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Biophys J, January 2000, p. 451-457, Vol. 78, No. 1
§ and
*Division of Medicine, Imperial College School of Medicine, Medical
Research Council Clinical Sciences Centre, Hammersmith Campus,
Du Cane Road, London W12 0NN, Division of
National Heart and Lung Institute, Imperial College School of Medicine,
Charing Cross Campus, St. Dunstan's Road, London W6 8RP,
Department of Anatomy and Developmental Biology, St.
George's Hospital Medical School (University of London), Cranmer
Terrace, London SW17 0RE, and §Office of Naval
Research International Field Office, 223 Old Marylebone Road,
London NW1 5TH, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Summary
REFERENCES
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We report a novel scanning ion conductance microscopy
(SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in
cell volume while retaining the cell functionality. The technique can
measure a wide range of volumes from 10
19 to
109 liter. The cell volume, as well as the volume of
small cellular structures such as lamelopodia, dendrites, processes, or
microvilli, can be measured with the 2.5 × 1020
liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed
during relatively long experiments. The SICM method can also be used
for rapid estimation of the changes in cell volume. These are important
when monitoring the cell responses to different physiological stimuli.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Summary
REFERENCES
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Regulation of cell volume is a fundamental
cellular homeostatic mechanism. Most cells are able to regulate their
volume. Normal cellular functions, such as secretion, ion transport
(the movement of ions is followed by a corresponding movement of
water), and a cell's adaptation to a changing osmotic environment,
often require complex reorganization of cell shape and volume (Hallows
et al., 1991
; Swanson et al., 1991; Hoffmann et al.,
1993; Alvarez-Leefmans et al., 1994; Valverde et al., 1996). To
investigate the physiology and pathology associated with cell volume
regulation, it is important to use an appropriate technique that allows
quantitative, high-resolution characterization of cell volume while
retaining the cell functionality. Currently, there are two major
approaches to assess cell volume. First, one that permits measurements
of relative changes in cell volume, and a second, that allows actual
measurements of cell volume. The most commonly used techniques of the
first approach are based on continuous monitoring of the intracellular
concentration of loaded reagents (fluorescent dyes or ions). The
concentration of these intracellular markers depends on the amount of
cell water, and thus changes when cell water volume is altered. Thus,
by continuous monitoring of the intracellular concentration of these
reagents/ions using quantitative fluorescence microscopy (Lee, 1989;
Crowe et al., 1995) or ion-sensitive microelectrodes (Alvarez-Leefmans et al., 1992), the relative changes in cell water volume can be measured. In some experimental condition, the relative changes in cell
volume can be assessed by a simple electrophysiological method for
continuous measurement of cell height (Kawahara et al., 1994). The
method is well suited for studying cell volume in cell monolayers where
cell expansion is mainly possible in the vertical direction. In this
method, a tip of a glass micropipette is placed on the cell surface so
that ion flow to the micropipette is partially restricted. Any changes
in cell volume induce changes in cell height that consequently
influence ion flow. Registering changes in ion flow allows estimation
of changes in cell height and volume.
The second approach for investigating cell volume includes methods that
allow actual measurements of cell volume. Impedance and
light-microscopy are commonly used methods. The impedance method allows
fast estimation of volume of a large number of cells (e.g., Nakahari et
al., 1990). However, the technique is limited to certain experimental
conditions, because it is only applicable to cells of nearly spherical
shape in suspension. Light microscopy methods, such as
video-enhanced contrast optical microscopy (Nakahari et al., 1990),
light microscopy with spatial filtering (Farinas et al., 1997), and
laser light-scattering system (McManus et al., 1993) have been used to
estimate cell shape and, consequently, cell volume. Some optical
microscopy methods have a good temporal resolution, and changes in cell
volume can be estimated in submillisecond scale (e.g., Meinild et al.,
1998). However, generally, these methods have limited spatial
resolution, and the cell plasma membrane cannot always be clearly
visualized. Currently, one of the most advanced ways of estimating cell
volume is by scanning laser confocal microscopy (SLCM). Image sets
consisting of very thin, serial optical sections across the cell can be
obtained and a three-dimensional model of an individual cell
constructed using digital image processing techniques (Guilak, 1994;
Zhu et al., 1994; Errington et al., 1997). However, even this method
has limitations. Photodynamic damage to the biological sample
remains a serious problem of SLCM (e.g., Saito et al., 1998). Special
requirements are needed for specimen preparation. Sample transparency
or specific experimental conditions when cell growth requires a special
substrate (e.g., a membrane filter) can limit SLCM resolution. The
method becomes more convoluted when long-duration observations of
living cell shapes are necessary.
New techniques, such as scanning probe microscopy (SPM)
(Binnig and Rohrer, 1982; Binnig et al., 1986; Hansma et al., 1989; Bard et al., 1991), have showed great potential for studying different samples, in some cases with atomic resolution. They also have proved to
be capable of producing high-resolution images of biological samples
and cell surfaces and providing important information on their
functions (Arkawa et al., 1992; Hansma and Hoh, 1994; Henderson et al.,
1992; Radmacher et al., 1992; Schoenenberger and Hoh, 1994). New
generations of SPM will provide ever-improving tools to study
biological samples. One of these, scanning ion conductance microscopy
(SICM), is capable of high-resolution imaging of living cells, and
direct visualization of changes in cell architecture (Korchev et al.,
1997a,b). We now report a new SICM technique for assessing cell volume,
including the volume of membrane surface protrusions.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Summary
REFERENCES
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Imaging system
A scanning ion conductance microscope
The sensitive probe of the SICM is a glass micropipette filled with electrolyte, which is connected to a high-impedance, head-stage current amplifier and mounted on a computer-controlled three-axis translation stage. The control electronics drive the translation stage to scan the specimen under the micropipette probe. The probe tip's position, in relation to the sample surface, strongly influences the ion current through the pipette
the ion current
declines as the tip-sample separation diminishes. The ion current
provides a signal for the feedback loop, which controls the vertical
axis of the positioning system and ensures that the sample and probe do
not make contact. To scan the sample, we have used a three-axis piezo
translation stage (Tritor 100, Piezosystem, Jena, Germany) with
100-µm travel distance in x, y, and
z directions. As a piezo driver, we have used a high-voltage
amplifier with high current output (System ENV 150, Piezosystem). The
control/data acquisition hardware and software are produced by East
Coast Scientific (Cambridge, England). The electronics consist of a
decoder, four digital-to-analog converters, and two analog-to-digital
converters. The DSP card (DSP32C PC, Loughborough Sound Images plc,
Loughborough, England) of a PC functions as a front-end controller and
provides digital feedback and scan control. The basic arrangement of
the SICM was described elsewhere (Korchev et al., 1997b).
The micropipettes were made from 1.00 mm outer diameter, 0.58 mm inner diameter glass microcapillaries (Clark Reading, Electromedical Instruments, UK) on a laser-based Brown-Flaming puller (model P-2000, Sutter Instrument Company, San Rafael, CA). We estimated the
tip radius of the micropipettes used in the present series of
experiments to be ~50 nm, and the cone angle of the tip to be
~1.5° (scanning electron microscopy data not shown). The
micropipettes and the bath were filled with the same solutions, usually
physiological or growth media. The measured micropipette resistance was
usually ~500 M
. The samples were usually placed on petri dishes,
glass coverslips, or membrane filters and imaged in the appropriate medium.
Cell volume calculation
Previously, we have demonstrated that SICM is extremely well suited for imaging living cell surfaces in physiological or growth media (Korchev et al., 1997a). Cell imaging with SICM involves raster
scanning. That is, the SICM image consists of a three-dimensional map
of the apical cell surface with a limited number of points, n, where each raster scan point represents a cell height.
Before assessing cell volume using the SICM, we have to measure the
vertical position (ZREF) of the probe
tip on the substrate where cells were grown (Fig.
1 A). This allows us to
calculate the real cell height
z(x, y) by subtracting
ZREF from the measured height value (Z(x,y)). This is
especially important when imaging a cluster of cells, and the acquired
image does not include a reference point to the substrate. Assuming
that basal cell membrane has a close contact with the substrate (e.g.,
glass coverslip, porous membrane) a cell volume
(VCell) can be estimated by
|
(1) |
5 µ3
(2.5 × 1020 liter) resolution. The
z(x, y) and
VCell values can be assessed with less
than 0.2% error.
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Calculation of surface characteristics
The cell surface area (SCell) can be calculated as a sum of areas (STriangle) of each triangle formed on the cell surface with coordinates of three adjacent SICM scan points,
|
(2) |
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(3) |
3 µ2 resolution for
SCell and a
102 µ resolution for RMS measurements.
Scanning laser confocal microscopy
To verify the SICM method of volume measurement, the following additional modifications were introduced to our existing SICM optical system (Korchev et al., 1997a,b) to enable simultaneous laser confocal
microscopy/SICM imaging of the same specimen. The current
control/data acquisition hardware and software (East Coast Scientific,
Cambridge, England) and the piezo-control system (Piezosystem) were
used to scan the specimen under the micropipette tip, or to perform
confocal microscopy scanning. The mechanical and piezoelectric translation stages (Tritor, Piezosystem) were added to the SICM for
coordinating optical and topographical imaging of the same area. The
excitation light source was provided by an LCM-F-6c laser diode (473-nm
wavelength, Laser Compact, Moscow, Russia) and a GPNT-02 laser diode
(532-nm wavelength, Lasertechnik, Bremen, Germany). The optical
recording system consisted of a Nikon Diaphot inverted microscope
(Diaphot 200, Nikon Corporation, Tokyo, Japan) equipped with
oil-immersion objective lOOX 1.3 NA, an epi-fluorescent filter block,
and a photomultiplier with a pinhole (D-104-814, Photon Technology
International, Surbiton, England).
The cell volume was estimated from a data set of thin serial SLCM
optical sections using digital image processing techniques described
elsewhere (Guilak, 1994; Zhu et al., 1994; Errington et al., 1997).
Relative changes in cell volume were assessed using fluorescence
methods described in detail elsewhere (Crowe et al., 1995).
Cell preparation
Renal tubular cells, A6 cell line
A single A6 cell line in the 117th passage derived from Xenopus laevis renal tubular cells was kindly provided by Dr. DeSmet (Belgium). All experiments were carried out between the 120th and 123rd passages. Cells were cultured as described previously (Sariban-Sohraby et al., 1984) on glass coverslips or on membrane filters (Falcon, Bedford, MA). Cells were grown and kept in a medium of a mixture of part modified Ham's F-12 and part Leibovitz's L-15, modified to contain 105 mM NaCl and 25mM
NaHCO3 (Gibco, Paisley, UK). The mixture
was supplemented with 10% fetal calf serum (Gibco), 1% streptomycin,
1% penicillin (Gibco). Cells were maintained at 28°C in an
atmosphere of humidified air plus 1% CO2. Cells
were passaged and used between days 3 and 4 when they were 80-90%
confluent. Aldosterone (Sigma, Dorset, UK) was added in a
concentration of 1.5 µM. For staining of actin cytoskeleton, A6 cells
were fixed using 4% formalin (Sigma) in phosphate-buffered saline
(PBS) for 10 min, followed by 0.1% Triton X-100 (Sigma) extraction for
5 min, rewashed three times with PBS and then stained by
Rhodamin-phalloidin (Sigma) (1:1000) 15 min at 37°C.
Ventricular myocytes
Ventricular myocytes were isolated from the hearts of 1- to 2-day-old rats after Iwaki et al. (1990). Cells were kept in a DMEM
medium (Gibco) with 15% fetal calf serum (Gibco), 1% streptomycin, 1% penicillin (Gibco), 1% NEAA (Gibco). 100 µg/ml G418
geniticin (Gibco) was added for inhibiting of fibroblast growth. Cells
were maintained at 37°C, in an atmosphere of humidified air plus 5% CO2. Cells were used after 2 days after plating.
Myocytes were cultured on glass coverslips. For fluorescent recording,
they were stained with acridine orange/ethidium bromide AM (Sigma) (1:100) for 20 min at 37°C in a medium containing a mixture of part
Leibovitz's L-15 (Gibco) and part buffer (115 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 1.2 mM CaCl2, 10 mM Hepes (Sigma)pH7.4, rewashed five times with the medium.
T47D breast cancer cells
The T47D cell line was originally isolated from the pleural effusion of a 54-year-old woman with ductal carcinoma of the breast. Cells were cultured in RPMI 1640 (Sigma) supplemented with 10% FCS (Sigma), 100 µg/ml streptomycin, 100 units/ml penicillin, and 2 mM glutamine (Sigma) and were maintained at 37°C in a humidified atmosphere containing 5% CO2. For use in experiments, cells were plated onto glass coverslips, grown in serum free RPMI 1640 for 24 h and stimulated with 30ng/ml FGF2.| |
RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Summary
REFERENCES
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Whole cell volume
We have used SICM to estimate the volume of single cells in a cell
monolayer. The kidney epithelial cell line A6 was grown on a glass
coverslip until the cells attained a dense monolayer. Because SICM
allows imaging both of fixed and of living cells, in this particular
experiment, the cells were fixed with 4% formaldehyde to abolish any
cellular dynamics for subsequent imaging by scanning confocal
microscopy. Thereafter, the cells were imaged with the SICM (Fig.
2 A). The cell boundaries are
clearly marked with denser microvilli. Because the cell boundaries are
easily visible, the cell volume of a single cell or a scanned cell
monolayer can be calculated using Eq. 1 assuming that cells are closely
attached to the substrate. The calculated volume of a 60 × 60 µ area of cell monolayer (Fig. 2 A) is equal to 36,637 µ3. The volume of the cells marked a and b is
4667 and 7171 µ3, respectively. The cell volume
was also assessed using scanning confocal microscopy (Guilak, 1994; Zhu
et al., 1994; Errington et al., 1997). We used a set of very thin
serial optical slices, similar to those shown in Fig. 2, B
or C across the same cells scanned by the SICM (Fig.
2 A) to confirm these calculations. Using digital image
processing, the shape of the cell monolayer was reconstructed, and cell
volume was estimated as 37,500 µ3 for the
scanned cell monolayer, and 5100 and 7600 µ3
for cells a and b (Fig. 2 B), respectively. The cellular
volumes estimated by these two methods are comparable, validating the SICM technique for volume measurement. The assumption that cells are
closely attached to the substrate is supported by confocal microscopy
data. Fig. 2 C illustrates a vertical slice across the cell
monolayer at the position indicated by the diagonal dotted double
arrowhead on the image in Fig. 2 B. The epithelial cells are attached to the substrate surface indicated by a white horizontal dotted line in Panel C, and that the cell-cell contacts are
arranged perpendicular to the substrate (Fig. 2 C).
Furthermore, the horizontal optical slice across the middle of the cell
monolayer (indicated by the red arrows in Figs. 2, A and
B) shows the lateral location of cell boundaries (Fig.
2 B) that closely matches the position of cell borders
marked as denser microvilli on the cell surfaces in the SICM image
(Fig. 2 A). Cell heights of ~3 µ assessed by scanning
confocal microscopy are also similar to those (~12 µ) measured by
SICM (see vertical scale in Fig. 2 A).
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The SICM method has several advantages over the scanning confocal
microscopy approach. It has a higher spatial resolution50-nm lateral
and 10-nm vertical resolution, compared with about 250- and 1000-nm
resolution of scanning confocal microscopy under optimal conditions.
The SICM technique does not induce photo damage to the biological
sample (compare with SLCM, e.g., Saito et al., 1998) and does not
require any preliminary preparation or staining of cells before
imaging. The transparency of the sample, or the focal length of the
objective, does not limit the SICM routines. The latter is important
when the cells are grown on a special material such as permeable
polymeric films, and when different growth mediums are required to
bathe the apical and basal sides of the cell monolayer. Figure
2 D illustrates the SICM image of living A6 cells growing
on a membrane filter. The volume of any imaged cell can be easily
estimated. For example, the volume of individual cells marked by green
arrows in Fig. 2 D is 3569 µ3
(left cell) and 2119 µ3 (right
cell).
Rapid estimation of volume
The SICM method can also be used for rapid estimation of the
changes in cell volume by measuring dynamic changes in cell height with
a 5 × 103-s temporal resolution. This is
extremely well suited for studying growing monolayers where cell
expansion is mainly in the vertical direction. The volume of an
isolated cell can also be estimated by this approach in conjunction
with light microscopy measurement of lateral cell dimensions. Figure
3 A shows single-cell height changes (SICM data) as a result of osmotic stress. In this experiment, a hypertonic solution was applied to a single cardiac myocyte. Osmotic
stress induced a 33% reduction in cell height without any significant
changes in cell length (light microscopy observation). The last is
probably due to the strong attachment of the cell to the substrate.
Considering a cylindrical shape and no length change of the myocyte, by
calculation, the 33% reduction in cell height (diameter) gives a 55%
decrease in cell volume. Figure 3 B also shows the
simultaneous measurements of the relative cell volume changes
(fluorescence measurements). These relative changes in cell volume were
assessed using fluorescence methods described in detail elsewhere
(Crowe et al., 1995). Osmotic stress induced a 52% reduction in cell
volume. The cell volume changes are comparable as assessed by both
methods.
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Changes in local cell volume and cell roughness
Dynamic changes of cellular volume are invariably accompanied by a complex rearrangement of cell shape. The SICM can provide important structural information allowing detailed quantitative analysis of highly local changes in cell volume. Volume, as well as surface characteristics of small cellular structures such as lamelopodias, dendrites, processes, or even microvilli, can be precisely measured. Figure 4 illustrates the development of microvilli on an A6 cell surface. During differentiation, A6 cells form a tight monolayer and increasing numbers of microvillus structures protrude through the apical cell membrane (compare Fig. 4, A and B). The volume of a single microvillus increases more than 10 times (0.141 and 0.188 µ3 in Panel A increasing to 1.190 and 2.993 µ3 in Panel B). Importantly, the volume of a single microvillus can be assessed with a resolution better than 0.1 femtoliter and less than 5% error. The volumes of all the microvilli in A (7.190 µ3) rises more than 10-fold compared to that in B (95.554 µ3).
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Cell volume changes are often accompanied by complex reorganization of cell surfaces, which are important in cell volume regulation. The SICM method can provide quantitative characterization of the cell surfaces. For example, cell surface area, and ruggedness characteristics can be estimated. If we compare the two SICM images in Fig. 4, A and B, the cell membrane ruggedness or roughness (expressed as RMS) increases by 3.67 times from 0.116 to 0.426 µ, and the apical cell surface area SCell by 2.40 times, from 90 to 217 µ2 per scanned region.
Moreover, both volume and surface characteristics can be simultaneously
and continuously assessed during relatively long experiments. This
could be important when monitoring the effects on cell dynamics of
physiological, pharmacological, or molecular interventions. For
example, we know that growth factor treatment of cells activate signal
transduction cascades leading to changes in cell morphology, motility,
and mitogenesis (Basilico and Moscatelli, 1992). Figure 5 illustrates the effect of FGF2 on T47D
cell surface characteristics and cell volume. After addition of FGF2,
the microvillus structures start to protrude through the cell membrane
(Fig. 5 B) significantly increasing relative cell surface
roughness (Fig. 5 A). This result correlates with previous
observations of increased polymerization of actin at the periphery of
the cell-forming protruding membrane ruffles after treatment of T47D
cells with FGF2 (Johnston et al., 1995). Both changes in F-actin
organization and increase of microvillus density are observed 10 min
after addition of FGF2. Two hours after addition of FGF2, the breast
cancer cells display an increase in cell volume and a decrease in cell
surface roughness (Fig. 5 A). The decrease in cell surface
roughness could be explained by stretching of the cell membrane during
cell volume increase. These observations may lead to clarification of
how a cell can preserve its integrity during volume changes to
maintain its functionality.
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Summary |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Summary
REFERENCES
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The SICM method has several advantages over currently used
techniques for the measurement of cell volume. First, it has a high
resolution (2.5 × 1020 liter) and a large
range of volume measurement from to 1019 to
109 liter. Second, the living material
investigated requires minimal preparation or staininga usually
laborious process with other techniques. Third, it has a fast response
time and can rapidly estimate cell volume changes, thus allowing
dynamic changes of the order of milliseconds, as well as over hours.
This includes an ability to assess complex rearrangement of cell shape
and changes in cell volume. Fourth, it can provide, in parallel with
cell volume changes, highly local structural information and regional membrane changes in volume, for example, dendritic processes. All the
foregoing lend themselves to, perhaps, the most important aspect. The
SICM can monitor changes in the cells after experimental interventions.
As examples of this, we have tried to choose several cell types to
represent a small spectrum of cells involved in volume regulation. The
A6 cell line shows, as part of its original function, aspects of volume
regulation. The heart cells demonstrate volume changes under osmotic
stress: a situation relevant to cell responses in ischemia and
reperfusion in the whole heart. The neoplastic breast cancer cells show
surface membrane changes related to abnormal growth under possible cell
signal stimulation. Finally, the scanning ion conductance microscope
can include additional cellular imaging with multifunctional probes or
other types of real-time microscopy, making it a potentially formidable
research tool.
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ACKNOWLEDGMENTS |
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Ventricular myocytes were kindly provided by Peter H. Sugden (National Heart and Lung Institute Division, Imperial College School of Medicine, London, U.K.). Our work is supported by the British Heart Foundation, the Office of Naval Research, University of London Central Research Fund, the Wellcome Trust, the Cancer Research Campaign, and the Garfield Weston Trust.
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FOOTNOTES |
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Received for publication 24 May 1999 and in final form 13 October 1999.
Address reprint requests to Yuri E. Korchev, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, 5th Flr. MRC Clinical Sciences Centre, Du Cane Road, London W12 0NN, U.K. Tel.: +44-181-383-2362; Fax: +44-181-383-8306; E-mail: y.korchev{at}ic.ac.uk.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Summary
REFERENCES
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and 
-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells.
J Biol. Chem
265:13809-13817[Abstract].
Biophys J, January 2000, p. 451-457, Vol. 78, No. 1
© 2000 by the Biophysical Society 0006-3495/00/01/451/07 $2.00
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-) and
membrane roughness (-
-) changes in the breast cancer cell (T47D
cell line) measured by SICM. The vertical arrow indicates the addition
of FGF2. Membrane roughness is expressed as RMS normalized to its
initial value (RMS0 at 0 time). (B) Two
single profiles across a breast cancer cell surface obtained at the
same position before and after 2 h of FGF2 treatment.