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# Laser gas-discharge absorption measurements of the ratio of two transition rates in argon

## I Introduction

We have recently developed a theory for particle-hole states of closed-shell atoms that is potentially able to predict energies and transition rates with high precision. Initially, this theory has been tested in neon Savukov et al. (2002); Savukov (2002). For transitions between neon excited states the accuracy of experiments is very good and experiments agree well with our theory. However, for heavier noble gas atoms, experiments are often prone to systematic errors and the precision is much lower. For example, many absolute transition rates of argon were based on high-pressure arc plasmas and depended on the assumption of local thermodynamic equilibrium of the upper state populations which often was satisfied very approximately (see discussion in Ref. A. Hirabayashi and Fujimoto (1987)). Lifetime measurements using different techniques or even the same technique often yield quite different results. In Ref. Wiese et al. (1989), a unified set of atomic probabilities for neutral argon is given. The data are obtained from extensive analysis of existing experiments and from new measurements of emission in the broad range 240-18000 nm. The data are included in the NIST data base. Errors of order 10% are quoted. Although our theory is able to reproduce NIST data, the difference between experiment and theory often exceeds quoted in Ref. Wiese et al. (1989) error bars. There are two possibilities: (i) the theory is less accurate and needs to be improved, (ii) experimental error bars are larger and critical, more precise measurements are needed. Therefore, the accurate measurements presented in this paper are well motivated.

We describe a new experimental technique for precise measurements of ratios of transition rates in gas discharges and detail an apparatus realizing this technique. We demonstrate our measurement of the ratio of two argon transitions and analyze systematic errors. Finally, we present a comparison with our theory and other theories and experiments.

## II An experimental technique for accurate measurements of transition rate ratios

The essential goal of the experimental technique is to measure the relative absorption rates for two different transitions that start from the same level. In general, the populations of the lower and upper states are needed for finding the cross-section $`\sigma _{if}`$ from the absorption coefficient $`\alpha _{if}`$,

$$\alpha _{if}=\left[N_i(g_i/g_f)N_f\right]\sigma _{if}$$
(1)
(here $`N_i`$ and $`N_f`$ are densities of atoms in the initial and final states, $`g_i/g_f`$ is the ratio statistical weights), but in some cases the upper state population can be neglected. This happens when the initial state is the ground or long-lived metastable state, and the final, upper states are short-lived and relatively unpopulated. In gas discharges, at low current densities, metastable levels are generally much more populated than any other non-metastable excited levels. Experimentally, we also observed states that have allowed decay modes. The $`\left[3p_{3/2}^14s_{1/2}\right]_1`$ and $`\left[3p_{1/2}^14s_{1/2}\right]_1`$ states, surprisingly, were populated almost as much as metastables at pressure of a few Torr. The observed lifetimes of these states are of the order of ten microseconds. The principal explanation for such a slow decay is radiation capture: many possibilities of emission and absorption of a photon occur before the photon can leave the discharge cell. The observation of long-lived species in discharges gives the possibility to use them as lower states for absorption measurements. The other excited levels (not belonging to the 4s group) quickly decay radiatively without radiation capture and are effectively depopulated in a pulsed discharge as soon as the discharge current disappears. Therefore, in upward transitions that start from the 4s levels, the condition $`N_i>>N_f`$ is satisfied, and the absorption becomes proportional just to the cross-section and the initial density. Thus, by measuring the absorption for two transitions starting from the same level, in our experiment $`\left[3p_{1/2}^14s_{1/2}\right]_1`$, we can find the ratio of cross-sections from the ratio of absorptions,

$$\frac{\sigma _{if_1}}{\sigma _{if_2}}=\frac{\alpha _{if_1}}{\alpha _{if_2}}.$$
(2)
For a Doppler broadened profile and for monochromatic laser light tuned to the maximum of the profile, the ratio of cross-sections is equal to the ratio of line strengths

$$\frac{\sigma _{if_1}}{\sigma _{if_2}}=\frac{S_{if_1}}{S_{if_2}}=\frac{S_1}{S_2}\text{.}$$
(3)
Therefore by measuring the ratio of absorptions for two transitions, we can find the ratio of line strengths $`S_1/S_2`$, the important parameter that can be compared with other experiments and with theory. The situation described above is ideal for precise measurements of transition rate ratios: it has been very closely reproduced in alkali vapor experiments, in which for Cs an accuracy of better than 0.1% is claimed by Rafac and Tanner (1998). However, in the case of noble gases, especially when we choose the starting level to be a non-metastable state, a more complicated analysis is required.

In order to satisfy the condition $`N_i>>N_f`$ for the lowest excited states, we use a pulsed rather than a DC discharge. In a DC discharge this condition is weaker, because radiatively decaying levels are constantly repopulated by collisions with hot electrons and by decay of higher levels. It is especially true for radiation-capturing levels. A further advantage of a pulsed discharge is the possibility for enhancing the signal by utilizing its time dependence. For example slow fluctuations in laser intensity can be excluded. In addition, the signal-to-noise ratio can be improved using averaging (128-scan in our experiment) on a digital oscilloscope. Without special efforts an absorption of order 0.1% can be easily detected. Such high sensitivity is useful for fast and precise tuning of the laser to centers of Doppler profiles which is needed if only one laser is available. The long-term stability of the pulsed discharge is also better: the pulsed discharge has substantially smaller sputtering and thermal effects, since the total charge from the discharge current is much smaller for a pulsed discharge.

The principal disadvantage of the pulsed discharge is the more complicated analysis of systematic errors, basically owing to the finite-time response of the detection system. The various factors that can influence the measurement of the ratio in pulsed discharges need careful analysis: we will consider systematic errors that arise from large absorption and inhomogeneity. When two transitions differ by a factor of at least three, a larger absorption is recommended for better signal-to-noise ratio. In such a case the linear relation between absorption and line strength may no longer hold, and instead Beer’s law must be used to derive the more general equation:

$$\frac{\mathrm{ln}(I_1/I_{10})}{\mathrm{ln}(I_2/I_{20})}=\frac{S_1}{S_2}.$$
(4)
Here $`I_1/I_{10}`$ and $`I_2/I_{20}`$ are the reductions in the intensity after passing the absorber for the first and the second transitions, respectively. This nonlinear equation can be the cause of several systematic errors.

One effect arises from a slow response of the detection system. This results in a reduction of actual height of the peak by an unknown coefficient $`\eta `$:

$$\frac{\mathrm{ln}(\eta I_1/I_{10})}{\mathrm{ln}(\eta I_2/I_{20})}=\frac{S_1}{S_2}.$$
(5)
If the equations were linear, this coefficient would drop out from the ratio, but for large and significantly different absorptions for the two transitions, it will cause a systematic error in the ratio. The other effect arises from inhomogeneity. So far we assumed that the absorption medium is homogeneous, but in gas discharges this is far from being true. The discharge produces densities of excited atoms with some transverse and longitudinal distributions. The longitudinal distribution does not cause any problem, since for two transitions the ratio at each small length of path stays the same, and eventually the ratio of logarithms of intensities in Eq. (4) will remain the same. The transverse distribution, however, will cause a deviation from the simple Beer’s law and will require the analysis of this distribution.

For a cylindrical discharge cell, which we use in our experiment, if $`r`$ is the distance from the axis, an integration over $`r`$ is needed,

$$I_{out}=I_{in}(r)\mathrm{exp}\left[\alpha (r)\right]2\pi r𝑑r\text{,}$$
(6)
where $`I_{in}(r)`$ is the input laser beam intensity, $`\alpha (r)`$ is the absorption coefficient, and $`I_{out}`$ is integrated over a detector area output intensity after an inhomogeneous discharge medium. For simplicity in this equation the change in absorption along the laser beam is ignored. It is difficult to find the spatial distribution accurately, although in some simple cases it can be predicted. In the case of a diffusion controlled cylindrical discharge, the distribution for the electron density $`n_e`$ and for excited state densities, has approximately the form of a Bessel function,

$$\alpha (r)n_eJ_0(2.4r/R)\mathrm{cos}(\pi z/L).$$
(7)
The expression for the electron density $`n_e`$ is taken from Ref. Raizer (1991), p. 67. Using the series expansion of the Bessel function for small argument, $`J_0(2.4r/R)1\frac{1}{4}(2.4r/R)^2`$ we can show that in our experiment with $`R=1.25`$ cm and $`r=0.2`$ cm, the maximum change in the absorption over the beam radius $`r`$ is 3.7% and the change in the integral for small $`\alpha (r)`$ is only 1.8%. Therefore the effect of the distribution is small. Moreover, if the absorption is not very large, the exponential can be expanded, and the linear dependence of the signal on the absorption coefficient can reduce further the effect of the distribution. If we examine NIST transition data nis  for neutral argon, we find many transitions in the range of tunable lasers such as a Ti:Sapphire laser, which can be used for the absorption experiments. However, in practice there are some restrictions: first, the pair of transitions has to start from the same metastable levels or radiation-capturing levels such as mentioned above. Second, the range is greatly restricted by the tunability of the laser: for example, our Ti:Sapphire laser has several mirror sets for several ranges of wavelengths. Because the change of mirrors takes many hours to complete, only one mirror set can be used for measurements of ratios at one time. As a result, for our currently installed mirror set, only two pairs of transitions were appropriate. However, our laser could not be tuned for one transition, and we ended up able to make measurements for only one pair of transitions: one transition at a wavelength 9227 Å and the other at 9787 Å.

## III Experimental arrangement

The optical and electrical diagrams of the experimental arrangement are shown in Fig. 1. The monochromatic 1W laser beam generated by the Ti:Sapphire laser is expanded by traveling 8 meters between two optical tables to the size of about 5 cm diameter. A variable aperture reduces both the beam’s size and its power. To avoid saturation of the transition, the beam intensity is further reduced using a 2% reflection off a glass plate. A chopper periodically interrupts the beam to give a 100% reference absorption. The beam is then directed through the discharge cell where it undergoes time-dependent absorption in the discharge initiated by HV pulses from the HV pulse generator. The optical path of the laser beam is terminated on the photodiode. The attenuator in front of the screened photodiode compartment is used to avoid any saturation effects in the detection system. The signal from the photodetector is amplified and sent to the digital oscilloscope for measurement of the absorption.

The laser is a commercial single frequency MBR-110 Ti:Sapphire laser, pumped with 7-15 W of CW Coherent argon ion laser. The MBR-110 laser can be tuned in a broad range, 700-1060 nm, but to cover all this range 6 different mirror combinations have to be installed.

The discharge cell is a quartz tube of diameter 2.5 cm and a length of 30 cm. Two standard electrodes were fused symmetrically in side-tubes near each end. The electrodes have large area to reduce unwanted sputtering from the cathode which can reduce the lifetime of metastables significantly and affect the population distribution in the discharge as we observed in detail in helium. Moreover, for the same purpose, an external ballast volume was added to the cell and a short-pulsed discharge was used instead of a DC discharge.

A high voltage pulse generator produces high-voltage (more than 10 kV to allow fast discharge breakdown) pulses each of a few microseconds. The circuit is based on a power transistor in the switching mode and high-voltage transformer. A rotating chopper is inserted into the optical path to produce a 100% normalization signal.

The detection system consists of a photodiode, an amplifier, and a digital oscilloscope. There is no special requirement on those elements except that the detection system should provide a linear response and the distortion of the waveform of the signal should be minimal. The first requirement is completely fulfilled if the laser intensity does not exceed the saturation limit for a particular diode. The second requirement is approximately satisfied: small distortion of the signal has been observed. This effect is analyzed in detail in the next section.

## IV Measurement and error analysis

To find the ratio of line strengths, we measure the peak absorption for two transitions and use the formula

$$\frac{\mathrm{ln}(1V_1/V_{10})}{\mathrm{ln}(1V_2/V_{20})}=\frac{S_1}{S_2}\text{.}$$

Here $`V_1`$ and $`V_{10}`$ are the voltages proportional to the maximum of a pulsed discharge absorption and to the chopper normalization absorption for the transition at $`\lambda =922.7`$ nm shown in Fig.2. Similar voltages for the second transition at $`\lambda =978.7`$ nm also shown in Fig.2. are $`V_2`$ and $`V_{20}`$.

Repeating measurements of absorption several times, we have obtained the ratio $`3.29\pm 0.04`$. A small error here indicates the excellent statistics. However, the result can still have systematic errors which need to be estimated and if possible be excluded.

The first type of systematic error can occur from a slow drift in discharge conditions. Though our pulsed discharge is very stable, to account for any slow drift, we alternate measurements of the absorptions for the two transitions.

The second possible systematic error can arise from the uncertainty in the wavelength when the laser is manually tuned to the resonances of the transitions by monitoring absorption peaks similar to shown in Fig. 2. If $`\epsilon _1`$ and $`\epsilon _2`$ are relative average shifts owing to tuning uncertainty, then the absolute systematic shift in the ratio $`\sigma (\epsilon _1,\epsilon _2)`$ is

$$\sigma (\epsilon _1,\epsilon _2)=\frac{\mathrm{ln}\left[(1\left(1\epsilon _1\right)V_1/V_{10})\right]}{\mathrm{ln}\left[\left(1\left(1\epsilon _2\right)V_2/V_{20}\right)\right]}\frac{S_1}{S_2}(\epsilon _2\epsilon _1)\text{,}$$
(8)
where this equation was simplified for small absorption. The tuning error is estimated to be about 1.5%.

The combined effect of the nonlinearity of absorption and finite-time response of the detection system leads to another systematic error. Suppose $`\epsilon `$ is the reduction in the peak height because of a slow time response. Then we can write for the $`S`$-ratio

$$\frac{\mathrm{ln}\left[1\left(1\epsilon \right)V_1/V_{10}\right]}{\mathrm{ln}\left[1\left(1\epsilon \right)V_2/V_{20}\right]}=\frac{S_1}{S_2}+\sigma $$

Comparing measurements of ratios at different discharge currents and, hence absorption, we find that the time responce error is about $`4\%`$. This error gives a systematic increase in the ratio by 4%, but the previous error due to tuning decreases the ratio slightly by 1.5% so that the overall systematic error is smaller. Finally, taking into account mentioned above statistical and systematic errors, we obtain the accuracy of the measurement of the ratio about 4%. The final value of our ratio is $`3.29\pm 0.13`$.

## V Comparison with CI+MBPT theory and the other measurements and calculations

The comparison of the ratio of line strengths is given in Table 1. Our measured ratio of line strengths $`3.29\pm 0.13`$ is in excellent agreement with the value 3.23 obtained from our mixed configuration-interaction and perturbation-theory calculations Savukov (2002). Both measured and theoretical ratios agree also with the ratio 2.89$`\pm `$15% calculated from the experimental transition rates of Ref. Wiese et al. (1989). Our results are close to semiempirical calculations by Lilly (1976). However, the ratio 4.62 from semiempirical calculations of Ref. Garstang and Blerkom (1965) based on a similar principle as calculations in Ref. Lilly (1976) disagree substantially with our result. One obvious problem with semiempirical approaches is that energies can not define completely the wave functions which are very sensitive to the accuracy of calculations. In addition, transition amplitudes have additional corrections which are not accounted by the effective Hamiltonian. For example, random-phase approximation (RPA) corrections are due to shielding by the atom of an external photon field. Those corrections are substantial in Ar and heavier noble-gas atoms and must be included to reach a good precision.

## VI Conclusion

We have measured an accurate ratio of line strengths for two argon transitions. For this measurement we developed a new experimental technique based on a pulsed discharge and a laser absorption measurement. This technique can be applied for measurements of other ratios in noble gas atoms. The agreement of our measurement and calculations is excellent.


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# Mechanically probing the folding pathway of single RNA molecules

## Model

We consider an experimental setup where the two ends of an RNA molecule are attached to a force- and extension-measuring device, e.g. an atomic force microscope or beads trapped by optical tweezers. For the sake of concreteness, we use the optical tweezer setup sketched in Fig. 1(a) as an example here. In an idealized theoretical model, the RNA molecule (in essence, a highly nonlinear elastic element for the present context) is connected in series with a linear harmonic spring, and the total extension is externally controlled, see Fig. 1(b). The linear spring models the force-measuring device. E.g., for optical tweezers, its harmonic potential approximates the potential of the optical trap, and the spring constant $`\lambda `$ is adjustable in the experiment through the laser intensity. The extension of the spring, $`R_s`$, corresponds to the position of the bead with respect to the minimum of the trapping potential, and is measured as a function of the total extension $`R_t`$. The average force acting on the RNA molecule and its average extension are

$`f`$ $`=`$ $`\lambda R_s,`$ (1)
$`R`$ $`=`$ $`R_tR_s.`$ (2)
Here, $`\mathrm{}`$ denotes a thermal average over all accessible conformations of the RNA molecule and the spring at fixed total extension $`R_t`$. Throughout this article, we assume that the pulling experiment is performed slowly enough, such that the RNA molecule can fully sample its conformational space on timescales which are short compared to the external timescale of the imposed stretching process. This limit corresponds to the quasi-equilibrium regime, where the work dissipated in the pulling process is negligible. The average $`\mathrm{}`$ then includes an average over all possible secondary structures of the molecule.

Partition function. To calculate the averages in Eqs. (1) and (2), as well as the averages of other observables to be introduced below, we first determine the partition function, $`Z(R_t)`$, of the RNA molecule and the spring at fixed total extension $`R_t`$. In previous work Gerland et al., (2001), we computed the partition function of the RNA molecule alone in the fixed distance ensemble. Here, we explicitly take the force-measuring spring into account; varying the spring constant $`\lambda `$ interpolates between the fixed distance ensemble for stiff springs (large $`\lambda `$) and the fixed force ensemble for soft springs (small $`\lambda `$). Intermediate spring constants effectively put the RNA molecule into a mixed ensemble. The explicit consideration of the spring in the model is necessary here, not only for a closer modeling of the actual experimental setup, but also from a theoretical point of view, since the force fluctuations diverge in the fixed distance ensemble and our aim is to study fluctuation measurements.

A model for an experimental setup such as the one sketched in Fig. 1(a) requires two separate parts, one that contains only information on the free energies for the secondary structures of the RNA sequence, and another which describes the polymer properties of ssRNA Gerland et al., (2001). The coupling between these two parts is through the total length of the exterior ssRNA segments of the molecule, i.e. the number $`m`$ of bases that actually “feel” the applied force, see Fig. 1(a). For every fixed total extension $`R_t`$, the system has to find a compromise between lowering the RNA binding energy by decreasing $`m`$, and gaining entropy (plus lowering elastic energy) by increasing $`m`$. The secondary structure part is given by the partition function, $`𝒬(m)`$, summed over all secondary structures of the RNA molecule with a fixed number $`m`$ of exterior single-stranded bases111We account for the width of stems within the exterior single strand by increasing the base count $`m`$ by three for each stem.. The polymer properties of ssRNA then enter through a function $`W_{\mathrm{tot}}(R_t;m)`$, which denotes the total end-to-end distance distribution of a ssRNA molecule of $`m`$ bases in series with the spring222For simplicity, we restrict the spring to be collinear with the end-to-end distance of the RNA molecule.. The total partition function, $`Z(R_t)`$ is then simply the convolution of $`𝒬(m)`$ with $`W_{\mathrm{tot}}(R_t;m)`$,

$$Z(R_t)=\underset{m}{}𝒬(m)W_{\mathrm{tot}}(R_t;m).$$
(3)
The detailed calculation of $`𝒬(m)`$ is described in Gerland et al., (2001). Briefly, the calculation takes the experimentally determined rules for the binding free energies of RNA secondary structures Walter et al., (1994) into account, but neglects tertiary structure effects and pseudoknots. Our method is based on a recursive method to calculate the partition function McCaskill, (1990) as implemented in the ‘Vienna RNA package’ Hofacker et al., (1994), but introduces an additional recursion relation for the calculation of $`𝒬(m)`$.

The total end-to-end distance distribution of a ssRNA molecule of $`m`$ bases in series with the spring can be expressed as

$$W_{\mathrm{tot}}(R_t;m)=\underset{0}{\overset{\mathrm{}}{}}dRW_{\mathrm{RNA}}(R;m)\frac{e^{\beta \lambda (R_tR)^2/2}}{\sqrt{2\pi /\beta \lambda }},$$
(4)
where $`W_{\mathrm{RNA}}(R;m)`$ denotes the distribution of the ssRNA molecule alone. We model the ssRNA as an elastic freely jointed chain, i.e., as a chain of segments with unrestricted relative orientations, whereas the segment length $`r`$ is constrained by a harmonic potential $`V(r)=\kappa (rb)^2/2`$. The average segment length $`b`$ corresponds to the Kuhn length of a non-interacting ssRNA chain. The number of segments of the elastic freely jointed chain used to represent an RNA molecule with $`m`$ bases is chosen as $`ml/b`$ where $`l`$ is the base to base distance of ssRNA. This yields $`W_{\mathrm{RNA}}(R;m)C\frac{h}{2\pi R}\left[q(h)\right]^{ml/b}e^{hR}`$, where $`C`$ is a normalization constant, $`q(h)=e^{h\widehat{𝐳}𝐫}`$, and $`h`$ is determined from $`R=\frac{mb}{l}\frac{}{h}\mathrm{log}q(h)`$ Gerland et al., (2001). Such a model has been shown to describe the elastic properties of ssDNA molecules Maier et al., (2000); Montanari and Mézard, (2001). Since we are not aware of the corresponding data for RNA, we use the DNA values for the polymer parameters as obtained by Montanari and Mézard, (2001) through fitting to the experiment of Maier et al., (2000), i.e. $`l=0.7`$nm, $`b=1.9`$nm, and $`(\kappa /k_BT)^{1/2}=0.1`$nm (we do not expect a large difference in the single strand properties of DNA and RNA, because of the high similarity between their chemical structures). We take the free energy parameters for RNA secondary structure as supplied with the Vienna package (version 1.3.1) at room temperature $`T=25^oC`$. The salt concentrations at which the free energy parameters were measured are $`[\mathrm{Na}^+]=1\mathrm{M}`$ and $`[\mathrm{Mg}^{++}]=0\mathrm{M}`$.

Observables. From the partition function (3) we get the total free energy $`F(R_t)=k_BT\mathrm{log}Z(R_t)`$, the average force $`f=F(R_t)/R_t`$, and through Eqs. (1) and (2) also the average extension $`R`$ of the RNA molecule. The equilibrium fluctuations in the extension of the RNA molecule are given by $`(\delta R)^2=R^2R^2=\left[\lambda ^2F/R_t^2\right]/\beta \lambda ^2`$. Using these relations, one can show333The derivation is straightforward after observing that
$$\frac{f}{R}=\frac{f}{R_t}/\frac{R}{R_t}=\frac{^2F(R_t)/R_t^2}{1(1/\lambda )^2F(R_t)/R_t^2}.$$
that the variance is related to the derivative of the force-extension curve as measured in the mixed ensemble through

$$(\delta R)^2=\frac{k_BT}{\lambda +f/R}.$$
(5)
This exact relation simply expresses the intuitive result that the extension of the molecule undergoes large thermal fluctuations in the flat regions of the force-extension curve.

To study the unfolding pathway, we introduce the weight $`x_m(R_t)`$ for the secondary structures with $`m`$ exterior open bases at a given value of $`R_t`$,

$$x_m(R_t)=\frac{𝒬(m)W_{\mathrm{tot}}(R_t;m)}{Z(R_t)},$$
(6)
which satisfy the normalization condition $`_mx_m=1`$. The binding probability of base $`i`$ and $`j`$ at given $`R_t`$ may then be expressed as

$$P_{ij}(R_t)=\underset{m}{}x_m(R_t)p_{ij}(m),$$
(7)
where $`p_{ij}(m)`$ denotes the base pairing probability in the ensemble of structures with fixed $`m`$, which can be calculated as described previously Gerland et al., (2001).

## Results

We first consider the P5ab hairpin, which is the simplest RNA molecule studied in the experiment of Liphardt et al., (2001), and is of a size that typically occurs as a structural subunit in larger RNA’s. Fig. 2(a) shows its native structure and the experimental FEC for P5ab with added linker, taken with permission from Liphardt et al., (2001). The superimposed solid line shows the FEC that results from our model as described above444The native structure of P5ab contains two non-standard G–A base pairs for which no quantitative information on the binding energies is available. We therefore follow the suggestion of Ref. Liphardt et al., (2001) and approximate the binding energy of G–A basepairs by replacing them with G–U pairs. with a spring constant of $`\lambda =0.2`$ pN/nm. Here, we modeled the RNA-DNA hybrid linker, which in the experiment connects the RNA molecule to the beads, as a worm-like chain (WLC) with an experimentally known length of $`L=320`$nm. We used the worm-like chain interpolation formula Bustamante et al., (1994)

$$f_{\mathrm{WLC}}(R)=\frac{k_BT}{l_p}\left(\frac{1}{4(1R/L)^2}+\frac{R}{L}\frac{1}{4}\right),$$
(8)
where the value for the persistence length, $`l_p=3.57`$nm and the (experimentally unknown) origin of the distance scale were obtained by fitting to the experimental FEC at low forces. The shape of the experimental FEC is well captured by the theoretical curve, with a characteristic “hump” indicating the opening of the hairpin (the force drop in the hump occurs, because the released single-strand creates “slack” in the polymer). However, the force at which the hairpin opens is overestimated by the theoretical model. To assess the origin of this discrepancy, we applied a simple salt correction to the RNA binding (free) energies SantaLucia, (1998) to account for the different ionic conditions between the single-molecule experiment ($`[\mathrm{Na}^+]=0.25`$M) and the standard conditions ($`[\mathrm{Na}^+]=1.0`$M). The resulting FEC (dashed line in Fig. 2a) yields near-quantitative agreement with the experiment, indicating that the difference in ionic conditions accounts for most of the discrepancy. The remaining discrepancy could be caused, for instance, by the freely jointed chain model for ssRNA or the unknown stacking energies for G–A basepairs. However, the good agreement we have achieved seems sufficient to justify the use of our model as a tool to study the general questions outlined in the introduction (in the following, we keep the salt concentration at the standard conditions).

The FEC’s in Fig. 2 contain useful information on the RNA molecule. For instance, the total binding free energy and the size of the hairpin can be read off by comparing to the FEC for a non-binding control sequence. Within our theoretical model, such control curves are obtained through a convolution of the WLC model (8) for the linker with the freely-jointed chain model for the single-stranded RNA. The resulting curves could also be used as a reference for the experimental data, although ideally the control curve should be measured, using e.g. an oligonucleotide with a homogeneous sequence (note that it suffices to record the control for a single sequence length, since it can be straightforwardly rescaled to an arbitrary length). Fig. 2(b) shows our theoretical FEC for P5ab both for a stiff spring ($`\lambda =0.2`$ pN/nm, solid line) and a soft spring ($`\lambda =0.01`$ pN/nm, dashed line), together with the control FEC (dotted) for a sequence of commensurate length. Since stretching the control sequence requires only work against entropic and elastic forces, while stretching of the P5ab hairpin requires additional work to break the basepairs, the area between the two FEC’s equals the total binding free energy of P5ab. As Fig. 2(b) shows, not only the region of the P5ab FEC where the basepairs are opened (the “hump” region) contributes to this area, but there is also a significant contribution from the initial part of the FEC. The initial part of the P5ab FEC corresponds to stretching the exterior single-stranded chain of the molecule, which is shorter than the full molecule and hence has higher entropic/elastic forces at the same extension. However, this additional entropic/elastic energy is “stored” in the molecule and is fully released when the hairpin is (adiabatically) unzipped, i.e. in the “hump” region. Note that as long as the experiment is performed in quasi-equilibrium, the area under the FEC is independent of the spring constant, and therefore the same in the fixed-force and the fixed-distance ensemble. Loosely speaking, the FEC in the fixed-force ensemble could therefore be considered as the ‘Maxwell construction’ of the FEC in the fixed-distance ensemble.

We now seek to characterize the unfolding process of the P5ab hairpin within our model. In the experiment of Liphardt et al., (2001), the time traces of the end-to-end extension at fixed forces around 14 pN show the characteristic pattern of a bistable system, i.e. the unfolding proceeds directly, without intermediates. To calculate theoretical time traces would require a full kinetic treatment of the RNA folding/unfolding process which is beyond the scope of the present article. Instead, we determine the amplitude of the equilibrium fluctuations in the end-to-end extension, $`\delta R`$, as a function of the mean force or extension. Experimentally, this amplitude would be determined as a time-average, $`\delta R^2=T^1_0^T𝑑t(R(t)\overline{R})^2`$, where $`R(t)`$ denotes the recorded time trace of the end-to-end extension and $`\overline{R}`$ its average. Our theoretical prediction is for the infinite time limit, $`T\mathrm{}`$, of this average. Note that the exact relation Eq. (5) directly links the equilibrium fluctuations to the derivative of the averaged FEC. In practice, where the averaging period $`T`$ is finite and limited to a few minutes by instrumental drift Liphardt et al., (2001), the amplitude $`\delta R`$ can be determined with much greater accuracy than the derivative of the time-averaged FEC, since taking the derivative strongly amplifies the statistical error. Therefore, the quasi-equilibrium FEC and the equilibrium fluctuations, $`\delta R`$, can effectively be regarded as two independent and complementary sources of information.

The inset of Fig. 2(b) shows the fluctuations $`\delta R`$ as a function of the average extension, $`R`$, in the range of extensions over which the hairpin opens. We observe a single peak of the fluctuations at the force/extension where the transition takes place, which is consistent with the two-state behavior found experimentally. Physically, this peak is caused by continual kinetic fluctuations between the open and the closed state of the hairpin.

Hairpin with intermediate. Next, we address the question whether the simple two-state behavior observed for P5ab is a generic property for hairpins of this typical size. The discussion of this question also serves as a preparation for our ensuing study of a larger RNA with more complicated structure. To this end, we modify the sequence of the P5ab hairpin slightly by replacing the two G–A basepairs with U’s on both strands, which leads to a small interior loop in the hairpin. This change does not significantly affect the FEC, see Fig. 3(a), however the fluctuations $`\delta R`$ take on a qualitatively different behavior, as shown in Fig. 3(b): For a soft spring, the $`\delta R`$-curve shows only a single peak as before, but a stiff spring yields two maxima with a pronounced minimum in between. At this minimum, the configurational distribution of the molecule is localized on an intermediate state, which strongly reduces the fluctuations in the extension. This is demonstrated explicitly in Fig. 4, which shows the probability distribution in $`m`$-space, i.e. $`x_m`$ as given by Eq. (6) (see caption for details).

The stiffness of the external spring therefore limits the attainable resolution of the measurements: At low resolution the hairpin appears as a two-state folder, while an intermediate state appears at higher resolution. This observation is in line with the comment of Fernandez et al., (2001) who discuss the experiment of Liphardt et al., (2001) by analogy to patch clamp experiments, where an intermediate state in the opening of an ion channel appears only at high resolution. While an increase in the spring constant increases the resolution, it decreases the amplitude of the fluctuations, which renders them harder to detect. For the present purpose, the optimal value for $`\lambda `$ results from a tradeoff between low resolution and low amplitude. Clearly, increasing $`\lambda `$ can increase the resolution only up to the point where the floppiness of the RNA plus linker becomes resolution limiting (this point corresponds approximately to $`\lambda =0.5`$ pN/nm for the hairpin sequences with linker discussed above). This point marks the optimal choice for $`\lambda `$, unless the spacial resolution of the experimental apparatus is restrictive already at smaller values of $`\lambda `$. The role of the spring constant in dynamic force spectroscopy measurements is discussed by Heymann and Grubmüller, (2000).

Intuitively, the fact that the appearance of the intermediate state depends on the spring constant is best understood by mapping the unzipping problem onto the problem of a particle with an attached spring in a random potential and coupled to a thermal bath (see Fig. 5). As the other end of the spring is moved in one direction, the particle performs a ‘stick-slip’ motion Bockelmann et al., (1998). With a stiff spring, the particle hops over short distances and tends to be localized, while the particle can make long jumps, sliding over many valleys, when the spring is soft.

RNA molecule with multiple structural subunits. Longer RNA molecules typically have a complex structure with many structural subunits interacting through weak tertiary contacts Tinoco and Bustamante, (1999). A typical example for a structural subunit is the P5ab hairpin studied above, which is indeed extracted from a larger RNA molecule, the self-splicing intron of Tetrahymena thermophila with a sequence of 419 bases (Genbank # J01235). The known secondary structure of this intron comprises 19 structural elements labeled P1, P2, P2.1, P3, etc. It was the first RNA molecule to be shown to have a catalytic activity and has since been studied in great detail Cech, (1993). Its active, i.e. self-splicing, conformation contains a pseudoknot, which is essential for the function. However, it also has a known stable and well characterized inactive conformation without pseudoknot Pan and Woodson, (1998); Zhuang et al., (2000). The minimum free energy structure obtained from the Vienna RNA Package (which supplies the basis to our model) is shown in Fig. 6. The overall structure, and almost all individual basepairs, are identical to the one of the inactive conformation as determined by Pan and Woodson, (1998) (we follow the labeling introduced there), which confirms that the RNA free energy rules describe the nucleotide interactions quite well. For the cyclized form of this intron, a very similar secondary structure was obtained by Jaeger et al., (1990).

For complex secondary structures such as the one shown in Fig. 6, it is interesting to study the physical process of structure formation, whereby e.g. a newly produced RNA molecule folds into its preferred structure(s). For instance, one may ask, in which order the structural elements in Fig. 6 form, i.e. what is the folding pathway? Mechanical single-molecule experiments offer a very controlled way to probe the folding process. In the quasi-equilibrium limit that we consider, the mechanical folding pathway is independent of (i) the direction, i.e. unfolding and refolding pathway are identical, and (ii) specific experimental parameters such as the spring constant $`\lambda `$. The latter is clear from the fact that the number of exterior unpaired bases, $`m`$, is a natural and well-defined reaction coordinate in the quasi-equilibrium limit. On average, the distribution $`x_m`$ will always shift to larger $`m`$ when $`R_t`$ is increased, and hence $`\lambda `$ can only affect the resolution of individual states in $`m`$-space, but not the average order in which they are visited.

How can one determine the (un)folding pathway in a single-molecule experiment? As we have seen, pulling on an isolated subunit produces a characteristic signature in the FEC, see Fig. 2. If every structural subunit in a larger RNA molecule would produce such a characteristic signature, one could directly read off the folding pathway from the FEC. However, our previous theoretical study predicted that the quasi-equilibrium FEC of RNA’s with many subunits will typically show no distinguishable signatures of individual subunits opening Gerland et al., (2001). This prediction is consistent with the experimental observation of smooth FEC’s for long ssDNA molecules Maier et al., (2000). Three physical effects are responsible for the smoothness: (i) the ‘floppiness’ of the external single-strand and the linkers, (ii) thermal fluctuations in the secondary structure, i.e. the contribution of suboptimal structures, and most importantly, (iii) the fact that changes in the extension of individual structural subunits can compensate each other, since only the total end-to-end distance is measured Gerland et al., (2001). As a result, quasi-equilibrium FEC’s cannot be used to study the folding pathway (or the secondary structure) of larger RNA molecules.

The smoothing mechanisms (ii) and (iii) can be strongly suppressed by performing the experiments at large pulling speeds which do not leave sufficient time for the molecule to sample the entire ensemble of different secondary structures with similar number of exterior unpaired bases, $`m`$, and free energy of folding. Hence, nonequilibrium FEC’s of larger RNA molecules should be very rugged and display signatures of individual structural elements opening; see also the discussion in Gerland et al., (2001). This was indeed observed recently in the Berkeley group (S. Dumont & I. Tinoco Jr., private communication). From the theoretical side, the calculation of nonequilibrium FEC’s is challenging, since the simple particle-in-a-landscape picture of Fig. 5 breaks down in nonequilibrium: the free energy landscape is no longer well-defined. The secondary structures that are accessible for a molecule at a given extension and within a given time window depend on the present structure of the molecule and the detailed folding kinetics. While important steps towards a kinetic theory of RNA folding have been taken Isambert and Siggia, (2000), there is currently insufficient experimental information available to construct a full theoretical model.

Here, we consider instead the equilibrium fluctuations in the end-to-end distance of the entire intron, and explore, within our theoretical model, how much information on the folding pathway can be obtained from this. While looking at the equilibrium fluctuations does not suppress any of the above-mentioned smoothing mechanisms, it does provide a greater resolution for the observation of structural transitions, as already discussed. We will see in the following that this enhanced resolution, which is dependent on the spring constant, is sufficient to reveal major steps of the unfolding process.

The quasi-equilibrium FEC of the intron is shown in Fig. 7(a). Here, we have not added an additional linker in the calculation, since the intron is already fairly large and flexible, so that the effect of a comparatively short and stiff double-stranded linker is negligible. As expected, the FEC displays no signatures of the secondary structure, mostly due to ‘compensation’ between different structural elements Gerland et al., (2001). As for the P5ab hairpin above, we compute the equilibrium fluctuations $`\delta R`$, which would correspond to the average standard deviation in an infinitely long experimental time trace $`R(t)`$. Obviously, our averaged $`\delta R`$ contains less information than experimental $`R(t)`$ traces. Nevertheless, as Fig. 7(b) shows, already the equilibrium fluctuations $`\delta R`$ display interesting sequence-dependent features, i.e. local maxima and minima. The minima can again be interpreted as intermediate states along the (un)folding pathway, and the maxima as structural transitions. Note that the central peak close to $`R=150`$ nm splits into two peaks as the stiffness of the spring is increased, indicating the appearance of a new intermediate on the (un)folding pathway. This illustrates again how the ability to resolve intermediate states depends on the stiffness of the force-measuring device. For the choice of the latter, a trade-off between resolution of intermediate structures and fluctuation amplitude has to be respected.

Within our theoretical model it is a simple matter to identify the structural transitions associated with the peaks in Fig. 7(b). To obtain a measure for the likelihood to find a given structural element at a given mean extension of the molecule, we have summed the basepair probabilities for all basepairs in the element and divided by the total number of basepairs in the intact element. Here, the basepair probabilities at a given extension are computed using Eqs. (67). For each of the labeled structural elements in Fig. 6, we plotted the normalized total basepair probabilities as a function of the mean extension at $`\lambda =0.02`$ pN/nm in Fig. 7(c) using a greyscale code (black corresponds to presence of the element with probability one and white to absence of the element). We observe that the opening of most structural elements is localized to a relatively small extension interval, however typically the opening of several elements takes place simultaneously. Roughly, the structural elements open in the following order: P7, Alt P3, P4, P5, P5a, P1, P2.1, P6a, P9.1, P9.1a, P9.2, P2, P9, P8, P6b, P5c, and P5b.

By simultaneous inspection of Fig. 7(b) and (c), we can assign particular peaks in the fluctuation curve to the opening of particular elements. For instance, the central peak is associated with the parallel opening of P9.1, P9.1a, and P9.2, whereas the most stable elements P5b, P5c open at the final “hump”. Generally, elements that free a lot of single strand upon opening, such as a stem-loop structure with a large loop (e.g. P2.1), are associated with a distinct peak in the fluctuation curve. This peak reflects the large difference in extension between the open and closed state of the element.

To obtain the information displayed in Fig. 7(c) in the experiment will be more laborious, but should be possible given the known secondary structure. Since at every mean extension (or force) only a small number (up to 3) structural elements open in parallel, one can expect a small number of characteristic plateaus in the experimental $`R(t)`$ traces. The identification of the plateaus with the corresponding elements can be obtained by cutting the molecule at several places between the known secondary structure elements and performing the same measurement on subsequences. A similar approach was taken for recent nonequilibrium experiments in the Berkeley group (S. Dumont & I. Tinoco Jr., private communication). Obtaining the mechanical (un)folding pathway for a moderately sized RNA with known secondary structure therefore appears feasible by measuring the fluctuations.

It is interesting to compare the mechanical unfolding studied so far with thermal unfolding. The thermal unfolding process of the group I intron can be studied theoretically with the Vienna RNA package Hofacker et al., (1994). Fig. 8(b) shows the fractional opening of all structural elements as in Fig. 7(c), but plotted against temperature. We observe that the individual stems show a less sharp transition from closed to open than in Fig. 7(c). As a result, the specific heat curve shown in Fig. 8(a) displays only a single broad peak for the unfolding of the secondary structure (the specific heat is obtained from the total folding free energy, $`\mathrm{\Delta }G`$, through $`T^2(\mathrm{\Delta }G)/T^2`$). This is consistent with UV absorption experiments Banerjee et al., (1993) that found only two broad peaks for the same RNA, where the low-temperature peak could be associated with melting of the tertiary structure and the high-temperature peak with the secondary structure.

From Fig. 8(b), a rough order for thermal opening of the structural elements can be discerned: P4, P5, P5a, P6a, P7, Alt P3, P1, P2.1, P9, P8, P9.1a, P9.2, P9.1, P6b, P5b, P2, P5c. No structural element in this thermal (un)folding pathway is more than four list positions away from its place in the mechanical (un)folding pathway discussed above. This suggests that the thermal and mechanical unfolding pathways, although different in detail, can still be used as reasonable priors for each other.

## Summary and Discussion

Single-molecule experiments are particularly useful when the molecule under study can take on several conformations, e.g. folding intermediates, because in these cases bulk measurements usually yield only average properties, while single-molecule measurements can characterize each of the different conformations. Here, we predicted experimental signatures of such folding intermediates in the quasi-equilibrium fluctuations of the end-to-end distance. We found that a local minimum in the fluctuations curve indicates a locally stable intermediate state in the average mechanical unfolding pathway (which is well-defined and identical to the (re)folding pathway). The number of local minima in the fluctuations curve can therefore serve as a lower estimate of the number of locally stable intermediate states in this pathway. Furthermore, these signatures could be used to reconstruct the (un)folding pathway of an RNA molecule with a known secondary structure by cutting the sequence at appropriate positions and individually probing different substructures of the molecule.

We also showed that the stiffness of the force-measuring device plays a crucial role in determining the resolution of the quasi-equilibrium fluctuations curve. If the device is too soft, some local minima can be lost, while a too rigid device has a low fluctuation amplitude and therefore a low signal-to-noise ratio. The optimal choice for the stiffness (see ‘Results’) leads to the best estimate for the number of locally stable intermediate states along the pathway and increases the degree to which the pathway could be reconstructed with the “cutting approach”. The dependence on the stiffness has a simple intuitive explanation within the statistical mechanics problem of a particle with an attached spring in a one-dimensional energy landscape (see Fig. 5): A stiff force-measuring device corresponds to a rigid spring, which tends to localize the particle in valleys of the landscape (i.e., locally stable intermediates) and forces it to make small jumps as it is pulled along the landscape. On the other hand, a soft spring allows the particle to spread out and make long jumps, effectively hiding the intermediates in the equilibrium curve.

We hope that this view, which also explains the smoothness of quasi-equilibrium FEC’s for large RNA’s Gerland et al., (2001), will be useful for the design and interpretation of future experiments. Of course, experiments typically record not only the average fluctuations, but the detailed time-traces of the end-to-end distance Liphardt et al., (2001). These traces probe the kinetics of structural rearrangements in RNA, and could provide even more detailed resolution of intermediate states (if the kinetics is slow enough to be resolvable in the experiment). However, we expect that it is still helpful to make the choice for the stiffness of the force-measuring device as discussed above, in order to limit the number of states that contribute to each of the timetraces.

Although single-molecule experiments on RNA have already produced remarkable results, there are many desirable future developments. For instance, it appears that the current approaches are not well-suited to measure the secondary structure of an unknown molecule (e.g., in the ’cutting approach’ one would not know where to cut and it would be too laborious to try all positions). Is there a direct way to mechanically measure the secondary structure? Also, could one detect signatures of pseudoknots or even tertiary interactions? We hope that theoretical models of the type presented here can be useful for the planning and design of new experimental approaches in the future.

Note. After completion of this work, we learned of a related theoretical study by S. Cocco, J.F. Marko, and R. Monasson (preprint, cond-mat/0207609). These authors present a kinetic model for the end-to-end distance fluctuations of stretched RNA molecules, which nicely complements our thermodynamic study. Very recently, A.F. Sauer-Budge, J.A. Nyamwanda, D.K. Lubensky, and D. Branton (preprint cond-mat/0209414) have experimentally studied the unzipping kinetics of double-stranded DNA with a short internal loop, and demonstrated the presence of an intermediate state comparable to the one discussed here for the modified P5ab hairpin.

Acknowledgments. We are grateful to S.M. Block, S. Dumont, H. Isambert, J. Liphardt, D.K. Lubensky, I. Tinoco, and S.A. Woodson for stimulating discussions. U.G. was supported in part by a fellowship from the ‘Deutscher Akademischer Austauschdienst’. R.B. and T.H. acknowledge support by the NSF through Grant No. DMR-9971456, DBI-9970199, and the Beckmann foundation.


text:
# GUT, Neutrinos, and Baryogenesis

## 1 Introduction

Flavor physics is going through a big revolution. Neutrino oscillation has been a big discovery, and new data on quark sector are pouring in. Given the excitement in flavor physics, I will discuss recent topics on baryogenesis and grand unification, and their connection to flavor physics in this talk.

## 2 Baryogenesis

Why there is only matter in Universe but no anti-matter is one of the big questions in cosmology and particle physics. Because of its fundamental importance, it is often quoted as one of the primary reasons to study CP violation in flavor physics.

First of all, the amount of baryon we have in our Universe had been quite well determined by Big-Bang Nucleosynthesis. The only free parameter in this theory is the amount of baryons that determines the rate of nuclear fusion process in early Universe. People often quote the baryon-to-photon ratio $`\eta =n_B/n_\gamma `$, namely the ratio of the baryon number over the number of photons in a fixed volume, because this quantity does not change as the Universe expands.111When a particle species freezes out, however, the ratio does change. The baryon-to-entropy ratio is constant across the thresholds. There was a period when different determinations of baryon-to-photon ratio did not agree with each other, and it was said to be a crisis. The problem has largely disappeared recently, and there emerged a consensus on the baryon-to-photon ratio. Let me quote two quantitative analyses:

$$\eta =\frac{n_B}{n_\gamma }=\{\begin{array}{cc}\left(4.7_{0.8}^{+1.0}\right)\times 10^{10}\hfill & \text{[1]}\hfill \\ (5.0\pm 0.5)\times 10^{10}\hfill & \text{[2]}\hfill \end{array}.$$
(1)
This determination of the baryon-to-photon ratio is also consistent with the analysis that combines power spectrum in cosmic microwave anisotropy, cluster mass, and large scale structure . Therefore, we know the baryon-to-photon ratio of the Universe with a good confidence.

When I mention the word “anti-matter” in a public talk, it causes a certain level of fear among the audience. It sounds something scary to them. And they are right. When the Universe was hot, at a temperature of 1 GeV, there were practically equal amount of matter and anti-matter. As the temperature decreased, anti-matter has annihilated with matter, leaving only radiation. However, at the level of one out of ten billions or so, there was an excess in the amount of matter over anti-matter, and this small excess is us. We have survived The Great Annihilation. This realization immediately leads to a question: “What caused a small excess in the amount of matter over anti-matter?” This excess is called the baryon asymmetry of Universe.

Sakharov pointed out that the small baryon asymmetry may be understood as a consequence of microphysics from a Universe with no asymmetry only if three conditions are satisfied:

1. Existence of process that violates the baryon number.
2. CP Violation.
3. Departure from thermal equilibrium.

The first requirement is obvious. If the Universe had no asymmetry as its initial condition, generation of baryon asymmetry is possible only if the baryon number can change. Then there may be a finite rate of a process that increases the baryon number $`\mathrm{\Gamma }(\mathrm{\Delta }B>0)0`$. If, however, CP were an exact symmetry, a process and its CP conjugate process would have the same rate. Because the baryon number is CP-odd, it would imply that $`\mathrm{\Gamma }(\mathrm{\Delta }B>0)=\mathrm{\Gamma }(\mathrm{\Delta }B<0)`$, and no baryon asymmetry can be generated. To make these rates different, CP violation is mandatory. Even so, thermal equilibrium, by definition, has the same rates for a process and its inverse process. Similarly to the CP conservation, it would also imply $`\mathrm{\Gamma }(\mathrm{\Delta }B>0)=\mathrm{\Gamma }(\mathrm{\Delta }B<0)`$, and no baryon asymmetry would be generated. Therefore, departure from thermal equilibrium is necessary to generate the baryon asymmetry.

It was once hoped that grand unified theories (GUTs) would provide the mechanism of generating baryon asymmetry, a.k.a. baryogenesis . GUTs indeed necessarily break baryon number. If a heavy particle from GUTs remain in the early Universe after the temperature drops below its production threshold, the leftover amount exhibits the departure from thermal equilibrium. Then their decay, if CP is violated, may preferentially produce baryons over anti-baryons, thereby generating baryon asymmetry. It is encouraging that such a decay asymmetry had been established, namely $`\epsilon ^{}0`$ or equivalently $`\mathrm{\Gamma }(K^0\pi ^+\pi ^{})\mathrm{\Gamma }(\overline{K}^0\pi ^+\pi ^{})`$.

However, the effect of electroweak anomaly changed the picture quite dramatically. The Standard Model actually violates $`B`$ . In the Early Universe when the temperature was above 250 GeV, there was no Higgs boson condensate and $`W`$ and $`Z`$ bosons were massless (so where all quarks and leptons). Therefore $`W`$ and $`Z`$ fields were just like electromagnetic field in the hot plasma and were fluctuating thermally. The quarks and leptons move around under the fluctuating $`W`$-field background. To see what they do, we solve the Dirac equation for fermions coupled to $`W`$. There are positive energy states that are left vacant, and negative energy states that are filled in the “vacuum” (Fig. 1). As the $`W`$-field fluctuates, the energy levels fluctuate up and down accordingly. Once in a while, however, the fluctuation becomes so large that all energy levels are shifted by one unit (Fig. 2). Then you see that one of the positive energy states is now occupied. There is now a particle! This process occurs in the exactly the same manner for every particle species that couple to $`W`$, namely for all left-handed lepton and quark doublets. This effect is called the electroweak anomaly. Therefore the electroweak anomaly changes (per generation) $`\mathrm{\Delta }L=1`$, and $`\mathrm{\Delta }q=1`$ for all three colors, and hence $`\mathrm{\Delta }B=1`$. Note that $`\mathrm{\Delta }(BL)=0`$; the electroweak anomaly preserves $`BL`$.

Because of this process, the pre-existing $`B`$ and $`L`$ are converted to each other to find the chemical equilibrium at $`B0.35(BL)`$, $`L0.65(BL)`$ . In particular, even if there was both $`B`$ and $`L`$, both of them get washed out if $`BL`$ was zero.

Given this problem, there are now two major directions in the baryogenesis. One is the electroweak baryogenesis , where you try to generate $`B=L`$ at the time of the electroweak phase transition so that they do not get washed out further by the electroweak anomaly. The other is the leptogenesis , where you try to generate $`L0`$ but no $`B`$ from neutrino physics well before the electroweak phase transition, and $`L`$ gets partially converted o $`B`$ due to the electroweak anomaly.

### 2.1 Electroweak Baryogenesis

It appears at the first sight that the baryogenesis is possible in the Minimal Standard Model. First, the baryon number is violated as we discussed above. Second, CP is also violated in the Standard Model. Third, if the phase transition of electroweak symmetry breaking is first order, the coexistence of broken and unbroken phases at the phase transition is a departure from equilibrium. Then all three conditions by Sakharov are satisfied. The question is if enough baryon asymmetry can be generated quantitatively . There are at least two big problems in the Standard Model. The first problem is the order of phase transition. The first order phase transition is possible only if the Higgs boson is relatively light, $`m_H\begin{array}{c}<\hfill \\ \hfill \end{array}60`$ GeV. Above this mass, the phase transition becomes second order, and there is no departure from equilibrium. The LEP bound on the Higgs boson has excluded this possibility. The other problem is the size of CP violation. In the Standard Model, any CP violating effects must be proportional to the so-called Jarlskog parameter, $`J=\mathrm{}(\text{det}[M_u^{}M_u,M_d^{}M_d])`$. At the phase transition temperature $`T_{EW}v`$, the dimensionless quantity that characterizes the size of CP violation is $`J/v^{12}10^{20}10^{10}`$. Unless there is a mechanism of tremendous enhancement by ten orders of magnitude, the resulting baryon asymmetry would be too small.

The Minimal Supersymmetric Standard Model (MSSM) can go around both problems. The first order phase transition becomes a possibility again if one of the scalar top quark is very light, $`m_{\stackrel{~}{t}_R}\begin{array}{c}<\hfill \\ \hfill \end{array}160`$ GeV, despite the LEP bound. There is also a new CP violating phase $`\mathrm{}(\mu ^{}M_2)`$ in the chargino sector which could in principle be order one. See Fig. 3 and the caption for the mechanism of the baryogenesis.

However, the model is getting cornered; the available parameter space is becoming increasingly limited due to the LEP constraints on chargino, scalar top quark and Higgs boson . This is because the LEP bound on the lightest Higgs boson requires a large radiative correction, and hence a large scalar top quark mass. Because we need a light right-handed scalar top quark to achieve the first order phase transition, only the left-handed scalar top quark can be raised above TeV for this purpose. $`\mathrm{tan}\beta `$ also needs to be large to raise the Higgs mass. However, the CP violation is a relative phase between $`\mu `$ and $`M_2`$ in the chargino mass matrix

$$\left(\begin{array}{cc}M_2& \sqrt{2}m_W\mathrm{cos}\beta \\ \sqrt{2}m_W\mathrm{sin}\beta & \mu \end{array}\right),$$
(2)
and the phase becomes unphysical as $`\mathrm{cos}\beta 0`$ ($`\mathrm{tan}\beta \mathrm{}`$). Therefore, $`\mathrm{tan}\beta `$ cannot be large to retain enough CP violation, causing a tension with the requirement of heavy enough Higgs mass. Moreover, the constraints from electric dipole moments are quite severe if the relative phase between $`\mu `$ and $`M_2`$ is order unity.222After my talk, the constraints from electric dipole moments had been shown to be even more severe . What it means is that we are supposed to find a right-handed scalar top quark, charginos “soon” with a large CP violation in the mass matrix.

It is important to ask if there is any interesting consequence of this scenario in CP violation on B systems. It turns out, however, that there is no new CP violation in B systems from the relevant phase for the electroweak baryogenesis in the MSSM .333It is quite possible that other models of electroweak baryogenesis lead to new CP violation in $`B`$-physics. The most important effect is the contribution to $`B_{d,s}`$$`\overline{B}_{d,s}`$ mixing. Surprisingly, the new CP violating phase does not appear in the diagram, and hence the mixing amplitude has the same phase as in the Standard Model. There is, however 20–30% enhancement in the mixing amplitude. Such an enhancement cannot be established now because of the theoretical uncertainty in the $`B`$-parameter. However lattice calculations are expected to reduce the uncertainty down to 5% level in the near future, and this enhancement may be seen experimentally. It would require a complicated analysis. In the case of $`B_s`$ mixing, the Standard Model prediction must be improved with a better determination of $`V_{cb}`$ (and hence $`V_{ts}`$ through unitarity) and a better calculation of the $`B`$-parameter. In the case of $`B_d`$ mixing, we used to determine $`V_{td}`$ from the mixing. For the purpose of extracting a new contribution in the mixing, we have to determine $`V_{td}`$ by alternative method. By an improved determination of $`V_{ub}`$ combined with measurement of angles from tree-level processes, $`V_{td}`$ can in principle be determined without relying on the $`B_d`$ mixing, and an enhancement in the mixing may be established. In order to truly establish the model, we would like to see the new CP violating phase in the chargino mass matrix. An electron-positron linear collider would be the best approach.

### 2.2 Leptogenesis

In leptogenesis, you generate $`L0`$ first. Then $`L`$ gets partially converted to $`B`$ by the electroweak anomaly. The question then is how you generate $`L0`$. In the original proposal , it was done by the decay of a right-handed neutrino (say $`N_1`$), present in the seesaw mechanism, with a direct CP violation. At the tree-level, a right-handed neutrino decays equally into $`l+H`$ and $`\overline{l}+H^{}`$. At the one-loop level, however, the interference between diagrams shown in Fig. 6 cause a difference in the decay rates of a right-handed neutrino into leptons and anti-leptons as

$`{\displaystyle \frac{\mathrm{\Gamma }(N_1l_iH)\mathrm{\Gamma }(N_1\overline{l}_iH)}{\mathrm{\Gamma }(N_1l_iH)+\mathrm{\Gamma }(N_1\overline{l}_iH)}}`$
$`{\displaystyle \frac{1}{8\pi }}{\displaystyle \frac{\mathrm{}(h_{1j}h_{1k}h_{lk}^{}h_{lj}^{})}{|h_{1i}|^2}}{\displaystyle \frac{M_1}{M_3}}.`$ (3)
Once the right-handed neutrinos are produced in early Universe, their long lifetime would allow them to decay out of equilibrium, thereby generating an asymmetry between leptons and anti-leptons. The lepton asymmetry then is partially converted to baryon asymmetry thanks to electroweak anomaly. Much more details had been worked out in the light of recent neutrino oscillation data and it had been shown that a right-handed neutrino of about $`10^{10}`$ GeV can well account for the cosmic baryon asymmetry from its out-of-equilibrium decay .

There is some tension in the supersymmetric version because of cosmological problems caused by the gravitino, which prefers a low reheating temperature after inflation. But a supersymmetric problem has a supersymmetric solution. It can be circumvented using the superpartner of right-handed neutrino that can have a coherent oscillation after the inflation . Leptogenesis can work.

Can we prove leptogenesis experimentally? Lay Nam Chang, John Ellis, Belen Gavela, Boris Kayser, and myself got together at Snowmass 2001 and discussed this question. The short answer is unfortunately no. There are additional CP violating phases in the heavy right-handed neutrino sector that cannot be seen by studying the light left-handed neutrinos.444If you believe in a certain scenario of supersymmetry breaking, low-energy lepton-flavor violation can carry information about CP violation in the right-handed neutrino sector . However, such connection depends on the assumptions in the origin of supersymmetry breaking. For example, even two-generation seesaw mechanism is enough to have CP violation that can potentially produce lepton asymmetry, unlike the minimum of three-generations for CP violation in neutrino oscillation. However, we decided that if we will see (1) electroweak baryogenesis ruled out, (2) lepton-number violation e.g. in neutrinoless double beta decay,555There is, however, a realistic model of leptogenesis without lepton-number violation . and (3) CP violation in the neutrino sector e.g., in very long-baseline neutrino oscillation experiment, we will probably believe it based on these “archaeological” evidences.

## 3 Grand Unified Theories

As we have discussed, baryogenesis may not be a good motivation for GUTs any more. However, there are still many reasons to consider GUTs seriously to answer some of the big questions in the Standard Model. For example, electric charges are quantized in the unit of $`e/3`$ among quarks and leptons, while the electromagnetic $`U(1)`$ gauge invariance does not require such quantization. Similarly, the non-trivial anomaly cancellation and seemingly random hypercharge assignments hint at deeper organizing principles behind the quantum numbers of quarks and leptons. Three forces are seemingly unrelated, but they are all based on the gauge principle. Also philosophically, unified description of all forces has been a dream since Einstein. GUTs address these questions beautifully.

The quarks and leptons are unified in GUT multiplets. In the case of $`SU(5)`$ group, 5 multiplet includes a lepton doublet and a right-handed down quark of three colors, and 10 multiplet includes a quark doublet and a right-handed up quark of three colors, and right-handed charged lepton.

Phenomenologically, the original non-supersymmetric $`SU(5)`$ GUT no longer works because the gauge coupling constants measured precisely at LEP and SLC extrapolated to higher energies do not meet at a point. However with the MSSM particle content, running of the gauge couplings changes and they do meet within a percent level accuracy at $`2\times 10^{16}`$ GeV. Therefore, the Minimal supersymmetric $`SU(5)`$ GUT appears a success phenomenologically.

On the other hand, another prediction of GUTs than the gauge coupling unification, namely the proton decay, has not been observed. In fact, the limit from SuperKamiokande $`\tau (p\overline{\nu }K^+)>6.7\times 10^{32}`$ yr implies the mass of the color-triplet $`SU(5)`$-partner of the Higgs boson to be heavier than $`7.6\times 10^{16}`$ GeV, while the coupling unification requires it to be well below $`10^{16}`$ GeV (Fig. 8). Even with the so-called “decoupling” limit where first- and second-generation squarks are assumed to be heavy, the lower bound comes down only to $`5.7\times 10^{16}`$ GeV. Therefore, the Minimal supersymmetric $`SU(5)`$ GUT is now excluded .

Unfortunately, the prediction of the proton lifetime is sensitive to the ugly part of GUT model building, namely how to accommodate observed quark and lepton masses, which the minimal model gets wrong by a factor of three, and how to keep only the doublet Higgs light while making color-triplet partner heavy (“triplet-doublet splitting problem”). Expanded particle content at the GUT scale also makes the prediction more uncertain . Some fine-tuning can suppress the decay rate . A recent proposal of breaking $`SU(5)`$ using extra dimension on a one-dimensional orbifold $`S^1/Z_2`$ eliminates this mode of proton decay . Therefore, one cannot say that supersymmetric GUTs are excluded. In other words, proton decay may be just around the corner. It should be remembered that proton decay is a truly unique window to physics at $`>10^{15}`$ GeV scale, and it is worth pursuing anyway. Some other supersymmetric GUT models predict $`pe^+\pi ^0`$ mode close to the experimental limit, including flipped $`SU(5)`$ model  and orbifold GUT .

In the absence of direct signal (proton decay), it is natural to look for other indirect effects of GUT. It has been well studied that quark-lepton unification causes flavor-changing effects among leptons through top quark Yukawa coupling, giving rare phenomena such as $`\mu e\gamma `$, $`\mu e`$ conversion at experimentally accessible rates . Recent discovery of $`\nu _\mu \nu _\tau `$ atmospheric neutrino oscillation with large mixing angle may also give observable $`\tau \mu \gamma `$ etc at near future experiments .

I would like to advertise yet another interesting signal of GUT in $`B`$-physics . Take the large mixing between $`\nu _\mu `$ and $`\nu _\tau `$. In $`SU(5)`$ GUT, they are in the same multiplets as $`s_R`$ and $`b_R`$, respectively. Therefore, it is natural to expect a large mixing between $`s_R`$ and $`b_R`$. Such mixing drops out completely from CKM matrix because it keeps track of mixing only among left-handed quarks that participate in the charged-current weak interaction. However, in presence of supersymmetry, a large mixing between $`\stackrel{~}{s}_R`$ and $`\stackrel{~}{b}_R`$ would lead to observable effects. For example, supersymmetric contribution to $`B_s`$ mixing may well be comparable to the standard model contribution even with squarks at 1 TeV. Supersymmetric penguin contribution to $`bs\overline{s}s`$ allows direct CP violation and may give different “$`\mathrm{sin}2\beta `$” in $`B_d\varphi K_s`$ from that in $`B_dJ/\psi K_s`$.666Recently both BABAR and BELLE collaborations announced discrepancy between two measurements of “$`\mathrm{sin}2\beta `$” at two standard deviation each .

## 4 Models of Flavor

The most pressing question on the origin of flavor is what distinguishes flavor. Three generations of quarks and leptons have exactly the same quantum numbers. But then, how come that they have so different masses and small mixings? When we learn quantum mechanics, we see that states with similar quantum numbers have similar energies, and they mix greatly. Due to some reason, three generations do not follow this common wisdom. Why?

One way to answer this question, developed very actively in the past few years, is the concept of explicitly broken flavor symmetry. The idea is simple: there must a new (but hidden) quantum number that distinguishes different flavors. Because of the difference in the flavor quantum number, particles in different generations have very different masses and mix little. According to Nöther’s theorem, a quantum number means a symmetry, and hence flavor symmetry. The flavor symmetry must allow top quark Yukawa coupling because it is a coupling of order unity which we consider natural. Other Yukawa couplings are very small. They are forbidden by the flavor symmetry, and are generated only when the symmetry breaks. The smallness of the Yukawa couplings are controlled by the smallness of symmetry breaking. The mixings are also forbidden by the flavor symmetry, and are induced by the symmetry breaking.

To make the discussion more concrete, let us study the following $`SU(5)`$-like flavor quantum number assignment. I choose a simple $`U(1)`$ charge, and I assign

$`\begin{array}{ccc}\mathrm{𝟏𝟎}_1(+2),& \mathrm{𝟏𝟎}_2(+1)& \mathrm{𝟏𝟎}_3(0)\\ \mathrm{𝟓}_1(+1),& \mathrm{𝟓}_2(+1),& \mathrm{𝟓}_3(+1).\end{array}`$ (6)
The subscripts refer to the generations, and the charges shown in parentheses. With this assignment, the only allowed Yukawa coupling is the one for the top quark. Other Yukawa couplings are forbidden. Next I assume that the $`U(1)`$ symmetry is broken by a small parameter $`ϵ0.05`$ that carries the charge $`1`$. Then it becomes possible to fill in all elements of the mass matrices,

$`M_u`$ $``$ $`\left(\begin{array}{ccc}ϵ^4& ϵ^3& ϵ^2\\ ϵ^3& ϵ^2& ϵ\\ ϵ^2& ϵ& 1\end{array}\right),`$ (10)
$`M_d`$ $``$ $`\left(\begin{array}{ccc}ϵ^3& ϵ^3& ϵ^3\\ ϵ^2& ϵ^2& ϵ^2\\ ϵ& ϵ& ϵ\end{array}\right),`$ (14)
$`M_l`$ $``$ $`\left(\begin{array}{ccc}ϵ^3& ϵ^2& ϵ\\ ϵ^3& ϵ^2& ϵ\\ ϵ^3& ϵ^2& ϵ\end{array}\right).`$ (18)
The symbol $``$ emphasizes that we cannot predict precise numbers based on this simple idea, but only the order of magnitudes suppressed by powers of $`ϵ`$. A simple approximate prediction out of this charge assignment is that

$`m_u:m_c:m_tm_d^2:m_s^2:m_b^2`$ (19)
$`m_e^2:m_\mu ^2:m_\tau ^2ϵ^4:ϵ^2:1,`$
which is phenomenologically acceptable. Note that up quarks are doubly hierarchical compared to the down quarks and charged leptons. Mixing angles are also predicted, $`V_{cb}V_{ts}ϵ`$, $`V_{ub}V_{td}ϵ^2`$, which work quite well, and $`V_{us}V_{cd}ϵ`$ is a little bit too small but not crazy.

What about neutrinos? Indeed, recent data on neutrino oscillations have shed considerable insight into the flavor symmetry. The MNS matrix suggested by atmospheric, solar (LMA solution), and reactor data has the form

$`(e\mu \tau )\left(\begin{array}{ccc}\mathrm{𝑙𝑎𝑟𝑔𝑒}& \mathrm{𝑙𝑎𝑟𝑔𝑒}& \mathrm{𝑠𝑚𝑎𝑙𝑙𝑖𝑠ℎ}\\ \mathrm{𝑙𝑎𝑟𝑔𝑒}& \mathrm{𝑙𝑎𝑟𝑔𝑒}& \mathrm{𝑙𝑎𝑟𝑔𝑒}\\ \mathrm{𝑙𝑎𝑟𝑔𝑒}& \mathrm{𝑙𝑎𝑟𝑔𝑒}& \mathrm{𝑙𝑎𝑟𝑔𝑒}\end{array}\right)\left(\begin{array}{c}\nu _1\\ \nu _2\\ \nu _3\end{array}\right),`$ (27)
$`{\displaystyle \frac{\mathrm{\Delta }m_{12}^2}{\mathrm{\Delta }m_{23}^2}}=0.01\text{}0.2.`$
The mass hierarchy is not very large, especially after taking square roots. All angles are large except $`|U_{e3}|\begin{array}{c}<\hfill \\ \hfill \end{array}0.15`$, but even this constraint is not particularly strong compared to $`U_{e2}0.4`$, $`U_{\mu 3}0.7`$. It has been a big surprise to all of us that the pattern is so different from quarks and charged leptons.

I actually find the neutrino masses and mixings very natural. In view of the question I posed earlier, if three quantum mechanical states share the same quantum numbers, their energies (masses) are expected to be similar, and their mixings unsuppressed. We have been so much used to hierarchical masses and small mixings over many decades, but what is surprising is not the neutrinos but other quarks and leptons rather! I view the observed pattern of neutrino masses and mixings a great confirmation of our naive intuition.

In terms of flavor quantum numbers, all we need to do then is to assign the same quantum numbers to three generations of neutrinos. Indeed, the charge assignment (6) was motivated by this requirement to obtain

$$M_\nu \left(\begin{array}{ccc}1& 1& 1\\ 1& 1& 1\\ 1& 1& 1\end{array}\right).$$
(28)
Here, the overall suppression of $`ϵ^2`$ is dropped because the overall mass scale of neutrino masses is probably determined by other physics such as seesaw mechanism.

But you may wonder if such an argument may only produce largish mixing angles, but never dramatically maximal mixing as in atmospheric neutrino oscillations. It turns out that a maximal mixing is in a sense the most natural angle. Using a simple Monte Carlo of random neutrino mass matrices, you can see that there is a peak in the distribution at $`\mathrm{sin}^22\theta _{23}=1`$. Actually, you can understand this distribution based on purely group-theoretic consideration. The unique invariant measure (Haar measure) of $`SU(3)`$ MNS matrix gives this distribution.

We called this simple fact “anarchy” . Because there is no quantum number to distinguish three generations of neutrinos, neutrino mass matrix lacks any particular structure. That alone explains large mixing angles and small hierarchy. Even though $`\mathrm{sin}^22\theta _{13}`$ appears indeed small compared to the obtained distribution, if you get three quantities $`\mathrm{sin}^22\theta _{23}`$, $`\mathrm{sin}^22\theta _{12}`$, and $`\mathrm{\Delta }m_{12}^2/\mathrm{\Delta }m_{23}^2`$ right with one outlier, I find it perfectly reasonable. The anarchy then predicts the LMA solution, testable at KamLAND, and $`\theta _{13}`$ just below the bound. CP violation is also $`O(1)`$. This is the prefect scenario for long-baseline neutrino oscillation experiments.

## 5 Conclusion

Flavor physics is going through an amazing period. Just to name a few important points I covered in this talk, (1) Electroweak baryogenesis is getting cornered, (2) Leptogenesis is gaining momentum, and (3) Neutrinos provide new insight into the origin of flavor. The good news is that we will obtain more data, including rare decays and possible deviations from the Standard Model, to hopefully pin down the flavor symmetry and eventually its dynamical origin.

## Acknowledgements

I thank the organizers for inviting me to the exciting workshop and also for the patience waiting for my manuscript. This work was supported in part by the DOE Contract DE-AC03-76SF00098 and in part by the NSF grant PHY-0098840.