Objective Morphological Quantification of Microscopic Images Using a Fast Fourier Transform (FFT) Analysis

Curr Protoc Essent Lab Tech, 2013 · DOI: 10.1002/9780470089941.et0905s07 · Published: January 1, 2013

Medical Imaging Bioinformatics Research Methodology & Design

Simple Explanation

The staining intensity of immunohistochemistry (IHC) depends on multiple factors, such as the concentration of both primary and secondary antibodies, incubation periods, temperature, tissue quality, and reagent purity. Microscopy settings and the perception of individuals viewing the images adds additional variability. These factors create an unnecessary variability in the inter-laboratory, and even intra-laboratory, quantification of IHC. 2-D Fast Fourier Transformation (FFT) has been used to quantify microscopic structures related to elastin networks in the tunica media of blood vessels.

Study Duration

Not specified

Participants

Adult rats

Evidence Level

Not specified

Key Findings

1
FFT allows for an objective quantification of photomicrographs based on morphology, which reduces the subjectivity associated with pixel-intensity methods.
2
Alignment for both cell types was observed in the cortex of the sectioned tissue; values for the ratio to the mean orthogonal angle were calculated at 8.7 and 12.6 for oligodendrocytes and neurons, respectively.
3
There was either nominal or no alignment observed in the thalamus; values for the ratio to the mean orthogonal angle were calculated at 1.9 and 0.8 for the oligodendrocytes and neurons, respectively.

Research Summary

Quantification of immunohistochemistry (IHC) and immunofluorescence (IF) using image intensity depends on a number of variables. These variables add a subjective complexity in keeping a standard within and between laboratories. Fast Fourier Transformation (FFT) algorithms, however, allow for a rapid and objective quantification (via statistical analysis) using cell morphologies when the microscopic structures are oriented or aligned. Quantification of alignment is given in terms of a ratio of FFT intensity to the intensity of an orthogonal angle, giving a numerical value of the alignment of the microscopic structures.

Practical Implications

Objective Quantification

FFT allows for objective quantification of photomicrographs based on morphology, reducing subjectivity associated with pixel-intensity methods.

Reproducibility and Standardization

FFT offers reproducibility and standardization between immunohistochemistry runs.

Versatile Application

FFT can be used on biological tissues showing alignment on a straight line, such as cell cytoskeleton, blood vessels, neuronal circuitry, and connective and muscle tissue fibers.

Study Limitations

1
Inability to analyze microscopic structures that align but do not describe a straight line.
2
Quantitative results serve to supplement the information in original images.
3
Some subjectivity in placement of the feather mask in Adobe Photoshop software and the Oval in NIH ImageJ software.

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