NOTE: Most
digital cameras can be zoomed – both optically and digitally. A nice
feature, but one that is best avoided for scaling purposes. For the CP995 I
follow the procedure recommended by Aaron
Messing, using just enough zoom to fit the rectangular capture zone
(3:2) within the field of view. My master is based on this
arrangement.
Making the
Master File
Step 1
- To get things started you will need to create the master template, on to
which your scale bar "collection" will be pasted. This can be done by
imaging an empty field of view in bright field. The magnification setting is
not important. You are creating a “blank slate” having the same dimensions,
and resolution, as your microimages. You can also do this via your graphics
software by creating a new file of appropriate width and height, and
resolution ("New Image" in PSP). Name and save the file (set-up a separate
folder for this exercise, and save to this folder).
Step 2
- Place the stage micrometer on the stage, and focus using the lowest
magnification. Take a picture of the micrometer. Repeat this for each
objective. I recommend you name the file to match the objective (and Optovar
magnification changer settings if you use one). For example, your 4x would
be “4.jpg”, if you use an Optovar you can add the setting, for example
“4125.jpg”.
Step 3
- Open your low power micrometer image. PSP has a text entry option, as do
most graphics programs; this is used to create the bar. Activate this
option. Key in the length as underlined text (you will need to play with
font size to make it visible). The underlined text should appear on the
image. To extent the line below the text to the right length use the line
key. As the background will be white you can select any text colour –
I recommend red or black. Once this is done select “okay” and the line and
text should appear on the micrometer image. You can save, or delete, this
image as it is no longer needed.
Step 4
- Open your master template file and select the text option. With PSP this
will bring up the last entry, the one you just created – which is fine. Now
select okay and the line and text will be inserted on to your master
template image. (As the picture of my master illustrates, I chose to add a
header above the scale bars indicating the respective objective - there are
three bars under each objective that match my Optovar settings). Place the
bar where you want it and save the file. Repeat this process for each
micrometer picture, pasting the line and text on to the master until you
have covered the magnification range (be sure to save every change to the
master). Avoid placing the bars close together as the insertion process will
be easier if there is adequate free space around each bar.
The image
below (click for a full size view) illustrates the process, the last box
would be your master template.
Creating a scale bar image
Inserting a
Scale Bar
Open the
master template and select the appropriate scale bar. Employ the “cropping”
tool to outline the bar and text. Now crop BUT do not close the file.
Instead, copy the cropped scale bar image. In PSP this is done clicking the
right hand mouse key and choosing “copy”. Close the file (do not save) and
open the image that needs the scale bar. In PSP, clicking the right mouse
button, and selecting “paste as new layer”, will insert the bar on to this
image. Without closing this option move the scale to the top or bottom
corner and select save (with PSP this will permanently embed the bar in the
image – so you may want to save it under another name).
Here are a
few examples of the final product (Note - I now use a white background,
rather than clear, as this avoids having the scale bar "disappear" in the
image if the text colour is the same as your picture):
Questions are
welcome.
All the
best,
Ron
Neumeyer
