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ARTICLE IN PRESS Ultramicroscopy 108 (2008) 635–641 www.elsevier.com/locate/ultramic 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. ARTICLE IN PRESS A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641 636 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 ARTICLE IN PRESS A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641 637 0 nm 50 100 150 200 0 200 400 600 nm 0 12 25 topography (nm) 38 0 nm 50 100 150 200 0 200 400 600 nm -75 -72 -69 -66 -63 -61 deflection amplitude (dBm) 0.12 0.1 20 0.08 15 0.06 10 Absorbance Vibration amplitude of the tip (nm) 25 -58 0.04 5 0.02 0 1800 1750 1700 1650 1600 1550 1500 1450 1400 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 ARTICLE IN PRESS A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641 638 0 nm 250 500 750 0 500 -50 1000 nm 0 50 100 topography (nm) 1500 150 0 nm 250 500 750 0 500 -80 1000 nm 1500 -75 -70 -65 deflection amplitude (dBm) 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. ARTICLE IN PRESS 0 0 1 1 m m A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641 2 3 4 4 1 2 µm 3 4 1.0 0 0 1 1 1.5 2.0 2.5 µm 3.0 3.5 4.0 m m 2 3 0 639 2 2 3 3 0 1 2 3 0 µm 1 2 3 µm 0.0 0.00 0.5 1.0 1.25 1.5 m m 2.0 2.50 2.5 3.0 3.5 3.75 4.0 4.5 0.00 1.25 2.50 µm 3.75 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 µm 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). ARTICLE IN PRESS A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641 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. 0 250 nm 640 500 750 1000 0 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 ARTICLE IN PRESS A. Dazzi et al. / Ultramicroscopy 108 (2008) 635–641 641 Time –1e-9 Surface Temperature [K] Deformation Deplacement [m] x10-4 Maxi: 349.5 1.26 1.24 1.22 1.2 1.18 1.16 1.14 1.12 1.1 1.08 1.06 1.04 1.02 1 0.98 0.96 0.94 0.92 0.9 0.88 0.86 0.84 0.82 0.8 0.78 0.76 0.74 0.72 0.7 0.68 345 340 335 330 325 320 315 310 305 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 300 4.5 Mini:299.962 -4 x10 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.