Inactivation of viruses by coherent excitations with a low power visible femtosecond laser
© Tsen et al; licensee BioMed Central Ltd. 2007
Received: 10 April 2007
Accepted: 05 June 2007
Published: 05 June 2007
Resonant microwave absorption has been proposed in the literature to excite the vibrational states of microorganisms in an attempt to destroy them. But it is extremely difficult to transfer microwave excitation energy to the vibrational energy of microorganisms due to severe absorption of water in this spectral range. We demonstrate for the first time that, by using a visible femtosecond laser, it is effective to inactivate viruses such as bacteriophage M13 through impulsive stimulated Raman scattering.
Results and discussion
By using a very low power (as low as 0.5 nj/pulse) visible femtosecond laser having a wavelength of 425 nm and a pulse width of 100 fs, we show that M13 phages were inactivated when the laser power density was greater than or equal to 50 MW/cm2. The inactivation of M13 phages was determined by plaque counts and had been found to depend on the pulse width as well as power density of the excitation laser.
Our experimental findings lay down the foundation for an innovative new strategy of using a very low power visible femtosecond laser to selectively inactivate viruses and other microorganisms while leaving sensitive materials unharmed by manipulating and controlling with the femtosecond laser system.
Modern biochemical and pharmaceutical methods for inactivating or altering the functionality of viruses and other microorganisms are not only partially successful but also evoke problems of drug resistance and clinical side effects. New methods are therefore desirable, in particular for treating diseases such as HIV and other blood-borne viral diseases.
One experimental technique toward this goal is microwave absorption. However, a major drawback for excitation through microwave absorption is that water absorbs severely in the vibrational frequencies of capsids of viruses which are typically in the range of 30 GHz to 300 GHz [1, 2]. Thus, it is extremely difficult to transfer microwave excitation energy to the vibrational energy of microorganisms. To overcome this difficulty, it is essential to transfer the excitation sources from the microwave range to, say, the visible range in which water is transparent.
Impulsive Stimulated Raman Scattering (ISRS) has been shown to be a viable way of producing large-amplitude vibrational modes in molecules in liquid solution as well as lattice vibrations in solid state systems . In this paper, we report for the first time the use of a visible femtosecond laser system to excite a coherent acoustic Raman-active vibrational mode (which is associated with vibrations of viral capsids) in M13 phages through ISRS to such a high-energy state as to lead to their inactivation. Our work demonstrates a new method of manipulating, controlling, and inactivating unwanted microorganisms. It suggests that the basic principles of impulsive coherent excitation using a laser optical system can represent a general way to selectively alter the function of viruses and potentially other microorganisms through the property of their mechanical acoustic excitations. In addition, since structural change due to the mutation of microorganisms leads to very minimal variation of the vibrational frequency of their capsids, damage caused to viruses and/or other microorganisms through vibration of their mechanical structures likely would not be immune to simple mutation of cell surface receptors and the same treatment procedure remains valid, our approach would thus not evoke problems of drug resistance and as a result would be applicable to drug resistant strains of microorganisms.
Sample and Experimental setup
The M13 bacteriophage samples used in this work were purchased from Stratagene.
The activity of M13 bacteriophages was determined by plaque counts. In brief, M13 bacteriophages with nominally prepared 1 × 103 pfu or 5 × 102 pfu were added into a tube of soft agar at 70°C containing 0.3 ml of bacteria culture. Here, "nominally prepared" means that we prepare/dilute the M13 bacteriophage samples based on the pfu concentration specified by the manufacturer upon purchasing. The mixture was mixed well by vortexing and then poured onto a LB agar plate immediately. The plate was swirled well in order to spread the mixture over the entire plate evenly. The mixture on the agar plate was incubated for 8–16 hr. The plaques formed on the plate were counted.
All data are expressed as mean ± SD. Student's t-test was used for comparison of group with 5% as significant level.
Experimental results, analysis and discussions
where I is the power density of the excitation laser; α is the polarizability of the medium; R is the displacement away from the equilibrium intermolecular distance; δα/δR is proportional to the Raman scattering cross section; ω0 is the angular frequency of the excited coherent vibrational mode; τ L is the FWHM of the pulse width of the excitation laser; m is the molecular mass; n is the index of refraction; and c is the speed of light.
Dependence of the status of M13 bateriophages on pulse width (The laser power density is kept at 64 MW/cm**2)
Pulse width (fs)
(Yes or No)
From Eq. (1) it is clear that larger Raman cross sections, higher laser power densities, as well as lower vibrational frequencies, would contribute to bigger excited vibrational amplitude. In fact, for a moderate Raman scattering cross section, a sufficiently low vibrational frequency and a reasonable excitation power density, the amplitude of the vibrational displacement in the 0.01 to 1Å could be achieved through ISRS.
In our previous Raman scattering experiments [13, 14], we have reported the observation of low frequency (≅ 8.5 cm-1) vibrational mode of M13 bacteriophages, which is shown to be associated with the axial torsion vibrations of the capsid of M13 bacteriophage. This particular Raman-active mode was observed because of relatively low damping rate resulting from the nature of its vibrations, i.e., it vibrates along the axis of the cylindrical-shape M13 bacteriophage. Therefore, we believe what is happening is that under our current ultrashort pulse laser excitation experiments with M13 bacteriophages, the amplitude of this mode has been coherently excited by ISRS to the extent that leads to their inactivation. To further support this explanation, we have carried out similar experiments with M13 bacteriophages by varying the power density as well as pulse width of the excitation laser.
We have also found that within the statistical error of the experiments there is almost no observable inactivation of the M13 bacteriophages if the pulse width of the excitation laser is longer than about 800fs while the power density of the excitation laser remains constant at ≅ 64 MW/cm2 (this power density corresponds to an ultrafast laser with 100 fs pulse width and 40 mW of average power). The experimental results are summarized in Table 1. According to Eq. (1), if the laser power density remains constant, the amplitude of vibrational displacement excited by an ultrashort laser decreases with the increasing laser pulse width. As a matter of fact, Eq. (1) predicts that the amplitude of vibrational displacement excited by an ultrashort laser having a pulse width of 700 fs with power density of ≅ 64 MW/cm2 approximately equals to that by an excitation laser having 100 fs pulse width but with power density 50 MW/cm2. Therefore, the experimental results in Table 1 are consistent with the predictions by Eq. (1) and with the power-density results of Fig. 4. We also notice that because the laser energy per pulse in the case of 800 fs or longer pulse width is actually higher than that in the case of 700 fs or shorter, the experimental results in Table 1 rule out the possibility that our observed inactivation of M13 bacteriophages is due to transient, micro-thermal effects that might develop at the laser focused volume.
We notice that viruses have been known to be inactivated by the ultraviolet photons with wavelengths around 250 nm. There is a possibility that our excitation laser might be generating 212.5 nm through the nonlinear property of the sample system; as a result the produced ultraviolet photons inactivate the M13 bacteriophages. However, this possibility can be ruled out. Because if the excitation laser with a pulse width of 100 fs and power density of 64 MW/cm2 inactivated the M13 bcteriophages by the generated ultraviolet photons, then a laser with longer pulse width but the same power density would have produced more ultraviolet photons and would have been able to inactivate the M13 bacteriophages as well. This is in contradiction with the experimental results of Table 1. Thus, it is unlikely that the nonlinearly generated ultraviolet photons (if there are any) play a significant role in the inactivation of M13 bacteriophages observed in our current experiments.
Finally, we note that partly because our experimental results on power density and pulse width dependences are consistently explained by ISRS based upon the assumption that single laser pulse inactivates the M13 bacteriophage and partly because the fact the pulse separation produced in the laser system is as long as 13 ns, we believe that one single pulse in our visible femtosecond laser is capable of inactivating the M13 bacteriophage.
We have shown that microorganisms such as bacteriophage M13 can be inactivated by a very lower power visible femtosecond laser system through ISRS. Our work demonstrates a new strategy for manipulating, controlling, and inactivating unwanted microorganisms. It suggests that the basic principles of impulsive coherent excitation using a laser optical system can be a general way to selectively alter the function of viruses and potentially other microorganisms through the property of their mechanical acoustic excitations. Furthermore, since damage caused to viral organisms through vibration of their mechanical structures likely would not trigger an immune response to simple mutation of cell surface receptors and the same treatment procedure remains valid, our approach would thus not evoke the problems of drug resistance. As a result, it would be applicable to drug resistant strains of microorganisms as well.
This work is supported by the National Science Foundation under Grant No: DMR-0305147. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, or the U.S. Department of Defense.
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