Adding a scale bar to images taken with the Leica Fluorescent Microscope

Adding a Scale Bar to Images Taken with the Leica Fluorescent Microscope

Using Built in Software (Leica Application Suite) more convenient for a couple images

Take an image Aquire tab -> Acquire Image

Switch to Process tab

Select your image from the dashboard at the bottom of the screen

Switch to Calib. Sub-tab

Click Manual (Draw Line)

Enter the pixels and length for the objective you used according to the chart below:

Lens MagnificationPixelsLengthUnit of Length

Leica 5x107200m

Leica 10x107100m

Leica 20x438200m

Leica 40x431100m

Leica 63x680100m

Select microns as the unit

Click Apply

Switch to Annotate sub-tab

In the Scale Bar tab, click Show

You can now click and drag the scale bar to place it wherever you please

You may also change the length by selecting User Length and typing a new value

Format the font and colors in the font box

In the Actions box, click Merge

Right click on the large image, click Save As

Save the image with scale bar to you folder.

Using ImageJ Easier for many images

Save all images with the magnification in the name or on the folder!!! (ex: 5x, 10x)

Download ImageJ: http://rsb.info.nih.gov/ij/download.htmlInstall ImageJ

Open the first picture file into ImageJ: ctrl+O (or) file -> open

Set Scale: Analyze -> Set Scale

Enter pixel and distance values and unit of length according to table below:

Lens MagnificationDistance in PixelsKnown DistanceUnit of Length

Leica 5x107200m

Leica 10x107100m

Leica 20x438200m

Leica 40x431100m

Leica 63x680100m

Check the Global length box!

Click: Analyze -> Tools-> Scale Bar

Enter in the desired length of scale bar in the Width in box

Format the bar with the following boxes

You can now quickly add scale bars to your other pictures:

Open image

Click: Analyze -> Tools-> Scale Bar

Format scale bar

If you clicked the global length box, you will not have to set the scale for each image until you edit an image that used a different objective.

If you do not know the Distance in pixel or known distance

Get a measuring standard slide (or mm ruler for the dissecting microscopes).

Take a picture, record the objective magnification as well as any projector lenses between the objective and camera.

Open picture in ImageJ

Zoom in on the standardization lines with the magnifying glass tool (10th icon on tool bar)

Use the line tool (5th icon on tool bar) and draw a line along the known length of the standardization lines (hint: hold shift to constrain the line to right angles)

Go to Set Scale: Analyze -> Set Scale

The value in distance in pixels is your Distance in Pixels and the standardization length is the known distance

Additional Pixel/Length Tables (Note: there are multiple lenses! The projector lens sits in the collar adapter right beneath the camera.)

Olympus Compound Microscope

lens 1 objectivelens 2 projectorDistance in PixelsKnown Distance (m)

42.5750.00666.67

43.3829.68550.00

45826.04366.67

102.5791.01283.33

103.3748.03200.00

105839.06150.00

202.5742.00133.33

203.3746.10100.00

205742.0266.67

402.5783.0070.00

403.3801.0953.33

405749.1533.33

1002.5757.0026.67

1003.3765.0220.00

1005742.0013.33

WILD Dissecting Microscope

lens 1 screw onlens 2 bottomlens 3 objectivelens 4 projectorDistance in PixelsKnown Distance (m)

0.5162.52.1825.00

0.5163.32.3020.00

0.51651.7310.00

0.51122.51.7610.00

0.51123.32.2810.00

0.511252.457.00

0.51252.52.146.00

0.51253.31.944.00

0.512552.123.00

0.51502.52.163.00

0.51503.31.932.00

0.515051.461.00

none162.50.342.00

none163.30.452.00

none1650.682.00

none1122.50.682.00

none1123.30.912.00

none11251.372.00

none1252.51.532.20

none1253.31.761.90

none12551.681.20

none1502.51.811.30

none1503.31.861.00

none15051.680.60