Inactivation of a virus by ultrashort pulsed lasers
We take M13 as an example for demonstration. Figure shows plaque forming units (pfu) as a function of laser power density for M13 bacteriophages excited by a near-infrared Ti-sapphire cw mode-locked laser [4
] The intriguing feature of these assay results is the rapid cut-off of the pfu of M13 bacteriophages at around 60 MW/cm2
. A similar feature (which is not shown here) is also found when a visible USP laser is used for inactivation. This unique feature of inactivation upon laser power density indicates the emergence of a new virus inactivation mechanism for M13 bacteriophages by the irradiation of USP lasers – impulsive stimulated Raman scattering (ISRS) – which is elucidated below.
Number of pfu as a function of laser power density for M13 bacteriophages excited by a near-infrared Ti-sapphire cw mode-locked laser. See text for discussions.
The atomic force microscope (AFM) images from the control and laser treated M13 bacteriophage samples provide an important clue for the inactivation mechanism. The AFM images of a M13 bacteriophage sample before and after the visible USP laser irradiation are shown in Figure (a) and (b), respectively [10
]. The relatively smooth worm-like features having a diameter of about 6 nm and about 850 nm in length in Figure (a) revealed the presence of M13 bacteriophages in the control. Figure
(b) showed, in contrast to Figure (a), the appearance of many small structures which were about 6 nm in diameter after laser irradiation. As discussed later, these small structures were consistent with the size of individual α-helix protein units of which the protein capsid of the M13 bacteriophage is composed. As a result, these small structures are attributed to individual α-helix protein units of the M13 bacteriophage. In addition, some zigzagged worm-like features (encircled by artificially drawn black curves for the sake of clarity) were observed. The fact that its length was about 850 nm and that it was in a zigzagged structure indicated that these zigzagged structures were naked viral genomic DNAs from M13 bacteriophages. The observation of the naked DNAs in the laser-irradiated M13 bacteriophage sample indicated that irradiation of the visible USP laser severely altered the structural integrity of the protein shell of the M13 bacteriophages, potentially causing the DNA to “leak out”.
Figure 2 Atomic Force Microscope images of M13 bacterioaphages (a) without laser irradiation and (b) with laser irradiation by a visible femtosecond laser. For clarity, the black curves in (b) were drawn to encircle the bare DNAs. See text for discussions (with (more ...)
By taking into account the size of small structures about 6 nm in diameter in the AFM images of M13 bacteriophages after USP laser irradiation in Figure (b), the resolution of the tip of AFM used in the imaging, and the actual size of the α-helix protein unit which forms the capsid of a M13 bacteriophage, we have found that the small structures observed in Figure (b) are consistent in size with those of the α-helix protein units of the capsid of M13 bacteriophages. This analysis further supports our conclusion that USP laser irradiation under our experimental conditions does not damage individual protein units in M13 bacteriophages.
Figure shows the result from agarose gel electrophoresis on single-stranded DNAs from M13 bacteriophages (control) and from M13 bacteriophages irradiated with a visible USP laser [10
]. The laser-irradiated M13 bacteriophage sample showed a single dark band similar in width to and located at the same position as that of the control sample. Therefore, these experimental results indicated that, within experimental uncertainty, irradiation of a visible USP laser caused no severe structural change of single-stranded DNAs of M13 bacteriophages. In other words, the gel electrophoresis results of Figure on the single-stranded DNAs of M13 bacteriophages indicate that irradiation of a visible USP laser does not significantly alter the structure of single-stranded DNA.
Figure 3 Gel electrophoresis experiments on single-stranded DNAs of M13 bacteriophages (control) and the laser-irradiated M13 bacteriophages after treatment with the visible femtosecond laser, operated at 425 nm, at a repetition rate of 80 MHz, average power of (more ...)
The luminescence, excitation, and circular dichroism (CD) spectra from amino acids of proteins are very sensitive to the structural changes of proteins. Therefore, these optical characterization methods were employed to detect the primary and secondary structural changes of proteins before and after the visible USP laser irradiation. Figures
(a)(b)(c) show our preliminary results for bovine serum albumin (BSA) proteins in buffer solution with and without irradiation with an USP laser [10
]. In Figure (a), the excitation spectrum corresponded to the broad structure centered around 280 nm. The luminescence spectrum represented the broad peak around 340 nm. Each spectrum contained 4 curves in which two of them were control and two were laser-irradiated samples, as indicated. The two control samples and two laser-irradiated samples had 60 μM, 300μM of BSA proteins, respectively. For clarity, the spectra shown were normalized to the concentration of BSA proteins. In Figure (b), the far UV CD contained four curves, in which two of them were control and two were laser-irradiated samples. The two control samples and two laser-irradiated samples had 60μM, 300μM of BSA proteins, respectively. For clarity, the spectra shown were normalized to the concentration of BSA proteins. In Figure (c), the near UV CD included four curves in which two of them were control and two were laser-irradiated samples. The two control samples and two laser-irradiated samples had 60 μM, 300 μM of BSA proteins, respectively. For clarity, the spectra shown were normalized to the concentration of BSA proteins. The experimental results show that, within experimental uncertainty, the luminescence, excitation spectra and circular dichroism of BSA proteins remained practically the same before and after the laser irradiation, indicating minimal or no structural changes in BSA proteins after irradiation with a visible USP laser. Therefore, these experimental results on the optical characterization of BSA proteins suggest that there is virtually no structural change in BSA proteins upon USP laser irradiation. Because BSA is primarily made up of α-helix proteins, and the capsid of a M13 bacteriophage is mostly composed of α-helix protein units, these results suggest that the visible USP laser irradiation will not damage the individual protein units that comprise the protein capsid of M13 bacteriophage.
(a): Excitation and luminescence spectra of BSA proteins; (b): Far UV circular dichroism spectra of BSA proteins; (c): Near UV circular dichroism spectra of BSA proteins (with publisher’s permission).
Thus, the AFM images of Figure together with the DNA gel electrophoresis results of Figure and optical results of BSA proteins of Figure are consistent with our model: that irradiation with a USP laser alters the structural integrity of the protein capsid of M13 bacteriophages by disrupting weak interactions between proteins without damaging either the viral genomic single-stranded DNA or the individual protein units of M13 bacteriophage capsid.
Irradiation with an intense ultrashort pulsed laser such as a femtosecond laser can deposit laser energy onto the protein capsid of a viral particle by the excitation of low-frequency acoustic vibrations on the capsid of a virus. This process, known as impulsive stimulated Raman scattering (ISRS), has been used to deposit laser energy to solid state systems as well as to biological molecules [13
The ISRS process can be understood as follows:
The vibrational mode of a macromolecule such as a virus excited by the laser is represented by normal coordinate Q. If we ignore dispersion in the index of refraction and assume that the incident electric field from the excitation laser is not depleted by the stimulated scattering, the equation of motion for Q can be written as [21
is the angular frequency of vibration,
is the damping constant and
is the impulsive driving force produced by the excitation laser and is described next.
The electric field
of the laser induces a polarization on the molecule due to its polarizability α
, where for simplicity we neglect the tensor properties of α
. The polarizability has a static part that produces elastic Rayleigh scattering, and a part that is modulated by the oscillating displacement Q. It is this modulated contribution that produces the Raman effect and the ISRS process in the macromolecule. The polarizability α
, expanded in a Taylor series in Q, is
higher order terms in Q (2); where α0 is the zero order term
is the first order term resulting in the first order Raman scattering processes;
is the second order term, etc.
The potential energy stored in an induced polarization is
. If we keep up to the first order term and neglect the second order and higher order terms in the polarization expansion in Eq, (2), the generalized driving force
on the right hand side of Eq. (1) becomes
Equation (1) with
given by Eq. (3) can be solved by using Green’s function method to determine the normal coordinate Q(t) [13
]. In particular, for excitation by a single-beam ultrashort laser having a pulse width of
, and intensity
, assuming small damping, the displacement is
Of greatest importance in
is the amplitude
of the displacement away from the equilibrium position of the molecule produced by ISRS process, which is given by [13
is the peak intensity of the excitation laser,
is the polarizability derivative proportional to the amplitude of the Raman scattering cross section, n
is the index of refraction, c the speed of light, and
the permittivity of the dielectric medium.
Therefore, in this ISRS process, the deposited laser energy on the protein capsid of a viral particle is proportional to the square of the laser intensity and to the Raman scattering cross section. If the deposited laser energy or the amplitude of the excited resonance mode on the capsid of a viral particle is large enough, it can break the weak links (for example, hydrogen bonds or hydrophobic contacts) between the proteins, damage to the capsid of the virus occurs, leading to the viral inactivation.
In the ISRS process, operated in near-infrared/visible wavelength range to which water is transparent, one way of selective killing of microorganisms is by varying the laser power density; the other way of selective killing of microorganisms in biological systems is by controlling the range of spectral content of an ultrashort pulsed laser. For a transform-limited pulsed laser, by using Heisenberg uncertainty principle, it is equivalent to controlling the laser pulse width. The presence of the factor
in Eq. (4) indicates that in order to excited significantly large amplitude
of a vibrational frequency
in a microorganism for damaging effect, the excitation laser pulse width
has to be chosen so that
. Because each microorganism has its own characteristic resonance vibrational frequency
, by choosing the proper pulse width of an ultrashort pulsed laser, the amplitude of this resonance mode can be excited so high as to damage and inactivate the microorganism.
We note that cw (continuous wave) laser cannot excite the resonance mode
of a microorganism through an ISRS process. Because
for a cw laser, Eq. (4) therefore indicates that the amplitude of the excited vibrational mode is zero. A Q-switched laser cannot excite the resonance mode
of a typical microorganism through ISRS process either. This is because each microorganism has a characteristic resonance vibrational frequency
which typically is in the range of 100 GHz;[24
] for example, helix-shaped M13 bacteriophage is around 300 GHz [27
] and icosahedral viruses of 30 nm in size like murine norovirus is around 65 GHz [24
] and if we use a viral frequency of 100GHz and the fact that a typical Q-switched laser has a pulse width of about 100 nanosecond, from Eq. (4), the factor
becomes vanishingly small. Therefore, the amplitude of vibrations a Q-switched laser will excite is negligibly small.
The rapid switch from non-inactivation to inactivation at the laser power density of 60 MW/cm2 shown in Figure for M13 bacteriophages can be explained by the ISRS process. When the laser power density is small (<60MW/cm2), the excited amplitude of vibration on the capsid of M13 bacteriophage is not large enough to break the weak links and no inactivation is observed; however, as the laser power density increases to and beyond 60 MW/cm2, the excited amplitude of vibration becomes large enough to break the weak links on the capsid of the M13 bacteriophage, leading to the inactivation of M13 bacteriophage.
To further support our argument that viral particles are inactivated by the irradiation of USP lasers through an ISRS process, we show experimental results of the inactivation of M13 bacteriophages as a function of laser pulse widths/spectral widths in Table [4
] while the laser intensity is kept constant. The abrupt change from inactivation to no inactivation observed in the experiments when the pulse width of the laser changes from 500 fs to 800 fs is consistent with the prediction of Eq. (4) by using the Raman mode frequency of 10cm−1
which was measured by Raman spectroscopy for M13 bacteriophages [27
Dependence of the status of M13 bacteriophage on laser pulse width
Therefore, schematically, this is what is happening in our model for USP laser inactivation of viruses such as the M13 bacteriophage: The electric field from a femtosecond laser produces an impulsive force through the induced charge polarization on the virus, as shown in Figure (A). This mechanical impact coherently excites Raman-active vibrational modes on the capsid of the virus, as depicted in Figure (B). Figure (C) demonstrates that if the pulse width/spectral width and intensity of the USP laser are appropriately chosen, the vibrational modes can be excited to such high energy states as to break off the weak links on the capsid of the virus, damaging/disintegrating the capsid and leading to the inactivation of the virus.
Figure 5 Diagrams showing how the M13 bacteriophage is inactivated by an USP laser. (A) The electric field from a femtosecond laser produces an impulsive force through the induced charge polarization on the virus; (B) The resultant mechanical impact coherently (more ...)
Inactivation of bacteria by ultrashort pulsed lasers
We take Salmonella typhimurium
as an example.
To obtain insight into the inactivation mechanisms, we have performed inactivation of a mutant Salmonella typhimurium
by a visible USP laser. The mutant is deficient in RecA proteins which are responsible for the repair of damaged DNA. In other words, the mutant is very sensitive/vulnerable to the damage of DNA. Figure [10
] shows the inactivation of both the wild-type and mutant Salmonella typhimurium
by a visible USP laser as a function of the laser fluence. In general, the log – load reduction factor at a given laser dose has be found to be higher for the mutant than for the wild strain. In particular, our experimental results indicate that by using the USP laser, with laser dose of about 800 J/cm2
, a log - load reduction factor of about 5 for mutant Salmonella typhimurium
was observed; however, by employing the same laser parameter, a log-kill factor of only 0.5 for the wild Salmonella typhimurium
was found. Because the only difference between these two strains of Salmonella typhimurium
is the RecA proteins which are in charge of the repair of damaged DNA, these experimental results indicate that irradiation of a visible USP laser causes DNA damage and subsequent inactivation of the Salmonella typhimurium
Figure 6 Log-kill factor as a function of laser fluence for the wild, mutantSalmonella typhimurium,as indicated (with publisher’s permission).
Figure demonstrates our preliminary results for isolated double-stranded DNAs in buffer solution before and after irradiation by a visible femtosecond laser, as detected by the agarose gel electrophoresis method [10
]. The control sample (labeled No. 1) revealed the presence of three dark bands corresponding to circular, linear, and super-coiled double-stranded DNA, respectively. Sample No. 2 showed that stirring the sample slightly changed the relative darkness of the bands. On the other hand, the laser-irradiated sample (labeled No. 3) showed that the relative darkness of the three bands was greatly altered. These data suggest that the effects of visible femtosecond laser irradiation primarily caused relaxation of the supercoiled double-stranded DNA to produce relaxed circular double-stranded DNA. Because forced changes in the supercoiling status of DNA can disrupt cellular metabolism, which can lead to the death of the cell, one mechanism which can contribute to the inactivation of Salmonella typhimurium
by the irradiation of a visible USP laser is relaxation of supercoiled DNA in the bacteria.
Figure 7 Gel electrophoresis experiments on double-stranded DNAs. #1 is the control without magnetic stirring showing the presence of super-coiled, linear and circular DNAs; #2 is another control with magnetic stirring; #3 is the laser-irradiated sample with magnetic (more ...)
It has been known that photo-stimulation of endogenous intracellular porphyrin molecules in the bacteria by continuous wave visible light irradiation may result in the production of reactive oxygen species (ROS), predominantly singlet oxygen, and consequently, damage to the DNA and the death of bacteria [30
]. Therefore, the other mechanism which can contribute to the inactivation of Salmonella typhimurium
by a visible USP laser is the photo-production of ROS.