GUI overview

The theatRICS graphical interface is organized into several analysis tabs. Each tab is focused on a specific workflow and shares a common logging, progress, and export system.

General interface features

Across the application, the GUI provides:

Tabbed workflows for different analysis tasks
A status bar showing the current task state
A progress bar for long-running computations
A Results & Logs tab for textual output and session tracking
A Cancel Running Task button for stopping active processing jobs
A Restart button for restarting the application
A Toggle Dark Mode button for changing the visual theme

Long-running calculations are executed in background worker processes so that the interface can remain responsive.

Image Simulation

The Image Simulation tab generates synthetic image stacks for testing and benchmarking RICS-style analyses.

Main inputs

Image shape (pixels)
width and height of the simulated frames
Number of cores
number of CPU workers used for simulation
Number of frames
Pixel dwell time (µs)
Pixel size (nm)
Brightness (kHz)
Number of particles
Diffusion coefficients in X and Y
Rotation
Background
PSF sigma (pixels)
Simulation type
one of:
- isotropic
- anisotropic
- anisotropic_rotated
Output path

Actions

Run Simulation
Load Existing

Display

The simulation display shows:

first frame
last frame
time-averaged image
average intensity trace over time from a centered crop

Typical use

This tab is useful for:

validating fitting procedures
testing anisotropic diffusion scenarios
generating known-reference data

RICS Export

The RICS Export tab computes correlation maps and uncertainty maps from image stacks.

Main inputs

Input file
a single image stack
Input folder
used for batch analysis
Channel to use
Crop factor
Window size (odd)
Correct drift

Actions

Export RICS
Load RICS

Display

The RICS export display can show:

raw image preview
drift-corrected image preview
RICS correlation map
uncertainty map
3D surface view of the RICS map

Outputs

Typical outputs include:

correlation TIFF
uncertainty TIFF
optionally corrected intermediate data

Typical use

This is the first analysis step when starting from microscopy image stacks.

RICS Fitting

The RICS Fitting tab fits diffusion models to exported RICS maps.

Main inputs

RICS map file
Input folder for batch fitting
Results file
Pixel size (nm)
Pixel dwell (µs)
Line time (ms)
PSF size XY (µm)
PSF aspect ratio
Crop factor fast
Crop factor slow
Diffusion model
Channel
Optional 1D Fast Axis Fit

Diffusion map subsection

The same tab also contains a Diffusion Map Fitting Parameters section with:

Input file for diffusion map
Window size
Offset
Channel

Actions

Run 2D/3D Fitting
Generate Diffusion Map

Display

The fitting display can show:

3D RICS data surface
fitted model surface
residual surface
fast-axis cross section
slow-axis cross section
optional 1D fit residuals
optional diffusion map / brightness map / number map

Outputs

Typical outputs include:

fit summary CSV
NPZ fit arrays
exported SVG plots

Typical use

This tab is used after RICS export to estimate diffusion coefficients and model quality.

SFCS

The SFCS tab performs perpendicular scanning FCS analysis.

Main inputs

Input file
Channel
Bleach correction
Number of cores

Actions

Correlate

Display

The SFCS display can show:

original frame crop
aligned frame crop
intensity trace
bleach-corrected intensity trace
autocorrelation curve G(τ)
uncertainty band, if available

Outputs

Typical outputs include:

correlation curves
exported SVG figures
logged summary values

Typical use

Use this tab when analyzing perpendicular scanning FCS line scans rather than image-based RICS data.

FCS Export

See Workflow: FCS Export.

The FCS Export tab supports:

PicoQuant PTU files (via tttrlib) in standard single-channel mode, PIE mode, or plain two-channel cross-correlation mode
Zeiss ConfoCor3 / LSM980 .raw files (custom binary format)

Correlation pair selection (DD / AA / DA)

For two-channel measurements the user can independently enable or disable:

DD — channel 1 / donor autocorrelation
AA — channel 2 / acceptor autocorrelation
DA — channel 1 × channel 2 cross-correlation

Single-channel files always compute their own autocorrelation regardless of these checkboxes.

Corrections available per file type

Correction	PTU	Zeiss .raw
Wohland SD uncertainty	✓	✓
Afterpulsing subtraction	✓	✓
Bleach/drift correction	✓	✓
Burst removal	✓	✗
FLCS background correction	✓	✗
PIE mode	✓	✗

PIE mode, FLCS background correction, and burst removal are automatically disabled in the GUI when a .raw file is selected, since these require TCSPC micro-time information not present in Zeiss .raw files.

FCS Fitting

The FCS Fitting tab fits correlation curves exported as CSV files using a selection of FCS diffusion and blinking models.

Supported modes

Single-file fitting
Recursive batch fitting over all matching files in subfolders of a selected folder

Main inputs

Single CSV
Batch folder
Batch file pattern
for example:
- *_xy_intensity_trace_correlation.csv
Model
Tau min (s)
Tau max (s)
PSF radius (µm)
PSF aspect ratio
Experiment T (°C)

Calibration-only inputs

For calibration models (model names containing Cal), the GUI additionally shows:

Given D (µm²/s)
Given D temp (°C)

These fields are hidden for non-calibration models.

Model-dependent initial parameters

The tab contains a dynamic Initial parameters editor:

only parameters relevant to the selected model are shown
default values are taken from the legacy fitting code
user-modified values are preserved while switching models

Examples of model-dependent parameters include:

N
tau diffusion
delta
F_Blink
offset
f1
rho_D
rho_B
Gamma
Alpha
G0
tau characteristic decay
MEMFCS parameters

Actions

Run Fit

Display

The FCS fit display shows:

observed correlation and fitted model
weighted residuals
iMSD or a log-log correlation representation depending on model
histogram of residual values

Outputs

Per file, the GUI exports:

SVG figure
fitted correlation CSV
iMSD CSV where applicable

For batch mode:

a single summary CSV is written in the outer selected folder

Typical use

This tab is intended for fitting precomputed correlation curves rather than directly processing images.

FRAP

The FRAP tab analyzes fluorescence recovery after photobleaching from CZI image files containing circular ROI annotations.

Supported modes

Single-file analysis
Batch analysis over matching FRAP files in subfolders

Main inputs

Single CZI
Batch folder
Pattern
for example:
- *FRAP*.czi
Imaging bleach correction
Fallback pixel size (µm)
Initial D (px²/frame)
D lower bound
D upper bound

Automatic processing steps

The FRAP workflow automatically:

reads image frames from the CZI file
extracts frame interval from metadata
extracts pixel size from metadata when available
reads circular ROI annotations
detects the bleach frame
identifies the control ROI
normalizes FRAP traces using the control ROI
fits each FRAP ROI using the selected FRAP model

Display

The FRAP display shows:

raw intensity traces before normalisation
control-normalised traces with fitted recovery curves
bleach-corrected fit representation
residual traces

Outputs

For each file, the software writes:

*_FRAP_raw_data.xlsx
*_FRAP_summary.xlsx
*_FRAP_overview.svg

Typical use

This tab is used for ROI-based FRAP analysis from annotated CZI time series.

ICS

The ICS tab performs temporal Image Correlation Spectroscopy on TIFF image stacks, computing block-wise normalised correlation values to track changes in molecular density or aggregation state over time.

Main inputs

Single TIFF: a single TIFF image stack
Batch folder: a parent folder containing sample subfolders
File pattern: filename pattern for batch mode, for example *.tiff
Block length (frames): number of frames per temporal block
Frame skip (lag τ): temporal lag between correlated frames
Bin frames: optional frame binning before block analysis
Threshold multiplier: controls which pixels are included via MIP-based masking
Save block images: optionally save per-block diagnostic TIFFs

Actions

Run ICS

Display

The ICS display shows a 2×2 overview figure:

mean G ± SD vs block number
normalised G vs block number
mean intensity image of the last block
G map of the last block

Typical use

Use this tab to:

track changes in molecular clustering or aggregation over time
compare normalised correlation across experimental conditions
batch-process multiple samples and export a combined summary

AFM

The AFM tab loads JPK QI atomic force microscopy height images and performs interactive line-profile analysis for measuring condensate heights and wetting angles on membrane surfaces.

Main inputs

JPK file: a .jpk-qi-image or .jpk file
Channel: height channel to load (height_trace, height_retrace, adhesion_force_trace, stiffness_trace)

Profile parameters

Fit points: number of data points used to fit the wetting angle slope, adjusted with a real-time slider (3–60 points)
Smooth window: Savitzky-Golay smoothing window length
Smooth poly order: Savitzky-Golay polynomial order
Profile points: number of interpolated sample points along the line
Threshold fraction: fraction of peak height used to define contact points

Actions

Load File: loads and displays the height image
Reset last: removes the most recently drawn profile
Clear all: removes all profiles
Save results: exports all profile arrays, summary CSV, and SVG figure

Interaction

After loading a file, draw a line profile by clicking two points on the AFM image:

Click the start point on the bare membrane on one side of the object
Click the end point on the bare membrane on the other side

The line must cross the object. The two clicked heights define the linear baseline.

Display

The AFM display contains two panels:

Left: height image with false-colour map, drawn profile lines, and click markers
Right: line profile showing raw data, baseline, corrected profile, contact point markers, tangent lines, and wetting angle title

Profiles listbox

Completed profiles are listed with:

peak height in nm
FWHM in nm
mean wetting angle in degrees

Click a profile to display it in the right panel.

Typical use

Use this tab to measure:

condensate height above a supported lipid bilayer
wetting angles at the condensate-membrane contact line
FWHM and contact width of surface-adsorbed objects

Vesicle Finder

The Vesicle Finder tab detects vesicles (such as GUVs) in CZI images or time series, allows interactive selection, and exports either square crops or unrolled membrane strips.

Supported modes

Single-frame detection: detect vesicles in frame 0 and display interactively
Batch cropping: export selected vesicles across all selected frames
Membrane straightening: unroll the membrane ring into a flat strip for intensity analysis

Detection methods

hough: Hough circle transform, best for fluorescence membrane images
hough_transmitted: gradient-based Hough, best for transmitted-light images
weighted_intensity: peripheral intensity maximization (improved and modified from Kohyama et al. 2022), robust for both image types
cellpose: deep learning segmentation with optional shape filtering and circle fitting
otsu: simple threshold-based fallback

All spatial parameters in µm

Pixel size is read automatically from CZI metadata. All radius, distance, and margin parameters are entered in µm and converted to pixels internally.

Display

Left panel: raw image with detected vesicle overlays (circles or contours)
Right panel: segmentation label map

Membrane straightening

For each detected vesicle with a known radius, the software can unroll the annular membrane region into a flat strip. The output shows:

the straightened membrane image (frame 0)
a heatmap of intensity vs position along membrane vs time
total membrane intensity vs time

Debug images

Enabling Save debug images writes intermediate processing images to a debug folder alongside the CZI file. This is useful for diagnosing detection failures.

Outputs

Square crop TIFFs per selected vesicle (all frames)
Straightened membrane TIFFs per vesicle
Intensity profile CSVs per vesicle
Total intensity CSV per vesicle
Overview SVG figure

Results & Logs

The Results & Logs tab collects textual output from all workflows.

Features

scrolling log view
Clear Log
Save Results
Export All Plots
Save Session
Load Session

Typical use

Use this tab to:

review processing history
inspect warnings and failures
save text output
store and restore GUI parameter states

Notes on responsiveness and background processing

Most heavy computations in the GUI are executed in separate worker processes. This design allows:

progress tracking
cancellation support
batch processing
reduced UI blocking during long-running fits or exports

However, very frequent figure redraws or per-file updates in large batch jobs can still reduce responsiveness. For large batches, the software may update only logs and progress during processing and refresh the full display at the end.

Typical analysis routes

Simulate → export → fit

Generate synthetic data in Image Simulation
Compute correlation maps in RICS Export
Estimate diffusion parameters in RICS Fitting

Image-based experiment

Export RICS from microscopy data
Fit RICS maps
Generate diffusion maps if required

Scan-correlation experiment

Correlate line-scan data in SFCS
Optionally fit exported FCS-style curves in FCS Fitting

Recovery experiment

Analyze photobleaching time series in FRAP
Inspect mobile fraction, half-time, and diffusion outputs