2.1.

Procedure for Atomic Force Microscopy Imaging

Biomolecules such as protein, DNA, and RNA can be easily captured on the aminopropyltriethoxysilane (APTES) modified surface using glutaradehyde. The laser-irradiated viral particles were immobilized on the APTES mica and imaged under atomic force microscopy (AFM).^{9, 10, 11, 12}

2.2.

APTES Mica Preparation

Fresh cleaved mica was placed in a desiccator with 30 μl APTES (99%, Sigma-Aldrich, St. Louis, Missouri) and 10 μl N,N-diisopropylethylamine (99%, distilled, Sigma-Aldrich) in the bottom. The desiccator was then purged with argon for 3 min, and the mica was allowed to remain in the APTES vapor for 1 h to achieve good modification.

2.3.

Sample Immobilization

One hundred microliters of 2 μm glutaradehyde (grade I, Sigma-Aldrich) was deposited onto the APTES mica surface for 10 min, and the surface was then washed gently with distilled water. After that, 200 μl of virus samples with concentration ranging from 1×10⁶ to 1×10⁸ particles per millimeter were pipetted onto the glutaradehyde-treated mica surface and allowed to incubate for 40 to 80 min. The mica surface, now containing the immobilized sample, was then gently rinsed with distilled water and dried with nitrogen.

2.4.

Imaging

Imaging were carried out with a PicoPlus 2500+ AFM [molecular imaging; now 5500 AFM (N9410S) from Agilent] equipped with a Si3N4 cantilever (AppNano SPM) with a spring constant range from 25 to 75 N/m and the resonance frequency around 300 kHz.

2.5.

Procedure for Protein Structure Evaluation

BSA (≥99% purity) was purchased from Sigma-Aldrich (St. Louis, Missouri).

BSA solutions (34 and 340 μm) were prepared in 10 mm phosphate buffer (pH = 6.0). Concentrations were determined by UV absorbance at 280 nm (ɛ = 43,824 m⁻¹ cm⁻¹) using a Cary50 UV-Vis spectrophotometer (Varian). After laser irradiation, aliquots from the two samples were diluted to a final concentration between 1.4 and 2 μm to avoid signal saturation in subsequent measurements. Two identical samples that were not laser irradiated, but otherwise underwent identical treatment, were also diluted to a final concentration between 1.4 and 2 μm. The circular dichroism and fluorescence of all four samples were measured.

2.6.

Fluorescence Spectroscopy of BSA

Fluorescence spectra were measured in a 1 cm quartz cuvette (Hellma QC) using a QuantaMaster 40 spectrofluorometer (Photon Technology International) with a 0.5 mm bandpass. Excitation and emission spectra were measured from 250 to 325 nm (λ_em = 336 nm) and 283 to 450 nm (λ_ex = 281 nm), respectively, and buffer subtracted.

2.7.

Circular Dichroism Spectroscopy of BSA

Circular dichroism (CD) spectra^{13, 14} of the samples were measured in a 1 mm quartz cuvette (Starna Cells) using a Jasco J-710 spectropolarimeter (Jasco Company) with a 1 nm bandwidth. For each sample, four spectra taken between 250 and 190 nm with a 0.2 nm pitch at a 50 nm/min scan speed were averaged and then buffer subtracted.

Spectra were rescaled by the samples’ respective concentrations, determined by the UV absorbance values, before data were analyzed.

2.8.

Procedure for Gel Electrophoresis Experiments

Single-stranded DNA from M13 bacteriophages was purchased from New England Biolaboratory (Ipswich, Massachusetts). The double-stranded DNA was from an ASU-homemade plasmid which contains mouse mammary tumor virus (MMTV). All the DNA samples with 10× nondenaturing dye were loaded to the gel wells of a 0.8× agrose gel that was prepared in 1× tris-borate-ethylenediamine tetra-acetic acid buffer. The gel was run at room temperature at constant voltage at 80 V for 1.5 h.¹⁵

2.9.

Statistical Analysis

All data are expressed as mean ± standard deviation. The significance was detected with P<0.05 using Student's t-test.

3.1.

Inactivation of EMCV, M13 Bacteriophage, and Salmonella Typhimurium by a Visible Femtosecond Laser

Figures 2, 3 show the pfu on a logarithmic scale as a function of fluence on a logrithmic scale for EMCV and M13 bacteriophage samples, respectively. Log-kill factors of 3 and 5 have been found for EMCV and M13 bacteriophage, respectively, with 1 h of laser exposure time.

Fig. 2

Log-kill factor as a function of the log laser fluence for the femtosecond laser-irradiated, CW laser-irradiated EMCV, as indicated.

Fig. 3

Log-kill factor as a function of the log laser fluence for the femtosecond laser-irradiated, CW laser-irradiated M13 bacteriophages, as indicated.

Figure 4 shows the cfu on a logarithmic scale as a function of fluence on a logarithmic scale for Salmonella typhimurium. A log-kill factor of about 6 was observed with 1 h of laser exposure time, suggesting that visible femtosecond laser irradiation can very effectively inactivate the bacteria.

Fig. 4

Log-kill factor as a function of the log laser fluence for the femtosecond laser-irradiated, CW laser-irradiated Salmonella typhimurium, as indicated.

We have found that log (pfu)/log (cfu) versus log (fluence) in Figs. 2, 3, 4 are nonlinear.

3.2.

Atomic Force Microscope Images of M13 Bacteriophages

Figure 5 shows atomic force microscope pictures of M13 bacterioaphage without laser irradiation [Fig. 5a] and with laser irradiation [Fig. 5b] by a visible femtosecond laser. The relatively smooth worm-like features having a diameter of about 6 nm and about 850 nm in length in Fig. 5a revealed the presence of M13 bacteriophages in the control. Figure 5b showed, in contrast to Fig. 5a, appearance of a lot of small structures which were about 6 nm in diameter after laser irradiation. As discussed later, these small structures were consistent in size with the size of individual α − helix protein unit of which the protein shell of the M13 bacteriophage is composed. As a result, these small structures are most likely individual α − helix protein units of the M13 bacteriophage. In addition, some zigzagged worm-like features (encircled by artificially drawn black curves) were observed. The facts that its length was about 850 nm and it was in zigzagged structure indicated that these zigzagged structures were bare DNAs from M13 bacteriophages. The observation of the bare DNAs in the laser-irradiated M13 bacteriophage sample indicated that irradiation of the visible femtosecond laser severely alters the structural integrity of the protein shell of the M13 bacteriophages.

Fig. 5

Atomic force microscope picture of M13 bacterioaphages (a) without laser irradiation and (b) with laser irradiation by a visible femtosecond laser. For clarity, the black curves were drawn to encircle the bare DNAs. See text for discussions.

3.3.

Effects of Irradiation of a Visible Femtosecond Laser on Proteins and DNAs

To elucidate the possible inactivation mechanisms for viruses and bacteria by a visible femtosecond laser, we have tested the effects of irradiation of a visible femtosecond laser on the structure of BSA proteins, double-stranded DNA from homemade plasmid, and single-stranded DNA from M13 bacteriophages.

The luminescence, excitation spectra, and CD 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 the proteins before and after the laser irradiation. Figures 6a, 6b, 6c show our preliminary results for BSA proteins in buffer solution with and without irradiation with a visible femtosecond laser. In Fig. 6a, 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 and, 300 μm of BSA proteins, respectively. For clarity, the spectra shown were normalized to the concentration of BSA proteins. In Fig. 6b, the far ultraviolet 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 60 and 300 μm of BSA proteins, respectively. For clarity, the spectra shown were normalized to the concentration of BSA proteins. In Fig. 6c, the near ultraviolet 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 and 300 μm of BSA proteins, respectively. For clarity, the spectra shown were normalized to the concentration of BSA proteins. The experimental results indicated that, within experimental uncertainty, the luminescence, excitation spectra, and circular dichroism of BSA proteins remained practically the same before and after the laser irradiation, indicative of minimal structural change of BSA proteins by the irradiation of a visible femtosecond laser.

Figure 7 shows the effect of visible femtosecond laser irradiation on the single-stranded DNA from M13 bacteriophages, as detected by the agarose gel electrophoresis method. The control sample revealed the presence of a dark band from the single-stranded DNA of M13 bacteriophages. The laser-irradiated sample showed a single dark band similar in width to, and located in the same position, as that of the control sample. Figure 8 shows the agarose gel electrophoresis results on the single-stranded DNAs from the M13 bacteriophages (control) and the laser-irradiated M13 bacteriophage sample. The dark band, interpreted to be from the single protein units of the laser irradiated M13 bacteriophage sample, is out of scale and is, therefore, not shown here. Again, 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 the experimental uncertainty, irradiation of a visible femtosecond laser caused no denaturing or severe structural change of the single-stranded DNAs of M13 bacteriophages.

Fig. 7

Gel electrophoresis experiments on single-stranded DNA of M13 bacteriophages before and after the visible femtosecond laser irradiation, as indicated, for 1 h.

Fig. 8

Gel electrophoresis experiments on single-stranded DNAs of M13 bacteriophages (control) and the laser-irradiated M13 bacteriopahges after the visible femtosecond laser irradiation for 1 h. For clarity, on the laser irradiated sample, an additional band resulting from the α − helix protein units of M13 bacteriophages, which appears on a different scale, is not shown.

Figure 9 demonstrates our preliminary results for double-stranded DNA in buffer solution before and after irradiation by a visible femtosecond laser, as detected by the agarose gel electrophoresis method. 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 altered greatly. These data suggest that the effects of visible femtosecond laser irradiation primarily relaxed the super-coiled double-stranded DNA or linear double-stranded DNA to the circular double-stranded DNA.

Fig. 9

Gel electrophoresis experiments on double-stranded DNA from an ASU-homemade plasmid which contains MMTV in buffer solution before and after the visible femtosecond laser irradiation, as indicated, for 1 h.

4.1.

Insight into the Inactivation of a Virus by a Visible Femtosecond Laser

The gel electrophoresis results of Figs. 7, 8 on the single-stranded DNAs of M13 bacteriophages, indicate that irradiation of a visible femtosecond laser does not alter the structure of single-stranded DNA. In fact, this interpretation is consistent with the observation of the bare, undamaged DNAs in our AFM images of Fig. 5b for the laser-irradiated M13 bacteriophage sample.

The experimental results on the optical characterization of BSA proteins (Fig. 6) suggest that there is practically no structural change of BSA proteins upon laser irradiation. Because the capsid of a M13 bacteriophage is mostly composed of α − helix protein units, these results suggest that visible femtosecond laser irradiation under our experimental conditions most likely will not damage the individual protein unit of which the protein shell of M13 bacteriophage is made up.

Fig. 6

(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.

By taking into account the size of small structures about 6 nm in diameter in Fig. 5b, 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 M13 bacteriophage, we have found that the small structures observed in Fig. 5b are consistent in size with those of the α − helix protein units of the capsid of M13 bacteriophages. This estimate further supports our conclusion that visible femtosecond laser irradiation under our experimental conditions does not damage individual protein units in M13 bacteriophages.

Therefore, the AFM images of Fig. 5, together with the DNA gel electrophoresis results of Figs. 7, 8 and optical results of BSA proteins of Fig. 6, demonstrate that irradiation of a visible femtosecond laser alters the structural integrity of the protein shells of M13 bacteriophages by breaking their weak links without damaging either the single-stranded DNAs or the basic protein units of M13 bacteriophages.

Deposition of laser energy onto the capsid of M13 bacteriophages can cause the breaking of weak links such as hydrophobic contacts and hydrogen bonds of the capsids. As a matter of fact, it has been estimated¹⁶ that simultaneous (or within a 100 fs time interval) deposition of a sufficient number of photons (of the order of 100) on the capsid of cowpea chlorotic mottle virus (CCMV) could break the hydrogen/hydrophobic bonds on the capsid of CCMV, leading to the subsequent disintegration of the capsid of CCMV into trimers.

Based on the above arguments, the inactivation of M13 bacteriophages by a visible femtosecond laser is most likely due to the deposition of laser energy onto the capsids of M13 bacteriophages. Possible mechanisms that can account for our experimental observations for the inactivation of M13 bacteriophages are discussed in Secs. 4.1.1, 4.1.2, 4.1.3.

4.2.

Insight into the Inactivation of Bacteria by a Visible Femtosecond Laser

To obtain insight into the inactivation mechanisms, we have performed inactivation of a mutant Salmonella typhimurium by a visible femtosecond 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 and mutant Salmonella typhimurium by a visible femtosecond laser as a function of the laser fluence. In general, the log–load reduction factor at a given laser dose is higher for the mutant than for the wild strain. In particular, our experimental results indicate that by using the visible femtosecond 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 stains 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 femtosecond laser causes DNA damage and subsequent inactivation of the Salmonella typhimurium.

Fig. 10

Log-kill factor as a function of laser fluence for the wild, mutant Salmonella typhimurium, as indicated.

It has been established that^{29, 30, 31, 32, 33, 34} photostimulation of endogenous intracellular porphyrin molecules in the bacteria by continuous wave visible light irradiation results in the production of reactive oxygen species, predominantly singlet oxygen, and consequently, damage to the DNA and the death of bacteria. We believe that this is the dominant mechanism for the inactivation of Salmonella typhimurium by a visible femtosecond laser.

We have also carried out absorption experiments on Salmonella typhimurium. Our results indicated that, on average, deposited laser energy for a Salmonella typhimurium is about 1.1×10⁻¹⁶ J/pulse or 1.1×10⁻⁹ J/cm², indicating that the shock wave generation mechanism²⁸ is not the inactivation mechanism for Salmonella typhimurium.

4.3.

Inactivation of Viruses and Bacteria by a Continuous-Wave Laser at a Wavelength of 425 nm

We have also performed inactivation of M13 bacteriophages, EMCV, and Salmonella typhimurium as a function of laser fluence with an InGaN CW laser operated at 425 nm. The results are shown in Figs. 2, 3, 4, respectively. Because a CW laser is expected to inactivate viruses and bacteria dominantly through linear absorption process, comparison of inactivation between a CW visible laser and the visible femtosecond laser operated at the same wavelength provides valuable information on the inactivation mechanisms. For example, the substantial differences of inactivation between a CW laser and a femtosecond laser in Figs. 2, 3 reflect significant inactivation of M13 bacteriophages and EMCV due to a nonlinear two-photon Raman scattering process. On the other hand, Fig. 4 shows that inactivation between a CW laser and a femtosecond laser is very similar for Salmonella typhimurium, indicating that the linear absorption process is the primary mechanism for the inactivation of Salmonella typhimurium by a visible femtosecond laser.

4.4.

Two-Photon Absorption Process

Based on the assumption that the two-photon absorption cross section is 10⁻⁵⁰ cm⁴ s photon⁻¹ for a typical molecule, the excitation laser power density is about 100 MW/cm², and for the laser parameters used in our experiments, we estimate that the number of ultraviolet (UV) photons at wavelength λ = 2.12.5 nm produced in the sample by the excitation laser through two-photon absorption process is about 15. This number of generated UV photons is too small to contribute to the inactivation of M13 bacteriophages, EMCV, and salmonella. Therefore, we believe the two-photon absorption process cannot account for the inactivation of M13 bacteriophages, EMCV, or salmonella bacteria observed in our experiments.