The holographic parameters of purple membrane-polyacrylamide films obtained from a mutant form of Halobacterium salinarum (originally Halobacterium halobium) were measured. The synthesized films have an absorption of around 2.5 at 532 nm and a pH of 8.65. The results show that diffraction efficiencies of about 1.2 % (measured at 633 nm) can be achieved with writing intensities in the range of 200–400 mW/cm2 (532 nm), and these values remain constant after saturation. Pump-probe experiments were also used to measure the M state lifetime and our PM films were found to have the lowest M state lifetime described at this pH.
©2003 Optical Society of America
The need for improved materials in many technological applications has been the focus of much research and different design strategies have been adopted. An interesting option is to employ biological materials, making use of their improved properties due to natural evolution. One of these biological materials is the photochromic retinal protein bacteriorhodopsin (BR), which is contained within the purple membrane (PM) of members of the haloarchaea species, usually encountered in hypersaline environments. Under these extreme conditions BR is advantageous for the survival of these microorganisms, acting as a light-driven proton pump, transforming light energy into chemical energy by a mechanism which has been described previously [1, 2], with a high quantum efficiency.
BR containing materials have been used for many applications in optical image processing, such as optical memories [3, 4, 5], optical phase conjugation , real time holography [7, 1, 8, 2], spatial light modulators [9, 10], real-time optical Fourier processing , novelty filters , edge enhancement , all optical switching  and holographic interferometry . The applications and the biochemical and photophysical properties of BR have been summarized in various reviews, such as references [3, 5].
The main problem, or perhaps the main advantage, of biological species is that their natural evolution differs depending on the surrounding medium, and this may result in their properties changing. In this paper we present the preliminary results of the characterization of PM films, obtained from a mutant form of Halobacterium salinarum (originally Halobacterium halobium) with constitutive production of bacteriorhodopsin , as the holographic recording material. We analyze various important parameters for these kinds of materials, such as evolution of transmittance, the M state lifetime and the resulting amplitude and refractive index modulation.
1.1. Culture medium and growth conditions
H. salinarum MP was grown in 10 litres of a culture medium containing 250 g/l NaCl, 20 g/l MgSO4.7H2O, 3 g/l trisodium citrate.2H2O, 2 g/l KCl, 10 g/l oxoid bacteriological peptone, supplemented with 0.1 ml of a trace metal solution. The trace metal solution was composed of 1.32 g ZnSO4.7H2O, 0.34 g MnSO4.H2O, 0.78 g Fe(NH4)(SO4).6H2O and 0.14 g CuSO4.5H2O dissolved in 200 ml of 0.1 N HCl. The pH was adjusted to 7.3 with NaOH 1M and HCl 1M. Cells were grown at 37°C with aeration for 10 days in the dark.
For the purple membrane purification, the cells were concentrated by tangential flow filtration with 0.45 µm pore size filters (MILLIPORE) and harvested by centrifugation at 4°C for 15 min. at 16000 g (JLA 16.25 rotor BECKMAN). Purple membrane was isolated using the procedure described by Oesterhelt and Stoeckenius , then suspended in distilled water and finally lyophilised (TELSTAR) for conservation.
1.2. Preparation of BR-acrylamide films
The polymer matrix used for films saturated in BR was an acrylamide solution (40 %). This solution was made by mixing acrylamide and N,N’-methylene-bis-acrylamide in a ratio of 36.7:1 (w/w). 2.5 ml of a concentrated purple membrane suspension were mixed with the acrylamide solution to obtain a final concentration of 20 %. The gel solution was prepared by mixing the acrylamide-PM solution with the polymerization catalyst ammonium persulfate 0.05 % (w/w) and initiator N,N,N’,N’-tetramethylethylenediamine (TEMED) 1 µl, resulting in a pH of 8.65. Immediately after preparation, the gel solution was poured into two rectangular glasses (7.5×8 cm2) separated 1.5 mm. After polymerization, the glasses were removed and the PM-film was covered with two gel drying cellulose films and held firmly in a drying cell. The film was dried at room temperature for 48 hours and stored in the dark. The optical spectra of the resulting film (thickness 330 µm) is shown in Fig. 1. It has an absorption higher than 2.5 in the 500–600 nm range and an absorption ≈1 at 633 nm. It is also important to note that the film has a good homogeneity.
1.3. Experimental set-up
A standard symmetric transmission holographic set-up was employed (Fig. 2). A beam from a frequency doubled Nd:VO4 laser operating at a wavelength of λirr =532 nm was divided by a beam splitter into two collimated coherent beams which were overlapped at the PM film at an angle of θ =13.8° with respect to the normal of the film (the angles are in the air), resulting in a spatial frequency of 1100 lines/mm. The beam ratio was fixed to 1:1 and the holographic gratings recorded were monitored by a third beam operating at λR =632.8 nm from a He-Ne laser. The incidence of the reconstruction beam was fixed at the Bragg angle, using an intensity of about 80 µW/cm2 to minimize the photobleaching at this wavelength. Vertical polarization was used for the writing and reading beams. In order to measure the variation in transmittance at 633 nm when the PM film is illuminated with a single beam at 532 nm, we used the previously described set-up turning off one of the irradiation beams.
2. Results and discussion
Before giving a complete description of the experimental results obtained it is important to briefly explain the photochromic properties of the PM. The photocycle of BR is composed of seven states with lifetimes from picoseconds to seconds. Since in this paper we worked with a cw laser, the states considered can be reduced to a maximum of four due to the time scale used. These are the so-called B, M, N and O states. The cycle starts at the B state ( =570 nm, see Fig. 1), which upon illumination is converted into the M specie that have a maximum absorption of around 410 nm  and a lifetime that is greatly influenced by the medium conditions, especially by the pH. The photocycle is closed by the return to the B state, via an intermediate N and O states, with a maximum absorption located at 560 and 640 nm respectively . The stability and population of the N and O states depend on the lifetime and population of the M state.
Figure 3 shows the variation in transmittance at 633 nm when the PM film is illuminated at 532 nm with three different intensities from 100 to 400 mW/cm2. As can be seen, for the two lower intensities, the curves behave like those obtained with normal photochromic materials. For these two intensities, the photobleaching can be explained by a two level model involving the B and M states. The curve obtained for the higher intensity presents a decrease in transmittance before the saturation value. This response may be explained by the implication of the N and O states of the photocycle, which have an absorption wavelength near 633 nm.
As mentioned above, the population of the N and O states depend on the stability of the M state, which is an important parameter of the material. We measured the M state lifetime using the same procedure as Downie et al. , fitting the decay curve of the transmittance at 633 nm when the laser beam at 532 nm was turned off. As in reference , the curve was better adjusted when more than one exponential function were used. To be specific, we probed to fit the curve with the functions:
Where ΔT f is the final transmittance modulation during exposure, ΔT is the transmittance decay after irradiation and n=1, 2. The effective lifetime was defined as
In the top right-hand corner of Fig. 3, the experimental data and the fitted transmittance decay curve are shown. It can be observed that better results are obtained for the two exponential curves with a regression coefficient of 0.9932 than for the exponential decay curve with a regression coefficient of 0.9651. The effective lifetime obtained with the two exponential curves is 0.23 seconds, which is a value one order of magnitude lower than that obtained with the wild-type PM in references  and  for films with a similar pH (>8).
Figure 4 shows the diffraction efficiency (DE) at a wavelength of 633 nm versus the exposure time for three different recording intensities (I0=100 mW/cm2, I0=200 mW/cm2 and I0=400 mW/cm2). As can be seen, the curves of diffraction efficiency are correlated with the transmittance curves. The lowest value corresponds to the lowest intensity, while for the medium intensity the DE exhibits a saturation value of around 1 % and no decrease is observed. This is in contrast to the results previously observed for PM films , in which DE decreases after the optimum exposure time (the diffraction efficiencies of the wild-type PM were similar to ours). The maximum diffraction efficiency was observed for the high intensity curve, whose final DE is around 1.2 %, and no significant decrease was observed in this case either. Regarding to the optimum exposure energies, it can be concluded that our films need more energy than that described in the bibliography for other PM films . This may be explained by the low lifetime of the M state, which retards the stable formation of the hologram. Also, as in reference , the decay time of the hologram is faster than that of the pump-probe experiment. The time-scale of this signal is out of the range that could be measured with our instruments (0.03 s).
If we compare the transmittance modulation with the final DE for the three intensities employed, it can be deduced that the recorded holograms are mixed (amplitude and phase), since the total DE can not be explained by a pure amplitude hologram. The phase component of the obtained gratings is explained using the Kramers-Kronig equation, which predicts a variation in the real component of the refractive index when the absorption change (imaginary component of the refractive index). These results allow us to calculate the resulting refractive index modulation for the three writing intensities. For this purpose we used Kogelnik’s expression for mixed amplitude and phase gratings at bragg angle in volume holograms (Eq. (3)), using the measured transmittance variations in the amplitude modulation.
In Eq. (3) α 1 and n1 are the amplitude and refractive index modulation, d is the material thickness, θ is the reconstruction angle inside the material (a refractive index of n=1.535 at 633 nm  was used in the Snell correction) and λ is the reading wavelength. The holographic parameters obtained are shown in table 1. The magnitude order of the values is similar to that previously described in BR films  for wild-type BR films of the same absorption. A tendency to reach saturation as the intensity increases is observed for both, n1d and α 1, but is more important in the case of the refractive index modulation, which varies by less than 8% between the holograms recorded at 200 and 400 mW/cm2. As a consequence of these results, the optimal writing intensity can be fixed (532 nm) for our PM films in the range of 200–400 mW/cm2. Last, it is important to comment that the material is reversible, and several cycles has been probed without material fatigue.
In conclusion, we measured the holographic parameters of purple membrane films obtained from a mutant form of Halobacterium salinarum and compared them with those previously described for other PM materials.We synthesized films with a pH=8.65 and absorption of about 2.5. The results show that our PM films have the lower M state lifetime described at this pH (0.26 s), with diffraction efficiencies similar to those measured for the wild-type BR (around 1.2 %) and higher exposure energies, which may be attributed to the M state lifetime. It is also observed that with recording intensities from 100 mW/cm2 to 400 mW/cm2 the diffraction efficiency remains constant after reaching the maximum and does not decrease as was described in the case of other PM films.
The authors acknowledge support from projects MAT2000-1361-C04-03 and MAT2002-01690 of Ministerio de Ciencia y Tecnología of Spain, CTDIB/2002/134 of Consellería de Innovación y Competitividad de la Generalitat Valenciana and Bras de Port S.A..
References and links
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