Scale Bars

For the epifluorescent microscopes and the Spinning disk its best to add scale bars in Image J. The image will
need to be calibrated first so this image is converted from pixels to microns. Always save your image without
the scale bar first as when making your figure you may wish to increase to alter it.

Image J:

First set the scale of the image to microns instead of pixels.

To do this go to Analyse, Set Scale:

NB. We have already calculated the camera pixel to micron information for you. The excel file can be found
on the Z-drive. See below an example of the information collected from the Timelapse microscope. Make
sure you select the correct microscope and objective:

Fill out the information in the Set Scale box, this will vary depending on the microscope and objective used:

If you tick the Global option, it will set the scale for all images currently open

Once you have set the scale, you can then add a scale bar. Go to Analyse, Tools
and Scale Bar

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 The ScaleBar Plus dialogue will appear:

Decide the size of scale bar you want, as well as its colour, location and whether or
not you want text:

Once you are happy, remember to save your image File, Save As, Tiff

A general note about the scale of your images

The scale stays the same if you crop an image. So if 100m = 103 pixels in A, 100m = 103 pixels in B

A
B

C
The scale will change if you resize an image (C)
e.g. From 1024 x 1024 to 512 x 512
In this image your scale with be half as large.

There are two approaches to this. Either note the factor by which you have
reduced your image size and then alter the scale bar accordingly when you
make it.
Or have the scale bar on the image when your reduce it. (easier)

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Zen
In the tabs at the bottom of the image, select Overlay. Click on the ruler icon and draw a scale bar of desired
length on the image

You can change the colour and thickness of the line as desired

If you tick the Show all box on the right hand side of the Overlay tab, more options will appear

Here you can change the size and style of the font, and adjust the position of the scale bar within the image
using the x and y coordinates

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Click elsewhere in the image to hide the selection markers at the ends of the scale bar

To save the scale bar (or any overlays you have made), use the Save option in the Overlay tab. You can also
load previously saved scale bars by clicking the Load button. This way you do not need to re-create your
scale bar each time. NB. File type should be OVL

To export the image with the scale bar, choose File, Export, and Full resolution of the image window

Alternatively, you can use the line tool to make a scale bar.
Select the Line tool and make sure the Measurement
option is ticked

Draw a line of desired length, at an angle of 0 degrees

Un-tick the Measurement box to remove the text, then

alter the colour or thickness of the line, and align in the
X and Y as before

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 StereoInvestigator

1. To add a scale bar to your image go to Image menu and select Add Scale bar

a. The scale bar dialogue will appear. Choose the size colour and
location of your scale bar. Once you are happy with the settings press apply.
You will need to save your image with the scale bar on it. (File Save Image). If
you want to alter the brightness and contrast you can do this in the Image,
Image Adjustment menu

Other image processing software

It is possible to draw scale bars on your images using other image processing software such as Photoshop,
Adobe Illustrator or Inkscape etc.

The main principal is to draw a line of a known length and add this to all your images.

If you know how long your line should be in pixels you can just make a new image in any package of the
appropriate length in pixels x 5 or 10 pixels wide. Select the area in the new image, copy and then paste this
image onto any of the images you need annotating. The Zeiss software will tell you how many pixels wide
your image is.

To calculate the no pixels in a micron the formula is as follows: Microns/Pixels = No of microns in 1 pixel

Alternatively, use the information in our Camera micron to pixel excel file, and work out how long to draw
your scale bar in pixels for it to be the desired length in microns. Then just draw a line that long.