This notebook is part of smFRET burst analysis software FRETBursts.
This notebook shows how to implement Burst Variance Analysis (BVA) (Torella 2011) using FRETBursts.
For a complete tutorial on burst analysis see FRETBursts - us-ALEX smFRET burst analysis.
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from fretbursts import *
sns = init_notebook(apionly=True)
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# Tweak here matplotlib style
import matplotlib as mpl
mpl.rcParams['font.sans-serif'].insert(0, 'Arial')
mpl.rcParams['font.size'] = 12
%config InlineBackend.figure_format = 'retina'
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url = 'http://files.figshare.com/2182601/0023uLRpitc_NTP_20dT_0.5GndCl.hdf5'
download_file(url, save_dir='./data')
full_fname = "./data/0023uLRpitc_NTP_20dT_0.5GndCl.hdf5"
d = loader.photon_hdf5(full_fname)
loader.alex_apply_period(d)
d.calc_bg(bg.exp_fit, time_s=50.1, tail_min_us='auto', F_bg=1.7)
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d.burst_search(m=10, computefret=False, ph_sel=Ph_sel(Dex='DAem'))
d.calc_fret(count_ph=True, corrections=False)
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ds = d.select_bursts(select_bursts.naa, th1=30, computefret=False)
ds1 = ds.select_bursts(select_bursts.size, th1=30, computefret=False)
ds_FRET = ds1.select_bursts(select_bursts.S, S1=0.25, S2=0.85, computefret=False)
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dx=ds_FRET
alex_jointplot(dx)
We define a function to compute $s_E$:
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def bva_sigma_E(n, bursts, DexAem_mask, out=None):
"""
Perform BVA analysis computing std.dev. of E for sub-bursts in each burst.
Split each burst in n-photons chunks (sub-bursts), compute E for each sub-burst,
then compute std.dev. of E across the sub-bursts.
For details on BVA see:
- Torella et al. (2011) Biophys. J. doi.org/10.1016/j.bpj.2011.01.066
- Ingargiola et al. (2016) bioRxiv, doi.org/10.1101/039198
Arguments:
n (int): number of photons in each sub-burst
bursts (Bursts object): burst-data object with indexes relative
to the Dex photon stream.
DexAem_mask (bool array): mask of A-emitted photons during D-excitation
periods. It is a boolean array indexing the array of Dex timestamps
(`Ph_sel(Dex='DAem')`).
out (None or list): append the result to the passed list. If None,
creates a new list. This is useful to accumulate data from
different spots in a single list.
Returns:
E_sub_std (1D array): contains for each burst, the standard deviation of
sub-bursts FRET efficiency. Same length of input argument `bursts`.
"""
E_sub_std = [] if out is None else out
for burst in bursts:
E_sub_bursts = []
startlist = range(burst.istart, burst.istop + 2 - n, n)
stoplist = [i + n for i in startlist]
for start, stop in zip(startlist, stoplist):
A_D = DexAem_mask[start:stop].sum()
assert stop - start == n
E = A_D / n
E_sub_bursts.append(E)
E_sub_std.append(np.std(E_sub_bursts))
return E_sub_std
Next we prepare the data for BVA:
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ph_d = ds_FRET.get_ph_times(ph_sel=Ph_sel(Dex='DAem'))
bursts = ds_FRET.mburst[0]
bursts_d = bursts.recompute_index_reduce(ph_d)
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Dex_mask = ds_FRET.get_ph_mask(ph_sel=Ph_sel(Dex='DAem'))
DexAem_mask = ds_FRET.get_ph_mask(ph_sel=Ph_sel(Dex='Aem'))
DexAem_mask_d = DexAem_mask[Dex_mask]
and call the bva_sigma_E function:
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n = 7
E_sub_std = bva_sigma_E(n, bursts_d, DexAem_mask_d)
Finally, we make a KDE plot of the 2D distribution E_sub_std versus the burst FRET efficiency:
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plt.figure(figsize=(4.5, 4.5))
x = np.arange(0,1.01,0.01)
y = np.sqrt((x*(1-x))/n)
plt.plot(x, y, lw=2, color='k', ls='--')
im = sns.kdeplot(ds_FRET.E[0], np.asfarray(E_sub_std),
shade=True, cmap='Spectral_r', shade_lowest=False, n_levels=20)
plt.xlim(0,1)
plt.ylim(0,np.sqrt(0.5**2/7)*2)
plt.xlabel('E', fontsize=16)
plt.ylabel(r'$\sigma_i$', fontsize=16);
plt.text(0.05, 0.95, 'BVA', va='top', fontsize=22, transform=plt.gca().transAxes)
plt.text(0.95, 0.95, '# Bursts: %d' % ds_FRET.num_bursts,
va='top', ha='right', transform=plt.gca().transAxes)
plt.savefig('BVA.png', bbox_inches='tight', dpi=200, transparent=False)
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x, y = ds_FRET.E[0], np.asfarray(E_sub_std)
hist_kws = dict(edgecolor='k', linewidth=0.2,
facecolor=sns.color_palette('Spectral_r', 100)[10])
g = sns.JointGrid(x=x, y=y, ratio=3)
g.plot_joint(sns.kdeplot, cmap='Spectral_r', shade=True, shade_lowest=False, n_levels=20)
g.ax_marg_x.hist(x, bins=np.arange(-0.2, 1.2, 0.025), **hist_kws)
g.ax_marg_y.hist(y, bins=50, orientation="horizontal", **hist_kws)
x1 = np.arange(0,1.01,0.01)
y1 = np.sqrt((x1*(1-x1))/n)
plt.plot(x1, y1, lw=2, color='k', ls='--')
g.ax_joint.set_xlim(0,1)
g.ax_joint.set_ylim(0,np.sqrt(0.5**2/7)*2)
g.ax_joint.set_xlabel('E', fontsize=16)
g.ax_joint.set_ylabel(r'$\sigma_i$', fontsize=16);
g.ax_joint.text(0.05, 0.95, 'BVA', va='top', fontsize=22, transform=g.ax_joint.transAxes)
g.ax_joint.text(0.95, 0.95, '# Bursts: %d' % ds_FRET.num_bursts,
va='top', ha='right', transform=g.ax_joint.transAxes)
plt.savefig('BVA_joint.png', bbox_inches='tight', dpi=200, transparent=False)