COMMENTARYBlackwell
Oxford,Genes
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1356-9597
79September
Blackwell
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Cells
Science,
2002
Science
LtdLtdSpectral imaging fluorescence microscopy
SpectralT
Haraguchi
imaging
et al.fluorescence microscopy

Tokuko Haraguchi1,2, Takeshi Shimi1,2, Takako Koujin1, Noriyo Hashiguchi1 andYasushi Hiraoka1,2,*
1
CREST Research Project, Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku,
Kobe 651-2492, Japan2
Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama,Toyonaka, Osaka 560-0043, JapanThe spectral resolution of fluorescence microscope images in living cells is achieved by using a
confocal laser scanning microscope equipped with grating optics. This capability of temporaland spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; forexample, those associated with fluorescence resonance energy transfer (FRET). Using the spectral
imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescentdyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate(FITC) and green fluorescent protein (GFP).

Since the discovery of the jellyfish green fluorescentprotein (GFP) and its colour variants, multiple wavelength fluorescence imaging has become a useful tool instudies of cell biology. For imaging multiple wavelength
fluorescence images, computer-controlled microscope
systems that automatically switch optical filters duringobservation have been developed (Hiraoka et al. 1991;
Haraguchi et al. 1999). While such microscope systems
can collect fluorescence images at multiple wavelengths,
fluorescence emissions with a spectral overlap can cause
cross-talk between the emitted signals, thus limiting the
choice of fluorescent dyes.
Here we introduce a microscope technique that is
capable of resolving the spectra of fluorescence images
without switching optical filters; the fluorescence
spectra are obtained as a series of images as a functionof fluorescence wavelength. This capability of spectral
resolution makes it possible to unmix the cross-talk between
overlapping fluoresence emissions. It also provides aunique capability for detecting spectral changes influorescent indicators; for example, an indicator for
fluorescence resonance energy transfer (FRET). Suchtechniques have been devised by the use of an acoustooptic tunable filter (AOTF) or a grating (Wachman et al.
1997; Hanley et al. 2000; Tsurui et al. 2000; Ford et al.
2001; Lansford et al. 2001).
*Correspondence: E-mail: yasushi@crl.go.jp© Blackwell Science Limited

The hardware design we used is an implementation of
the previously reported technology of two-photon
imaging spectroscopy (Lansford et al. 2001) to a commercial single-photon confocal scanning microscope;
the technology is applicable to a single-photon or
multiphoton fluorescence microscope. In this spectral
imaging microscope system (LSM510 META; Carl Zeiss,
Jena, Germany), fluorescence spectra are resolved using
grating optics; resolved fluorescence spectra at each pixel
are detected by an array of 32 photomultiplier tubes(PMT) placed at the confocal plane, and recorded on a
pixel-by-pixel basis during scanning to generate a setof images, each corresponding to the fluorescence wavelength resolved at 10 nm intervals. The maximum rangeof wavelength is 380–720 nm, resolved to 32 divisions.

Spectral resolution of fluorescence images
We first demonstrate that the use of grating optics allows
us to obtain multiple-wavelength images without switching optical filters. An example of spectral imaging, or
imaging spectroscopy, is shown in Fig. 1A. A mixtureof four fluorescent beads emitting light of different
wavelengths (515 nm, 560 nm, 605 nm and 645 nm; seelegend to Fig. 1) was observed. Figure 1A shows a setof 20 spectral images ranging from 485 to 688 nm, andFig. 1B shows selected images at 417 nm, 570 nm,
602 nm and 656 nm. Figure 1C shows spectra for eachof the four beads. These beads have distinct spectral
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Figure 1 Spectral imaging. (A) Spectal
images of fluorescent beads. Four different
fluorescent beads of 1 µm diameter weremixed: wavelengths for excitation/emission
were: 490/515 nm, 530/560 nm, 580/
605 nm and 625/645 nm (Molecular Probes, Eugene, OR). Beads were excited by
488 nm and 543 nm light from an argon
laser, and 633 nm light from a helium-neonlaser. A series of images were obtainedon Zeiss LSM510 META using a waterimmersion objective lens (C-Apochromat
40×/NA = 1.2) and a dichroic mirror (HFT
UV/488/543/633). (B) Images of beads
at selected wavelengths. (C) Spectra of thebeads. (D) Colour separation of the four
fluorescent beads.

profiles, and can thus be separated without further
processing. Figure 1D shows a pseudocolour representation of the beads.
This capability of spectral imaging makes it possible toresolve the fluorescence images of dyes that have a large
spectral overlap of fluorescence emission.The fluorescence
spectra of an observed image is a linear combinationof the spectra of multiple fluorescent dyes used to stain
the specimen. By knowing the fluorescence spectra ofeach of the dyes, observed mixed spectra can be unmixed
to each component; this calculation is generally called‘linear unmixing’ (Lansford et al. 2001). Such processingof the observed image on a pixel-by-pixel basis produces
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a set of spectrally resolved images. Figure 2 shows anexample of colour separation of GFP and fluorescein
isothiocyanate (FITC) in fixed HeLa cells. Because thefluorescence of GFP and FITC is separated by only 7 nm
at their emission peak, images of GFP and FITC are
not resolved using a standard filter combination for the
GFP/FITC wavelength. A set of spectral images wereobtained for these cells (Fig. 2A), and the fluorescence
spectra of GFP and FITC measured at the regions
labelled 1 and 2, respectively (Fig. 2B). Using these spectra
as a reference spectrum for each dye, the observed
spectral images were processed to resolve FITC and
GFP images by a linear unmixing algorithm (Fig. 2C).
© Blackwell Science Limited

Spectral imaging fluorescence microscopy

Figure 2 Colour separation of GFP and
FITC. HeLa cells expressing GFP-fused
histone H2B (Kanda et al. 1998) were
fixed with ice-cold methanol for 15 min;microtubules were stained with the anti-α
tubulin antibody TAT1 and FITC-labelled
secondary antibody. The specimen was
excited by 488 nm light from an argon laser.
(A) A series of images were obtained using
a water-immersion objective lens (CApochromat 40×/NA = 1.2) and a dichroic
mirror (HFT488). (B) Regions in theimage that were stained with only one ofthe dyes were selected and the spectrum
measured at this selected region and usedas the reference spectrum for the dye inquestion. (C) Images were processed bya linear unmixing algorithm using thereference spectrum of each dye. The
merged image with pseudocolours is shownin the bottom panel.

Figure 3 shows another example of the colour separationof GFP and yellow fluorescent protein (YFP) in HeLa cells(see legend to Fig. 3 for details).Thus, the linear unmixing
demonstrates a colour separation of fluorescence images
with a large spectral overlap such as GFP and FITC, or
GFP and YFP, as an extreme example.

Spectral images of living cells
Spectral imaging provides an opportunity for rapidly
recording multiple wavelength images with no needfor switching optical filters. This capability is especially
useful in time-lapse observations of living cells. The
spectral imaging of living cells produces a temporal seriesof spectral images, as shown in Fig. 4A. In this example,
the nuclear membrane was stained with lamin B receptor(LBR) protein with cyan fluorescent protein (CFP)(Haraguchi et al. 2000), and protein import into the
nucleus was detected using YFP fused to the protein
STAT1 (signal transducer and activator of transcription).
© Blackwell Science Limited

STAT1 is usually distributed in the cytoplasm, and enters
the nucleus following treatment with γ-interferon (Köster
& Hauser 1999; Sekimoto et al. 1997). This example
demonstrates the spectral and temporal resolution offluorescently stained living cells; the linear unmixing of
the images clearly separated the CFP staining of thenuclear membrane and YFP staining of STAT1 (Fig. 4B).

Detection of spectral changes
The capability of this system for the temporal andspectral resolution of fluorescence microscope images
provides a unique opportunity for detecting spectralchanges of fluorescence in living cells. Figure 5 demonstrates an example of spectral change of a dye inducedby changes in the intracellular calcium concentration in
HeLa cells. In this example, we used a split version ofyellow chameleon-2 (YC2.1) as a FRET-based calcium
indicator dye (Miyawaki et al. 1999). Split YC2.1 generates
FRET by interaction between calmodulin (CaM) andGenes to Cells (2002) 7, 881– 887

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Figure 3 Colour separation of GFP and
YFP. HeLa cells expressing GFP-emerin and
YFP-SUMO1 were excited by 458 nm lightfrom an argon laser. (A) A series of images
were obtained using a water-immersion
objective lens (C-Apochromat 40×/NA =1.2) and a dichroic mirror (HFT488). (B) The
regions in the image that were stained with
only one of the dyes were selected and thefluorescence spectra of each dye measured at
the selected regions were used as a reference
for spectra of each of the dyes. (C) Images
were computationally processed by a linear
unmixing algorithm using the reference
spectrum of each dye; the merged image withpseudocolours is shown in the right panel.

M13 in the presence of Ca2+ (Fig. 5A). To detect such aspectral change associated with FRET, CFP-CaM and
M13-YFP were introduced into living HeLa cells, andthe calcium uptake to the cells was induced by the addition of ionomycin. Images were obtained as a temporalseries of spectral images; fluorescence intensity isdisplayed in Fig. 5B as a pseudocolour representation.
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The fluorescence of YC2.1 changed its spectra shortly
after the addition of ionomycin (Fig. 5B,C). In Fig. 6C,
fluorescence intensities at an exitation wavelength of
484 nm and at 527 nm are plotted as a function of time;
the acceptor signal (at 527 nm) increased and the donor
signal (at 484 nm) decreased following the induction,
indicating that FRET occurred. In spectral imaging,© Blackwell Science Limited

Spectral imaging fluorescence microscopy

Figure 4 Time-lapse spectral images of living
cells. (A) Living HeLa cells expressing CFP
fused with LBR and YFP fused with STAT1.
Cells were excited by 458 nm and 477 nm
light from an argon laser. Images were obtained using an oil-immersion objective lens(Plan-Apochromat 63×/NA = 1.4) and adichroic mirror (HFT488). Live observation
was carried out at 37 °C upto 24 h in 5 minintervals. A set of spectral images at eachtime-point are displayed in each column as afunction of fluorescence wavelength, andcolumns of spectral images are displayed fromleft to right as a function of time. Numbers
at the top of each column represent time inminutes after treatment with γ-interferon.
(B) Selected images computationally processed by linear umixing; 10 min before and
10 min after addition of γ-interferon.

spectral changes can be measured directly at multiple
wavelengths, as shown in Fig. 6A and B, whereas thefluorescence intensity measured conventionally at only
two wavelengths is used to estimate FRET efficiency.
Thus, spectral imaging can take full advantage offluorescent indicator dyes that undergo spectral changes,
and will provide wider applications for such fluorescentindicators.

Concluding remarks
Spectral imaging solves some of the technologicallimitations that complicate multiple-wavelength imaging.
In this manner, efficient multiple colour image acquisition is achieved by the use of grating optics to resolve
fluorescence spectra. Using spectral imaging, it is now© Blackwell Science Limited

possible to detect the spectra of fluorescence microscope
images, which has not been possible before. This
capability is useful for efficiently resolving spectral
cross-talk by linear unmixing computation.
Spectral imaging is best appreciated as a tool for
detecting spectral changes in fluorescence, for example,
those of FRET indicators. This capability will also
eliminate problems associated with autofluorescence.
Fluorescence staining is often interfered with by the
autofluorescence of cells or tissues—especially in plants.
Spectral imaging and linear unmixing can remove autofluorescence by separating its spectra from the spectraof fluorescently stained organelles and cell structuresof interest.
Finally, it should be mentioned that the adventof spectral imaging will provide an impetus for the
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Figure 5 FRET
imagesofyellow
chameleon-2. (A) A split version of YC2.1 iscomposed of two separate portions of CFPtagged calmodulin (CFP-CaM) and YFPtagged M13 (YFP-M13) as a donor and an
acceptor of FRET, respectively (Miyawaki
et al. 1999). The calmodulin portion (CaM)
and the M13 portion interact with eachother in the presence of Ca2+ to generate
FRET. (B) Living HeLa cells expressing
CFP-CaM and YFP-M13 were observed
using a water-immersion objective lens
(C-Apochromat 40×/NA = 1.2) and a dichroic
mirror (HFT488); excitation light of wavelength 458 nm was from an argon laser.
(C) Ratio image of fluorescence intensity at
528 nm to that at 495 nm is displayed for
before and after the addition of ionomycin.
Basically the same results were obtainedusing a joint version of YC2.1 (CFP-CaMM13-YFP) which generates FRET by its
conformational change in the presence ofCa2+ (Miyawaki et al. 1999).

development of a new class of fluorescent dyes. Currently
available fluorescent dyes are not necessarily optimal forspectral imaging. Wavelength selection by optical filters
requires fluorescent dyes with different wavelengths forboth excitation and emission. On the other hand, forspectral imaging, dyes excited by the light of a single
excitation wavelength and emitting light of separate
wavelengths will be more preferable, allowing a single
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laser to excite multiple dyes simultaneously. Dyes thatchange their spectra under certain conditions (e.g.
phosphorylation and dephosphorylation events, molecular interactions or conformational changes of proteins)are also awaited for applications in spectral imaging
to detect changes in the intracellular environment. Thismethodology could therefore prove useful for the realtime detection of biochemical reactions inside single cells.
© Blackwell Science Limited

Spectral imaging fluorescence microscopy
work was supported by a grant from the Japan Science andTechnology Corporation to Y.H.

References

Figure 6 Spectral change of yellow chameleon-2. (A) Fluorescence spectra of split YC2.1 before (open circle) and after(filled circle) ionomycin treatment. (B) FRET spectra, subtracting
fluorescence spectra before ionomycin treatment from that after
ionomycin treatment. (C) Fluorescence intensity at 488 nm and528 nm is plotted as a function of time. Time 0 indicates the
addition of ionomycin.

Acknowledgements
We are grateful to Dr Yoshihiro Yoneda (Osaka University) for
DNA plasmids encoding STAT1, and Dr Atsushi Miyawaki (Brain
Science Institute, RIKEN) for the yellow chameleon dyes. This© Blackwell Science Limited

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