1. Introduction

Recently, there have been rapid developments in the field of information technology, resulting in the need to generate, store, and transport a large amount of information while ensuring data security, an important issue in today’s digital age. To meet future demands in information technology, femtosecond laser pulse processing [1–11] offers a powerful tool for developing new high-capacity devices because it allows fabrication of three-dimensional (3-D) structures inside a wide range of transparent materials. In particular, multilayered 3-D optical bit recording is a promising technique for next-generation computing systems because it offers a large recording capacity by stacking many recording layers without increasing the recording density per layer [4, 7, 12–15]. Our goal is to realize optical data storage in a human fingernail for highly secure data transportation that does not suffer from problems such as theft, forgery, or loss of recording media [16, 17]. Processing of structures inside a human fingernail by a femtosecond laser has been investigated, the available recording density has been estimated from the processed feature size, and 3-D optical data recording inside a fingernail has been demonstrated [16]. The drastic changes of morphology on a fingernail surface ablated with a femtosecond laser pulse according to the focusing position have also been investigated [17].

In this paper, we describe a suitable readout method for the bit data stored in a human fingernail. We demonstrate an increase in fluorescence intensity compared with the surrounding auto-fluorescence intensity at a structural change produced by a focused femtosecond laser pulse inside a human fingernail. The spectrum of the increased fluorescence coincides with the auto-fluorescence spectrum of a fingernail and that of pure keratin. The increased fluorescence intensity is also observed in a heated fingernail. It is suggested that the increased fluorescence is a result of a local denaturation of keratin protein caused by the femtosecond laser pulse irradiation. The increased fluorescence effect is very useful for reading out the bit data recorded inside a human fingernail. We also demonstrate that three-dimensionally-arranged structural changes can be read out with little cross-talk by making use of the increased fluorescence. Furthermore, we demonstrate that fluorescence can be observed for up to 6 months, corresponding to the time required for a fingernail to grow from root to tip.

2. Experimental procedure

An optical system for recording bit data inside a human fingernail is composed of a femtosecond laser system and an optical microscope, as shown in Fig. 1. The femtosecond laser system is composed of a mode-locked Ti:sapphire laser (Spectra Physics, Tsunami) pumped by a diode-pumped solid state continuous-wave green laser (Spectra Physics, Millenia), and a multi-kilohertz pulsed Ti:sapphire regenerative amplifier (Spectra Physics, Spitfire) pumped by a diode-pumped Nd-YLF laser (Spectra Physics, Merlin). A femtosecond laser pulse with a central wavelength of 800 nm and a pulse width of less than 100 fs (FWHM) is introduced into the optical microscope. The optical microscope system (Olympus, IX70) has a computer-controlled three-axis motorized stage. In most experiments, a 40×objective (numerical aperture (NA)=0.55) is used. A sample on the motorized microscope stage is observed with a charge coupled device (CCD) image sensor under transmitted illumination. The recording depth Z is defined as the distance moved along the optical axis by the microscope stage. The zero depth is determined by microscope observation of the sample surface. When the focusing position is inside the sample, Z is positive. The irradiation pulse energy E_p described in this paper is the product of the energy measured at the entrance of the microscope and the transmittance of the microscope system, including transmittance of the objective. The transmittance of the microscope is 0.49. The sample is a small piece of human fingernail whose size is about 2×2×0.4 mm³, and its surface is polished with abrasive lapping films (#1000~#10000). The surface polish reduces the required pulse energy for processing because the scattering and the distortion of the wavefront are decreased. [16].

The optical setup for reading out the bit data is a fluorescence microscope (Nikon, ME600) consisting of a xenon arc lamp as an exciting light source, filter blocks, and a 60×objective (NA=0.95). Each of the filter blocks consists of an excitation filter (EX), which is a band-pass filter passing wavelengths from λ^(EX)1 to λ^(EX)2, a dichroic mirror (DM), which is a high-pass filter with a cut-off wavelength λ(DM), and a barrier filter (BA), which is a high-pass filter with a cut-off wavelength λ^(BA). Three kinds of filter blocks, that is, an ultraviolet (UV) excitation set with λ^(EX)₁=330 nm, λ^(EX)₂=380 nm, λ^(DM)=400 nm, and λ^(BA)=420 nm, a blue excitation set with λ^(EX)₁=450 nm, λ^(EX)₂=490 nm, λ^(DM)=505 nm, and λ^(BA)=520 nm, and a green excitation set with λ^(EX)₁=510 nm, λ^(EX)₂=560 nm, λ^(DM)=575 nm, and λ^(BA)=590 nm, are used. The spatial distribution of the fluorescence and the spectrum of a small area of the fingernail are observed with a CCD image sensor and a spectrometer (Hamamatsu, PMA-11 C7473-36), respectively.

 

Fig. 1. Femtosecond laser processing system.

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3. Results and discussions

When the femtosecond laser pulse is focused inside a material, molecules are subjected to multi-photon ionization and optical field ionization at a local volume where the laser pulse is focused. Consequently, the ionized molecules repulse each other, and a microexplosion occurs, which causes a structural change in the material. Figure 2 shows transmission-illumination microscope observations of three bit arrays recorded inside a human fingernail. These bit arrays were recorded with E_p =0.485 µJ at (a) Z=40 µm, (b) Z=60 µm, and (c) Z=80 µm. The bit diameter was about 3.1 µm. Under these recording conditions, the bit spacing was 5 µm in the transverse direction and 20 µm in the vertical direction. This gives a corresponding recording density of 2 Gbit/cm³.

Figure 3 shows a side view of the structural changes formed inside a human fingernail sample. The structures were formed at Z=100 µm by irradiation of a single femtosecond laser pulse with (a) E_p =0.25 µJ, (b) E_p =0.49 µJ, (c) E_p =0.98 µJ, (d) E_p =2.0 µJ, and (e) E_p =3.9 µJ, respectively. A pair of the structure indicated by (a) to (e) was formed by two times of single irradiation of a focused pulse with the same energy at the different position. The pulse was irradiated from the upper side in Fig. 3. The structures indicated by (a), which are barely visible, have a diameter of 1 µm, measured from the light intensity profile in this picture. The structures indicated by (b) have a straight line with a maximum diameter of 2.4 µm. The diameter is small compared with the diameter measured from the top view picture like as shown in Fig. 2. With increasing E_p , the length of the straight-line structure becomes longer, as shown in (c) and (d). It is expected that around the center of the straight-line structure, the short parallel line at the side of the straight-line is processed by the second peak of the diffraction pattern formed by the circular aperture of the objective lens [17]. The focusing position of the peak of the laser pulse is at that point. Consequently, the linear shape below the focal point is formed by a self-trapping mode of the propagating pulse. With further increasing E_p , the structures at the focal point change to a conical shape, indicated by (e). This conical shape is formed at the position where the energy density of the focused laser pulse was over the threshold energy density. The multiple focusing lines forming the cone originate from inhomogeneity of the fingernail.

 

Fig. 2. Multilayered bit arrays at (a) Z=40 µm, (b) Z=60 µm, and (c) Z=80 µm. The scale bar indicates 10 µm.

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Fig. 3. Side view of the structures formed in a human fingernail with (a) E_p =0.25 µJ, (b) E_p =0.49 µJ, (c) E_p =0.98 µJ, (d) E_p =2.0 µJ, and (e) E_p =3.9 µJ. The scale bar indicates 10 µm. The figure is made of two images that were obtained with adjusting the observation focus to the structures in each images.

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In investigating a suitable readout method of the recorded bit data inside a human fingernail, we discovered increased fluorescence at the structural changes formed in the fingernail compared with the auto-fluorescence of the fingernail. Figure 4 shows a fluorescence image of a side view of the structures formed in a human fingernail. This image was taken at the same place shown in Fig. 3. The increased fluorescence of the structure formed by irradiation of a single femtosecond laser pulse with E_p =0.25 µJ was not observed, as indicated in (a). The positions where the increased fluorescence occurred, indicated by (b) to (e), coincided with the positions of structural changes, which are dark regions in Fig. 3. In the profile of the side view, shown in the bottom of Fig. 4, the fluorescence from the bits indicated by (d) and (e) are stronger than the background autofluorescence, and the fluorescence from the bits indicated by (b) and (c) are not sufficiently high for the back ground. In the top view, however, the bits recorded with the same energy of 0.49 µJ can be observed, because the fluorescence is summed up along the optical axis (See Fig. 8.).

 

Fig. 4. Fluorescent image of a side view of the structures formed in a human fingernail. The scale bar indicates 10 µm. The profile is the intensity distribution along a white line in the image. The figure is made of three images that were obtained with adjusting the observation focus to the structures in each images.

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A transmission-illumination image and a fluorescence image of the bits recorded with changing focal position are shown in Figs. 5(a) and 5(b), respectively. The bit at the left side in the pictures was recorded at Z=-2.0 µm, and at 0.5-µm intervals in the Z direction. The pulse energy E_p was 0.49 µJ. The bit at the right side was recorded at Z=13.5 µm. The blue-excitation filter set was used in the fluorescence observation. The pictures were taken with the focal point set inside the fingernail; therefore, the structure (pit) on the fingernail surface was slightly out-of-focus. In the fluorescence image of Fig. 5(b), increased fluorescence intensity was observed at Z≥6.0 µm. Atomic force microscope (AFM) observation images at Z=5.5 µm and 6.0 µm are shown in Fig. 5(c) and (d), respectively. Surface height of AFM images is indicated in gray-scale as shown in the bottom right of Fig. 5. At Z<6.0 µm, that is, where increased fluorescence was not observed, a pit was always observed at the processing point in the AFM observation, as shown in Fig 5(c). The pit is formed by ablation of protein in the fingernail. The focal spot has longitudinal length. Therefore the pit can be formed while Z is positive. On the other hand, at Z≥6.0 µm, increased fluorescence was observed because the bits were recorded inside the fingernail. These results show that protein molecules modified with an intense laser pulse exist inside the fingernail. A swell with a height of 738 nm, shown in Fig. 5(d), was formed by melting of the material caused by the high energy concentration of the focused laser pulse, the pressure wave caused by an internal explosion, and solidification after cooling. From these experiments, it was found that the protein molecules modified by laser pulse irradiation exhibited the increased fluorescence effect.

Figure 6(a) shows the fluorescence spectrum of the processed area of a fingernail and the auto-fluorescence spectrum obtained with the UV excitation set. The solid curve and the dashed curve show the fluorescence spectra of the non-processed area and the processed area of the fingernail, respectively. Both measurements were performed on circular areas with a diameter of about 17 µm. The distance between both areas was about 47 µm. The fluorescence spectrum of the processed area increased over the entire observed wavelength range compared with the auto-fluorescence. From estimating the intensity ratio of the fluorescence spectrum of the processed area by dividing its value by the value of auto-fluorescence at each wavelength, indicated with the dotted curve, it is clear that both spectra are almost identical. The spectrum of the auto-fluorescence was also identical to that of pure keratin, which is the main component of a human fingernail. Similarly, increased fluorescence intensity and identical spectra were also observed with use of blue and green excitation sets. The strongest fluorescence was obtained when the UV excitation set was used, but the highest contrast was obtained when the blue excitation set was used. Consequently, this indicates that blue excitation is most suited for the readout of the bit data inside fingernails.

 

Fig. 5. Bits recorded by changing focusing position. (a) A transmission-illumination image and (b) a fluorescence image. Atomic force microscope observation images of (c) a pit (Z=5.5 µm) and (d) a swell (Z=6.0 µm). The scale bar indicates 10 µm in (a) and (b), and the side length of figures (c) and (d) is 7.5 µm.

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Fig. 6. (a) The fluorescence spectra from auto-fluorescence of a fingernail (solid curve) and from the structure formed by femtosecond laser pulse irradiation (dashed curve), and the ratio of fluorescence intensity of the structure and the auto-fluorescence (dotted curve). (b) The fluorescence spectra from fingernails heated at various temperatures (dashed, dotted, dash-dotted, and dash-dot-dotted curves) and a fingernail kept at room temperature (RT, solid curve).

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We consider that the increased fluorescence effect is caused by denaturation of the keratin protein by femtosecond laser pulse irradiation. Figure 6(b) shows the fluorescence spectra of heated fingernails measured with the UV excitation set. The samples were five pieces of human fingernail obtained by dividing a single piece of fingernail. One of the five samples was kept at room temperature, and the others were heated in a drying oven at 60, 120, 180, and 240 °C for 30 minutes, respectively. The fluorescence intensity of the heated sample became stronger as the temperature increased, and the sample heated at 180 °C had a fluorescence about 5 times as strong as the sample kept at room temperature. No noticeable change of the spectral distribution was observed. The fingernail sample heated at 240 °C was carbonized, and the fluorescence intensity decreased drastically. These results suggest that the increased fluorescence effect at the formed structure is caused by a local denaturation of the keratin protein by the focused femtosecond laser pulse.

This increased fluorescence effect is very useful for reading out the bit data formed in a fingernail with a reflection-type optical system. Using this effect, we have demonstrated readout of a 3-D optical memory fabricated inside a human fingernail. Figure 7 shows the fluorescence readout observed with the fluorescence microscope using the blue excitation set. The bit layers were recorded in the same transverse position at different depths of (a) Z=40 µm, (b) Z=60 µm, and (c) Z=80 µm, respectively. The pulse energy E_p was 0.49 µJ. Each bit layer could be read out without crosstalk. The pictures shown in Figs. 7(a), (b), and (c) were taken 1 day after the recording. Figure 8 shows the fluorescence readout image of the same layer shown in Fig. 7(a) taken 172 days after the recording. The readout image is still very clear at 172 days, which is generally the limit for using a fingernail as memory because the fingernail is completely replaced by growing in about 6 months.

 

Fig. 7. Fluorescence images of 3 bit planes recorded inside human fingernail at (a) Z=40 µm, (b) Z=60 µm, and (c) Z=80 µm. Images were taken 1 day after recording. The scale bar indicates 10 µm.

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Fig. 8. Fluorescence image taken 172 days after recording. The scale bar indicates 10 µm. The profile is the intensity distribution along a white line in the image.

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4. Conclusion

We have demonstrated an increased fluorescence intensity at the structural change inside a human fingernail produced by a focused femtosecond laser pulse. The fluorescence intensity was higher than the surrounding auto-fluorescence intensity of the fingernail. The structural changes, whose geometrical shape drastically depends on the irradiated pulse energy, are observed as a dark region by using a microscope with transmission illumination. The increased fluorescence intensity was observed in the dark region. The spectrum of the increased fluorescence coincided with the auto-fluorescence spectra of the fingernail. The increased fluorescence intensity was also observed in a fingernail heated in a drying oven. It is suggested that the increased fluorescence of the structure is a result of a local denaturation of the keratin protein caused by heat generated by the femtosecond laser pulse irradiation.

We demonstrated that the increased fluorescence of the structure is useful for reading out three-dimensionally recorded data inside a human fingernail. We recorded three bit planes inside a human fingernail. We demonstrated that three bit planes can be read out with little cross-talk by using fluorescence readout. Furthermore, we demonstrated that fluorescence can be observed for up to 6 months, corresponding to the time required for a nail to grow from root to tip. Under these recording conditions, a recording density of 2 Gbit/cm³ is achievable. When the recording performed on an accessible volume of 5×5×0.1 mm³, the recording capacity of the data is 5 mega bits.