sako and Uyemura, this issue).
In addition, the development of several imaging technologies based on fluorescence microscopy has made it possible to examine the dynamic behaviors and interactions in
living cells. Fluorescence resonance energy transfer (FRET)
provides a novel approach for monitoring dynamic proteinprotein interactions in living cells. Fluorescence recovery
after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and fluorescence correlation spectroscopy (FCS) can measure the mobility of fluorescently-labeled proteins in living cells.
This series of reviews introduce powerful new technologies for live cell imaging: time-lapse fluorescence imaging
(Swedlow and Platani, this issue), Nipkow-disc confocal
microscopy (Nakano, this issue), TIRFM (Sako and Uyemura, this issue), spectral imaging (Hiraoka et al., this
issue), and new fluorescent probes (Miyawaki, this issue).
They will highlight current technological achievements and
limitations to provide an insight into the future possibilities
of the imaging technology.
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