# Plot Profile in ImageJ: line scans, wide lines and intensity profiles

> Plot Profile in ImageJ: draw a line, press Ctrl K, set the line width to average noise, copy the values, plot two channels and profile a rectangle.

Published 2026-11-06 by LabCanvas team. https://www.labcanvas.com/blog/imagej-plot-profile

![ImageJ Plot Profile of a straight line across the nuclei sample: one peak per nucleus, two touching nuclei as a double peak, the List, Data, More and Live buttons, and the Data menu opened](https://www.labcanvas.com/blog/imagej/imagej-plot-profile-window.webp)

To plot an intensity profile in ImageJ, draw a line across the structure with a line tool and choose **Analyze › Plot Profile** (Ctrl K): a window plots the pixel values against distance along the line, in µm if the image is calibrated. Click **List** for the numbers, **Data »** to copy or save them, and **Live** to make the plot follow the line as you move it; set a wider line (double-click the line tool) to average noise across it.

A line profile, or line scan, is the simplest quantitative look at an image: the values along one path, nothing more. Below: the plot window explained, line width with a real example, profiles of two channels, profiles of a rectangle, and the other profile tools in ImageJ. Every picture is ImageJ 1.54f on our synthetic sample images, not real lab data.

## How do I plot a line profile in ImageJ (or Fiji)?

Plot Profile is part of ImageJ 1.x, so these steps are the same in Fiji.

1. **Open the image.** Check the scale with **Image › Properties…**: with a pixel size the x-axis reads in µm, without it in pixels.
2. **Pick a line tool** in the toolbar: **Straight**, **Segmented** (click points, double-click to finish) or **Freehand**. Right-click the tool to switch between them.
3. **Draw the line** across what you want to scan. Zoom in first if the ends matter; one pixel is a lot on a small structure.
4. **Analyze › Plot Profile**, or Ctrl K. The plot opens in its own window.
5. **Read or export the values** with the buttons under the plot.

![ImageJ Plot Profile window for a line across the nuclei sample, with the Data menu opened](https://www.labcanvas.com/blog/imagej/imagej-plot-profile-window.webp "ImageJ 1.54f on the nuclei sample: a 97.5 µm line across four nuclei, Analyze › Plot Profile. Each nucleus is a peak; two touching nuclei give two peaks with a dip between them. The Data » menu is shown opened.")

### What do List, Data, More and Live do?

| Button | What it does |
|---|---|
| **List** | Opens a table of Distance and Gray Value, one row per point along the line |
| **Data »** | **Save Data…** (a CSV file), **Copy 1st Data Set**, **Copy All Data**, **List (Simple Headings)**, **Add from Table…**, **Add from Plot…**, **Add Fit…** |
| **More »** | **Set Range…**, **Reset Range**, **Set Range to Fit All**, **Zoom to Selection**, **Axis Options…**, **Legend…**, **Contents Style…**, **High-Resolution Plot…**, **Freeze Plot**, **Plot Defaults…** and a few more |
| **Live** | The plot redraws whenever you move the line, drag an end or change the slice or channel |

Two of these save time every day. **Live** turns placing a line scan into a few seconds of nudging the line until the peak is centred. **Set Range…** fixes the axes, so plots from several images can be compared by eye; set it once for all profiles in a figure.

The number of points equals the length of the line in pixels: a 300-pixel line gives about 300 values, spaced one pixel apart. Values between pixel centres are interpolated (the **Interpolate** box in **Edit › Options › Plots…**, ticked by default).

## How do I set the line width, and why does it matter?

A one-pixel line samples one pixel at each point, so every bit of noise shows. A wider line averages the pixels across its width at each point along it. The signal along the line stays; the noise shrinks roughly with the square root of the width.

To set it:

- **Double-click the line tool** in the toolbar. That opens **Image › Adjust › Line Width**, a small window with a slider.
- Or **Edit › Options › Line Width…** and type the width in pixels.
- For a line already drawn, press **y** (**Edit › Selection › Properties**) and change its width there.

A wide line shows on the image as a band, so you can see exactly which pixels are averaged.

![Two ImageJ profiles across HRTEM lattice fringes, one with a 1-pixel and one with a 30-pixel line, and the Line Width dialog](https://www.labcanvas.com/blog/imagej/imagej-plot-profile-line-width.webp "ImageJ 1.54f on the synthetic HRTEM sample of a gold nanocrystal 0.02 nm per pixel. The same 4.8 nm line across the fringes, 1 pixel wide top and 30 pixels wide bottom. The wide line gives clean, even fringes: 20 peaks, 19 spacings over 4.46 nm, d = 0.235 nm.")

That example is a real measurement in miniature. On the 30-pixel profile we found the peaks with ImageJ's Array.findMaxima: the first at 0.22 nm and the last at 4.68 nm along the line, 19 spacings apart, so the lattice spacing is \(4.46 \div 19 = 0.235\ \mathrm{nm}\), the Au (111) spacing built into the sample. The 1-pixel line gives 0.234 nm here too, but its peaks wander up and down from 1,581 to 2,437, and on a noisier image it would miss or split peaks. The whole method is in [d-spacing from HRTEM lattice fringes](https://www.labcanvas.com/blog/d-spacing-hrtem-lattice-fringes).

How wide? As wide as the structure stays straight and uniform across the line. On fringes, the line must run exactly across them: a tilted wide line averages neighbouring fringes into each other and flattens the peaks. On a cell edge or a membrane, a width of 3 to 10 pixels is typical; on a filament, keep the width close to the filament's own width or you average in background.

## How do I plot a profile of two channels?

**Analyze › Plot Profile** in ImageJ 1.54f plots only the channel that is selected in a multichannel image. To compare channels you have two options:

- **Step through the channels with Live on.** Move the channel slider; the plot redraws for each channel, one at a time.
- **Plot both in one graph with a short macro.** It reads the profile of each channel along the current line and draws them together:

```
Stack.setChannel(1); p1 = getProfile();
Stack.setChannel(2); p2 = getProfile();
getPixelSize(unit, pw, ph);
x = newArray(p1.length);
for (i = 0; i < x.length; i++) x[i] = i * pw;
Plot.create("Both channels", "Distance (" + unit + ")", "Gray value");
Plot.setColor("green"); Plot.add("line", x, p1);
Plot.setColor("magenta"); Plot.add("line", x, p2);
Plot.setLegend("Channel 1\tChannel 2", "top-right");
Plot.show();
```

Run it with **Plugins › New › Macro**, paste, and **Run**. It respects the line width you set.

![ImageJ: Plot Profile of one channel and a macro plot of both channels along the same line on the two-channel sample](https://www.labcanvas.com/blog/imagej/imagej-plot-profile-two-channels.webp "ImageJ 1.54f on the synthetic two-channel sample 0.1 µm per pixel, a 30 µm line, 3 pixels wide. Top: Plot Profile shows only channel 1. Bottom: the macro plots both. At about 11.5 and 14.3 µm the green and magenta peaks coincide; at about 16.5 and 17.7 µm they are 1.2 µm apart.")

A two-channel line scan is a good way to show colocalisation in a figure: peaks that coincide, and peaks that do not, are easy to read. It is not a colocalisation measurement. A line samples a few hundred pixels picked by hand, so it can show whatever you choose to show. For numbers, use Pearson's and Manders' coefficients over the whole cell (see [colocalisation analysis](https://www.labcanvas.com/blog/colocalization-analysis-pearson-manders)), and use the line scan to illustrate them.

The channels must be aligned for any of this to mean anything. A chromatic shift of 1 or 2 pixels between channels moves every peak; check it on multicolour beads before you trust a 100 nm offset.

## What does Plot Profile do with a rectangle?

Draw a rectangle instead of a line and **Plot Profile** gives a **column average**: each point along the x-axis is the mean of one column of the rectangle. A long, thin rectangle across a row of bands or cells is in effect a very wide line, with every pixel in it used.

![ImageJ profile of a rectangle across the top row of bands in a western blot sample, with the Plots options dialog](https://www.labcanvas.com/blog/imagej/imagej-plot-profile-rectangle.webp "ImageJ 1.54f on the synthetic western blot sample: a 620 × 28 pixel rectangle across the eight target bands. Each band is a dip dark bands on a light background, deeper from lane 1 to lane 8. Edit › Options › Plots… holds the Vertical and Fixed y-axis scale options.")

For a profile that runs **down** the rectangle (a row average, for example along one lane of a gel), either hold **Alt** as you press K, or tick **Vertical** under Profile Plot Options in **Edit › Options › Plots…**. The same dialog has **Fixed y-axis scale** with a Minimum and Maximum Y, which keeps every profile on the same axis; that matters when you show profiles side by side.

On a blot, remember that bands are dark: lower values mean more protein. ImageJ's **Analyze › Gels** commands handle lanes and band areas for you; see [western blot quantification](https://www.labcanvas.com/blog/western-blot-quantification-imagej).

## Other profile tools in ImageJ

- **Segmented and freehand lines** follow a curved structure (an axon, a cell edge); the x-axis is the distance along the path. Combined with a time-lapse and **Image › Stacks › Reslice**, that is a [kymograph](https://www.labcanvas.com/blog/kymograph-imagej).
- **Image › Stacks › Plot Z-axis Profile** plots the mean of a selection through the slices or frames of a stack: a time course, or intensity against depth.
- **ROI Manager › More » › Multi Plot** plots the profiles of several line selections stored in the ROI Manager on one graph.
- **Analyze › Surface Plot** shows the whole image as a 3D landscape, the same idea in two dimensions.

## Pitfalls

- **An uncalibrated image.** The x-axis reads in pixels. Set the scale first (**Analyze › Set Scale…**).
- **A one-pixel line on noisy data.** Peaks and widths come out ragged. Widen the line, along the structure, not across a gradient.
- **A wide line across a curved or tilted structure** blurs it. Keep the band perpendicular to edges and fringes.
- **Saturated pixels.** A flat-topped peak at the maximum value (255, 4095 or 65535) is clipped; its true height is unknown.
- **Comparing profiles with auto-scaled axes.** Fix the y-range (**More » › Set Range…** or Fixed y-axis scale).
- **Measuring on an RGB or flattened copy.** Profile the raw image, not the version with an overlay burnt in.
- **One hand-picked line as evidence.** Report how lines were placed, and show several.

## Reporting it

A methods sentence to copy:

> Line intensity profiles were measured in [Fiji (ImageJ 1.54f)] with Analyze › Plot Profile along [straight] lines [3] pixels ([0.3] µm) wide, drawn [perpendicular to the membrane through the centre of each cell]; values were exported and plotted in [software].

Show profiles with fixed axes, give the line width in the legend, and mark the line on the image panel. Cite ImageJ or Fiji.

## An easier way: line profiles in LabCanvas

The same line scan works in the browser, with nothing to install, in [LabCanvas Image Analysis](https://www.labcanvas.com/analysis.html), and a multichannel image needs no macro.

1. Click **Open an image** (a TIFF keeps its scale and channels), or the two-channel sample on the start screen.
2. Pick the **Line** tool (L) and draw across the structure.
3. Open **Measure › Profile**: the plot is live as you draw and move the line, with **one curve per channel**. Set **Line width** to average across the line; hovering over the plot marks the point on the image.
4. **Add to results** for the numbers, then **Copy table**, or **Send to Graphs** with the **XY** option for a figure-ready profile.

:::grid
![Line intensity profile in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/profile.webp "Line intensity profile: Green and magenta values along a 28.56 µm line Line 1, 3 px wide, one curve per channel.")
![Lattice spacing from HRTEM in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/em-hrtem.webp "Lattice spacing from HRTEM: Profile across the lattice fringes of a gold nanocrystal: 10 spacings over 2.353 nm, so d = 0.235 nm, the Au 111 spacing.")
:::

On the HRTEM gold sample, the Profile panel finds 10 spacings over 2.353 nm, d = 0.235 nm; ImageJ's 30-pixel profile above gave the same 0.235 nm from 19 spacings over 4.46 nm.

Why it is a good choice for line scans:

- **Nothing to install**: it runs in the browser on any computer or tablet.
- **Every channel at once**, from the raw values, with no macro.
- **Straight into Graphs**: the XY profile becomes a plot with axis labels and styling, and the table carries a methods sentence to copy.
- **Free with a LabCanvas account**; downloading the CSV is part of LabCanvas Pro. The measurements are checked in [more than 4,000 automated tests](https://www.labcanvas.com/image-analysis-validation).

ImageJ and Fiji are stronger when you need hundreds of profiles placed by a macro, or profiles on files in a maker's format that only Bio-Formats reads. [Open Image Analysis](https://www.labcanvas.com/analysis.html) and try a line scan on the two-channel sample. ImageJ and Fiji are independent open-source projects; LabCanvas is not affiliated with them.

## References

- Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. *Nature Methods* 9, 671–675. [doi:10.1038/nmeth.2089](https://doi.org/10.1038/nmeth.2089)
- Schindelin J, Arganda-Carreras I, Frise E, et al. (2012). Fiji: an open-source platform for biological-image analysis. *Nature Methods* 9, 676–682. [doi:10.1038/nmeth.2019](https://doi.org/10.1038/nmeth.2019)

## Frequently asked questions

### How do I plot an intensity profile in ImageJ?

Draw a line with the Straight, Segmented or Freehand line tool, then choose Analyze › Plot Profile (Ctrl K). A plot window opens with distance along the line on the x-axis (in calibrated units if the image has a scale) and the pixel values on the y-axis. Click List for the numbers.

### How do I make a wider line for Plot Profile?

Double-click the line tool in the toolbar (it opens Image › Adjust › Line Width) or use Edit › Options › Line Width…, and type a width in pixels. Plot Profile then averages the pixels across that width at each point along the line, which smooths noise. Press y (Edit › Selection › Properties) to change the width of a line you have already drawn.

### How do I copy the Plot Profile values to Excel?

In the plot window, click Data » and choose Copy 1st Data Set (or Copy All Data), then paste. Or click List to open a table of distance and value and copy from there; Data » Save Data… writes a CSV file.

### Can ImageJ plot a profile of several channels at once?

Not with Analyze › Plot Profile in ImageJ 1.54f: it plots the channel that is selected. Step the channel slider with Live on, or run a short macro that reads getProfile() for each channel and adds them to one plot (shown on this page).

### What does Plot Profile do with a rectangle?

It plots a column average: each point is the mean of one column of the rectangle, so a wide rectangle averages a lot of noise away. For a row average (a profile running down the rectangle), hold Alt as you press K, or tick Vertical in Edit › Options › Plots….

### What is the Live button in the plot window?

With Live on, the plot follows the selection: move the line or its ends, or change the slice or channel, and the profile redraws. It is the quickest way to place a line scan across a structure.

## Related links

- [d-spacing from HRTEM lattice fringes](https://www.labcanvas.com/blog/d-spacing-hrtem-lattice-fringes): Count fringes along a wide line profile
- [Colocalisation: Pearson vs Manders](https://www.labcanvas.com/blog/colocalization-analysis-pearson-manders): When a line scan is not enough
- [Rolling-ball background subtraction in ImageJ](https://www.labcanvas.com/blog/rolling-ball-background-subtraction): Profiles before and after
- [Western blot quantification in ImageJ](https://www.labcanvas.com/blog/western-blot-quantification-imagej): Lane profiles and band areas
- [How to make a kymograph in ImageJ](https://www.labcanvas.com/blog/kymograph-imagej): A profile along a line, frame after frame
- [Image Analysis: the complete guide](https://www.labcanvas.com/blog/image-analysis-complete-guide): Every tool and setting in the app