Reading µs-ALEX data from Photon-HDF5 with h5py

In this notebook we show how to read a µs-ALEX smFRET measurement stored in Photon-HDF5 format using python and a few common scientific libraries (numpy, h5py, matplotlib). Specifically, we show how to load timestamps, build an alternation histogram and select photons in the donor and acceptor excitation periods.

See also a similar notebook using pytables instead of h5py.



In [1]:

    
from __future__ import division, print_function  # only needed on py2
%matplotlib inline
import numpy as np
import h5py
import matplotlib.pyplot as plt

1. Utility functions

Here we define an utility function to print HDF5 file contents:



In [2]:

    
def print_children(group):
    """Print all the sub-groups in `group` and leaf-nodes children of `group`.

    Parameters:
        data_file (h5py HDF5 file object): the data file to print
    """
    for name, value in group.items():
        if isinstance(value, h5py.Group):
            content = '(Group)'
        else:
            content = value[()]
        print(name)
        print('    Content:     %s' % content)
        print('    Description: %s\n' % value.attrs['TITLE'].decode())

2. Open the data file

Let assume we have a Photon-HDF5 file at the following location:



In [3]:

    
filename = '../data/0023uLRpitc_NTP_20dT_0.5GndCl.hdf5'

We can open the file, as a normal HDF5 file



In [4]:

    
h5file = h5py.File(filename)

The object h5file is a pytables file reference. The root group is accessed with h5file.root.

3. Print the content

Let's start by taking a look at the file content:



In [5]:

    
print_children(h5file)









    



acquisition_duration
    Content:     600.0
    Description: Measurement duration in seconds.

description
    Content:     b'us-ALEX measurement of a doubly-labeled ssDNA sample.'
    Description: A user-defined comment describing the data file.

identity
    Content:     (Group)
    Description: Information about the Photon-HDF5 data file.

photon_data
    Content:     (Group)
    Description: Group containing arrays of photon-data.

provenance
    Content:     (Group)
    Description: Information about the original data file.

sample
    Content:     (Group)
    Description: Information about the measured sample.

setup
    Content:     (Group)
    Description: Information about the experimental setup.

We see the typical Photon-HDF5 structure. In particular the field description provides a short description of the measurement and acquisition_duration tells that the acquisition lasted 600 seconds.

As an example let's take a look at the content of the sample group:



In [6]:

    
print_children(h5file['sample'])









    



buffer_name
    Content:     b'TE50 + 0.5M GndCl'
    Description: A descriptive name for the buffer.

dye_names
    Content:     b'Cy3B, ATTO647N'
    Description: String containing a comma-separated list of dye or fluorophore names.

num_dyes
    Content:     2
    Description: Number of different dyes present in the samples.

sample_name
    Content:     b'20dt ssDNA oligo doubly labeled with Cy3B and Atto647N'
    Description: A descriptive name for the sample.

Finally, we define a shortcut to the photon_data group to save some typing later:



In [7]:

    
photon_data = h5file['photon_data']

4. Reading the data

First, we make sure the file contains the right type of measurement:



In [8]:

    
photon_data['measurement_specs']['measurement_type'][()].decode()









    Out[8]:





'smFRET-usALEX'

Ok, tha's what we espect.

Now we can load all the timestamps (including timestamps unit) and detectors arrays:



In [9]:

    
timestamps = photon_data['timestamps'][:]
timestamps_unit = photon_data['timestamps_specs']['timestamps_unit'][()]
detectors = photon_data['detectors'][:]



In [10]:

    
print('Number of photons: %d' % timestamps.size)
print('Timestamps unit:   %.2e seconds' % timestamps_unit)
print('Detectors:         %s' % np.unique(detectors))









    



Number of photons: 2683962
Timestamps unit:   1.25e-08 seconds
Detectors:         [0 1]

We may want to check the excitation wavelengths used in the measurement. This information is found in the setup group:



In [11]:

    
h5file['setup']['excitation_wavelengths'][:]









    Out[11]:





array([  5.32000000e-07,   6.35000000e-07])

Now, let's load the definitions of donor/acceptor channel and excitation periods:



In [12]:

    
donor_ch = photon_data['measurement_specs']['detectors_specs']['spectral_ch1'][()]
acceptor_ch = photon_data['measurement_specs']['detectors_specs']['spectral_ch2'][()]
print('Donor CH: %d     Acceptor CH: %d' % (donor_ch, acceptor_ch))









    



Donor CH: 0     Acceptor CH: 1



In [13]:

    
alex_period = photon_data['measurement_specs']['alex_period'][()]
donor_period = photon_data['measurement_specs']['alex_excitation_period1'][()]
offset = photon_data['measurement_specs']['alex_offset'][()]
acceptor_period = photon_data['measurement_specs']['alex_excitation_period2'][()]
print('ALEX period:     %d  \nOffset:         %4d      \nDonor period:    %s      \nAcceptor period: %s' % \
      (alex_period, offset, donor_period, acceptor_period))









    



ALEX period:     4000  
Offset:          700      
Donor period:    [2180 3900]      
Acceptor period: [ 200 1800]

These numbers define the donor and acceptor alternation periods as shown below:

$$2180 < \widetilde{t} < 3900 \qquad \textrm{donor period}$$$$200 < \widetilde{t} < 1800 \qquad \textrm{acceptor period}$$

where $\widetilde{t}$ represent the (timestamps - offset) MODULO alex_period.

For more information please refer to the measurements_specs section of the Reference Documentation.

5. Plotting the alternation histogram

Let start by separating timestamps from donor and acceptor channels:



In [14]:

    
timestamps_donor = timestamps[detectors == donor_ch]
timestamps_acceptor = timestamps[detectors == acceptor_ch]

Now that the data has been loaded we can plot an alternation histogram using matplotlib:



In [15]:

    
fig, ax = plt.subplots()
ax.hist((timestamps_acceptor - offset) % alex_period, bins=100, alpha=0.8, color='red', label='donor')
ax.hist((timestamps_donor - offset) % alex_period, bins=100, alpha=0.8, color='green', label='acceptor')
ax.axvspan(donor_period[0], donor_period[1], alpha=0.3, color='green')
ax.axvspan(acceptor_period[0], acceptor_period[1], alpha=0.3, color='red')
ax.set_xlabel('(timestamps - offset) MOD alex_period')
ax.set_title('ALEX histogram')
ax.legend(loc='center left', bbox_to_anchor=(1, 0.5), frameon=False);

6. Timestamps in different excitation periods

We conclude by showing, as an example, how to create arrays of timestamps containing only donor or acceptor exitation photons.



In [16]:

    
timestamps_mod = (timestamps - offset) % alex_period
donor_excitation = (timestamps_mod < donor_period[1])*(timestamps_mod > donor_period[0])
acceptor_excitation = (timestamps_mod < acceptor_period[1])*(timestamps_mod > acceptor_period[0])
timestamps_Dex = timestamps[donor_excitation]
timestamps_Aex = timestamps[acceptor_excitation]



In [17]:

    
fig, ax = plt.subplots()
ax.hist((timestamps_Dex - offset) % alex_period, bins=np.arange(0, alex_period, 40), alpha=0.8, color='green', label='D_ex')
ax.hist((timestamps_Aex - offset) % alex_period, bins=np.arange(0, alex_period, 40), alpha=0.8, color='red', label='A_ex')
ax.set_xlabel('(timestamps - offset) MOD alex_period')
ax.set_title('ALEX histogram (selected periods only)')
ax.legend(loc='center left', bbox_to_anchor=(1, 0.5), frameon=False);



In [18]:

    
#plt.close('all')



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