Workflow: AFM profile analysis

The AFM tab loads atomic force microscopy height images from JPK .jpk-qi-image files and performs interactive line-profile analysis for measuring condensate or particle heights and wetting angles on membrane surfaces.

Overview

The AFM workflow supports:

Loading JPK QI image files
Displaying the height image as a false-colour map
Drawing one or more line profiles interactively by clicking two points on the image
Automatic baseline correction using the two clicked endpoint heights
Savitzky-Golay smoothing of the raw profile
Detection of contact points (where the object meets the membrane)
Fitting of wetting angles (contact angles) on both sides
Measurement of:
- peak height above the membrane baseline
- FWHM (full width at half maximum)
- contact width (distance between left and right contact points)
Export of all profiles and measurements to CSV and SVG

Input requirements

A JPK QI image file (.jpk-qi-image or .jpk)
The AFMReader Python package must be installed

Install AFMReader:

pip install AFMReader

Loading a file

Open the AFM tab.
Under JPK file, click Browse and select a .jpk-qi-image file.
Choose the Channel to load:
- height_trace
- height_retrace
- adhesion_force_trace
- stiffness_trace
Click Load File.

The height image is displayed as a false-colour map using the afmhot colormap. Axis units are in µm.

Drawing a line profile

After loading a file:

Click anywhere on the AFM image to place the start point.
- The start point should be placed on the bare membrane (flat reference surface) on one side of the object.
Click again to place the end point.
- The end point should also be on the bare membrane on the other side.
- The line must cross the object (condensate or particle).

The two clicked heights define the linear baseline representing the membrane plane. The software:

extracts the height profile along the line
applies Savitzky-Golay smoothing
subtracts the linear baseline
detects the contact points
fits the wetting angles
computes measurements
updates the display

Coordinates of each click are logged in the Results & Logs tab.

You can draw multiple profiles on the same image. Each new profile is added to the profile list and displayed.

Profile parameters

Fit points

Number of data points used to fit the slope at each contact point.

Adjusted with a slider (3–60 points). The current value is shown next to the slider.

Changing this slider immediately recomputes the wetting angles for the currently selected profile without re-extracting the profile.

Smooth window

Window length for Savitzky-Golay smoothing (must be odd and greater than smooth poly order).

Default: 15

Smooth poly order

Polynomial order for Savitzky-Golay smoothing.

Default: 3

Profile points

Number of interpolated sample points along the line.

More points give finer spatial resolution along the profile.

Default: 300

Threshold fraction

Fraction of the peak height used to define the contact points.

A contact point is the first index from each side where the baseline-corrected height exceeds:

threshold = threshold_fraction × peak_height

Default: 0.05 (5% of peak height)

Display panels

The AFM display contains two panels:

Left panel: height image

Shows the loaded AFM height image with:

false-colour map (afmhot colormap)
colorbar in nm
axis units in µm
drawn profile lines (cyan) labeled with profile numbers
click markers (+) for placed points

Right panel: line profile

Shows the most recently selected profile with:

raw height data (blue, semi-transparent)
linear baseline (brown dashed)
baseline-corrected height (blue, solid)
filled region above zero
contact point markers (circles) at the membrane level
tangent lines at each contact point
data points used for slope fitting (colored circles)
title showing computed angles

The profile panel title displays:

θ_left = X.X°  |  θ_right = X.X°  |  θ_mean = X.X°

Profile list

Completed profiles are listed in the Profiles listbox with:

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

Click a profile in the listbox to display it in the right panel.

Wetting angle computation

The wetting angle is defined as the angle between:

the membrane plane (horizontal, after baseline correction)
the tangent to the object surface at the contact point

For each contact point:

n_fit_points data points are selected starting at the contact point going into the object (inward direction).
A line is fitted through these points using numpy.polyfit.
The wetting angle is:

θ = arctan(|slope|)   [degrees]

The mean wetting angle is:

θ_mean = (θ_left + θ_right) / 2

Measurements

For each profile, the following quantities are reported:

Measurement	Description
`peak_height_nm`	Maximum baseline-corrected height in nm
`fwhm_nm`	Full width at half maximum in nm
`contact_width_nm`	Distance between left and right contact points in nm
`theta_left_deg`	Wetting angle at the left contact point in degrees
`theta_right_deg`	Wetting angle at the right contact point in degrees
`theta_mean_deg`	Mean wetting angle in degrees

Actions

Reset last

Removes the most recently drawn profile from the list and redraws the image and profile display.

Clear all

Removes all profiles and resets the display.

Save results

Opens a folder selection dialog and saves:

<stem>_profile<n>.csv per profile
Full profile arrays: distance_nm, height_raw_nm, height_smoothed_nm, baseline_nm, height_corrected_nm
<stem>_AFM_summary.csv
One row per profile with all scalar measurements
<stem>_AFM_overview.svg
Vector export of the current AFM display figure

Outputs

For an input file sample.jpk-qi-image, saving to a folder produces:

<selected folder>/
    sample_profile1.csv
    sample_profile2.csv
    ...
    sample_AFM_summary.csv
    sample_AFM_overview.svg

`*_profile<n>.csv`

Full numerical arrays for one profile:

Column	Contents
`distance_nm`	Distance along the profile in nm
`height_raw_nm`	Raw sampled height in nm
`height_smoothed_nm`	Savitzky-Golay smoothed height in nm
`baseline_nm`	Linear baseline in nm
`height_corrected_nm`	Baseline-corrected height in nm

`*_AFM_summary.csv`

One row per profile with columns:

Profile (number)
peak_height_nm
fwhm_nm
contact_width_nm
theta_left_deg
theta_right_deg
theta_mean_deg
start_px_x, start_px_y (click coordinates)
end_px_x, end_px_y

`*_AFM_overview.svg`

Vector export of the current two-panel figure.

Typical use

The AFM tab is designed for measuring the height and wetting properties of condensates or particles sitting on a supported lipid bilayer or other flat membrane surface.

Typical workflow

Load the height image.
Identify a condensate of interest.
Draw a profile crossing the condensate, placing both endpoints on the flat membrane surface on either side.
Inspect the profile panel:
- verify the baseline looks flat on both sides of the object
- verify the contact points are at the base of the object
- adjust Fit points with the slider to change which part of the object edge is used for angle calculation
- adjust Threshold fraction if contact points are placed at the wrong height level
Repeat for additional condensates or profiles.
Click Save results.

Optimising contact point detection

If the contact points are placed too high on the object:

decrease Threshold fraction (e.g. from 0.05 to 0.02)

If the contact points are placed on noise rather than the object edge:

increase Threshold fraction (e.g. from 0.05 to 0.1)

Optimising the wetting angle

If the fitted slope includes flat membrane rather than the object edge:

decrease Fit points

If the fitted slope is noisy because too few points are used:

increase Fit points

The slider provides real-time feedback without requiring a new profile extraction.

Troubleshooting

“AFMReader is not installed”

Install the required package:

pip install AFMReader

File will not load

Check:

the file is a valid JPK QI image
the selected channel exists in the file
the file is not corrupted or still being written

Height image looks inverted or all-black

Try a different channel (e.g. height_retrace instead of height_trace).

No contact points found

This can happen if:

the profile does not cross the object (both endpoints are on the same side)
the object is too flat relative to the noise level
the threshold fraction is too high

Try:

redrawing the profile to cross the object more centrally
decreasing the Threshold fraction

Wetting angle looks wrong

Check that:

both click points are on the flat membrane (not on the object)
the profile crosses the full width of the object
the Fit points slider is not set too high (including flat membrane in the slope fit)

Profile is very noisy