2. Photon-HDF5 format definition

2.1. Overview

A Photon-HDF5 is a HDF5 file with a predefined structure for timestamp-based data.

A screen-shot of a typical Photon-HDF5 file opened in HDFView is shown here:

HDView screenshot showing a Photon-HDF5 file

The previous figure shows the 5 main groups contained in a Photon-HDF5 file. Of these, /photon_data and /setup contains the raw data and all the information needed for the analysis. A schematic overview is shown in the next figure:

Photon-HDF5 structure: photon_data, setup

The remaining 3 groups provide additional metadata not necessary for data analysis:

Photon-HDF5 structure: sample, identity, provenance

A brief description of these 3 metadata groups follows:

  • /identity: Information about the data file.
  • /provenance: Information about the original data file (when the Photon-HDF5 file has been converted from another format).
  • /sample: Description of the measured sample.

2.2. Root-level parameters

The root node (“/”) in a Photon-HDF5 file contains the following fields:

  • /acquisition_duration: (float) the measurement duration in seconds.
  • /description: (string) a user-defined measurement description.

In addition, the root node has the following attributes which distinguish a Photon-HDF5 file from other HDF5 files:

  • format_name: must contain the string “Photon-HDF5”
  • format_version: (string) the Photon-HDF5 format version.

2.3. Photon-data group

This section describes the layout and fields in the /photon_data group. Note that only the kind of data is specified (i.e. scalar, integer array, float array), but no data type size is mandated. For arrays, the most commonly used data-type is indicated.

Mandatory fields:

  • timestamps: (array) photon timestamps. Typical data-type int64.

  • timestamps_specs/
    • timestamps_unit: (float) timestamp units in seconds.

Optional if there is only 1 detector, otherwise mandatory:

  • detectors: (array of integers) Detector pixel IDs for each timestamp. Typical data-type uint8.

When the dataset contains TCSPC or nanotime information (i.e. arrival time of each photon with respect to a laser pulse), the following fields must be present:

  • nanotimes:(array of integers) TCSPC nanotimes. Conventionally the time axis direction is the “natural” direction, i.e. lifetime decays look correctly oriented in time. For more details see Nanotimes time direction. Typical data-type uint16.

  • nanotimes_specs/
    • tcspc_unit: (float) TAC/TDC bin size (in seconds).
    • tcspc_num_bins: (integer) number of TAC/TDC bins.
    • tcspc_range:(float) (optional) full-scale range of the TAC/TDC (in seconds). This a derived field equal to tcspc_unit * tcspc_num_bins.

Finally, if the data come from a simulation, /photon_data may contain:

  • particles: (array of integers) a particle ID (integer) for each timestamp. Typical data-type uint8.

2.3.1. Measurement specs

The optional /photon_data/measurement_specs group contains additional information allowing unambiguous interpretation of the data for each specific type of measurement.

  • measurement_type: (string) the type of the measurements. Valid names are:

    • “smFRET” (1 excitation color, 2 detection colors)
    • “smFRET-usALEX” (2 excitation colors, 2 detection colors)
    • “smFRET-usALEX-3c” (3 excitation colors, 3 detection colors)
    • “smFRET-nsALEX” (2 excitation colors, 2 detection colors)

    New names can be created for different kind of measurements and we encourage users to submit new name requests.

The measurement_type field describes the type of measurement saved within the file. It is an important field allowing software packages reading and saving Photon-HDF5 files to perform consistency checks (see also Measurement type).

For μs-ALEX, 2, 3 or N colors:

  • alex_period: (integer or float) duration of one complete excitation alternation period expressed in timestamp units. The alternation period is equal to alex_period * timestamps_unit.

For ns-ALEX (or lifetime with no alternation):

  • laser_repetition_rate: (float) excitation laser pulse repetition rate in Hertz.

For 2-color (or more) μs-ALEX and ns-ALEX (optional):

  • alex_offset: (scalar) [μs-ALEX only] Time offset (in timestamps units) to be applied to the timestamps array before computing the μs-ALEX histogram. It is assumed that the μs-ALEX alternation histogram is the histogram of (timestamps - alex_offset) MOD alex_period.
  • alex_excitation_period1: (array with an even-number of integer elements, normally 2) start and stop values identifying the excitation periods for the first wavelength in /setup/excitation_wavelengths (which is the shortest wavelength). In smFRET experiments with 2-colors excitation this field defines the donor excitation period. See also Wavelengths and spectral band order and note below.
  • alex_excitation_period2: (array with an even-number of integer elements, normally 2) start and stop values identifying the excitation periods for the second wavelength in /setup/excitation_wavelengths. In smFRET experiments with 2-colors excitation this field defines the acceptor excitation period. See also Wavelengths and spectral band order and note below.

For 3 (or more) colors alternated or interleaved excitation:

  • alex_excitation_period3: (array with an even-number of integer elements, normally 2) start and stop values identifying the excitation periods for the third wavelength in /setup/excitation_wavelengths. See also Wavelengths and spectral band order and note below.
  • etc...

Note

For μs-ALEX, both alex_excitation_period1 and alex_excitation_period2 are 2-element arrays and are expressed in timestamps_units. For ns-ALEX (also known as PIE), they are arrays with an even-number of elements, comprising as many start-stop nanotime pairs as there are excitation periods within the TAC/TDC range. In this case the values are expressed in nanotimes_units.

For more details see Definition of alternation periods.

2.3.1.1. Detectors specs

Within measurement_specs, the detectors_specs/ sub-group contains all the pixel ID–detection channel associations, i.e. spectral bands, polarizations or beam-split channels.

When a measurement records more than 1 spectral band, the fields:

  • spectral_ch1
  • spectral_ch2
  • etc...

specify which detector pixel is employed in each spectral band. When the measurement records only 1 spectral band these fields may be omitted. The spectral bands are strictly ordered for increasing wavelengths. For example, for 2-color smFRET measurements spectral_ch1 and spectral_ch2 represent the donor and acceptor channel respectively.

If a measurement records more than 1 polarization states, the fields:

  • polarization_ch1
  • polarization_ch2

specify which detector pixel is used for each polarization. When the measurement records only one polarization, these fields may be omitted.

When the detection light is split into 2 channels using a non-polarizing beam-splitter the fields:

  • split_ch1
  • split_ch2

specify which detector pixel is used in each of the “beam-split” channels.

All previous fields are arrays containing one or more Detector pixel IDs. For example, a 2-color smFRET measurement will have only one value in spectral_ch1 (donor) and one value in spectral_ch2 (acceptor). A 2-color smFRET measurement with polarization (4 detectors) will have 2 values in each of the spectral_chX and polarization_chX fields (where X=1 or 2). For a multispot smFRET measurement, in each photon_dataN group, there will be spectral_chX fields containing the donor/acceptor pixels used in that spot (see Multi-spot measurements).

2.4. Setup group

The /setup group contains information about the measurement setup. This group can be absent in some files, an example being a file containing only detector dark counts, for which the following fields do not necessarily have a meaning. When setup is present, the following 7 fields are mandatory:

  • num_pixels: (integer) total number of detector pixels. For example, for a single-spot 2-color smFRET measurement using 2 single-pixel SPADs as detectors this field is 2.
  • num_spots: (integer) the number of excitation (or detection) “spots” in the sample. This field is 1 for all the measurements using a single confocal excitation volume. When not applicable, for example under wide-field illumination with 2-D imaging detectors, this field is omitted.
  • num_spectral_ch: (integer) number of distinct detection spectral channels. For example, in a 2-color smFRET experiment there are 2 detection spectral channels (donor and acceptor), therefore its value is 2. When there is a single detection channel or all channels detect the same spectral band, its value is 1.
  • num_polarization_ch: (integer) number of distinct detection polarization channels. For example, in polarization anisotropy measurements, its value is 2. When there is a single detection channel or all channels detect the same polarization (including when no polarization selection is performed) its value is 1.
  • num_split_ch: (integer) number of distinct detection channels detecting the same spectral band and polarization state. For example, when a non-polarizing beam-splitter is employed in the detection path, its value is 2. When no splitting is performed, its value is 1.
  • modulated_excitation: (boolean) True (or 1) if there is any form of excitation modulation either in the wavelength space (as in μs-ALEX or PAX) or in the polarization space. This field is also True for pulse-interleaved excitation (PIE) or ns-ALEX measurements.
  • lifetime: (boolean) True (or 1) if the measurements includes a nanotimes array of (usually sub-ns resolution) photon arrival times with respect to a laser pulse (as in TCSPC measurements).

The remaining fields are optional:

  • excitation_wavelengths: (array of floats) list of excitation wavelengths (center wavelength if broad-band) in increasing order (unit: meter).
  • excitation_cw: (array of booleans) for each excitation source, this field indicates whether excitation is continuous wave (CW), True, or pulsed, False. The order of excitation sources is the same as that in excitation_wavelengths and is in increasing order of wavelengths.

The following fields are optional and not necessarily relevant for all experiments. If the associated information is irrelevant or not available, these fields are omitted.

  • excitation_polarizations: (arrays of floats) list of polarization angles (in degrees) for each excitation source. The order of excitation sources is the same as in excitation_wavelengths and is in increasing order of wavelengths.
  • excitation_input_powers: (array of floats) excitation power in Watts for each excitation source. This is the excitation power entering the optical system.
  • excitation_intensity: (array of floats) excitation intensity in the sample for each excitation source (units: Watts/meters²). In the case of confocal excitation this is the peak PSF intensity.
  • detection_wavelengths: (arrays of floats) reference wavelengths (in meters) for each detection spectral band. This array is ordered in increasing order of wavelengths. The first element refers to detectors_specs/spectral_ch1, the second to detectors_specs/spectral_ch2 and so on.
  • detection_polarizations: (arrays of floats) polarization angles for each detection polarization band. The first element refers to detectors_specs/polarization_ch1, the second to detectors_specs/polarization_ch2 and so on. This field is not relevant if no polarization selection is performed.
  • detection_split_ch_ratios: (array of floats) power fraction detected by each “beam-split” channel (i.e. independent detection channels obtained through a non-polarizing beam splitter). For 2 beam-split channels that receive the same power this array should be [0.5, 0.5]. The first element refers to detectors_specs/split_ch1, the second to detectors_specs/split_ch2 and so on. This field is not relevant when no polarization- and spectral-insensitive splitting is performed.

2.5. Sample group

The /sample group contains information related to the measured sample. This group is optional.

  • num_dyes: (integer) number of different dyes present in the samples.
  • dye_names: (string) comma-separated list of dye or fluorophore names (for example: "ATTO550, ATTO647N")
  • buffer_name: (string) a user defined description for the buffer.
  • sample_name: (string) a user defined description for the sample.

2.6. Identity group

The identity/ group contains information about the specific Photon-HDF5 file.

The following fields are mandatory (and automatically added by phconvert):

  • creation_time: (string) the Photon-HDF5 file creation time with the following format: “YYYY-MM-DD HH:MM:SS”.
  • software: (string) name of the software used to create the Photon-HDF5 file.
  • software_version: (string) version of the software used to create the Photon-HDF5 file.
  • format_name: (string) this must always be “Photon-HDF5”
  • format_version: (string) the Photon-HDF5 version string (e.g. “0.4”)
  • format_url: (string) A URL pointing to the Photon-HDF5 specification document.

The following fields are optional:

  • author: (string) the author of the measurement (or simulation).
  • author_affiliation: (string) the company or institution the author is affiliated with.
  • creator: (string) the Photon-HDF5 file creator. Used when the data was previously stored in another format and the conversion is performed by a different person than the author.
  • creator_affiliation: (string) the company or institution the creator is affiliated with.
  • url: (string) URL that allow to download the Photon-HDF5 data file.
  • doi: (string) Digital Object Identifier (DOI) for the Photon-HDF5 data file.
  • funding: (string) Description of funding sources and or grants that supported the data collection.
  • license: (string) License under which the data is released. Many journals and funding agencies require or suggest “CC0” (or equivalently “Public Domain”) for the data.
  • filename: (string) Photon-HDF5 file name at creation time. This field saves the original file name even if the file is later on renamed on disk.
  • filename_full: (string) Photon-HDF5 file name (including the full path) at creation time.

2.7. Provenance group

The provenance/ group contains info about the original file that has been converted into a Photon-HDF5 file. If the file is directly saved to Photon-HDF5 there is no previous “original” file and in this case the provenance group may be omitted. Also, if some information is not available the relative field may be omitted.

  • filename: (string) File name of the original data file before conversion to Photon-HDF5.
  • filename_full: (string) File name (with full path) of the original data file before conversion to Photon-HDF5.
  • creation_time: (string) Creation time of the original data file.
  • modification_time: (string) Time of last modification of the original data file.
  • software: (string) Software used to save the original data file.
  • software_version: (string) Version of the software used to save the original data file.

2.8. Additional notes and definitions

2.8.1. Detector pixel IDs

A detector pixel ID (or simply pixel ID) is the “name” of each pixels and is usually a single integer. Pixels are normally numbered incrementally, but not necessarily so. In other words, a file containing data taken with 2 single-point (pixel) detectors could have the first detector labeled “4” and the second detector labeled “6”. In some cases (when using detector arrays) the pixel ID can be a n-tuple of integers. This allow to specify, for each pixel, the module number and the X, Y location, for example. Therefore, an array of pixel IDs can be either a 1-D column array or a 2-D array. In either cases, each row identifies a pixel.

2.8.2. Beam-split channels

When the emitted light path is split in 2 or more detection paths by using a non-polarizing beam splitter the measurement has so called beam-split channels. The fields split_ch1 and split_ch2 contains the list of Detector pixel IDs for each beam-split channel (see Detectors specs).

Beam split channels can receive same or different (depending on whether the beam splitter is 50-50). The fractional power of each beam split channel can be saved in the field detection_split_ch_ratios in the Setup group.

2.8.3. Wavelengths and spectral band order

In Photon-HDF5, by convention, all the excitation wavelengths and detection spectral bands are ordered in increasing order: from the shortest to the longest wavelength. This ordering is strictly followed and removes any ambiguity in defining first, second, etc... wavelength or spectral band.

For examples, for μs-ALEX and ns-ALEX (or PIE) the excitation wavelengths (in /setup/excitation_wavelenths) are ordered as

  1. donor excitation wavelength,
  2. acceptor excitation wavelength

Similarly, the donor (or acceptor) excitation period range is defined by /photon_data/measurement_specs/alex_excitation_period1 (or /photon_data/measurement_specs/alex_excitation_period2).

Finally the donor (or acceptor) Detector pixel IDs number is defined in /photon_data/measurement_specs/detectors_specs/spectral_ch1 (or /photon_data/measurement_specs/detectors_specs/spectral_ch2).

2.8.4. Definition of alternation periods

2.8.4.1. Note for μs-ALEX

The fields alex_offset, alex_excitation_period1 and alex_excitation_period2 define the excitation period for each excitation source. The alternation histogram is the histogram of the following quantity:

A = (timestamps - alex_offset) MODULO alex_period

Note that alex_offset must be a value that shifts the timestamps in a way that the resulting alternation histogram has uninterrupted excitation periods for each excitation source. It can be thought as the delay between the start of the timestamping and the start of the alternation modulation. In most cases this is just an empirical parameter depending on the specific setup.

Photons emitted during the donor period (or, respectively, acceptor period) are obtained by applying the condition:

  • (A >= start) and (A < stop)
μs-ALEX alternation histogram with marked excitation ranges.

Alternation histogram showing selection for the donor and acceptor periods.

2.8.5. Measurement type

Each measurement_type has an associated set of mandatory fields which must be present to ensure that all information needed to unambiguously interpret the data is present. For example, for a 2-color smFRET measurement, a software package creating a file should check that the association between detector-pixel and donor or acceptor channel is present. If some necessary field is absent, the software package should warn the user in order that this information is added before saving the file.

2.8.6. Nanotimes time direction

In typical TCSPC measurement the start and stop inputs are inverted, i.e. the start is triggered by the photon and the stop by the the laser sync. This allows to start TAC or TDC measurements only when a photon is detected and not after each laser sync pulse. However, due to this experimental configuration, the resulting raw TCSPC histogram looks inverted along the time axis, with the nanotimes of photons emitted shortly after a laser pulse being larger than the nanotimes of photons emitted much later.

By convention, the Photon-HDF5 format requires nanotimes to be properly oriented. In other words, when a nanotimes time axis inversion is needed, this correction needs to be performed before the data is saved into a Photon-HDF5 file. As a corollary, TCSPC histograms computed directly from nanotimes from Photon-HDF5 files are always properly oriented, regardless of the way the nanotimes were acquired.

2.8.7. Multi-spot measurements

Multi-spot measurements are simply handled by having multiple photon_data groups, one for each excitation spot. The naming convention is the following:

photon_data0
photon_data1
...
photon_data10
...
photon_data100

Note that the enumeration starts from zero and there is no zero filling. Each photon_dataN group will have a complete measurement_specs sub-group so that it can effectively treated as a single-spot measurements when reading the file. As a result, even if the measurement_type field is not expected to change for different spots, it will be replicated inside each photon_dataN group.