## Measuring DNA Contour Lengths

> This is an example of how to measure the lengths of DNA contours on images acquired using an atomic force microscope (AFM). It requires ImageJ 1.30 or later.

> Figure 1. AFM image of 538 bp DNA fragment deposited on mica.

> In this example, we measure the length of the DNA contour in the lower right corner. The field width of the image is 500nm. The image is from M. Lysetska, et al., "UV light-damaged DNA and its interaction with human replication protein A: an atomic force microscopy study", Nucleic Acids Research, 2002, Vol. 30, No. 12 2686-2691 (nar.oupjournals.org/cgi/content/full/30/12/2686), used with permission.

> Figure 2. Set Scale Dialog Box.

> Use the Analyze/Set Scale dialog to define the spatial scale. Enter the image width in the "Distance in Pixels" field, enter the field width in the "Known Distance" field, enter "nm" as the "Unit of Length", then click "OK".

> Figure 3. Zoomed and spatially calibrated image.

> Use the magnifying glass tool to zoom in on the DNA contour to be measured, in this case, the one in the lower right corner of the image. To zoom out, right-click or alt-click with the magnifying glass tool.

> Figure 4. ImageJ toolbar with segmented line tool selected.

> This is the ImageJ toolbar. Use the segmented line tool (sixth tool from the left) to outline the DNA contour. The three tools on the right end of the toolbar are tool macros.

> Figure 5. Selected DNA contour.

> Use the segmented line tool to create a line selection that outlines the DNA contour. To finish outlining, right-click, double-click or click in the box at the starting point. The line selection can be adjusted by clicking and dragging the the tiny black and white "handles" along the outline.

> Figure 6. Results Table.

> Finally, use the Analyze/Measure command to measure the length of the DNA contour, in this case 181nm. Measurements can be transferred to a spreadsheet by right-clicking in the "Results" window, selecting "Copy All" from the popup menu, switching to the spreadsheet program, and then pasting.

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