Customer Solutions

Quantitative Analysis of Cytoskeletal Fiber Orientation

Author(s):

Yoshigi Masaaki, University of Utah

Industry:

Life Science

Product:

LabVIEW, Vision

The Challenge:

Building a versatile, robust, and interactive image processing tool to quantify and display intracellular cytoskeletal fiber orientation in the fluorescent microscopic image of cells.

The Solution:

Using 2D-FFT algorithm and image processing tools in IMAQ Vision library to build a program (VI), so you can adjust brightness of the image, click around individual cells, analyze fiber orientation and alignments within the cell, display analysis graphs, and export the data as an ASCII file.

Introduction
Cytoskeletal fibers are filamentous proteins found in any kind of cell. Cytoskeletal fibers relate to cell shape, cell differentiation, and cell migration. Remodeling and rearrangement of cytoskeletal fibers is a major topic in molecular and cellular biology. With recent molecular biology techniques, quantifying the amount of cytoskeletal fiber protein is possible. However, we can qualitatively assess alignment and arrangement of the cytoskeletal fibers in the cell by visual impression. We have developed a versatile, robust, and interactive VI to quantify intracellular cytoskeletal fiber orientation. We used IMAQ Vision library and built a more robust image processing tool than NIH Image.

Fiber Orientation Analysis VI
We grew vascular smooth muscle cells on a glass cover slip using standard cell culture techniques. We stained cytoskeletal fibers of the vascular smooth muscle cells using fluorescent antibodies. And, we used IMAQ PXI-1408 inserted into a PXI-1000 chassis with a PXI-8156 embedded controller to capture images.
The Fiber Orientation Analysis (FOA) VI can capture the fluorescent microscopic image and store it in TIF format. For postprocessing, we can select the cell to analyze and then click around the region of interest (ROI). The fiber orientation analysis VI uses ROI functions that eliminate unnecessary image area, and the Centroid function to calculate the gravity center of the ROI. We can choose images of 256 by 256, 512 by 512, and 1024 by 1024 resolution interactively and perform 2D-FFT.

The brightness of the original image can vary depending on the antibody reactions or the selection of fluorescent dyes. With the VI, we can select several brightness adjustments. Mean brightness or modal brightness calculated using the Histogram function in IMAQ Vision is the basis of brightness standardization. When we use the 2D-FFT function in IMAQ Vision, the filamentous structure in the image decomposes into frequency and amplitude information. So, we see a definitive peak at high frequency in the amplitude spectrum, with directional infomation of the stripe.

2D-FFT Results
We used the high-frequency-in-the-center representation to view the 2D-FFT results. If the cell has complete parallel cytoskeletal fibers, 2D-FFT displays a bright single band that is perpendicular to the cytoskeletal fibers. If the cell has completely random cytoskeletal fiber orientation, the 2D-FFT displays a round distribution pattern. To quantify directivity of the fiber orientation, the FOA.VI rotates an imaginary straight line 180° around the center of the 2D-FFT image, then calculates the integral of the line profile on the probe line. This operation uses Line Profile and Get Angles in IMAQ Vision. The VI then plots the integral of pixel values on the probe line with angles on the x-axis. When the cells have three clusters of cytoskeletal fibers with three different directions, the distribution plot has three major peaks. The distribution plot, thus, tells us which orientation is the major cytoskeletal fiber orientation with highly quantitative values. We can then quantify cell shape and differentiation status based on cytoskeletal fiber orientation.

Conclusion
Progress in recent biomedical research has been highly dependent on engineering techniques. The accuracy, speed analysis of versatility, and robustness are the key features of the novel bioengineering analysis techniques. The LabVIEW and IMAQ Vision library have great potential to pioneer new types of image processing tools for biomedical research. The fiber orientation analysis VI is a unique tool for quantifying cytoskeletal fiber orientation with ease and providing unprecedented information for cellular biology.

For more information, contact:

Masaaki Yoshigi

Pediatrics, University of Utah

50 N. Medical Drive

Salt Lake City, Utah 84132

Tel: (801) 585-0663

Fax: (801) 581-4920

E-mail: masaaki.yoshigi@hsc.utah.edu

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