Workflow: FRAP analysis

This tab performs FRAP (Fluorescence Recovery After Photobleaching) analysis on CZI image files containing circular ROI annotations.

The implementation uses a 2D diffusion model with an infinite reservoir based on the Soumpasis recovery formulation, with optional correction for imaging bleach.

Overview

The FRAP workflow supports:

  • Single-file analysis of one *.czi file

  • Batch analysis of a folder containing multiple FRAP CZI files

  • Automatic extraction of:

    • frame interval

    • pixel size (if available from metadata)

    • ROI geometry

  • Automatic identification of:

    • bleach frame

    • control ROI

    • FRAP ROIs

  • Embedded display of:

    • raw ROI intensities

    • fitted recovery curves

    • bleach-corrected fit

    • residuals

  • Export of:

    • raw data Excel file

    • summary Excel file

    • SVG overview figure

Input requirements

The FRAP tab expects a CZI file containing:

  • time-resolved image data

  • circular ROI annotations in metadata

  • at least 2 ROIs

    • one ROI is treated as the control

    • the remaining ROIs are treated as FRAP ROIs

If fewer than 2 valid ROIs are found, the analysis fails.

Single-file analysis

  1. Open the FRAP tab.

  2. Under Single CZI, click Browse and select a CZI file.

  3. Set the analysis parameters:

    • Pattern (used only in batch mode)

    • Imaging bleach correction

    • Fallback pixel size (µm), optional

    • Initial D (px²/frame)

    • D lower bound

    • D upper bound

  4. Click Run FRAP.

The analysis result is displayed inside the GUI and exported next to the input file.

Batch analysis

  1. Under Batch folder, click Browse and select a root folder.

  2. Set the filename pattern, typically:

    *FRAP*.czi

  3. Set analysis parameters as above.

  4. Click Run FRAP.

The software recursively searches the selected folder for matching CZI files and analyzes each one.

During batch analysis:

  • the progress bar is updated

  • the embedded display can update to the most recently finished file

  • all results are written next to the individual files

Model

The implemented FRAP model is based on Soumpasis (1983) for circular bleach regions.

Recovery term

The recovery function uses:

  • a circular bleach radius R

  • a diffusion coefficient D

  • the Soumpasis expression involving modified Bessel functions

With imaging bleach correction

If Imaging bleach correction is enabled, the model includes an additional exponential decay term for acquisition-related bleaching.

This version fits:

  • x_0 — bleach frame position

  • R — ROI radius

  • F_0 — pre-bleach signal

  • f_bl — bleached fraction

  • f_mob — mobile fraction

  • D — diffusion coefficient

  • t_b — imaging bleach decay constant

Without imaging bleach correction

If imaging bleach correction is disabled, the exponential decay term is omitted.

This version fits:

  • x_0

  • R

  • F_0

  • f_bl

  • f_mob

  • D

Control normalisation

The control ROI is used to correct for global intensity loss unrelated to recovery.

For each frame:

  1. The control trace is normalised by its pre-bleach mean.

  2. Each FRAP trace is divided by the normalised control trace.

  3. This corrects for illumination drift and global photobleaching.

This control-normalised trace is then used for fitting.

Automatic detection steps

Bleach frame

The bleach frame is detected automatically from the largest intensity drop across all ROI traces.

Control ROI

The ROI with the smallest bleach drop is chosen as the control ROI.

FRAP ROIs

All remaining ROIs are treated as FRAP ROIs.

Display panels

The embedded Matplotlib display contains four panels.

1. Raw intensity (pre-normalisation)

Shows:

  • control ROI trace

  • FRAP ROI traces

  • bleach time marker

2. Normalised fit

Shows:

  • control-normalised FRAP traces

  • fitted recovery curves

  • optional imaging bleach baseline term

3. Bleach-corrected fit

Shows:

  • bleach-corrected recovery

  • fitted mobile fraction curve

  • estimated half-time markers

4. Fit residuals

Shows:

  • residual trends after bleaching

  • a smoothed residual trace per FRAP ROI

Parameters in the GUI

Pattern

Filename pattern used in batch mode, e.g.

*FRAP*.czi

Imaging bleach correction

Enable or disable the imaging-bleach model term.

Recommended: enabled for most acquisitions.

Fallback pixel size (µm)

Used only if the CZI metadata does not contain pixel size information.

If left blank and metadata is missing:

  • diffusion is still fitted in px²/frame

  • diffusion in µm²/s cannot be reported

Initial D (px²/frame)

Initial guess for diffusion coefficient.

D lower bound / D upper bound

Bounds for the fitted diffusion coefficient in px²/frame.

Output files

For an input file such as:

sample1_FRAP.czi

the software writes:

Per file

  • sample1_FRAP_raw_data.xlsx

  • sample1_FRAP_summary.xlsx

  • sample1_FRAP_overview.svg

Description

*_FRAP_raw_data.xlsx

Contains:

  • frame index

  • time

  • raw ROI intensities

  • control-normalised traces

  • fitted model traces for FRAP ROIs

*_FRAP_summary.xlsx

Contains:

  • ROI geometry

  • fitted parameters

  • mobile fraction

  • immobile fraction

  • diffusion coefficient in px²/frame

  • diffusion coefficient in µm²/s (if pixel size known)

  • recovery time constant

  • half-time

  • optional imaging bleach time constant

  • warnings

*_FRAP_overview.svg

Vector graphic export of the embedded FRAP display figure.

Batch summary

The FRAP analysis code also supports condition-level aggregation from summary Excel files.

This is based on:

  • condition folders

  • replicate subfolders

  • *_FRAP_summary.xlsx files inside replicates

From these, the analysis can compute:

  • mean half-time

  • mean mobile fraction

  • mean diffusion coefficient

  • outlier filtering using MAD-based robust z-scores

If implemented in the GUI workflow, this can be extended to generate:

  • FRAP_condition_summary.png or a GUI-based figure export.

Fit outputs and interpretation

Mobile fraction

f_mob is the estimated fraction of the bleached population that is mobile and able to recover.

Immobile fraction

Computed as:

1 - f_mob

Diffusion coefficient

Reported as:

  • D (px²/frame)

  • D (µm²/s) if pixel size is available

Half-time

Recovery half-time:

t½ = τ · ln(2)

where τ is estimated from the fitted diffusion parameters.

Warnings

The fitter may mark results as potentially unreliable, for example if:

  • D is at the lower fit bound

  • the estimated half-time is much longer than the acquisition duration

These warnings are written to the summary Excel file.

Troubleshooting

No ROIs found

The CZI file must contain circular ROI annotations in metadata.

Less than 2 ROIs

At least one control ROI and one FRAP ROI are required.

Pixel size missing

If pixel size cannot be read from metadata and no fallback is provided:

  • fitting still runs

  • D (µm²/s) is reported as unavailable

Poor fit quality

Try:

  • enabling imaging bleach correction

  • adjusting the initial diffusion guess

  • widening or tightening D bounds

  • checking whether the bleach ROI and control ROI are correctly annotated

Batch folder finds no files

Check the filename pattern, for example:

*FRAP*.czi

and ensure the selected folder contains matching files in subdirectories if recursive search is expected.