FLIMKit

Input Files

Analysis: Single FOV Z-stack

Data file *

Z-stack folder *

A folder of region_zX.ptu slices, fitted as one FOV

LAS X export

Instrument Response Function (IRF)

IRF / machine IRF path

Uses the LAS X export entered in Input Files above

Fitting Parameters

Fit model: n-exp Gaussian dist. Lorentzian dist. n-exp tail

Components: 1 2 3

Components: 1 (unimodal) 2 (bimodal)

Fitting mode: Full Fast(fast = no FLIM image)

Fit window (ns) to

Output prefix

Tail fit: no IRF used, fitted past the decay peak

Masking & Thresholding

Apply cell mask (Cellpose-SAM)

Intensity threshold (min photons/px):

Apply Coates pile-up correction (recommended if pile-up > 5%)

Time-varying bg PTU

Input Files

XLIF metadata *

PTU tile directory *

Base output dir *

A sub-folder named after the ROI will be created inside

Rotate tiles 90° CW (recommended for FLIM microscope)

Pipeline

Stitch tiles only Stitch then fit full ROI Per-tile fit [recommended - fits each tile independently] Multidimensional series [z-stack and/or timelapse tiles]

Instrument Response Function (IRF)

IRF / machine IRF path

Uses an LAS X export (browse to one above)

Fitting Parameters

Fit model: n-exp Gaussian dist. Lorentzian dist. n-exp tail

Components: 1 2 3

Components: 1 (unimodal) 2 (bimodal)

Per-pixel fitting [REQUIRED FOR ROI ANALYSIS]

Export τ-weighted map Export intensity-weighted map

Export amplitude-weighted map Save individual component maps

Lifetime display (ns) to (blank = auto)

Intensity display to (blank = auto)

Fit window (ns) to

Tail fit: no IRF used, fitted past the decay peak

Masking & Thresholding

Intensity threshold (min photons/px):

Apply Coates pile-up correction

Time-varying bg PTU

Tile Registration

Phase-correlation registration (fixes stage Y/X drift)

Max shift (px)(increase if drift > 120px)

Per-Tile IRF Directory (optional)

IRF XLSX dir

One <tile_name>.xlsx per tile; leave blank to use the IRF method above

Multidimensional Series

Pool decay every N timepoints(1 = every timepoint)

One consensus lifetime is fitted across the whole series, so amplitudes stay comparable between timepoints.

Batch Processing

Multi-Tile ROI Fit Single FOV Fit Timelapse Fit

Input / Output

XLIF folder *

PTU folder *

Output base dir *

Save beside input folder

IRF

Machine IRF (.npy) *

Fitting Parameters

Fit model: n-exp Gaussian dist. Lorentzian dist. n-exp tail

Components: 1 2 3

Components: 1 (unimodal) 2 (bimodal)

Fit window (ns) to

Tile Registration

Phase-correlation registration

Max shift (px)

Masking

Intensity threshold (min photons/px):

Apply Coates pile-up correction

Time-varying bg PTU

Image Export

Lifetime image (uint16 TIFF) Component RGB TIFF Intensity TIFF

Raw maps (.npy) Individual component maps

4D stacks - (T, H, W) .npy per map (timelapse only)

Lifetime colour scale (ns) to (blank = auto)

Gamma(0.4 = boost dim tissue; 1.0 = linear)

Intensity display max(blank = auto 99th percentile)

Lifetime weighting

Timelapse - Reference Lifetimes

Fix reference τ values (skip pooled-decay fit)

τ₁ τ₂ τ₃

Pool all positions for a shared reference τ

Compute bound fraction α₂/(α₁+α₂)

Source Data

PTU/XLSX folder *

Builder uses matching <name>.ptu + <name>.xlsx pairs.

Build Settings

Align anchor

Reducer

Output

Output directory *

Base filename

Input Mode

New PTU file Resume session (.npz)

PTU file *

IRF XLSX

Machine IRF

XLSX takes priority if both supplied

ChannelFrequency (MHz)

Channel and frequency are only needed if FLIMKit asks for them.

Session (.npz) *

Display Options

Min photons (fraction)Max cursors

Find Peaks

Smooth σThreshold

FRET Analysis

Donor τ (ns)

Acceptor τ (ns)(blank = donor-only)

Donor fretting

Expert Settings

Optimizer Cost function DE population DE max iter LM restarts Binning (NxN) CPU workers Min photons/px Channel filter IRF FWHM (ns) IRF alignment IRF shift ±bins Fit start (ns) Fit end (ns) Exclude bands

Align measured IRF peak to the decay rising edge Free τ per pixel (slower) Pile-up in the model Background in the model Free t0 (tail fit only)

File & Session

ROI Analysis

	Name	Type	τ_mean	τ_med	τ_sd	Photons	σ_photons

ROI fit: τ_min τ_max

Mode: Ellipse Polygon

Radius Minor/major

Phasor filter

Cursor fit: τ_min τ_max

Machine IRF

Build a machine IRF to see the plot here.

Parameter	Value	Unit

Export Images...

Include scale bar Include ROI annotations

PNG OME-TIFF OME-Zarr