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 LengthLeica 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 LengthLeica 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