Cavity-Enhanced Observation of Conformational Changes in BChla

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1 Cavity-Enhanced Observation of Conformational Changes in BChla Dirk Englund Summer Undergraduate Research Fellowship 2001 California Institute of Technology October 25, 2001 Abstract This research aims to devise a tool for observing molecular dynamics at the single-molecule level. A light cavity made from two mirrors is excited using a single-mode laser. Positioned between the mirrors is a very dilute solution of the bacteriochlorophyll-a (BChla) in acetone, which absorbs negligibly at the wavelengths of light used to excite the cavity. All other sources of absorption are minimized so that BChla has the greatest possible effect on the signal output. The cross section of the laser beam is minimized and the solution diluted enough to make the event of more than a few BChla molecules floating in the beam unlikely. The immediate goal is to be able to detect single molecules inside the beam. This will most likely require shot-limiting the cavity output, using heterodyning methods. Since the absorptive properties of the solute depends on its conformation, this technique could be used to illuminate how and when the solute molecule undergoes conformational changes. Ultimately, the goal is to observe conformational changes in BChla while it is embedded in the cell membrane. In the even more distant future, this method could be used to observe in real time many types of organic molecules as they undergo conformational changes. It could provide information on the dynamics of many types of organic molecules, which, combined with knowledge obtained through crystallography and direct mutagenesis, would greatly improve our understanding of many kinds of organic complexes and the processes in which they take part. 1 Introduction The goal of this research is to devise a method to observe how single molecules transition between different electronic configurations after the absorption of quanta of energy. Traditional methods are not applicable for this goal. Light microscopes, for example, lack the resolution, while electron microscopes lack the speed. We take a new approach. A solution of BChla in acetone is placed between the two mirrors of a Fabry-Perot cavity (see Fig. 1.) The cavity is excited with planar Gaussian modes. The BChla absorbs at certain frequencies. Depending on the amount of absorption, the light intensity inside the cavity, and hence the beam output intensity, decreases. This process would be nothing new if it were carried out for a large ensemble of molecules. The difficulty arises, however, because we are interested in observ- 1

2 Figure 1: Resonance Cavity: (a) BChla in ground state absorbs light, while (b) BChla in excited state does not. ing the absorptions and ensuing conformational changes for a single molecule, because only at the single-molecule level can we observe many of the molecule s unfavorable or short-lived states, which are averaged out in a large ensemble. Moving to the single-molecule level creates several challenges. First we need to ensure that the number of BChla molecules inside the cavity beam is small. To do this, we need to minimize the cavity beam volume and use a small concentration of BChla. The other major challenge is to allow for very precise measurements of the cavity beam output intensity. This requirement is the more demanding since the noise in the detection of the output signal needs to be limited to shot-noise levels. In the current stage of the project, we limit ourselves to detecting the presence of a single BChla molecule in the cavity beam. This allows us to focus on a single absorption near 770 nm in vacuum for BChla dissolved in acetone (see Fig. 2). Absorption at this wavelength promotes an electron transition to the π excited state. At this point in the project, we have built a suitable resonance cavity as well as much of optical circuit for the light input to the cavity. To validate the concept, we have made some average absorption measurements of BChla in chlorophyll. 2 Materials and Methods 2.1 Absorption Measurements on BChla in Acetone Using First-Generation Cavity To determine what accuracies we require to detect the presence of single BChla molecules in the beam, we first need to know BChla s extinction coefficient in acetone. We measure it in two ways 1 : first with a commercial spectrometer 2, and then in the first-generation version of the resonance cavity. For the measurements in the resonance cavity, we used the optical setup sketched in Fig. 3. The electro-optic frequency modulator produces side-bands 1 The BChla used in this experiment was derived from Rhodopseudomonas spheroids, a photo-synthetic purple bacteria. It was partially purified. 2 UV VIS Spectrophotometer by Agilent Technology 2

3 Figure 2: Normalized Absorption Spectrum of BChla in acetone. [3] Figure 3: Setup for absorption measurements with first-generation cavity. A dilute solution of BChla in acetone fills the cavity. This cavity has a 1.0-cm mirror spacing; the mirrors have a finesse of 12,000±1000 (tested at nm) and a 1.0 m radius of curvature. to the predominant frequency in the input beam. As the frequency of the input beam is swept past the cavity resonance frequency, the sidebands produce resonance curves whose separation in time is used to scale the full width at half maximum of the main resonance peak, f fwhm. The overall absorption can then be calculated from δ i = 2π f fwhm f, where f is the resonance frequency spacing [1]. The absorption for BChla is determined by comparing the overall absorption of acetone to that of acetone and BChla. 2.2 Second-Generation Cavity Design As mentioned in sections 1 and 4.1, the new cavity has to meet several requirements: High Finesse 3

Figure 4: Second-Generation Resonance Cavity. high-finesse mirrors short mirror spacings mirror spacing adjustable by at least one wavelength ( 1µm).

4 Figure 4: Second-Generation Resonance Cavity. high-finesse mirrors short mirror spacings mirror spacing adjustable by at least one wavelength ( 1µm). Leak-Tight Seal for Acetone Easy Exchange/Flushing of Acetone Solution. access valves The new cavity, shown in Fig. 4, fulfills these requirements: Mirrors: finesse 100, ,000 at λ 770nm. mirror spacing as low as 50 microns piezo-electric cylinder for mirror spacing adjustments of ±1µm. Leak-Tight High Vacuum Seals 2 NPT Access Valves 3 Results The extinction coefficient of the samples of BChla used in our experiments was measured in two ways, as described in section 2.1. First, using the commercial spectrometer, we obtained a figure of mm 1 cm 1 ± at 770 nm. This value agrees with that found using the first-generation cavity. As the plot in Fig. 5 shows, the BChla acetone solution absorbs strongly at 770 nm, but is as transparent as pure acetone at the other wavelengths tested. At 770 nm, the extinction coefficient is ε BChla = 37 ± 10 mm 1 cm 1. 4

5 Figure 5: Plot of total internal fractional power loss per round trip in the cavity. The cavity is filled with (A) air, (B) acetone, (C) BChla in acetone. This absorption coefficient is sufficiently large to make single-molecule detection possible (Sec. 4.1), given that all noise is reduced to a minimum. Preparations for the single-molecule measurements are on track; the second generation cavity (Fig. 4) fulfills the requirements for the experiment (Sec. 2.2). 4 Discussion 4.1 Feasibility of Experiment The fundamental problem is whether a single BChla will absorb enough light to make a detectable difference in the cavity output beam intensity, I trans. A quick calculation shows what precision is required in the measurement of I trans. Suppose there are N BChla molecules in the beam. Then we require a fractional precision better than I trans I incident 1 η = I trans(n = 0) I trans (N = 1) I trans (N = 1) From [1], (1+R), where R is the ratio of the fractional absorption per 2 cycle of one BChla molecule to that of the mirrors, i.e. R = δ BChl 2δ mirror. From this, we obtain η = 2R + R 2 2R since R << 1, and thus η δ BChl δ mirror. Note that we neglected the losses in acetone in this calculation; acetone does not absorb in the frequency range we use. 5

6 From the Beer-Lambert Law, we have δ BChl = ln 10εCL, where C is the molar concentration C = (1/N A) V with V the beam volume. V can be approximated to be a cylinder whose diameter equals the waist size of the Gaussian resonance mode and whose length is the cavity spacing. For a cavity spacing of 40µm, using mirrors with 5cm radius of curvature, as well as ε = 50mM 1 cm 1 at 770 nm, these calculations yield η [2] 4.2 Noise Reduction Attaining such sensitivities is not easy, but possible if the output signal is detected near the shot-noise limit using hetero- or homodyning methods. Of course, other noise sources need to be minimized. We have done this as much as it is possible at this stage, using optical intensity stabilizers as well as an optical isolator. Since no transmitted light signal is available, the reflected signal must be used for measurements and alignment. Major noise sources include vibrations of the cavity, back-reflection through the setup, as well as noise in the laser input signal from the Ti-Saph laser. 5 Conclusions The research this summer indicates that single-molecule observations are possible if the reflection signal from the second-generation cavity is detected near the shot-noise limit. The extinction coefficient of BChla was measured with the first generation cavity. The result, 37 ±10 mm 1 cm 1, agrees with that measured using a commercial spectrometer. 6 Acknowledgments Special Thanks to the following for making this project possible: Caltech and Samuel P. and Frances Krown for providing an opportunity to do this research through the SURF program; Professor Hideo Mabuchi for serving as the mentor; and my lab partners Tim McGarvey and Michael Armen, without whom this project would have been only half as enlightening and fun. References [1] A. E. Siegman, Lasers, Oxford University Press (1986) [2] C. M. Borrego, et al., The molar extinction coefficient of bacteriochlorophyll e and the pigment stoichiometry in Chlorobium phaeobacteroides, Phtotosynthesis Research 60: (2-3) May [3] N. U. Frigaard, Light-harvesting structures in green sulfur bacteria, Ph. D. thesis, Odense University, Odense:

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