Morphometric evaluation and quantification using Fiji

Fiji is a free software which can be used for image analysis in anatomical studies which allows obtaining accurate measurements of fine structures for morphometric evaluation. Fiji has a user-friendly interface with no requirement of programming skills, hence it has gained a wide interest among basic scientists. Here, we describe a protocol for obtaining basic measurements in gross anatomical and radiological studies using Fiji.


Introduction
A myriad of studies in gross anatomy, radiological anatomy and developmental biology utilize measurements such as distance, angle, area, volume and curvature to describe morphology of different structures. The classical approach of obtaining these measurements was to use instruments such as calipers (1), measuring tapes (2), set squares (3) and goniometers (4). Often magnifying glasses were used to aid measurements since fine structures could not be accurately resolved by the naked eye (5). This approach had several limitations when measuring curvilinear distances, angles and areas which necessitated indirect measuring strategies such as using threads (3). Moreover, since the measurements were obtained real-time, it was laborious to revisit the specimens and obtain new measurements for subsequent analyses and revisions of the studies.
These shortcomings could be easily overcome by utilizing image analysis software for morphometric evaluation in anatomical studies. Fiji (previously known as ImageJ) is one such popular software used by the basic scientists worldwide for image analysis. Fiji was originally developed by the National Institute of Health (NIH) for analysis of histological images (6). It is an open source (free) software which is constantly being modified and enhanced by the users and universally accepted by high impact journals inclusive of Nature, Cell and Science (6). Fiji has a user-friendly interface with no need of knowledge in programming languages and coding. It allows obtaining a wide array of measurements to analyze structural morphology encompassing linear and curvilinear distances (7), angles (7), area (8), and volume (9). Measurements can be obtained accurately after magnifying images (10) which increases reproducibility. Large scale image analyses such as detection and counting of vascular (11) and neural (12) networks can be conducted using automated 'macros' and freely available extensions of Fiji. Moreover, there is a growing interest in analyzing 3-dimensional datasets based on cadaveric and radiological cross-sections, which could be easily achieved by using this software (10,13).
The objective of this paper is to introduce Fiji for anatomical and radiological studies. Here, we describe a protocol for obtaining basic measurements such as distance, angle and area.

Setting up Fiji environment
Fiji can be downloaded from the site (https://imagej.net/Fiji/Downloads). Once extracted, Fiji can be launched by clicking on the Fiji/ImageJ.exe icon. Figure 1 shows the Fiji interface. The software can be updated by selecting Help  Update.

Getting sources of data
The structures which need to be measured are required to be photographed with a scale (ruler or a caliper) kept at the same plane of the specimen (Figure 2A). Having a fixed distance between the camera and the specimen will make image analysis faster. Care should be taken to orient the specimen perpendicular to the camera lens.

Importing images to Fiji
Fiji supports a variety of file formats inclusive of JPEG, PNG, GIF, TIFF, BMP, DICOM. They can be imported by selecting File  Open. Alternatively, the images can be dragged and dropped on to the interface.

Calibration
If the images have been obtained at a fixed distance, the images can be calibrated to obtain the subsequent measurements. First, a line should be drawn across the scale (Figure 2A) using the Line Selection tool ( Figure 1B). Then the scale should be set up by selecting Analyze  Set Scale. In the dialog box ( Figure 2B) the "known distance" should be entered. The calibration is applied to all the opened images if the "global" option is selected. The settings can be applied by clicking OK.

Distance -linear and curvilinear
Once the scales are set (see under calibration), the distances can be measured by drawing a line between the structures of interest using the Line Selection tool ( Figure 1B). The actual distance is now displayed in the status bar as the "length". The measurement can also be obtained by clicking Analyze  Measure (or Ctrl+M) and scrolling to the "length" tab ( Figure 2C), which can be subsequently copied to a database (eg: Excel, SPSS). The curvilinear distances can be measured in a similar way using the "segmented line selection" tool ( Figures 2D and 2E). The segmented line tool can be selected by right clicking on the line selection tool and selecting the segmented line option ( Figure 2D).

Angle
Angle tool ( Figure 1B) is used for measuring angles. The intended angle needs to be indicated by selecting three points ( Figure 2F).
The measurement can be obtained by clicking Analyze  Measure, and scrolling to the "angle" tab.

Area
The region of the structure can be first selected using area selection tools ( Figure 1B). The measurement can subsequently be obtained by selecting Analyze  Measure and scrolling to the "area" tab.

Advanced analyses and image editing
A variety of other measurements in biological image analysis such as curvature, skewness, kurtosis, center of mass, perimeter, shape descriptors can be obtained by Fiji, which is beyond the scope of this article. A scale bar can be added to the images using Fiji for publication purposes. Fiji also allows a variety of options for image editing such as sharpening, smoothening, contrast enhancement. However, care should be taken to apply the same parameters/modifications for the entire dataset in order to prevent "image manipulations".

Conclusions
Fiji is an open source software which allows a variety of image analysis options for anatomical research which is widely accepted by the scientific community.