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
Unit of
Magnification Pixels Length
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
Distance in
Known
Unit of
Magnification
Pixels
Distance
Length
107
200
Leica 5x
µm
Leica
Leica
Leica
Leica
Check the
107
10x
438
20x
431
40x
680
63x
Global length box!
100
200
100
100
µm
µm
µm
µm
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
lens 2
objective
projector
4
4
4
10
10
10
20
20
20
40
40
40
100
100
100
2.5
3.3
5
2.5
3.3
5
2.5
3.3
5
2.5
3.3
5
2.5
3.3
5
Distance in
Known Distance
Pixels
(µm)
750.00
666.67
829.68
550.00
826.04
366.67
791.01
283.33
748.03
200.00
839.06
150.00
742.00
133.33
746.10
100.00
742.02
66.67
783.00
70.00
801.09
53.33
749.15
33.33
757.00
26.67
765.02
20.00
742.00
13.33
WILD Dissecting Microscope
lens 1
lens 2
screw on
bottom
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
none
none
none
none
none
none
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
lens 3
lens 4
Distance
objective projector in Pixels
2.18
6
2.5
2.30
6
3.3
1.73
6
5
1.76
12
2.5
2.28
12
3.3
2.45
12
5
2.14
25
2.5
1.94
25
3.3
2.12
25
5
2.16
50
2.5
1.93
50
3.3
1.46
50
5
0.34
6
2.5
0.45
6
3.3
0.68
6
5
0.68
12
2.5
0.91
12
3.3
1.37
12
5
Known
Distance
(µm)
25.00
20.00
10.00
10.00
10.00
7.00
6.00
4.00
3.00
3.00
2.00
1.00
2.00
2.00
2.00
2.00
2.00
2.00
none
none
none
none
none
none
1
1
1
1
1
1
25
25
25
50
50
50
2.5
3.3
5
2.5
3.3
5
1.53
1.76
1.68
1.81
1.86
1.68
2.20
1.90
1.20
1.30
1.00
0.60