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## NanoLocz User Guide <img src="https://github.com/George-R-Heath/NanoLocz/assets/90329395/36d664a6-38e2-4405-b5cc-a962093cf13b" width="30">

<a href="installation.html">Installation</a> - - <a href="getting_started.html">Getting Started</a> - - <a href="saving_data.html">Saving Data</a> \
<a href="image_levelling.html" style="color: red;">Level</a> - - 
<a href="particle_detection.html" style="color: lightblue;">Detect</a> - - 
<a href="fine_align.html" style="color: orange;">FineAlign</a> - - 
<a href="localize.html" style="color: pink;">Localize</a>\
<a href="area_analysis.html" style="color: limegreen;">Area Analysis</a> - - <a href="keyboard_shortcuts.html">Keyboard Shortcuts</a>\
<a href="simulation_afm.html" style="color: orange;">Simulation AFM</a> - - <a href="general_use.html">YouTube Video Tutorials</a> 

# Image Levelling

![leveling](https://github.com/George-R-Heath/NanoLocz/assets/90329395/2be0667b-9a0c-437e-95a2-c77f6eb34f9e)

### Manual Leveling
**Plane Leveling:**\
To remove tilt or bow in images:
* Set the x and y <a style="color:orange"> Plane</a> values to perform polynomial fitting to all the x and/or y values in each image.
* Plane fitting is previewed in real-time.
* Press the 'Accept' button to apply.
* Tip: X and Y values of 1 will remove linear tilt, values of 2 or more can remove image bow.
  
**Line Leveling:**\
To remove height variations between scan lines:
* Set the x and y <a style="color:limegreen"> Line</a> values to perform polynomial fitting to each image line in x and/or y.
* Line fitting is previewed in real-time.
* Press the 'Accept' button to apply.
* Tip: use '-Med' to remove the median of each line in x.
  
**Line or Plane Leveling with a Mask:**\
To level images and movies that contain a variety of height levels requires certain regions (referred to as masked regions) to be excluded from the polynomial fits. Typically the included region should be a surface which is expected to be level and has a high coverage (>50%).
The options to generate a mask are:
1) Using the upper and lower threshold values on the height histogram
2) Manually drawing using the **Draw Mask** button
3) Using the 'Mask with z-scale' to mask values outside of the colormap scale
4) Automatically using the Otsu method
5) Automatically using the **Fit** button (fits a Gaussian to all the values in the movie and using 1.5σ (standard deviation) as the threshold values.)
* To level with a mask, line leveling or plane leveling is then performed as before but fitting is performed using only the non-masked values.
* Press the 'Accept' button to apply.

### Automatic Leveling
Leveling typically requires multiple iterations, to achieve this automatically several pre-set leveling routines are implemented in NanoLocz. For example the ‘Iterative Holes’ and ‘Iterative Peaks’ routines which use the following routine: 
1.	Subtract a plane fit in x and y with 2nd order polynomial then subtract the median line in x.
2.	Fit a Gaussian to all the heights in the movie and set a mask threshold to
z > 1.5σ (Iterative Peaks) or z <-1.5σ (Iterative Holes) to generate a mask.
3.	Repeat leveling step 1. with the threshold mask applied.
4.	Generate new mask by repeating step 2.
5.	Subtract a plane fit in x and y with 2nd order polynomial then subtract a 1st order polynomial line in x with the threshold mask applied.
   
‘Iterative Holes’ and ‘Iterative Peaks’ automatic leveling routines are designed for image sets with a fraction of lower features such as membrane defects (Iterative Holes) or a fraction of objects with higher features such as features on a mica surface (Iterative Holes). Other iterative leveling routine in NanoLocz include basic leveling followed by Otsu mask leveling or leveling followed by two leveling iterations with Gauissan fitted masks for z outside the range of +/-1.5σ

## YouTube video on Leveling + Area Analysis
[![YouTube NanoLocz Leveling + Area Analysis](https://img.youtube.com/vi/R5D6T04M7Rc/0.jpg)](https://www.youtube.com/watch?v=R5D6T04M7Rc)