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 Magnification / Pixels / Length / Unit of Length
Leica 5x / 107 / 200 / µm
Leica 10x / 107 / 100 / µm
Leica 20x / 438 / 200 / µm
Leica 40x / 431 / 100 / µm
Leica 63x / 680 / 100 / µm

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.html

Install 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 Magnification / Distance in Pixels / Known Distance / Unit of Length
Leica 5x / 107 / 200 / µm
Leica 10x / 107 / 100 / µm
Leica 20x / 438 / 200 / µm
Leica 40x / 431 / 100 / µm
Leica 63x / 680 / 100 / µm

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 objective / lens 2 projector / Distance in Pixels / Known Distance (µm)
4 / 2.5 / 750.00 / 666.67
4 / 3.3 / 829.68 / 550.00
4 / 5 / 826.04 / 366.67
10 / 2.5 / 791.01 / 283.33
10 / 3.3 / 748.03 / 200.00
10 / 5 / 839.06 / 150.00
20 / 2.5 / 742.00 / 133.33
20 / 3.3 / 746.10 / 100.00
20 / 5 / 742.02 / 66.67
40 / 2.5 / 783.00 / 70.00
40 / 3.3 / 801.09 / 53.33
40 / 5 / 749.15 / 33.33
100 / 2.5 / 757.00 / 26.67
100 / 3.3 / 765.02 / 20.00
100 / 5 / 742.00 / 13.33

WILD Dissecting Microscope

lens 1 screw on / lens 2 bottom / lens 3 objective / lens 4 projector / Distance in Pixels / Known Distance (µm)
0.5 / 1 / 6 / 2.5 / 2.18 / 25.00
0.5 / 1 / 6 / 3.3 / 2.30 / 20.00
0.5 / 1 / 6 / 5 / 1.73 / 10.00
0.5 / 1 / 12 / 2.5 / 1.76 / 10.00
0.5 / 1 / 12 / 3.3 / 2.28 / 10.00
0.5 / 1 / 12 / 5 / 2.45 / 7.00
0.5 / 1 / 25 / 2.5 / 2.14 / 6.00
0.5 / 1 / 25 / 3.3 / 1.94 / 4.00
0.5 / 1 / 25 / 5 / 2.12 / 3.00
0.5 / 1 / 50 / 2.5 / 2.16 / 3.00
0.5 / 1 / 50 / 3.3 / 1.93 / 2.00
0.5 / 1 / 50 / 5 / 1.46 / 1.00
none / 1 / 6 / 2.5 / 0.34 / 2.00
none / 1 / 6 / 3.3 / 0.45 / 2.00
none / 1 / 6 / 5 / 0.68 / 2.00
none / 1 / 12 / 2.5 / 0.68 / 2.00
none / 1 / 12 / 3.3 / 0.91 / 2.00
none / 1 / 12 / 5 / 1.37 / 2.00
none / 1 / 25 / 2.5 / 1.53 / 2.20
none / 1 / 25 / 3.3 / 1.76 / 1.90
none / 1 / 25 / 5 / 1.68 / 1.20
none / 1 / 50 / 2.5 / 1.81 / 1.30
none / 1 / 50 / 3.3 / 1.86 / 1.00
none / 1 / 50 / 5 / 1.68 / 0.60