Live Cell DNA Labeling and
Techniques for the discrimination and location of
DNA, chromatin architecture, chromosomes, nuclear
superstructures, and cell nuclei in their various forms
through the cell cycle are of increasing interest in the
biosciences and the generation of automated screening
systems. The appropriate selection of a DNA-labeling
dye may also be important for tracking events in
microscale devices (e.g., lab on a chip). Here the
critical issue is the transmittance advantage of
longer wavelengths using external laser excitation or
an incorporated source (e.g., a red laser diode within
an optical biochip).
Advanced microscopy methods such as multiphoton
excitation laser scanning (MPLSM) are now
becoming more generally accessible, as they provide
significant advantages in live cell studies. Although it
is apparent that MPLSM technology will evolve, providing
more convenient and adaptable systems, the
principles discussed here will still apply. Currently,
MPLSM instrumentation usually consists of a tuneable
femtosecond pulsed infrared (IR) laser attached to
a scanning microscope with IR-compatible optics
to ensure efficient transfer of long wavelength light
(690-1000nm). Such a laser configuration provides
virtually simultaneous delivery of two photons to a
focal point of the microscope objective lens, achieving
volume limited excitation by two or more photons of
any fluorophor molecule with appropriate absorption
characteristics. The advantages of this approach compared
to confocal laser scanning microscopy (CLSM)
are numerous. Because photons are only emitted from
the "in focus" plane, all the photons can be collected
via a nondescanning route, including those photons scattered by the sample and usually rejected at the confocal
aperture. This provides a massively improved
efficiency of emission light collection, an important
consideration in photon-limited systems (White and
Errington, 2002). Importantly, infrared light penetrates
biological material further than shorter wavelengths
and the potential damaging effects of ultraviolet
(UV)-visible wavelengths are avoided. Reviews of the
subject and information on two-photon fluorescence
excitation cross sections are available (e.g., Denk et al.
1995; Williams et al.
, 2001; Albota et al.
, 1998). However,
a pragmatic outcome of the combined advantages of
MPLSM is a resurgence of the use of W-excited fluorescent
probes, although these have pitfalls for use
with GFP-based fluorophors (see later). Two-photon
fluorescence absorption and emission spectra have
been obtained for DNA probes relevant for live cell
work. The technique of multi-photon excitation of a
fluorophore, using a pulsed laser light source, provides
solutions to several problems associated with
continuous wave excitation via single photon absorption.
Typically for multi-photon excitation the sample
is illuminated with light of a wavelength which is
approximately twice (or three times) the wavelenghth
of the absorption peak of the fluorophore in use
(Errington et al.
, 2005) (Bestvater et al.
, 2002; Smith et
al., 2000; van Zandvoort et al.
, 2002) and a selection is
shown in Table I.
DNA offers a highly attractive substrate for chemical
and biophysical probe interactions and there are
excellent references for the chemical descriptions and
cytometric applications of a wide range of DNA dyes
(Darzynkiewicz et al.
, 1990; Latt and Langlois, 1990;
Waggoner, 1990). The forms of interaction range from
covalent binding, inter- and intrastrand cross-linking,
adduct formation, to ternary complex formation with DNA-binding proteins. Additionally, major or minor
groove residence are potentially stabilized through
hydrogen bonding and van der Waals forces, phosphate
group interactions, and various levels of intercalation
between the base pairs. A restricted number of
agents are now available for DNA labeling in live cell
systems and MPLSM. The most frequently used are
UV-excitable fluorochromes such as the bisbenzimidazole
dyes Hoechst 33258 and Hoechst 33342, and the
agent DAPI (4', 6-diamidino-2-phenylindole). Cellpermeant,
acridine orange displays metachromatic
staining of nucleic acids that precludes its convenient
use as an exclusively DNA-discriminating probe. The
introduction of cell-permeant cyanine SYTO nucleic
acid stains (Frey, 1995; Frey et al.
, 1995) has provided
many different reagents with a wide range of spectral
characteristics. These agents passively diffuse through
the membrane of most cells and can be excited by UV
or visible light but stain RNA and DNA in both live
and dead eukaryotic cells. Such dyes would be
accessible to two-photon excitation (Table I). Their
extremely low intrinsic fluorescence, with quantum
yields typically less than 0.01 unbound (>0.4 when
bound to nucleic acids), reduces the need to remove
extracellular dye prior to imaging. It has been stressed
that the highly versatile SYTO dyes do not act exclusively
as nuclear stains in live cells and that they should
not be equated in this regard with compounds such as
DAPI or the Hoechst 33258 and Hoechst 33342 dyes.
At the other end of the fluorescence spectrum,
DRAQ5, a DNA-binding anthraquinone derivative,
can provide sufficient discrimination of cellular DNA
content in multiparameter fluorescence studies.
Anthraquinones are a group of synthetic DNAintercalating
agents (Lown et al.
, 1985) but are only
weakly fluorescence (Bell, 1988). DRAQ5 is a modified
anthraquinone with enhanced DNA affinity and intracellular
selectivity for nuclear DNA (Smith et al.
2000). DRAQ5 appears to achieve its live cell nuclear
discrimination by its high affinity for DNA. Excitation
at the 647-nm wavelength, close to the Exλmax
, produces a fluorescence spectrum extending from 665-nm out to
beyond 780-nm wavelengths. Thus the emission spectrum
is beyond that of fluorescein, phycoeryrthrin,
Texas Red, Cy 3, and, perhaps most importantly, EGFP.
(Waggoner, 1990) DRAQ5 enters cells and nuclei
rapidly, the broad excitation spectrum of DRAQ5
means that flow cytometric applications can utilize
excitation wavelengths down to 488nm (Smith et al.
2000); two-photon excitation is also permissible at
wavelengths >1047nm. The two-photon dark excitation
region of DRAQ5 (720-860nm) permits detection
of low-intensity intracellular fluorescence of other
probes by MPLSM and then definition of nuclear location
by CLSM. This article provides simple procedures
for two DNA-specific probes for living cells and their
use in MPLSM studies. These dyes sit at either end of
the excitation spectrum, Hoechst 33342 (UV excitable)
and DRAQ5 (far red excitable), and therefore offer
maximum flexibility in multiprobe analyses.
II. MATERIALS AND
DRAQ5 (molecular weight 412.54) dye is supplied as
an aqueous stock solution of 5mM
Ltd) and can be diluted in aqueous buffers or added
directly to full culture medium. DRAQ5 is stable at
room temperature as a stock solution but can be stored
routinely at 4°C although freezing of the stock solution
should be avoided. 33342 Hoechst (H-3570; termed here
Hoechst 33342) has a molecular weight of 615.99 in the
trihydrochloride/trihydrate form and is supplied by
Molecular Probes Inc. as a 10-mg/ml stock solution in
water in a light-excluding container. It is routinely
stored at 4°C without freezing. With both DNA dyes,
appropriate safety information is available from the
suppliers. Because both agents have the potential to
damage DNA, they should be considered both cytotoxic and potentially mutagenic. Currently available
information indicates that although they are not listed
as carcinogens by the National Toxicology Program
(NTP), the International Agency for Research on Cancer
(IARC), or the Occupational Safety and Health Administration
(OSHA), the dyes should be treated as such.
Mitotracker Orange CMTMRos (M7510) is supplied by
Molecular Probes in 20 × 50-µg aliquots, stored at-20°C Before use a stock solution is made by the addition of 50
gl dry dimethyl sulfoticle, which can be subsequently
frozen; however, repeated freeze-thawing is not recommended.
Zinquin ethyl ester [Zinquin E, [2-methyl-8- (4-methylphenylsulfonylamino) quinolinyl] oxyacetic
acid ethyl ester] (Alexis Corporation, Nottingham, UK)
is stored as a 5 mM
stock solution in ethanol at 4°C The
HEPES buffer (1M
) (Sigma; H-0887) filter-sterilized
solution is stored at 4°C Stock preparations of 8%
paraformaldehyde (Sigma; P 6148) are made up in
phosphate-buffered saline (PBS) lacking calcium and
magnesium and are kept in aliquots of 20ml at -20°C All live cell work is conducted with cells seeded onto
Nunc Lab-tek (#1 coverglass) chambered wells, appropriate
for high-resolution imaging and recommended
when handling cytotoxic agents.
The flow cytometry system is a FACS Vantage cell
sorter (Becton Dickinson Inc., Cowley, UK) incorporating
two lasers: (1) a Coherent Enterprise II laser
simultaneously emitting at multiline UV (350- to 360-
nm range) and 488-nm wavelengths and (2) a Spectra
Physics 127-35 helium-neon laser (maximum 35-mW
output) emitting at 633 nm with a temporal separation
of about 25g from that of the primary 488-nm beam
for the UV or red lines. Light scatter signals are collected
as standard. The analysis optics are (i) primary
beam-originating signals analyzed at FL3 (barrier filter
of LP715nm) after reflection at a SP610 dichroic and
(ii) delayed beam-originating signals analyzed at FL4
(barrier filter of LP695nm for DRAQ5 emission or
DF660/20nm for Hoechst 33342 emission) or at FL5
(barrier filter of DF424/44 nm for Hoechst 33342 emission).
Forward and 90° light scatter are analyzed to
exclude any cell debris. Optical filters are originally
sourced from Becton Dickinson Inc. or Melles Griot
Inc.). All parameters are analyzed using CellQuest
software (Becton Dickinson Inc.).
Multiphoton excitation at wavelengths between 720
and 980 nm uses a laser-scanning microscope comprising
a 1024 MP scanning unit controlled by LaserSharp
software (Bio-Rad Cell Science Division) attached to a Zeiss Axiovert 135 (Carl Zeiss Ltd.). MPLSM mode
is achieved with a Verdi- Mira excitation source
(Coherent UK Ltd.). The Mira Optima 900-F (Coherent
UK Ltd.) is a simple, stable titanium:sapphire, KLM
mode-locking system with X-wave optics, a broadband,
single optics set tuning 700-1000nm, and a
purged enclosure. The continuous wave diode pump
laser (Verdi) operates at 5 W at 532nm. CLSM mode is
achieved using the same scanning unit and 488-, 568-,
and 647-nm lines of a krypton-argon laser. All images
are collected with either a ×63, 1.4 NA oil immersion
lens or a ×40 1.3 NA oil immersion lens. Typically, each
optical slice consists of 512 × 512 (x,y
resolution is approximately 1 µm). Multiphoton
excitation at 1047nm uses a similar BioRad
1024MP system incorporating an all solid-state excitation
source Nd:YLF laser (Coherent UK Ltd.) and
mode-locked femtosecond pulsed laser providing twophoton
excitation of DRAQ5 at a 1047-nm wavelength
at 15 mW with detection of fluorescence in the far red.
A. General Considerations
|FIGURE 1 Two-photon excitation (Exλ 750nm) of
Hoechst 33342 in live HeLa cells
(5µM x 60min). (Top) Main image
is a z-axis projection
of a series of optical slices (Emλ > 460 nm) revealing
intranuclear chromatin architecture and a display of
metaphase chromosomes in a mitotic cell.
(Bottom) The corresponding
z-axis slice for an x axis
intercepting the mitotic and three adjacent
Size bar: 10µm.
Hoechst 33342 is more lipophilic than Hoechst
33258 and is the form recommended for live cell
studies showing high affinity for A-T base pairs
through noncovalent binding in the minor groove of
DNA. The ligand shows fluorescence enhancement
upon binding. DRAQ5 is weakly fluorescent but has
high DNA affinity and intracellular selectivity for
nuclear DNA due to increased AT base pair preference
but with no apparent fluorescence enhancement upon
binding. When considering live cell labeling, the performance
status of the cell is the most critical issue for
achieving optimal staining. There is a subtle shift in the
violet to red bias in the emission spectrum of Hoechst
33342 upon DNA binding. A small (approximately
10 nm) red shift in peak wavelength also occurs upon
DRAQ5 binding. Nuclear staining is straightforward
and most cultured cells label well in full culture
medium (containing 10% fetal calf serum) supplemented
HEPES, reaching equilibrium
over some 5-60min for concentrations in the 1-20µM
range. Staining in phosphate buffers is less efficient.
Staining rate is enhanced at 37°C. The following procedures
are for generic uses of the dyes.
B. Two-Photon Excitation of Hoechst 33342
C. Two-Photon Coexcitation of
- Seed cells onto a sterile glass coverslip in a
culture dish (approximately 1 × 105 cells/25 cm2) or seed into a Nunc chamber coverslip. Incubate attached
cultures under standard conditions until analysis is
required. Add Hoechst 33342 directly to culture
medium at a final concentration of 5µM (3.1 µg/ml).
- For greater pH control conditions, especially
when manipulating cells out of 5% CO2 incubation,
supplement the culture medium with HEPES buffer
(final concentration 5-25 mM).
- After a 60-min incubation (minimum of 15 min)
at 37°C, wash coverslips gently in PBS to preserve
mitotic cell attachment. Mount coverslips in an
inverted position onto a microscope slide in PBS. To
avoid cell crushing, the edges of the coverslip can be
supported and sealed (e.g., using a piping of petroleum
jelly). To clean the noncell side of the coverslip,
the surface can be wiped (lens tissue).
- Imaging conditions and typical results are shown
in Fig. 1.
|FIGURE 2 Dual images of four live HeLa cells using two-
coexcitation (Exλ 800nm) of nuclear-located
Hoechst 33342 (5µM× 60min; left side Emλ 455/25nm)
and cytoplasmic Mitotracker
orange CMTMRos (5 ng/ml
× 5 min; right side Emλ 585/32 nm). Size
bar: 10 µm.
D. Hoechst 33342 Staining Kinetics and
Population Spectral Shift Analysis
- Generate cultures and label Hoechst 33342 as in
- A second cell-permeant probe may be introduced
concurrently with Hoechst 33342 staining or after
culture washing. Typically, Hoechst 33342-labeled cells
will lose approximately 25% nuclear fluorescence over
a 15- to 30-min postlabeling period in fresh medium.
Here we exemplify the signal separation of Hoechst
33342 and the probe Mitotracker Orange CMTMRos,
the latter introduced into the culture medium at
5ng/ml for the final 5-min period of Hoechst 33342
- Sample preparation is as in Section III,B, whereas
dual-imaging conditions and typical results are shown
in Fig. 2.
- Detach cultured cells by a standard trypsin/
EDTA method and resuspend at 2 × 105 cell/ml full
culture medium supplemented with 10 mM HEPES
and allow to equilibrate at 37°C prior to Hoechst 33342
addition (see Section III,B).
- Analyze single cell suspensions, in the presence
of the dye, by one-photon UV excitation flow cytometry
and acquire data for different dye uptake periods.
- The change of spectral emission with dye uptake
by mouse L cells is shown for populations of 104 cells
in Fig. 3. Cells showing a rapid, essentially immediate,
red shift are damaged and can be excluded by additional
electronic gating using light scatter changes.
|FIGURE 3 Flow cytometric contour plots of mouse L-cell populations
undergoing a violet-to-red spectral shift in fluorescence
during nuclear localization of Hoechst 33342 (10µM; 4 x 105 cells/ml) using one-photon excitation by multiline UV. The broad
fluorescence emission spectrum was monitored simultaneously in
the red region (Emλ 660/20nm) and the violet region (Emλ 405/
20nm) for 105 cells at the specified time points. Data show the rapid
development of violet fluorescence and the later red shift. Residual
levels of damaged cells not excluded by light scatter gating are
evident by their rapid red-shift pattern.
E. Two-Photon Excitation of Hoechst 33342
and Spectral Shift Analysis
- Generate cultures and label Hoechst 33342 as in
- Sample preparation is as in Section III,B with
dual-imaging conditions and typical results shown in
Fig. 4. The dual images of live mouse L cells show the
differences in emission by condensed chromatin in
metaphase cells lacking a nuclear envelope and interphase
|FIGURE 4 Dual images of live mouse L cells using two-photon
excitation (Exλ 750nm) of nuclear-located Hoechst 33342 (5µM× 60min) showing a red-biased fluoresence emission in metaphase
versus interphase cells (right side shows Emλ 585/32 nm and the left
side shows Emλ 405/35 nm). Size bar: 10 µm.
|FIGURE 5 Confocal image of live MCF-7 breast tumor
one-photon excitation (Exλ 647nm; Emλ
680/32nm) of nuclearlocated
DRAQ5 (10µM× 10min)
revealing nuclear architecture
and the presence of an
anaphase cell. (Left insert) Nuclear retention
of the dye
following preparation washing and conventional
paraformaldehyde fixation for the purposes of subsequent
colocalization analysis. (Right insert)
An example of
immunolocalization is shown for the
expression of nuclear-located
protein detected using a
secondary antibody conjugated to Alexa488
(Exλ 488 nm; Emλ 522/35 nm). Size bar: 10 µm.
F. One-Photon Excitation of DRAQ5 for Live
and Persistence in Fixed Cells
G. DRAQ5 Staining Kinetics and DNA
- Generate cultures and mount coverslips as in
- Cultures can be exposed to DRAQ5 by direct
addition to full culture medium (10µM × 10min)
prior to coverslip washing (optional) and mounting.However, the rapid staining kinetics of DRAQ5 (see
Section III,G) permit direct introduction of the dye to
already mounted samples and imaging immediately
for nuclear tracing.
- Figure 5 shows one-photon excitation images of
nuclear-located DRAQ5 and dye persistence following
standard 4% paraformaldehye fixation and the
sample subsequently processed for immunofluorescence,
which permits colocalization of an immunofluorescence
signal (Alexa 488) with nuclear structures.
- Prepare cells as in Section III,D and the conditions
given in Fig. 6.
- DRAQ5 far-red fluorescence is detectable within
seconds of dye addition to cell suspensions and can be
generated by both 488- and 633-nm wavelength onephoton
excitation with similar outcomes. The optical
configurations are shown in Fig. 6. The flow cytometry
protocol acquires fluorescence emissions with time and shows the ability to discriminate cells of different DNA contents.
|FIGURE 6 Flow cytometric analysis of the rapid cellular uptake of DRAQ5 demonstrating the ability to
discriminate DNA content using one-photon excitation at either Exλ 488nm (Emλ > 715nm) or Exλ 633nm
(Emλ > 695nm). Suspensions of live human B-cell lymphoma cells (4 × 105 cells/ml) were exposed to 20gM
DRAQ5 in complete medium supplemented with 10mM HEPES and incubated at 37°C for 600s. (Left)
Density dot plots for continuous event acquisition for sequential 488 nm then 633 nm excitation, monitoring
far red fluorescence commencing 40s after dye addition (total of approximately 1.2 × 106 cells acquired; background
unlabeled cells located in channel
< 25). Data show rapid equilibration of DNA staining, the discrimination
even at early staining times of relative DNA content, and the final equivalence of the content,
distributions (right) irrespective of blue or red line excitation.
H. Discrimination of the Intracellular
Location of Two-Photon Excited Fluorophors
- Generate cultures as in Section III,B and probe for
the expression of a given characteristic using a cellpermeant reporter (e.g., here using the UV-excitable
probe Zinquin-E for free zinc ions; Zalewski et al.,
1993). Following probe treatment, cells can be
processed for nuclear tracing using one-photon excitation
achieved rapidly by mounting of cells directly in
PBS containing 20 µM DRAQ5 (see Fig. 7).
- If two-photon acquisition of a DRAQ5 image is
sought, then wavelengths beyond 1 µm are required.
Figure 7 shows such acquisition (Exλ 1047nm) for
ethanol-fixed human lymphoma cells mounted in 20 µM DRAQ5 obtained using a 60× N.A. 1.4 oil objective,
a zoom of 1.9, and a Kalman filtering average of
|FIGURE 7 Sequential acquisition of a nuclear DNA signal (single-photon excitation of DRAQ5; Exλ 647nm; Emλ 680/32nm; 10µM× 10min; center) and the intracellular localization of a W-excitable fluorophor
(two-photon excitation of the intracellular Zn2+-sensitive reporting probe Zinquin-E; Exλ 780nm; Emλ >
460nm; left) in live human B-cell lymphoma cells. Images are dual-channel projections revealing bright
punctate Zinquin-Zn2+ complexes found to be exclusively cytoplasmic upon three-dimensional image reconstruction.
(Right) Two-photon excitation of nuclear-located DRAQ5 using a YLF mode-locked femtosecond
pulsed laser (Exλ 1047nm and far-red fluorescence) in ethanol-fixed human lymphoma cell cytocentrifuge
preparations mounted in 20µM DRAQ5 in phosphate-buffered saline. Size bar: 10µm.
- Probe selection issues include (i) excitation and
emission spectral characteristics, including differential
spectral shifts pertinent for multiparameter imaging;
(ii) dye "brightness" dictated by molar absorptivity,
quantum yield with fluorescent enhancement; and
(iii) advanced properties for more complex studies,
including lifetime signatures and molecular quenching
- Fluorescence lifetime microscopy (FLIM) provides
an important avenue for investigating the binding
of DNA probes to macromolecular structures.
The image contrast is concentration insensitive, but is
sensitive to the local environment. Multiphoton instruments
can be adapted to capture fluorescence decay
signals. Some dyes that cannot be used to differentiate
DNA/RNA using steady-state fluorescence do have
unique lifetime signatures when bound to these different
sites. Hence this provides a mechanism for
deriving the spatial map of RNA and DNA location
sites within a cell (Lakowicz et al., 1997; van Zandvoort et al., 2002).
- Investigators should always question both the
timing and the purpose of the staining within the
analysis procedures. The rapid staining of cells by
DRAQ5 is a significant advantage when tracking
events over short time frames and nuclear discrimination
can be achieved within seconds.
- MPLSM requires careful selection of optical
filters capable of efficient IR blocking.
- MPLSM requires a fundamental shift in experimental
design, as using a single (albeit well-chosen)
wavelength will excite multiple probes, and so good
separation of the emission wavelengths is absolutely
required. Thus the UV-excitable dyes are limited when
two-photon excitation of GFP-based fluorophors is
undertaken due to coexcitation and overlap of the
- Live cell labeling is subject to micropharmacokinetic
effects, which can vary with cell type. For example,
the ability of bisbenzimiazoles dyes to access
cellular DNA can be affected by dye effiux (e.g., via
a membrane-located p-glycoprotein encoded by the mdr-1 gene; Morgan et al., 1989), nuclear ejection
(Smith, 1988), and differences in chromatin compactness.
DRAQ5 is less subject to drug resistance effiux,
but at suboptimal concentrations, nuclear access may
restrict binding potential.
- Both dyes discussed can damage DNA. Benzimidazoles
can cause DNA strand breaks by photolytic
(UV) DNA strand cleavage or as a consequence of
ternary complex formation with intranuclear DNA
topoisomerases I and II. Anthraquinones can also
trap DNA topoisomerases but may also cause
DNA-protein cross-linking damage. Some cells appear
to be resistant to Hoechst 33342 cytotoxicity (e.g.,
HeLa), whereas others will undergo apoptosis readily
(e.g., p53 wild-type B-cell lymphoma). In the case of
DRAQ5, toxicity is clearly dose dependent (20nM-
20µM range), and the persistence of the agent makes
it unsuitable for long-term (>12h) tracking experiments
or viable cell sorting. The initial stress responses
to dye binding may be occult but result in progressive
cell cycle arrest or indeed modulation of sensitivity to
other agents under investigation.
This work was funded by Research Councils
(GR/s23483), BBSRC (75/E19292), AICR (00-292) and
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