Download Chemical mapping of the distribution of viruses into infected bacteria

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Ultramicroscopy 108 (2008) 635–641
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Chemical mapping of the distribution of viruses into infected bacteria
with a photothermal method
A. Dazzia, R. Prazeresa, F. Glotina, J.M. Ortegaa,, M. Al-Sawaftaha, M. de Frutosb
a
CLIO/LCP, Bat. 201 Porte 2, Univ. Paris-Sud, 91405 Orsay, Cedex, France
Laboratoire de Physique des Solides, Bat. 510, Univ. Paris-Sud, 91405 Orsay, Cedex, France
b
Received 3 April 2007; received in revised form 7 September 2007; accepted 10 October 2007
Abstract
We show that an infrared spectromicroscopy method based on a photo-thermal effect, is able to localize single viruses as well when
they are isolated and when they are located inside the bacteria they have infected. In this latter case, although the topography
performed by an AFM cannot image the viruses, the AFMIR is able to do so. In addition, we are able to determine different stages of the
bacteria infection.
r 2007 Elsevier B.V. All rights reserved.
PACS: 68.37.d; 68.37.Ps; 68.37.Uv
Keywords: Microscopy; Spectroscopy; Nanoscience; Cell imaging
1. Introduction
2. Set up and sample description
The combination of infrared spectroscopy and imaging is
a powerful tool [1] to identify and localize chemical species
by their ‘‘fingerprints’’ (i.e., infrared spectra). However, in
most cases, such as cell imaging, a high lateral resolution is
needed. This cannot be obtained with usual, far-field, optical
microscopy. Infrared near-field microscopy has been studied
by several authors [2–5]. However, in this case it is extremely
difficult to separate the information due to sample
topography and inhomogeneities (real part of the index of
refraction) from the information of interest: infrared
absorption (imaginary part of the index of refraction).
Recently, we have proposed a photothermal method,
AFMIR [6–8] that possesses the advantage of being sensitive
only to the sample absorption with a lateral resolution
o100 nm. In this paper, we show that the AFMIR is able to
detect object as small as viruses and, moreover, to
discriminate them from the rest of the biological material.
The AFMIR technique is based on the coupling between
a pulsed infrared laser (in our case a free-electron laser) and
an atomic force microscope. When a sample is illuminated
by the laser at the wavelength corresponding to one of its
absorption bands, it absorbs one part of the incident
energy and is heated almost instantaneously compared to
the AFM response time (typically o1 ms). The increase of
temperature creates a fast expansion of the object that
displaces the AFM tip (Fig. 1). The AFM cantilever starts
then to oscillate. By measuring the amplitude of oscillation
we get a signal, which is proportional to the absorption. By
recording this signal as function of wavelength of the laser,
we are able to make ultralocal infrared spectroscopy [6]. By
fixing wavelength, we can image the surface giving us
corresponding chemical mapping at nanometric scale [7,8].
The increase of temperature induces a stress inside the
object that creates a displacement of matter. The stress can
be written in a simple way for an isotropic object:
s ¼ aE DT,
Corresponding author. Tel.: +33 1 6446 8111; fax: +33 1 6446 8006.
E-mail address: [email protected] (J.M. Ortega).
0304-3991/$ - see front matter r 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultramic.2007.10.008
where a is the thermal expansion coefficient and E is the
Young modulus of the object.
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A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641
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Fig. 1. Experimental set-up. The thermal expansion of a sample induces a
displacement of the tip of an AFM cantilever, which is monitored by an
HeNe laser and a 4-quadrant detector.
separated by 16 ns. It is continuously tunable between
3000 and 80 cm1. Spectra can be taken within a factor of
two in wavelength within a few minutes, or more in case of
averaging. Access to another spectral range requires a
tuning of the machine, which takes typically an hour.
The sample was composed of Escherichia coli bacteria
and T5 phage (bacteria virus). Bacteriophages T5 st(0)
were produced from E. coli F and purified as described in
Ref. [10]. The final concentration of the phage stock was
evaluated to 1.8 1013 infecting phages/ml. Bacteria E. coli
F were grown in LB medium to the exponential growth
phase (3 108 cells/ml) and infected by phages with an
average multiplicity of 60. The infection was stopped
20 min after the beginning by adding chloramphenicol at a
final concentration of 50 mg/ml. Chloramphenicol is an
antibiotic inhibiting bacterial protein synthesis and consequently, blocking the phage multiplication. Infected
bacteria were centrifuged and the pellet was washed and
suspended three times in pure water to a final concentration of approximately 1.5 108 cells/ml. We have studied
two different types of samples: phages alone and bacteria
infected by bacteriophages. A drop of the solution was
deposited on the ZnSe prism and dried at the room
temperature. For the infected bacteria, as the infection was
stopped by chloramphenicol addition after only 20 min,
various stages of the virus development can be found inside
the cells [11].
3. Localization of isolated viruses
The displacement of the object is directly proportional to
the thermal expansion coefficient:
a DT ¼
u
,
R
where u is the displacement and R is the size of the object.
For example, for a bacterium of 1 mm radius and a DT
increase of 10 K1, the displacement u is 1 nm (taking the
thermal expansion coefficient to 104). This value is well
within the vertical sensitivity of an AFM (a few Å). In our
case, due to the resonance of the cantilever, the observed
displacement and thus the sensitivity are greatly enhanced.
Calculation of this effect is in progress. In practice, these
experiments were conducted by attenuating by a factor of
10 the incident laser power below the damage threshold of
the sample. We may assume that damage arises when the
temperature reaches 100 1C, since there is always some
water remaining inside dried samples. Therefore, the
temperature rise inside the sample can be estimated to be
of the order of 10 K and the measured displacement of a
ffi100 nm wide virus of about 0.1 nm.
The sample is deposited on a ZnSe prism that is
transparent in the mid infrared. The laser light is incident
with an angle to be propagative inside the sample and
evanescent in the air, allowing to protect the AFM tip from
the direct illumination.
The laser is the free-electron laser ‘‘CLIO’’ [9]. Its pulse
length is 9 ms, composed of about 600 micropulses
When the droplet of phage solution has evaporated, one
expects the viruses to have preserved their structure and
their DNA inside their protein envelope (capsid). To verify
this, we have studied the surface of the prism at two
different wave numbers: 1650 cm1 (amide I) characterizing the proteins of the capsid and 1080 cm1, which is the
maximum of the DNA band.
Fig. 2(a) and (b) show the topography and the
corresponding chemical mapping of a single virus, recorded
for the wavelength of 1650 cm1 (amide I). This wavelength
is situated in the absorption band of proteins. We can see
that the absorption signal of the phage (Fig. 2(b))
corresponds to its topography. There is no real detectable
lateral expansion due to the heating. Preliminary calculations indicate that this expansion should not be larger than
one nanometer. The signal magnitude is weak, because
proteins constitute only a small fraction of the phage head,
which is mainly constituted of DNA (about 70% of the
phage mass). However, the contrast with the background
(+6 dB) is sufficient to identify unambiguously the virus,
showing that this technique is really sensitive even for such
a small entity.
Topography and chemical mapping at 1080 cm1 (DNA
band) of several isolated viruses are represented in Fig. 3(a)
and (b). In this case, the AFMIR image is blurred
compared to the topography. These results indicate that
part of the phages have certainly been damaged and have
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Vibration amplitude of the tip (nm)
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wavelength (cm-1)
Fig. 2. Observation of a single virus by topography (a), and AFMIR (b) in the amide I absorption band (1650 cm1), and (c) typical AFMIR spectrum in
the amide region, of a small area of single cell (full line) recorded by the tip, compared with the far-field spectrum of a stockpile of them (dotted line).
lost their DNA when the droplet was dried, leading to
weaker and larger images of it. Therefore, the AFMIR
brings another information when compared to the AFM
topography.
4. Localization of viruses infecting bacteria
To localize phages inside bacteria, we have recorded a
series of chemical mappings centered on the DNA
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Fig. 3. Observation of a several isolated viruses by topography (a) and by AFMIR (b) in the DNA absorption band (1080 cm1).
absorption band. It is based on the fact that the virus is
mainly constituted of highly concentrated DNA. When a
bacteriophage T5 infects E. coli [11], it first binds on the cell
membrane and then injects its DNA through the membrane into the bacterium. This injection of the DNA is
followed by a full degradation of the host genome and by
the synthesis of many copies of the proteins and DNA
composing the phage. Phages capsids are assembled and
progressively filled with DNA. At the end of the infection,
the host cell contains about hundred of viruses that are
liberated by the bacteria explosion (lysis). When the sample
is dried, bacteriophages are not damaged and do not loose
their DNA due to the cellular environment that preserves
better their integrity. Therefore, due to the high DNA
concentration, the peak around 1080 cm1 is expected to
appear more intense at phages location. Mappings of
different bacteria correspond generally to different states of
infection. The most common situation, as described in
Ref. [11], consists in phages ‘‘being built’’, i.e., all capsids
having not yet filled with DNA. We will illustrate here
three infection stages: uninfected bacteria or in the first step
of infection (empty capsids), partially and largely invaded
with mature phages.
Fig. 4 describes the topography (left) and the corresponding chemical mapping (right), at DNA absorption
wavelength, for these three stages of infection. The color
bar of the chemical mapping pictures has been adapted to
have comparable contrast for each state.
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A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641
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Fig. 4. Topography (left) and the corresponding chemical mapping (right) of these three stages of infection: non-infected (upper images), heavily (middle),
only one phage visible (lower).
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The upper one shows that the DNA distribution of a
non-infected cell is homogeneous inside the bacteria.
Indeed, the DNA of the bacteria is spread all inside the
bacteria and is not concentrated in a particular region.
The middle figure shows the most advanced state of
infection since we can observe a very significant increase
of the AFMIR signal, compared to the topography, in
some area of the bacteria and a decrease in the other
parts. The topography picture exhibits also a wing at the
right side of the main part of the cell. This zone is too
small to be another bacteria, and it is likely that the
bacteria has partially exploded and separated in two
parts. Indeed, the infection tends to weaken the cell
membrane and the drying may have finished to damage
it. In practice, each time we have detected an advanced
infection state, the shape of the bacteria was not
perfectly cylindrical and exhibited a lot of deformations
and turgidities. This AFMIR image exhibits also a very
perceptible set of three virus (pointed by arrows) located
inside this wing: in this case they are spatially resolved,
contrary to the aggregates that can be seen in the other
part of the cell.
A state of partial infection is represented in the lower
figure. The AFMIR image exhibits one small hot spot
due to the presence of a single filled phage. Such a result
is rather rare since the development of the infection is
quite fast and most bacteria contain at least dozens of
viruses.
In addition, several isolated viruses appear outside the
bacteria on the topography and not on the AFMIR
images: this is due to the fact that these has expelled their
DNA and therefore cannot be seen at 1080 cm1, as
discussed in Section 3.
We have performed the local spectroscopy of the DNA
band of infected and non-infected cells (Fig. 5): these
spectra, normalized to the bacterium thickness determined
by topography, show that the absorption is much larger
when filled phages are present, as expected. It appears also
that these spectra fit well with the DNA band obtained by
an FTIR spectrometer on a thick layer of bacteria. This
illustrates how the AFMIR is able to make ultralocal
spectroscopy on a nanometric sample.
DNA (bacterium)
DNA (FTIR)
DNA (bacterium + T5)
Absorption (a.u.)
1200
1150
1100
1050
1000
950
wavenumber (cm-1)
Fig. 5. Spectrum, made by AFMIR, in the DNA band spectral region of
an infected (full line) and non-infected (dotted line) region of a bacterium.
These curves have been normalized to the thickness of each region
(measured by topography). An FTIR spectrum of an assembly of bacteria
is shown for comparison.
0
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nm
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5. Spatial resolution
On the absorption picture of an isolated virus (Fig. 2(a)),
its size appears to be only slightly larger than their usual
size (90 nm), demonstrating the excellent lateral resolution
of the AFMIR (o50 nm here). Alike AFM topography,
this value is determined by the convolution of tip curvature
(50 nm) with the object.
In the case of the buried virus, the resolution is different.
We have made a zoom (Fig. 6) in order to examine more
precisely its shape. Its diameter is around 200 nm, which is
noticeably larger than measured previously. In this case,
250
500
nm
750
1000
Fig. 6. Same as Fig. 2(b), but with a zoom on the virus to determine
lateral resolution.
the resolution is determined by the deformation of the
bacteria induced by the thermal expansion of the virus. A
simulation of the thermal expansion calculated by COMSOL is displayed in Fig. 7. The resulting apparent size is
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Time –1e-9 Surface Temperature [K] Deformation Deplacement [m]
x10-4
Maxi: 349.5
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-4
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Fig. 7. Simulation (COMSOL) of the bacterium deformation in the vicinity of the irradiated phage.
closed to the observed one, when the virus is not buried
very deeply (only a few nm from the surface). This example
illustrates that the AFMIR spatial resolution is not related
only to quality of the AFM tip but also to the thermomechanical coefficient of the studied materials and to the
geometry of the sample.
6. Conclusion
In this paper, we show the potentiality of the AFMIR
technique in cell imaging. The spatial resolution is linked to
the radius of the tip if the sample is isolated on the surface
(convolution effect) and to mechanical and geometrical
properties of the sample when it is buried. The sensitivity
and lateral resolution of AFMIR is reduced for objects
located below the surface, which is directly linked to the
near-field aspect of AFMIR technique and is unavoidable.
Nevertheless, we image viruses inside cells through their
infrared signature with a resolution of o200 nm. This
demonstrates the high potential of the AFMIR in chemical
mapping. The great advantages of infrared mapping is to
be non-destructive and to avoid the use of specific probes
as in fluorescence [12] or of AFM immunogold techniques
[13], which may modify the processes to be studied.
Experiments with cells immersed in water should also be
possible and are now envisioned.
References
[1] P. Dumas, G.L. Carr, G.P. Williams, Analysis 1 (2000) 68.
[2] A. Piednoir, F. Creuzet, C. Licoppe, J.M. Ortega, Ultramicroscopy
57 (1995) 282.
[3] R. Bachelot, P. Gleyzes, C. Boccara, Opt. Lett. 20 (1995) 1924.
[4] B. Knoll, F. Keilmann, Nature 399 (1999) 134.
[5] A. Dazzi, S. Goumri-Said, L. Salomon, Opt. Commun. 235
(2004) 351.
[6] A. Dazzi, R. Prazeres, F. Glotin, J.M. Ortega, Opt. Lett. 30 (18)
(2005) 2388.
[7] A. Dazzi, R. Prazeres, F. Glotin, J.M. Ortega, Infrared Phys.
Technol. 49 (2006) 113.
[8] A. Dazzi, R. Prazeres, F. Glotin, J.M. Ortega, Ultramicroscopy,
accepted for publication.
[9] J.M. Ortega, F. Glotin, R. Prazeres, Infrared Phys. Technol. 49
(2006) 133.
[10] M. Bonhivers, A. Ghazi, P. Boulanger, L. Letellier, EMBO J. 15
(1996) 1850.
[11] M. Zweig, H.S. Rosenkranz, C. Morgan, J. Virol. 9 (3) (1972) 526.
[12] R. Cubeddu, D. Comelli, C. D’Andrea, P. Taroni, G. Valentini,
J. Phys. D: Appl. Phys. 35 (2002) R61.
[13] Y. Arntz, L. Jourdainne, G. Greiner-Wacker, S. Rinckenbach,
J. Ogier, J.C. Voegel, P. Lavalle, D. Vautier, Microsc. Res. Tech.
69 (4) (2006) 283.