How to Quantify: Nanofiber Orientation

How to quantify nanofiber orientation on SEM images?


Electrospinning is one of the most promising nanotechnological processes in recent years, capable of producing polymer nanofibers efficiently and with great precision. These ultrafine fibers, often with diameters in the nanometer range, hold immense promise for various applications. However, not all nanofibers are created equal. Understanding and controlling their orientation is a critical aspect of harnessing their full potential.

Oriented nanofibers offer numerous advantages across various fields. They exhibit enhanced mechanical properties, making them valuable in applications where tensile strength and stiffness are paramount, such as in tissue engineering for ligaments or bone regeneration. In tissue engineering, oriented nanofibers facilitate cell alignment and tissue growth, making them invaluable for regenerative medicine. Additionally, their alignment enhances electrical conductivity, making them useful in applications such as flexible electronics, sensors, and conductive scaffolds. Moreover, oriented nanofibers are also efficient in filtration systems, enabling precise control over filtration efficiency and contaminant removal in air and liquid filtration.

How to do it

To determine the orientation of your nanofibrous product, all you need is an electron microscopic image of our fibers and the Fiji open source image evaluation software, which can be downloaded here.

After installing Fiji, run the program and open the image to be evaluated (File → Open). Your image should appear in a new window of Fiji. To determine the orientation of your nanofibers click on Analyze → Directionality in your menu bar and you should see the dialogue window presented on the right. In this window you can set your preferences on how the directionality of your sample should be calculated:


Fourier components:
This method is based on Fourier spectrum analysis. When you look at the Fourier transformation of an image, structures with that same orientation will create a repeating pattern rotated +90 degrees from the objects in the original image. This plugin divides the image into squares and calculates their Fourier power spectra. These are examined in polar coordinates, and the power for each angle is measured.

Local gradient orientation:

This is a local analysis approach. The image’s gradient is generated with a 5×5 Sobel filter and utilized to determine the local gradient direction. This orientation is then used to construct the histogram, with the square of the gradient norm placed in the appropriate bin. The square of the norm was kept so that the histogram has the same dimension as the Fourier analysis.


The number of bins (number of bars) used to build the orientation histogram.

Histogram start / end

Set the angles between which the program will perform the orientation check. In general, the default value (−90° → +90°) is perfectly suitable. 

Build orientation map

The application creates a colored map of your nanofibers, with the colors corresponding to the fiber orientation.

Display color wheel

This option displays a graph showing the relationship between the colors on the orientation map and the orientation angle.

Display table 

The application outputs the raw data it has gathered from your electron microscopic image in a separate window.

After setting your preferred parameters, hit the OK buttons, then after a few seconds, the application will display the requested information. Below we present electron microscopic images of an oriented and a randomly oriented nanofibrous sample and histograms characterizing the orientation of these samples.

Oriented nanofibers

Random nanofibers