DriftlessFLIP

A desktop application for time-domain fluorescence lifetime photometry with a PicoQuant TimeHARP 260 PICO DUAL. It acquires TTTR photon streams, turns them into per-sample decay curves, and derives intensity, mean photon emission time, and phasor coordinates — while keeping the raw photon records so any of those numbers can be recomputed later.

It runs without hardware. The GUI, CLI, and Python simulator default to a bundled, de-identified 15-minute excerpt of dLight3.8 from the recorded PFC channel, provided by Mohebi & Associates. Its real brightness and decay-shape dynamics are replayed through the normal T3 processing path at a compact, configurable photon rate. A fully synthetic exponential source remains selectable for engineering exercises (--source synthetic on the CLI, or SimulatorConfig(use_demo_data=False) in Python). See the real-data tutorial for source details and a walkthrough.

Ready for real experiments. The acquisition path has been validated with a physical TimeHARP 260 across multiple bench sessions and real recordings, including a sustained high-volume run. The simulator and automated suite cover the same processing and storage path. Before collecting irreplaceable data on a new computer, board, detector, or timing setup, verify that exact hardware profile and follow the deployment checks in DESIGN_PLAN.md section 7.

See it in action

Review a real recording

Explore a real dLight3.8 acquisition: intensity and MPET traces above, with the selected interval's decay and phasor below. The detector is labeled PFC in the recording metadata. The Simulator badge selects the next acquisition backend; the displayed recording was acquired on physical hardware.

Explore measured transients

This plot shows the measured 15-minute excerpt bundled with the simulator: brightness transients, raw MPET changes, and normalized decay curves from two intervals. Traces use one-second averages; raw MPET excludes background and afterpulse subtraction, so it differs from the corrected metrics in Review. During simulation, counts are scaled down while the measured brightness and decay evolution are retained.

Follow the real-data tutorial to try it, or consult the complete settings reference for individual controls. Click either image to view it at full size.

What it does

Acquisition

• Preview live signal without saving anything, to set discriminator levels and timing.
• Record for a fixed duration, or loop N repeats on a start-to-start interval.
• Start from software or from an external TTL trigger.
• Two detector channels, each with its own experiment-specific name such as DA in PFC.
• Four named TTL marker inputs, with automatic rising-to-falling pulse pairing.
• Free-text operator notes, timestamped from the moment you start typing.

Analysis

• Per-sample decay histograms, intensity, mean photon emission time, and first-harmonic phasor coordinates.
• Background subtraction from a separately recorded background, scaled for detector dead time so a busier detector is not over-corrected.
• Per-channel afterpulse correction across the complete decay curve.
• Single- and double-exponential fitting, convolved with a Gaussian instrument response and re-excited every laser period, with per-parameter fixed/floating control.
• Phasor plot with the universal semicircle and lifetime reference points.

Data you can trust later

• Bit-exact raw TTTR retained, so any derived quantity can be recomputed when an algorithm changes.
• Every recording writes raw HDF5, NWB, CSV, canonical metadata JSON, a run log, and a checksummed manifest, all cross-verified against each other.
• An interrupted recording can be recovered, reproducing the corrections the run was applying.
• Read-only import of supported legacy histogram recordings for comparison and analysis.

Installation for end users

Download DriftlessFLIP-<version>-Setup.exe from the corresponding GitHub Release, run it, and launch DriftlessFLIP from the Start Menu. The installer contains the application, Python runtime, and open-source dependencies; users do not need Conda, Python, or terminal commands. It installs per user and does not remove or overwrite recordings, profiles, or settings in Documents\DriftlessFLIP during upgrades or uninstall.

PicoQuant's driver and licensed TH260Lib64.dll remain separate installations and are never bundled. For hardware acquisition, obtain TH260Lib from PicoQuant or install PicoQuant's snAPI, which supplies the DLL, and keep the PicoQuant device driver installed. If automatic discovery does not find the DLL, select its installed location in DriftlessFLIP's TH260 DLL field. Simulator mode works without either component. Installer builds are currently unsigned, so Windows may show a publisher warning until code signing is added.

Installation for developers

Use 64-bit Python 3.11 or newer in a dedicated Conda environment. Clone this repository, open PowerShell in the clone, and run:

conda create -n driftlessflip python=3.12
conda activate driftlessflip
python -m pip install --upgrade pip
python -m pip install -e ".[dev]"

This editable installation uses the files in the clone, so code changes take effect without reinstalling. Run the tests with python -m pytest -q.

For a non-editable installation of the latest published Python package, use:

python -m pip install --upgrade driftlessflip

Developers must obtain the PicoQuant driver and licensed TH260Lib64.dll separately too; pip and Conda cannot install them. Install TH260Lib from PicoQuant or use the DLL supplied with snAPI. Keep it at the default path or select its actual location in the GUI's TH260 DLL field. Never commit the vendor DLL to this repository. Simulator mode works without the driver or DLL.

To create the application executable and Setup installer locally or through GitHub Actions, follow Building the Windows executable and installer. The short local build is:

python -m pip install -e ".[dev,packaging]"
.\scripts\build_windows_installer.ps1

The result is installer-dist\DriftlessFLIP-<version>-Setup.exe. The automation attaches that executable and its SHA-256 checksum to every published GitHub Release. Release tags must exactly match the application version as v<version>.

Getting started

Launch the interface:

driftlessflip gui

The normal workflow

1. Preview. Press Preview and watch the count rates, live traces, and decay curve. Nothing is saved. Adjust discriminator thresholds, channel offsets, and t0, then preview again — each preview re-applies the current settings. Recording stays disabled until at least ten completed samples pass the corrected-signal check.
2. Calibrate t0 against the live decay. In Hardware & calibration, press Fit decay curve and then Apply fitted t0. t0 is live-adjustable, so the running preview picks it up at once and MPET updates with it — no throwaway recording needed. Settings that reprogram the board, such as channel timing offsets, still need the preview stopped and restarted, because TH260Lib forbids changing them mid-measurement.
3. Check the rate. The count-rate readout is coloured against the pile-up and detector dead-time limits for your sync rate. If it reads over, reduce excitation power — never raise the discriminator threshold to bring the rate down. A higher threshold discards valid photon events rather than fixing pile-up, and costs signal-to-noise for nothing in return; lower power also reduces photobleaching and photodamage to the sample. A detected rate of roughly 2×10⁶–1.5×10⁷ counts/s is a reasonable operating range for this hardware; at the low end, aim for the signal to sit at least 10× above whatever the detector reads with no sample connected.
4. Record a background. Same settings, no signal, ten seconds is usually enough. Tick This run is a background measurement.
5. Validate, then record. Load the background as the reference and Preview again. Preview applies that background and warns if subtraction leaves too little positive signal. Stop a passing Preview to enable Start recording.
6. Review. Open the recording, integrate the decay over a time range, fit it, and read the phasor position.

From the terminal

driftlessflip preview --duration 10
driftlessflip simulate --output recordings --prefix bg --duration 10 --background
driftlessflip simulate --output recordings --prefix experiment --background-reference recordings\bg_<timestamp>
driftlessflip device-info
driftlessflip verify recordings\experiment_<timestamp>
driftlessflip import-legacy path\to\legacy-recording
driftlessflip recover recordings\experiment_<timestamp>

Key concepts

Preview is not a recording

Preview and recording share the device, so only one runs at a time. Preview exists for setup: it shows count rates, traces, and the summed decay curve you set CFD levels and t0 from, and it is bounded by a rolling window rather than the recording duration, so it can run indefinitely at fixed memory.

Preview cannot produce a recording: it cannot create a run folder, raw file, or manifest. It can read and verify the selected background. The Display selector on the Hardware & calibration tab switches every live graph between raw and background-subtracted values. In background-subtracted mode, the lifetime panel overlays the raw curve as a dashed line. While previewing, the control reads Previewing — not saving.

The interface requires a passing Preview for the current acquisition and correction settings. It evaluates completed samples only and requires at least 95% of corrected photon totals in each enabled channel to remain positive. Isolated negative lifetime bins are reported but do not fail an otherwise usable total; subtraction noise can make individual bins negative. Loading or clearing a background, or changing a setting that affects acquisition or correction, invalidates the result and requires another Preview. For legibility only, the background-subtracted live display clips those negative bins to zero and shows a warning with the number clipped. This never modifies the raw histogram, the signed corrected analysis, or any saved artifact.

What a recording contains

Each run creates one immutable folder named for its prefix and start time:

experiment_20260802T193052.123456Z/
  experiment_20260802T193052.123456Z.tttr.h5        raw TTTR records and applied corrections
  experiment_20260802T193052.123456Z.nwb            histograms and derived traces
  experiment_20260802T193052.123456Z_metrics.csv    per-sample, per-channel values
  experiment_20260802T193052.123456Z_metadata.json  canonical RunMetadata
  experiment_20260802T193052.123456Z_manifest.json  sizes and SHA-256 of every artifact
  experiment_20260802T193052.123456Z_run.log        structured event log

The raw TTTR stream and raw unsigned NWB histograms are authoritative. A selected background remains a separate verified recording; the experiment stores its identity, checksum, mean curve, and the correction parameters needed to reproduce subtraction. It does not overwrite raw photon counts with corrected or zero-clipped values. One versioned RunMetadata object is embedded in every artifact and cross-checked between them, so a package that verifies is internally consistent. driftlessflip verify re-checks that at any time.

Settings files and profiles

Two ways to keep a configuration, for two different jobs.

A settings profile is a named instrument setup kept between sessions in your home directory. It deliberately leaves out the fields that belong to one particular run — the recording prefix, the background reference, and whether the run is a background — so loading a routine setup cannot drag last week's background along with it.

A settings file is everything, written wherever you choose: hardware, analysis, experiment metadata, the background reference, output location and prefix included. Use Export all… and Import all… on the Acquire tab. It is indented, key-sorted JSON that you can read, hand-edit, diff between sessions, keep alongside a protocol, or send to a collaborator so they can reproduce your setup exactly.

Importing never repairs a file quietly. A file that is not a settings file, or that has a key the application does not know, is refused with the offending detail named. A file that loads but asks for something unavailable here — a background recording that has moved, a value outside its allowed range — is applied and the problem reported, so you can see and fix it rather than meeting it at record time.

The settings file is a setup document, not a provenance record: what a recording actually used is written inside that recording.

Where settings come from at startup

Everything DriftlessFLIP keeps for you lives in one visible folder:

Documents/DriftlessFLIP/
  default_settings.json     loaded at every start
  profiles/                 named instrument setups
  recordings/               where recordings go unless you point them elsewhere

It is deliberately not a hidden dotfolder: these files exist to be read, hand-edited, copied to a colleague, and handed over with a rig. Settings kept under ~/.driftlessflip by an earlier version are moved here automatically on first start, without overwriting anything already present.

On Windows the folder is found through the location Explorer itself uses, so a Documents folder redirected to OneDrive — the default on a new machine — is followed rather than guessed at. Because that can make for a deep path, and because a recording's prefix and timestamp appear both in its folder name and in every file inside it, DriftlessFLIP warns before arming if a run's longest path would exceed the 260-character Windows limit. That failure would otherwise land at finalisation, after the data had been collected.

DriftlessFLIP loads default_settings.json when it exists, so the application opens on your rig rather than on factory defaults. Press Set as default to write the current settings there. Having no such file is the ordinary first-run state. If the file exists but cannot be read, the application still starts, on built-in defaults, and says so in the status line and the Diagnostics tab — it will not fail silently and leave you believing your own settings are in force.

Three ways to load a setup, in the order you are likely to want them:

1. The default file, applied automatically at startup.
2. Import all…, for a settings file someone sent you or that you keep with a protocol.
3. From recording…, which rebuilds a setup from a past recording's own metadata.

The third has two forms. Next to Settings profile it loads a past recording's instrument settings into the current session while keeping your output location and recording name. Next to Settings file it writes that recording's configuration out as a settings file, prefix and background reference included, so a session can be turned into a reusable setup. The same thing without the interface:

driftlessflip export-settings recordings\experiment_<timestamp> --output my_rig.settings.json

Settings are read from the recording's canonical metadata JSON. The NWB file carries an identical copy of the same block, and both are checksummed in the manifest.

Background subtraction

Record a background with matching settings and no signal, then load it as the reference for an experiment. It is refused unless it matches on sample rate, lifetime binning, laser rate, sync configuration, and every per-channel discriminator and offset; the error names the field that differs. Duration may differ, since the correction uses the mean background per sample.

The measured background is scaled by the ratio of detector live fractions before subtraction, because a detector busy with signal photons collects less background than it did when measured alone. The background's identity and the correction actually applied are stored with the recording, so it stays reproducible even if the background file is later moved.

The live Raw / Background-subtracted display choice does not change acquisition or storage. Background-subtracted mode calculates a temporary display copy, clips negative bins to zero so the decay, intensity, MPET, and phasor remain visible, and warns whenever clipping occurs. The lifetime panel shows the clipped corrected curve in color and the raw curve dashed behind it. Raw mode shows only raw values. Signed corrected values—not the clipped display copy—remain the scientific analysis and quality-control source.

Which settings can change while acquiring

t0, the MPET window, and the afterpulse ratio are applied when metrics are recalculated, so they can be retuned during a preview with immediate effect. Everything else is locked while a measurement runs: TH260Lib forbids reprogramming the board mid-measurement, and the sample rate, lifetime binning, and output location fix buffer shapes and identity at start.

During a recording everything is locked, including those three, because metadata is captured when the recording starts and a later edit would let the stored configuration disagree with the derived outputs. Raw TTTR retention means any of them can be reapplied offline. For the reasoning behind exactly which settings fall on which side of that line — and how the raw T3 stream makes it possible — see SOFTWARE_REFERENCE.md section 3.

External TTL markers

The PICO DUAL marker inputs are Marker 1/pin 9, Marker 2/pin 4, Marker 3/pin 5, and Marker 4/pin 10. The acquisition trigger line is pin 13 and measurement status is output on pin 3, with ground on pin 12 or 14.

TH260_SetMarkerEdges selects one active edge per input, so a single input cannot capture both polarities. To measure pulse width, split the source to two inputs, set one rising and one falling, and give both the same Pulse signal name. Each rising edge is then matched to the next falling edge automatically.

The exact event table retains macro-sync count, recording-relative time, derived UTC timestamp, edge, input name and pin, source bitmask, and sample index. The per-sample external_marker_bits trace is only a summary — use the event table when timing or event multiplicity matters.

Interpreting the numbers

MPET is not a fitted lifetime. It is the photon-weighted mean arrival time within the configured analysis window, minus t0. Truncation by the laser period and the window shifts it below the exponential time constant, so the interface labels it mean photon emission time rather than lifetime. Use the fit when you want tau.

Microtime is nominal dtime * resolution, the bin's left edge, matching the TimeHARP convention. Bin centres exist only as a display axis. This means a calibrated t0 has to absorb the offset between a bin's left edge and the mean arrival time within it, which is why t0 is determined by fitting rather than assumed.

Phasor spans one full laser period, independent of the MPET window. A cropped window rotates and shortens the vector and moves even a pure single-exponential decay off the universal semicircle.

Corrections happen before windowing. Background and afterpulsing are removed across the complete decay curve, so changing the MPET window cannot change how much was subtracted.

Every one of these choices is recorded in the calculation_contract and algorithm_versions fields of each recording, so a file states the conventions it was computed under.

Current limitations

• Physical TimeHARP operation is validated and ready for experiments. A new acquisition computer, board, detector, or timing configuration still needs its own profile check; the supplied bench procedures make that verification reproducible.
• The board can enter an unresponsive state that neither DriftlessFLIP nor the vendor's own software can currently distinguish from "not connected." No software fix is known. If device-info or a board scan fails immediately after working moments earlier: close every process that might hold the device, and if it still won't open, power-cycle the computer (unplugging power for ~15 s before restart has been reported to help; this is a board/driver-level condition, not something DriftlessFLIP causes or can detect from software alone).
• Histogram memory is bounded before arming but allocated for the whole requested run, so a memory budget caps run length rather than removing the limit.
• The software FIFO handoff queue defaults to 256 chunks (at most 128 MiB) and is configurable from Acquire. A sustained full queue or a hardware FIFO-full flag stops acquisition and preserves a verified truncated prefix. Increasing the queue absorbs temporary storage/CPU stalls; it cannot reconstruct records the board already lost or compensate indefinitely when processing is slower than acquisition.
• NWB uses valid core types plus lossless canonical metadata; formal fibre-photometry extension mappings are not done.
• Unsupported legacy formats are refused rather than guessed.
• The default demo replays real dLight3.8 histogram dynamics, rescaled to the chosen median photon rate. It is demonstration data, not a biological reference or calibration. The optional synthetic source models a Gaussian instrument response, periodic re-excitation, afterpulsing, dead time, and structured background, but not a measured per-instrument IRF shape or timing jitter.

ROADMAP.md lists what remains and why.

Development

pytest

The suite covers T3 decoding against a hand-calculated golden stream, correction arithmetic, phasor geometry, fit parameter recovery, storage verification, recovery, overrun salvage, looping, and cross-validation against independent golden fixtures. GitHub Actions runs it on Python 3.11 and 3.12, on Ubuntu and Windows.

Documents

Development, inspiration, and license

DriftlessFLIP is developed by Mohebi and Associates.

The software was inspired by iFLIP2 from the Hong Lab.

Released under the MIT License. PicoQuant drivers and TH260Lib64.dll are separately licensed third-party components and are not distributed under it. Third-party manuals and journal articles are cited by link in DESIGN_PLAN.md, are not stored in this repository, and are outside the scope of the MIT license.