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com
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Monitoring simultaneous subcellular events in vitro

by means of coherent multiprobe fluorescence
DOUGLAS R. PLYMALE, JEFFREY R. HASKINS & FELIX A. DE LA IGLESIA
Pathology and Experimental Toxicology Department, Parke-Davis Pharmaceutical Research,
2800 Plymouth Road, Ann Arbor, Michigan 48105, USA
Correspondence should be addressed to F.A. de la I.; email: Felix.DeLaIglesia@wl.com

Advances in the development of fluorescent probe technology
have greatly facilitated the analysis of single cellular function
or single-mode microscopy. Existing approaches monitor
single probes or follow specific cellular events over time for the
purposes of dissecting complex molecular dynamics in living
cells1. In addition, fluorescent cellular probes are more fre-
quently used with fixed samples, yielding static information on
processes captured only at the time window when fixation of
cells or tissues had occurred. Fixation results in protein denatu-
© 1999 Nature America Inc. • http://medicine.nature.com
their native state, this approach will reveal relevant new in-
sights into normal, pathologic or toxic processes2.
Coherent multiprobe fluorescence probes and cellular functions
The probes were chosen based on their compatibility, or inher-
ent cell permeability, or whether they are readily internalized by
liver cells. A similar approach could be applied to other cell
types in culture, with a multiple-probe coherent algorithm of
fluorophores constructed to monitor a group of cytoplasmic or

ration, cross-linking and other interactions, with the subse- nuclear events3. Transfection or other similar procedures to in-
quent loss of dynamic signals that can only be captured in ternalize otherwise non-permeant probes cause substantial cel-
living cells. Techniques that would allow a tandem, coherent lular artifacts and have not been explored in this context. Many
approach using multiple probes in living cells would obviously fluorophores are available. However, few probes can be used
be preferable. An ideal system of compatible probes would concurrently, and the organelle and cellular targets that can be
allow simultaneous monitoring of various dynamic functions explored in liver cells limits the choice. Liver cells are ideal sub-
in a manner that was not feasible until recently. Here we jects of study because there is a variety of well-defined cytoplas-
describe a coherent system of fluorescent probes for tracking mic functions and organelle interactions. Many of these
multiple cellular functions in vitro closely spaced within a nar- functions or processes can be monitored by fluorophores with
row sampling period. The term ‘coherent’ applied to this sys- affinity for organelle integrity and disposition, ion transport, cy-
tem represents the systematic, coordinated and integral toskeleton assembly, and related enzyme/cofactor or transcrip-
connections between the probes used, their spectral character- tional activities1. Intracellular functions affected during the
istics and the instrumental mode of application to analyze si- development of toxicity provide convenient, useful targets for
multaneous target organelles or cellular functions with the simultaneous monitoring of multiple organelles (Table). In
acquired images stacked on register. Observing different cellu- the modality described here, a group of organelle responses can
lar events simultaneously within a narrow time window be followed in real time using quantitative fluorescence
increases the ability to discern interrelationships of processes microscopy and advanced digital imaging techniques. The
affecting cellular organelles from animals and humans. We simultaneous use of these fluorophores does not exclude the use
have studied specific organelle functions in vitro using live liver of micro-injected or transfected non-permeant probes in order
cells, a useful model system for observing cellular events in real to address specific functional hypotheses.
time. By analyzing the sequence of intracellular events closer to An example of a coherent multiprobe system that we tested
successfully included the parameters
Table Examples of coherent fluorescent probes for simultaneous monitoring of
of mitochondrial function, calcium
cellular processes
transport and cytoskeletal integrity
Group Cellular function Probes Spectral characteristicsa (Table, Group 1). Mitochondria have
Excitation Emission an important role in the regulatory
1 Intracellular free Ca2+ Fura-2 340/380b 510 pathways of energy supply, detoxifi-
Mitochondrial transmembrane potential MitoTracker Green 490 516 cation and cell survival. Many
Plasma membrane permeability/ Texas-Red phalloidin 591 608 processes leading to cell death are reg-
cytoskeletal Integrity ulated by cytoplasmic organelles,
2 Intracellular free Ca2+ Fura-2 340/380b 510 including mitochondria, which can
Ionic homeostasis BCECFc 450/505b 640/525b generate superoxide anion radicals
3 Ca2+ compartmentalization/distribution Fluo-3 506 526
and hydrogen peroxide4. Reduction
(mitochondrial & cytoplasmic) Rhod-2 552 581
4 Detoxification (glutathione) Monochlorobimane 380 480
of mitochondrial transmembrane
Lysosomal activity FITC-Dextrand 494 518 potential (∆ψm) is an early event, pre-
Mitochondrial transmembrane potential Rhodamine 123 507 529 ceding irreversible apoptotic and
5 Detoxification (glutathione) Monochlorobimane 380 480 necrotic events. Decreased ∆ψm is
Plasma membrane permeability/ FITC-phalloidind 496 516 associated with loss of mitochondrial
cytoskeletal integrity function, leading to disruption of
Mitochondrial transmembrane potential Tetramethylrhodamine 548 573 metabolic activity and lack of cell via-
a
Units in nanometers; bRatiometric probe; c2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF); dFluorescein bility5. Changes in ∆ψm can be mea-
isothiocyanate (FITC) conjugated to either dextran or phalloidin.
sured using cell-permeant, cationic,

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NEW TECHNOLOGY

Fig. 1 Multimode microscopy. The robotic stage auto-

matically scans the specimen in the x, y and z directions.
For this four-chambered slide images are taken from the
sequential fields in one pass (1–16) every 5 min for the
duration of the experiment. At each time point, the filter
wheel rotates through each filter optimally selected for
each probe and acquires an image with the CCD for each
probe in sequence. Inset, Spectral basis for multiprobe
fluorescence assay. The spectral curves at emission ( e) or
excitation (x) correspond to the probes used in the sys-
tem. The overlap of MitoTracker Green (MTe,MTx) with
Fura-2 (F2e) is not relevant to this study because MT
image capture is ahead of F2x, thus eliminating secondary
excitation artifacts. TR, Texas-Red phalloidin.

fluorescent probes that preferentially localize to

mitochondria. Mitochondria maintain electro-
chemical gradients across the inner membrane,
permitting incorporation and retention of these
probes. Thus, fluorescence decrements indicate
reduced or negative ∆ψm and, therefore, are a sig-
© 1999 Nature America Inc. • http://medicine.nature.com

nal of mitochondrial dysfunction6,7. For our initial

coherent, multiprobe combination, we used
MitoTracker to label mitochondria and followed ∆ψm changes.
This probe is similar to other ∆ψm-sensitive probes8,9, except that
MitoTracker has higher quantum efficiency, good photostabil-
ity and uneventful internalization, as well as retention by
healthy cells10.
Calcium is a chief regulator of cellular homeostasis, intracel-
lular messaging and cellular toxicity involving a variety of cellu-
lar and signaling processes as free cytosolic calcium11,12. The
intracellular traffic of free calcium ions can be monitored using
calcium-specific fluorescent probes. In a coherent multiprobe
real-time capture of fluorescence approach, we used the
calcium-binding probe Fura-2 (ref. 13).
The integrity of the cytoskeleton plays an active part in chief
cellular events. Components of the cytoskeleton coordinate cell
kinesis and cell shape, intracellular organelle trafficking, and
processes leading to cell division and cell death. Actin is a com-
ponent of the cytoskeleton, and the structure of the actin fila-
λ (nm)
ment network can be visualized using fluorescent-conjugated
probes in single-mode microscopy. For this purpose, we used
Texas Red-conjugated phalloidin, a cellular poison that binds to
polymerized actin and prevents the cycling of actin subunits14,15.
Phalloidins are normally cell-impermeant but injured hepato-
cytes internalize sufficient dye conjugate to allow discrete
visualization of the reticular architecture.
Multimode microscopy and coherent fluorescence analysis
The ability to monitor multiple cellular functions in real time is
predicated on a new generation of advanced light microscope
systems. The multimode microscope was developed initially at
the Center for Light Microscope Imaging and Biotechnology,
Carnegie Mellon University16. Multimode microscopy allows
the automated, rapid and sequential application of different
optical microscopy modes, including epifluorescence and differ-
ential interference contrast (DIC). The multimode microscope is

a Single probes
c Single probes
Average field intensity

Fig. 2 Photobleaching and

probe stability. To evaluate probe
stability in response to maximal
photodosage conditions, we cap-
tured sequential images from he-
patocytes loaded individually with
Fura-2, MitoTracker Green, or
b Coherent probes d Coherent probes Texas-Red phalloidin (a) or a co-
herent combination of the probes
(b). To evaluate photobleaching
Average field intensity

and probe stability over the in-

tended period of image capture,
we monitored cultures loaded
with the individual probes (c) or
the coherent combination (d)
over 2 h, collecting image sets
every 5 min. Average field inten-
sity values (± s.e.m.) of labeled
Exposure sequence Time (min) hepatocytes are shown.

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Validation of coherent multiprobe fluorescence assays

To use multiple probes simultaneously, we first had to define the
responses of each probe individually and under comparable ex-
perimental conditions. The set of conditions thus obtained pro-
vided the starting point for determining the effective sequential
∆ from t=0 min (%)

paradigm for the coherent multiple fluorescent probe combina-

Time (min.)
Fig. 3 Quantitation of multiprobe fluorescence in hepatocytes treated
with tacrine. Timecourse of near-simultaneous changes in fluorescence
from a multiprobe assay using rat hepatocytes with 1 mM tacrine in the
media. The image capture sequence at any time point is described in the
© 1999 Nature America Inc. • http://medicine.nature.com
tions. The first step defined optimization parameters such as dye
loading times, photobleaching and dye leakage over the time
and intensity of photoexposures for each individual probe.
These parameters were evaluated by loading isolated rat or
human hepatocytes with a single probe and applying the tools of
multimode microscopy in the same way as with the coherent
multiple probe setup. Preliminary experiments identified effec-
tive concentrations of Texas-Red phalloidin, MitoTracker Green
and Fura-2 that were not cytotoxic and gave adequate quantum
fluorescent responses, generally greater than 75% over the linear
range of the charge-coupled device (CCD) but not sufficient to
generate interfering autofluoroscence. To evaluate photobleach-
ing and probe stability over the intended period of image cap-
ture, multiple fields within cultures loaded with individual

text. Plasma membrane and cytoskeleton are tracked by Texas-Red phal-
loidin, mitochondrial transmembrane potential by MitoTracker Green and
intracellular calcium by Fura-2.
equipped with servos controlled by interfacing software linked
to a computer equipped with image capture and analysis pro-
grams. The mechanical, electrical, digital and optical compo-
nents of the multimode microscope have been optimized for
biological applications8 with further adaptations in our labora-
tory, including environmental control of cell preparations nec-
essary for the coherent multiprobe fluorescence system. The
robotic stage allows parallel tracking of multiple fields, both
within a single chamber and in multiple sample matrices such
as multiple-well chamber slides or plates (Fig. 1). Hence, this
multimode microscopy approach addresses inter- and intra-
sample variability and coherent corrections needed while con-
ducting these experiments.
probes were monitored over 2 h, collecting image sets every
5 min. To evaluate probe stability in response to maximal photo-
dosage conditions, sequential images were captured from hepa-
tocytes loaded with individual probes. All three dyes were stable
over the monitoring period and did not demonstrate significant
degradation based on the extent of photoexposure (Fig. 2). There
was also no evidence of probe-induced phototoxicity in the
experimental conditions described here.
We next evaluated multiple fluorophore interactions. Multiple
fields in cultures loaded with the probe combination were moni-
tored through acquisition of several multiprobe image sets. A
multiprobe image set is composed of a series of images captured
from each mode at each single time point. Parameters considered
when co-loading multiple probes included the loading sequence,
the loading interval for each probe, probe leakage, photobleach-
ing, emission-generated secondary excitation and residual phos-
phorescence. The spectral emission characteristics were also

Fig. 4 Multiprobe fluo-

rescence analysis. The
a b c d
power of the multimode
microscope is demon-
strated by analysis of
information from multiple
acquisition modes within a
single time series of a sin-
gle sample. For example,
images from Texas-Red
phalloidin (a and b),
MitoTracker Green (c and e f g h
d) and Fura-2 (e and f)
acquisition modes can
each be displayed in a
single color channel and
then coherently super-
imposed to create a
multicolor complemen-
tary image (g and h). This
allows spatial resolution
of the information on register from multiple fluorescence acquisition ial fluorescence (d) and moderate cytosolic calcium (f). White asterisks
modes. Analysis of images from the combined acquisition modes over indicate a group of altered cells with heavy accumulation of Texas-Red
time demonstrates the interrelationships of multiple cellular functions. phalloidin (b), little or no green (d) or blue (f), indicating significant cell
Control cells (a,c,e,g); tacrine-treated liver cells (b,d,f,h) were 1 h into injury or death. A comparison of the complementary images (g and h)
the 2-h exposure period. White arrows (right column) indicate a nor- shows the extent of loss of activity in the treated hepatocytes (h) com-
mal cell with no uptake of Texas Red phalloidin (b), green mitochondr- pared with the control (g).

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NEW TECHNOLOGY

Mitochondrial transmembrane potential, as

followed by MitoTracker Green fluorescence,
increased over the first 35 min and gradually
DIC decreased over the subsequent 60 min.
Membrane permeability (Texas-Red phal-
loidin) increased linearly within the same
35 min and remained essentially unchanged
for the subsequent 60 min. Calcium traffic,
tagged by Fura-2, showed progressive accumu-
lation in cells in parallel with the cell perme-
ability changes. At the end of the 2-h
experiment, some cells showed prominent
changes (Fig. 4), with appreciable loss of
Ca 2+ cytoskeletal integrity (Fig. 4b), loss of mito-
chondrial membrane potential (Fig. 4d) and
eventual loss of the excessively internalized
calcium. Viable cells showed no accumulation
of Texas-Red phalloidin, green mitochondrial
fluorescence and moderate cytosolic calcium.
Fig. 5 Time-lapse images of hepatocytes treated with melittin. Melittin (final concentration,
These results agree with laser-confocal
2 µM) was added to primary hepatocyte cultures loaded with Texas-Red phalloidin,
© 1999 Nature America Inc. • http://medicine.nature.com

cytometry results obtained with rhodamine

MitoTracker Green and Fura-2. Each timed set (5′, 25′ and 50′) shows paired images of hepa-
tocytes corresponding to transmitted DIC (top) and calcium-sensitive (bottom) wavelengths. 123 as a monoprobe for mitochondrial trans-
Arrows indicate cells for which substantial probe uptake precedes loss of viability. membrane potential7.
In hepatocytes exposed to melittin, the
plasma membrane lost integrity and the cy-
considered for compatibility. These parameters were evaluated toskeleton underwent extensive changes as the cells detached
with the multiprobe combination of Texas-Red phalloidin, from the plate. From the DIC images acquired over the 2-h time
MitoTracker Green and Fura-2. When loaded simultaneously, period, melittin caused substantial and extensive blebbing of the
the three probes were photostable over 2 h and at maximal con- plasma membrane in most cells examined (Fig. 5). The
ditions of maximal photodosage (Fig. 2). As with the individual multimode microscope facilitated the visualization of events
probes, phototoxicity was not seen when the probes were evalu- within the visible range in real time using DIC coupled with a
ated in combination. This robust protocol showed no single channel of fluorescence, which added a complementary
probe–probe interactions that substantially affected the spectral level of image information. Analysis of coherent images from the
properties of the individual probes. Fura-2 acquisition mode corresponding to the same sampling
times as the DIC image mode demonstrated that blebs contained
Coherent multiprobe fluorescence applications in hepatocytes cytoplasmic components, including calcium, and were released
After optimizing the multiple fluorophore combination of into the media. This observation indicates that high anion
Texas-Red phalloidin, MitoTracker Green and Fura-2, targeting concentrations were actively compartmentalized and reduced by
actin filaments, mitochondrial transmembrane potential and the cell in order to maintain homeostasis. The pinching-off
intracellular calcium, respectively, we used this coherent multi- process provides evidence of a mechanism whereby extempora-
probe fluorescence approach to monitor cytotoxic effects in live, neous membrane-bound cytoplasmic fragments containing en-
isolated hepatocytes. zymes are released into the media, an event that could explain
To exemplify this approach, we used three agents to study enzyme elevations in vivo. From the corresponding images
effects caused by chemical intervention. The first was tacrine acquired with the Texas-Red phalloidin acquisition mode, sub-
(1,2,3,4-tetrahydro-9-aminoacridine hydrochloride mono- stantial amounts of the dye conjugate were taken up and cells be-
hydrate), a cholinesterase inhibitor drug for the treatment of came considerably brighter, indicating profoundly increased cell
Alzheimer's disease. This drug induces release of cytoplasmic permeability (Fig. 5). Therefore, the dynamic range of the cap-
enzymes in hepatocytes in vitro and causes mitochondrial tured fluorescence represented an expression of cells progressing
dysfunction7. The second agent was melittin, a highly lytic towards death.
peptide derived from bee venom that binds to calmodulin17.
The final agent was human recombinant transforming growth Coherent fluorescent multiprobes: Future directions
factor β1 (TGF-β1), which induces apoptosis in cultured hepato- We have demonstrated the feasibility of coherent multiprobe
cytes18. Only results from trials with tacrine and melittin are fluorescence microscopy with a system of three cellular functions
described here. tracked simultaneously in live liver cells. This approach can be
In hepatocytes labeled with multiple probes and analyzed in extended by using other fluorophore combinations, increasing
real time, information from single acquisition modes was the number of probes or, ultimately, by applying more sensitive,
examined separately or collectively. Certain toxicants affect rapid and higher-throughput capacity systems for monitoring
cellular events at different times after exposure and require par- several more cell functions. It is possible that with improved sta-
ticipation of subcellular organelles either sequentially or some- bility of primary cell cultures and longer observation periods that
times concomitantly19,20. The timecourse of functional changes irreversible pathways or even compensatory or proliferative
in liver cells exposed to 1 mM tacrine for 2 h and using a responses will be recognized. Further probe combinations or a
coherent probe group (Group 1, Table) is shown in Fig. 3. larger number of coherent probes can be evaluated in a similar

354 NATURE MEDICINE • VOLUME 5 • NUMBER 3 • MARCH 1999

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manner and used to study interrelationships of different cellular
constituents and processes. The feasibility of these improvements
relies not only on an increased number of probes or a larger set of
fluorophores but also on advances in the hardware components.
We are now incorporating spectral analysis into the coherent
multiprobe approach by means of a Sagnac interferometer. The
application of spectral analysis in this setup will increase the
resolution of emission peaks separated by as little as 10–20 nm,
which will avoid the spectral overlap caused by wide bandpass
effects21. This approach will also allow the development of a larger
number of multiple coherent probes. A hardware limitation of
the multimode microscope is the time delay caused by the
mechanical positioning of the filters in the microscope beam
path. This limitation can be obviated by replacing the glass filters
and carriers with acousto-optical tunable filters with resulting
increased speed of the system22. This continuous solid-state
tunability will permit switching between multiple fluorescence
modes within milliseconds or less, allowing examination of
events at closer time intervals than the current system could
possibly attain. These increased efficiencies will facilitate moni-
© 1999 Nature America Inc. • http://medicine.nature.com
toring functional probes for enzyme inhibition or induction,
membrane or organelle synthesis and cytoplasmic motion.
Methods
Multimode microscopy. We used a microscope system modified from
that previously described8 (Fig. 1). The multimode microscope used here
consists of a robotic stage fitted with a controlled environmental chamber,
a series of dichroic filter sets housed within a motorized carrier, supple-
mental high-speed fluorescence and neutral density filter wheels, a high-
sensitivity CCD camera (Photometrics 12-bit Series 300 CH250, Tucson,
Arizona) and a video-rate camera (C2400 CCD; Hamamatsu, Hamamatsu
City, Japan), all attached to an inverted microscope (Zeiss Axiovert 135)
equipped with 100-W mercury and halogen light sources and high-
numerical aperture objective lenses.
Primary liver cell isolation and culture. Primary hepatocyte cultures were
prepared from rat livers perfused with collagenase as described7.
Hepatocytes were purified by differential centrifugation through a Percoll
gradient, washed and suspended in hepatocyte culture medium (HCM):
Leibovitz-15 with L-glutamine (Life Technologies) supplemented with
7.5% BSA (Life Technologies), 1% penicillin/streptomycin, 3 mg/ml pro-
line, 50 mg/ml galactose, 0.1% insulin-transferrin-selectin (ITS;
Collaborative Biomedical Products, Bedford, Massachusetts), 0.4 mg/ml
dexamethasone (Sigma), 8.4% sodium bicarbonate, and 0.1% trace ele-
ments (CuSO4, Fe(NO3)3, ZnSO4 and MnCl2). Hepatocyte viability was as-
sessed by Trypan Blue exclusion. Hepatocytes were plated into
four-chambered glass slides to a density of approximately 75,000 cells per
cm2, and incubated overnight at 37 °C in an environment of 5% CO2. Fresh
media was added to each chamber before the cells were exposed to the
fluorescence probe coherent combination.
Fluorescent probe loading. The fluorescent probes were loaded by
‘spiking’ each chamber with an appropriate volume of probe stock
solution. A 1-mM stock solution of Fura-2 and a 1-µM stock solution of
MitoTracker Green were prepared in DMSO. A stock solution of Texas-
Red-conjugated phalloidin (200 unit per ml) was prepared in methanol.
For single-probe experiments, media with the final probe concentration
was added to the culture chamber. After incubation at 37 °C, the cells were
washed once with fresh media and 0.5 ml was added to each chamber.
Preliminary experiments identified optimal probe concentrations and
loading times (not shown). For Fig. 4, Fura-2 was loaded for 30 min at a
final concentration of 5 µM, MitoTracker Green was loaded for
15 min at a final concentration of 5 nM, and 1 unit of Texas-Red phalloidin
per ml of media was loaded for 15 min. For the coherent probe combina-
tions, MitoTracker Green and Texas-Red phalloidin were added 15 min
after the addition of Fura-2. All other parameters and steps were identical
to those just described for single-probe loading.
NATURE MEDICINE • VOLUME 5 • NUMBER 3 • MARCH 1999
NEW TECHNOLOGY
Image acquisition and analysis. Under multimode microscopy, digital im-
ages were captured with sequential exposures of 0.05 s, 0.2 s, 1.0 s and 1.0
s for Texas-Red phalloidin, MitoTracker Green, Fura-2 (380 nm) and Fura-2
(340 nm), respectively. Illumination was provided by a 100-W mercury
lamp and 560/645, 480/530, 340 or 380/510 excitation/emission filters
for the same sequence of probes. These exposures consistently saturated
greater than 75% of the linear range of the 12-bit CCD detector. In coher-
ent labeling experiments, the order of acquisition for discrete field images
was transmitted/DIC, Texas-Red phalloidin, MitoTracker Green and Fura-2
at 340 nm and then at 380 nm (Fig. 1). A series of images using this filter
combination was sequentially acquired at each stage location every 5 min
over 2 h using the high-resolution, liquid-cooled CCD camera mounted on
the multimode microscope. Four fields from each chamber of a four-cham-
ber slide were photographed a total of 25 times in a single run, generating
400 images over the 2-h sampling period. For digital image analysis, the
average pixel intensities from the discrete fields were calculated using the
raw 12-bit images as sources and applying image analysis software (Media
Cybernetics, Silver Spring, Maryland).
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CORRECTION
In the New Technology article “Gene expression profiles of laser-
captured adjacent neuronal subtypes,” which appeared in the
January issue (Nature Medicine 5, 120; 1999), a company name
was misspelled. The correct spelling is Arcturus Engineering.
355