artículo optica II Alemañ

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Purple membrane-polyacrilamide films as

holographic recording materials

A. Fimia, P. Acebal, A. Murciano, S. Blaya, L. Carretero, M.

Ulibarrena, R. AlemanDepartamento de Ciencia y Tecnologa de Materiales. Universidad Miguel Hern andez, Av.

Ferrocarril s/n Apdo. 03202 Ed. Torrevaillo, Elx (Alicante) Spain

[email protected]

M. Gomariz, I. Meseguer

Departamento de Produccion Vegetal y Microbiologa. Universidad Miguel Hern andez, Av.

Ferrocarril s/n Apdo. 03202, Elx (Alicante) Spain

Abstract: 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 of8.65. 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

OCIS codes: (0090) Holography ; (2900) Holographic recording materials; (7330) Volume

holographic gratings

References and links

1. N. Hampp, A. Popp, C. Bruchle, and D. Oesterhelt, Diffraction efficiency of Bacteriorhodopsin Films for holog-

raphy containing bacteriorhodopsin Wildtype BRWT and its variants BRD85E and BRD96N, J. Phys. Chem. 96,

46794685 (1992).

2. J. D. Downie and D. T. Smithey, Measurements of holographic properties of bacteriorhodopsin films, Appl.

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683733 (1990).

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ory/interconnections, Opt. Eng. 39(11), 29642974 (2000).

5. N. Hampp, Bacteriorhodopsin as a photochromic retinal protein for optical memories, Chem. Rev. 100, 1755

1776 (2000).

6. O. Werner, B. Fischer, A. Lewis, and I. Nebenzahl, Saturable absorption, wave mixing and phase conjugation

with bacteriorhodopsin, Opt. Lett. 15, 11171119 (1990).

7. N. Hampp, A. Miller, C. Bruchle, and D. Oesterhelt, Properties of holographic media containing purple mem-

brane from Halobacterium halobium and its functional variants, GBF Monogr. 13, 377383 (1989).

8. F. Wang, L. Liu, and Q. Li, Readout of a real-time hologram on bacteriorhodopsin fiml with high diffraction

efficiency and intensity, Opt. Lett. 21, 16971699 (1996).9. R. Thoma, N. Hampp, C. Bruchle, and D. Oesterhelt, Bacteriorhodopsin films as spatial light modulators for

non-linear optical filtering, Opt. Lett. 16, 651653 (1991).

10. Q. W. Song, C. Zhang, R. Blumer, R. B. Gross, Z. Chen, and R. Birge, Chemically enhanced bacteriorhodopsin

thin-film spatial light modulator, Opt. Lett. 18, 13731375 (1993).

11. A. Panchangam, K. Sastry, D. Rao, B. DeCristofano, B. Kimball, and M. Nakashima, Processing of medical

images using real-time optical Fourier processing, Med. Phys. 28, 2227 (2001).

(C) 2003 OSA 15 December 2003 / Vol. 11, No. 25 / OPTICS EXPRESS 3438

#3314 - $15.00 US Received Novmeber 07, 2003; Revised December 05, 2003
mailto:[email protected]:[email protected]


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12. T. Okamoto, I. Yamaguchi, S. Boothroyd, and J. Chrostwiski, Novelty filter that uses a bacteriorhodopsin film,

Appl. Opt. 508511 (1997).

13. J. Joseph, F. J. Aranda, D. Rao, J. A. Akkara, and M. Nakashima, Optical fourier proccesing using photoinduced

dichroism in bacteriorhodopsin film, Opt. Lett. 21, 14991501 (1996).

14. P. Wu, D. R. amd B.R. Kimball, M. Nakashima, and B. DeCristofano, Enhancement of photoinduced anisotropy

and all-optical switching in Bacteriorhodopsin films, Appl. Phys. Lett. 81, 38883890 (2002).

15. A. Seitz and N. Hampp, Kinetic optimization of bacteriorhodopsin films for holographic interferometry, J.

Phys. Chem. B 104, 71837192 (2000).16. G. Juez and F. R. Valera, A mutant form of Halobacterium halobium with constitutive production of bacteri-

orhodopsin, FEMS Microbiol. Lett. 23(2-3), 167170 (1984).

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into red and purple membrane, Methods Enzymology 31, 667678 (1974).

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measuring the refractive index of bio-optical materials and its application to bacteriorhodopsin, J. Opt. Soc. Am.

B 12, 797803 (1995).

1. Introduction

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 ofthese 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 [6], real time holography [7, 1, 8,

2], spatial light modulators [9, 10], real-time optical Fourier processing [11], novelty filters [12],

edge enhancement [13], all optical switching [14] and holographic interferometry [15]. 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 naturalevolution 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 ofHalobacterium salinarum (originallyHalobacterium halobium)

with constitutive production of bacteriorhodopsin [16], 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 pep-

tone, supplemented with 0.1 ml of a trace metal solution . The trace metal solution was com-

posed 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 oC with aeration for 10 days in the dark.

For the purple membrane purification, the cells were concentrated by tangential flow filtration

with 0.45 m pore size filters (MILLIPORE) and harvested by centrifugation at 4oC for 15 min.at 16000 g (JLA 16.25 rotor BECKMAN). Purple membrane was isolated using the procedure




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described by Oesterhelt and Stoeckenius [17], 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 so-

lution 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 l, 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 m) is shown in Fig. 1. It has an absorption higher than 2.5 in the 500-600nm range and an absorption 1 at 633 nm. It is also important to note that the film has a good

homogeneity.

0

0.5

1

1.5

2

2.5

3

400 450 500 550 600 650 700

A

Wavelength (nm)

Fig. 1. Absorption spectra of the BR/acrylamide film (in the region of 400-700 nm)

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 irr=532 nm was divided bya beam splitter into two collimated coherent beams which were overlapped at the PM film at

an angle of=13.8o with respect to the normal of the film (the angles are in the air), resultingin 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 R=632.8 nm from a He-Ne

laser. The incidence of the reconstruction beam was fixed at the Bragg angle, using an intensityof about 80 W/cm2 to minimize the photobleaching at this wavelength. Vertical polarizationwas 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.




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2

HeNe

4NdVO

PM filmBS

M

M

PD

PD

PC

Fig. 2. Schematic representation of the holographic set-up employed (M: mirror, BS: beam

splitter, PD: photodetector, PC: personal computer).

2. Results and discussion

Before giving a complete description of the experimental results obtained it is important tobriefly explain the photochromic properties of the PM. The photocycle of BR is composed

of seven states[1] 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[1]. The cycle starts at the B state

(maxabs =570 nm, see Fig. 1), which upon illumination is converted into the M specie that havea maximum absorption of around 410 nm [1] 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 [1]. 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 transmit-

tance 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. [2], fitting the decay curve of the transmittance at 633 nm

when the laser beam at 532 nm was turned off. As in reference [2], 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:

T

Tf =

n

i=1 cie

ti

(1)

Where Tf is the final transmittance modulation during exposure, T is the transmittance

decay after irradiation and n=1, 2. The effective lifetime was defined as




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0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35 40

T(%)

Time (s)

0

0.2

0.4

0.6

0.8

1

0 0.5 1 1.5 2

Time (s)

Exp.F1F2

/

T

Tf

I = 100 mW/cm0

2

I = 200 mW/cm0

2

I = 400 mW/cm0

2

Fig. 3. Pump-probe curves at three different intensities. The top right-hand corner shows

the normalized transmittance decay curve after the pump beam was turned off.

e f f =

ni=1 cii

ni=1 ci

n

i=1

ci = 1 (2)

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 [1] and [2] 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/cm

2 and I0=400

mW/cm2). As can be seen, the curves of diffraction efficiency are correlated with the trans-

mittance 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 [2], 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 de-

scribed in the bibliography for other PM films [2]. This may be explained by the low lifetime

of the M state, which retards the stable formation of the hologram. Also, as in reference [2], 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 em-

ployed, 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 theobtained 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 modu-

lation for the three writing intensities. For this purpose we used Kogelniks expression[18] for

mixed amplitude and phase gratings at bragg angle in volume holograms (Eq. (3)), using the




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0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 20 40 60 80 100 120

DE(%)

Time (s)

I0=100 mW/cm2

I0=200 mW/cm2

I0=400 mW/cm2

Fig. 4. Diffraction efficiency versus exposure time for three different recording intensities

measured transmittance variations in the amplitude modulation.

= e2dCos

Sinh2

1d2Cos

+Sin2

n1dCos

(3)

In Eq. (3) 1 and n1 are the amplitude and refractive index modulation, d is the materialthickness, is the reconstruction angle inside the material (a refractive index of n=1.535 at 633nm [19] was used in the Snell correction) and is the reading wavelength. The holographicparameters obtained are shown in table 1. The magnitude order of the values is similar to

that previously described in BR films [2] for wild-type BR films of the same absorption. A

tendency to reach saturation as the intensity increases is observed for both, n 1d and 1, but ismore 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.

Table 1. Amplitude and refractive index modulation for the three intensities employed with

the BR films (the normalization to the maximum modulation is shown in italics).

I0 (mW/cm2) (104 m1) 1 (10

4 m1) n1d (m)

100 18.18 1.82 (0.27) 0.01485 (0.45)

200 18.18 5.9 (0.87) 0.03036 (0.92)400 18.18 6.82 0.033

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




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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.

Acknowledgements

The authors acknowledge support from projects MAT2000-1361-C04-03 and MAT2002-01690

of Ministerio de Ciencia y Tecnologa of Spain, CTDIB/2002/134 of Consellera de Innovacion

y Competitividad de la Generalitat Valenciana and Bras de Port S.A..



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artículo optica II Alemañ