Advances in protein-based three-dimensional optical memories

ELSEVIER BioSystems 35 (1995) 145-151

Advances in protein-based three-dimensional optical memories

Z. Chen*, D. Govender, R. Gross, R. Birge

WM. Keck Center for Molecular Electronics, Syracuse University, Syracuse, NY 13244, USA

Abstract

An oriented bacteriorhodopsin cube is optimized as a potential three-dimensional optical memory medium. Write/read capability is demonstrated by using the photovoltaic signal induced by two-photon absorption. Our results demonstrate that a two-photon induced photovoltage can he detected in a three-dimensional Bacteriorhodopsin (BR) cube as large as 1.6 x 1.6 x 1.6 cm3. The read/write speed, signal to noise ratio, and the laser damage threshold for the protein-based three-dimensional optical memory is examined.

Keywords: Optical memory; Molecular electronics; Biomaterials; Bacteriorhodopsin; Purple membrane

1. Introduction

Advances in semiconductor technology and computer architecture design have altered the nature of computing. Performance, once constrained by processor-limited environments, is becoming interconnect-limited. The overall throughput of many scientific and image analysis problems are determined more by RAM size and data-transfer bandwidth than by CPU and floating-point hard- ware speed. Present memory technologies such as magnetic disks, optical disks, and semiconductor memories, store information in a planar surface. The two-dimensional nature of the present storage devices limit the storage capacity and access bandwidth. Two-photon three-dimensional optical ad- dressing architectures offer significant promise for the development of a new generation of ultra-high

* Corresponding author, Biotechnology Division, US Army Natick RD&E Center, Natick, MA 01760, USA.

density random access memories (Parthenopoulos and Rentzepis, 1989; Birge et al., 1992). These types of systems read and write information by using two orthogonal laser beams to address an irradiated volume (l-50 pm3) within a much larger volume of a non-linear photochromic material. Because the probability that a molecule absorbs two photons is proportional to the square of the incident intensity, photochemical activation is limited to a first approximation to regions within the irradiated volume. Two-dimensional optical memories have a storage capacity that is limited to - l/1’, where ;1 is the wavelength, which yields approximately lo8 bits/cm2. In con- trast, three-dimensional memories can approach storage densities of 1/d3, which yields storage capacities in the range extending from 10” to lOI bits/cm3. In this paper, we report our research on the development of a two-photon three-dimensional optical storage medium based on the photochromic protein, bacteriorhodopsin.

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146 Z. Chen et al. / Bidystems 35 (1995) 145-151

2. Bacteriorhodopsin

Bacteriorhodopsin (BR) is the light transducing protein in the purple membrane of Halobacterium salinarium (Stoeckenius and Bogomolni, 1982; Birge, 1990). The purple membrane, which con- tains the protein bacteriorhodopsin in a lipid ma- trix (3:l (protein/lipid)), is grown by the bacterium when the concentration of oxygen be- comes too low to sustain the generation of ATP via oxidative phosphorylation. When the protein absorbs light in the native organism, it undergoes a complex photocycle which generates intermedi- ates with absorption maxima spanning the entire visible region of the spectrum (Fig. 1).

Bacteriorhodopsin exhibits an unusually large two-photon absorptivity due to the large change in dipole moment that accompanies electronic excitation. Indeed, the observed “‘BU* + ” +S, absorptivity (6 = 290 GM) is almost lo-fold larger than those observed and theoretically predicted

4

-

-

300 400 500 600 700

Wavelength (nm)

Fig. 1. Absorption spectra and photocycle of light-adapted bacteriorhodopsin.

for the isolated retinal chromophores (Birge and Zhang, 1990). Furthermore, bacteriorhodopsin exhibits a very high quantum efficiency for photoconversion into the blue-shifted M-state. Thus, laser wavelengths that are not absorbed under one-photon conditions can be used to stimulate bR o M photoconversion with high efficiency. The two-photon-induced photochromic behavior is summarized in the binary scheme below:

bR (State 0) (A,,, E 1140 nm)

2hw; Q, N 0.65 ,

iho. m , o 65 M (State 1) (A,,, = 820 nm), 52 .

where 0, and @ are the quantum efficiency of the forward and reverse reaction, respectively. We arbitrarily assign the ground state BR molecule to state 0 and the primary photochemical product M to state 1. The large two-photon absorption cross section and the high quantum efficiency for the photochromic transition indicate that two-photon absorption can be used to accomplish the ‘write’ operation. What remains to be resolved is how to reliably read the state of an arbitrary bit within the irradiated volume. Bacteriorhodopsin possess a very short-lived electronic excited state, and consequently, has a negligible quantum yield of fluorescence (Or = 10 -“). As a result, one cannot assign the state of the bit by monitoring the frequency of light emitted from molecules within an irradiated volume. A recent three-dimensional memory proposed and demonstrated by Parthenopoulos and Rentzepis (1989) utilizes fluorescence for state assignment. While the non- fluorescence property of bacteriorhodopsin may seemingly complicate state assignment, its absence lends the advantage that extraneous fluorescence- induced photochemistry can be effectively elimi- nated in regions outside the irradiated volume. One of the inherent advantages of bacteriorhodopsin as it relates to 3-D optical memories is the fact that the protein gives off a photoelectric signal that is characteristic of its photochromic state (bR vs. M). Readout in our BR-based memory system is accomplished by exploiting the inherent photoelectric properties of the BR protein. In this paper we report the fabrication of prelimi-

Z. Chen et al. / BioSystems 35 (1995) 145-151 141

two-photon irradiated

volume \

bsl

Fig. 2. Schematic diagram of the principal optical components of the proposed two-photon three-dimensional optical memory based on BR. Symbols and letter codes are as follows: (a) sealing polymer; (b) indium-tin-oxide conductive coating; (c) BK-7 optical glass; (d) SMA or OS-50 connector; and (e) Peltier temperature controlled base plate (0-20°C). AT, achro- matic focusing triplet; bs, beam stop; DBS, dichroic beam splitter; LD, laser diode; FL, adjustable focusing lens.

nary optical memory cubes based on bacteriorhodopsin and detection of two-photon-induced photovoltage from the BR memory cubes. In addition, we discuss the intensity-dependent nature of the observed photovoltage, the signal to noise ratio of the system, and the laser power damage threshold of the BR.

3. Three-dimensional optical memory

The schematic design of the two-photon three- dimensional optical memory is shown in Fig. 2. Bacteriorhodopsin, housed in a sealable cuvette, is oriented by applying a small electric field (20-40 V/cm) prior to polymerizing a solution containing protein and a mixture of acrylamide/bis-acrylamide monomers. Protein orientation is prerequi- site in observing a photoelectric signal from BR. A single bit write operation using the bR to M photoconversion (binary 0 + binary 1) is imple- mented by overlapping two orthogonal beams in

a region of the cube and simultaneously firing two laser pulses operating at 1140 nm. Similarly, M to bR photoconversion (binary 1 --, binary 0) is carried out by simultaneously firing two 820 nm lasers. In order to eliminate unwanted photochemistry along the laser axes, non-simultaneous firing of the additional lasers (not used in the original write operation) are carried out immedi- ately following the write operation. The clean pulse procedure is necessary since some photochemistry is initiated outside the irradiated volume. Fig. 3 shows the calculated probability of two-photon-induced photochemistry as a function of location relative to the center of the volume formed by the overlapped write beams. Analysis of Fig. 3 shows that photochemistry is predicted outside of the irradiated volume along the laser beam axes. Adjacent memory cells along the axes are transformed to (worst case) - 25% of the irradiated volume (desired memory cell) transfor- mation, and repeated read/write operations may irreparably destroy the contents of unaddressed memory cells after multiple write operations. This problem, however, is minimized by simple adjustment of either the clean pulse intensity, or laser pulse duration. A computer simulation showing the result of the cleaning operation in an irradiated volume is shown in Fig. 3 (calculated clean pulse figure). The model predicts that the clean operation serves to reduce unwanted photochemistry from - 25% to - 2%, the latter level being more than adequate to maintain data integrity outside of the irradiated volume. It is important to realize that the entire write operation, including subsequent cleaning pulses, can be completed in less than 50 ns.

As discussed above, a two-photon-induced photovoltage generated by oriented BR molecules within a radiated volume is used to readout the status of the bit stored in memory. By firing all four lasers simultaneously, the state of the irradiated volume can be probed by monitoring the polarity of the photovoltage. Careful adjustment of the relative intensities of the four lasers permits minimal disturbance of memory cells outside of the irradiated volume. A standard write operation is then performed to reset the memory cell to the correct state. This procedure serves to enhance

Fig. 3. The calculated probability of two-photon induced photochemistry as a function of location relative to the cent volume fort ned by the overlapped write beams and the effects of the clean pulse.

2. Chen et al. / BioSystems 35 (1995) 145-151

er of the


data integrity by reducing the risk that multiple read/write cycles along the axis occupied by the interrogated memory cell corrupt the data in that memory cell. As outlined above, the total read process can be completed in - 50 ns. Accord- ingly, the maximum serial data rate of the basic memory system is - 20 Mbits/s. The data rate throughput is limited only by the time required to move the cube to the next memory cell. This latency is determined by the extent to which the data to be read are contiguous and by the speed of the memory x, y, z translational actuators.

Although two-photon-induced photovoltages have been observed in oriented thin films of BR, photoelectric signals have never been measured for a cube larger than 1 cm3. Since the magnitude of the detected photoelectric signal is inversely proportional to the separation of the two capac- itive detection electrodes, the signal from a thick gel is expected to be much smaller in magnitude than those observed in thin films. We demonstrate in the following section that millivolt photoelectrical signals (unamplified) can be observed in a BR cube with dimensions as large as 1.6 x 1.6 x 1.6 cm3.

4. Experimental methods

Oriented cubes of bacteriorhodopsin are pre- pared by using standard electrophoretic methods (Varo, 1981). Briefly, purified purple membrane is first washed 5-6 times with doubly distilled water to reduce the conductivity of the solution. This process minimizes electrolysis of water at the elec- trode/solution interfaces. The purple membrane is then passed through a 5 pm pore size filter to remove particulate matter. Concentrated bacteriorhodopsin (l-2 mM) is mixed with 20% (w/w) acrylamide and 1% (w/w) N,N’-methylene-bis- acrylamide. The bacteriorhodopsin-acrylamide solution is degassed for 20 min and poured into the electrophoresis chamber. Polymerization of acrylamide is initiated using the catalyst ammonium persulfate and the initiator N,N,N’N’ te- tramethylethylenediamine (TEMED). The polymerization rate is adjusted to occur within 4-7 min by proper adjustment of the catalyst concentration. An electric field of 20 V/cm is applied to

the BR/acrylamide solution to orient the purple membrane sheets. The duration of the applied voltage is - 30 s and is timed to occur just prior to polymerization. After polymerization is com- plete, deionized water is poured into the chamber to cool the gel. The gel is then carefully removed from the electrophoresis chamber and bathed in 100 mM potassium chloride for 24 h. The gel slab is then cut into cubes that fit into the photoelectrical cell holder. The gel is sealed from the outside environment by applying a layer of silicon or epoxy to the top of the gel holder. This process is necessary to avoid dehydration and eventual de- naturation of the protein.

To measure the two-photon-induced photovoltage in a BR cube, a 1.06 ,um beam from a Q-switched YAG laser with a repetition rate of 10 Hz and a pulse width of 10 ns is used as the light source (DCR-2, Spectra Physics). A beam attenu- ator is used to modulate the laser light intensity without shifting the beam position. The BR cube is mounted on a precision x-y-z stage which is controlled via stepping motors interfaced with a microprocessor (ATS 302 from Aerotech). The photoelectrical signal is recorded with a 250 MHz bandwidth digital oscilloscope (HP 54510A). To prevent inductive pick up of electrical noise from the Q-switch of the Nd:YAG laser, the cell holder and all the electric cables are shielded with a conductive copper mesh.

5. Results and discussion

The measured two-photon-induced photovoltages for different laser pulse energies are shown in Fig. 4. All of the photovoltages measured were recorded without any signal amplifica- tion scheme. The results indicate that photovoltages are detectable at laser intensities as low as 15 mJ/cm2. Photovoltages as high as 2 mV have been measured at laser intensities of - 150 mJ/cm2. Noise in the signal stems primarily from inductive coupling of the high voltage Q-switch pulse of the exciting laser to the photoelectric cell. Copper mesh shielding proved to be a very effec- tive means in which to attenuate background noise. After shielding, the noise was reduced by a factor of four. Since the photovoltage is propor-

Z. Chen et al. 1 BioSystems 35 (1995) 145-151

I I I I I I I I,,

oriented

-60 -40 -20 0 20 40 60 80 100 120

Time Relative to Laser Pulse (ns) Fig. 4. Measured photovoltages induced by two-photon absorption from an oriented BR cube (1.6 x 1.6 x 1.6 cm3) for different laser energy densities.

tional to the number of photoactivated BR molecules, the signal to noise ratio of the memory is a function of the excitation light intensity. For laser pulse energy density of 150 mJ/cm*, the signal to noise ratio was found to be greater than 20. At 15 mJ/cm* the signal to noise ratio is reduced to four. The noise level floor is in the order of N 50 FV after shielding. The signal to noise ratio could be improved significantly if an amplifying circuit were built into the base of the cell and if the whole unit were shielded. Hewlett- Packard has generously supplied us with GaAs- based sample and hold circuits and this approach is currently under investigation.

The profile of the photoelectrical signal from the gel cube differs significantly from those derived from oriented dried films of bacteriorhodopsin. The photoelectrical signal from a

dried oriented BR film has two components: one fast (ns time scale) and one slow (ms time scale) (Ohno et al., 1983). However, only the fast com- ponent is observed in the gel cube. The half width at half maximum of the signal is less than 20 ns. For comparison, we also measured the one-photon-induced photovoltage excited by the 0.532 pm second harmonic beam of the Nd:YAG laser and the results indicate that the signal profile is identi- cal within experimental error to that observed via two-photon excitation. The fast two-photon-induced photovoltage indicates that the read speed of the memory is in the order of 30 ns. The write speed can also be derived from the recorded photovoltage signal. As is well known for the BR photocycle, once ground state BR molecules are photoactivated to the K-state, it will thermally decay to the M-state. Although the switching time between bR-M takes about 50 ,LLS, our photoelectrical signal results indicate that the actual laser pulse required to initiate the transition is less than 30 ns. Thus, the intrinsic limitation of the read/write speed is 30 ns. This speed is higher than all other optical access methods currently proposed. Hence, the speed of the bacteriorhodopsin-based three-dimensional memory is limited by optical access time rather than by the material response.

Finally, we also measured the damage threshold of BR. BR is a robust molecule and the photo- cyclicity has been measured to be better than lo6 (Chen et al., 1991). However, the laser damage threshold has not been evaluated. Since the two- photon process depends quadratically on the laser intensity, relative high peak power laser pulses must be used. The laser damage threshold is an important parameter for evaluating the future efficacy of the protein-based memory. We measured the damage threshold of the BR cubes. For a 532 nm green beam of the YAG laser, perma- nent bleaching of the protein is observed when the peak intensity of the laser is greater than 2.5 MW/cm*. However, for 1.06 pm beam of the YAG laser, no damage is observed for laser peak intensity as high as 10 MW/cm*. From these results we conclude that laser-induced denatura- tion of the memory medium can be ignored for the write/read intensities investigated.


6. Conclusion

We have successfully constructed an oriented bacteriorhodopsin cube for three-dimensional optical storage. Write/read capabilities were demonstrated by using the photovoltaic signal induced by two-photon absorption. Although photovoltaic signals have been previously observed in thin films of the oriented BR, our results demonstrate that such signals can be detected in a three-dimensional cube as large as 1.6 x 1.6 x 1.6 cm3. The intrinsic limitation of the material response to the optical read process is determined from the two-photon signal to be less than 30 ns. The write/read speed of the memory, therefore, is limited by the access time rather than by the material response.

References

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Birge, R.R., Gross, R.B., Masthay, M.B., Stuart, J.A., Tallent, J.R. and Zhang, C.F., 1992, Nonlinear optical properties of bacteriorhodopsin and protein based two-photon three- dimensional memories. Mol. Cryst. Liq. Cryst. Sci. Tech- nol. Sec. B. Nonlinear Opt. 3, 133-147.

Birge, R.R. and Zhang, C.F., 1990, Two-photon spectroscopy of light adapted bacteriorhodopsin. J. Chem. Phys. 92, 71787195.

Chen, Z., Lewis, A., Takei, H. and Nabenzahl, I., 1991, Bacteriorhodopsin oriented in polyvinyl alcohol films as an erasable optical storage medium. Appl. Opt. 30, 5188- 5196.

Ohno, K., Govindjee, R. and Ebrey, T.G., 1983, Blue light effect on proton pumping by bacteriorhodopsin. Biophys. J. 43, 251-254.

Parthenopoulos, D.A. and Rentzepis, P.M., 1989, Three-dimensional optical storage memory. Science 245, 843-845.

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Advances in protein-based three-dimensional optical memories

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