Leica INM-100 Microscope Extended Depth of Focus Instructions

Leica INM-200 Microscope Extended Depth of Focus
Matthew Batchelor, Meyer Instr.
Roger Robbins, UTD
This instruction paper describes a satellite program that works with the Leica
INM-100 microscope and the Image Pro software to automate the collection of
component images required for the Extended-Depth-of-Focus feature.
This feature is a software means of obtaining a high magnification image over
topology deeper than the depth of focus of the objective lens. It achieves this by
taking multiple images through the depth of the topology and then recombining all
these images into a single image composed of just the in-focus portions of all the
component images.
This automation software requires that the operator find the feature of interest and
input the upper and lower stage positions associated with the bottom and top of the
image focus range, declaring the lens depth of focus, (given later in a table), and then
pushing the acquire button. The software then automatically steps the z-axis (stage
height) through the topology range and captures images at the appropriate focus.
These images are then fed to the Image pro software and composed into a single
image using just the in-focus portions of all the component images. A clever routine
allows the user to view all the component images in a timed series movie just like an
operator would see if he were to move the stage through the range of focus.
1) Turn on all microscope and computer equipment
2) Start the Image-Pro Plus software by clicking on the icon below in the lower left
corner of the Windows screen.
3) Start the INM-Focus Software by clicking on the INM-Focus.exe icon also
located in the lower left hand corner of the Windows screen.
4) Your computer screen should look something like this:
5) If the INM-Focus software is communicating properly with the INM200
microscope, there should be a number displayed in the CURRENT Z POSITION
frame in the dialog box. If this is not showing properly, see the next step.
6) If you do not see a position on the OPERATE tab in the CURRENT Z POSITION
section, and if pressing UPDATE does not produce an updated position, then
communications with the scope is not working properly. Switch to the SETUP
tab. There are a number of settings which change the way that INM-Focus
interacts with the COMM (Serial) Port, the operating system, and Image-Pro. If
changes are made to these settings, press the STORE SETUP IN INMFOCUS.INI button, close the INM-FOCUS software and restart it. This allows
the changes to be stored on disk and then used by the next run of the software.
The content of the INM-FOCUS.INI file that is stored by the settings shown above, is
shown in the following screen:
This file is located immediately adjacent to the INM-FOCUS.EXE program file
7) When the INM-FOCUS software is properly communicating with the microscope,
check the PREVIEW IMAGE ON checkbox and Image-Pro Plus will turn on its
PREVIEW so that you can set the focus, exposure, and framing for your sample.
The image should look something like the following:
If the image is too dark (as it is here), right click on the image select AUTO
EXPOSURE + DYNAMIC (shown below) and the computer will automatically
find an appropriate exposure.
After the proper exposure is found, turn off dynamic auto exposure or your results
will be affected by the changes in the exposures during your image capture.
8) Adjust the focus on your microscope so that the stage is at the highest (max z)
position and then press the UPDATE button to capture the stage z position. Note
that the highest stage z position occurs when you are focused on the bottom
portion of your image.
While the software is communicating with the microscope, there will be a YEILD
symbol in the upper right corner of the INM-FOCUS dialog box. Please do not
push any buttons while this symbol is being displayed. Sending multiple
commands to the microscope simultaneously could yield unexpected results!
9) When the Maximum Z value has been captured, press the SET button to record
the Current Z Position as the TOP (MAXIMUM Z).
10) Change the focus on the scope so that the stage is at its minimum position,
(highest point in the image), update the current position with the UPDATE button,
then transfer that position to the BOTTOM (MINIMUM Z) section of the dialog
box with the appropriate SET button.
11) The dialog box automatically calculates the thickness of the sample by subtracting
the BOTTOM from the TOP, (Note the arithmetic symbols to the left of the
dialogue. The THICKNESS is displayed in um.
The dialog box defaults to assuming that the DEPTH OF FIELD for the microscope
objective is 1um. In the example shown above, with a THICKNESS of 2.95 um and
a DOF of 1 um, the system calculates that the NUMBER OF IMAGES as 4: One at 0
um, 1 at 1 um, 1 at 2 um, and 1 at 3 um. You can override this number. In the
following example I have changed the DOF to .5 um, and you can see that the
NUMBER OF IMAGES has changed to 7.
12) You can press the GO TO buttons in the TOP (Maximum Z) and BOTTOM
(Minimum Z) sections of the dialog box to confirm your settings. You can also
manually enter numbers into these sections of the dialog box.
PLEASE NOTE! There is no error checking on the numbers you enter. It is
possible for you to request a stage position which would cause the sample to
smash into contact with the microscope objective, possibly causing serious
damage. It is MUCH better to position the stage using the focus knob, update the
software, and then set the limits of travel.
13) Once the TOP and BOTTOM locations have been set, you can use buttons shown
below to manually step through the focus levels.
14) When you are satisfied with the settings, you can capture all of the images using
the CYCLE FOCUS section of the dialog box. With the radio button labeled
CAPTURE selected, press the START button and the software will:
- Travel to the point you designated as BOTTOM
- Pause for the amount of time designated
- Capture the first image into a new image frame in Image-Pro Plus
- Travel to next focus point
- Pause for the amount of time designated
- Capture the next image into the frame created for the first image
- Continue until all images are captured
While images are being captured, the dialog box will look something like this:
When the cycle finishes, the following message box will be displayed:
If for some reason you need to stop the CYCLE FOCUS routine, you can press the
STOP button. Within a couple of seconds, the routine should stop and you should see
the following message:
15) If you want to make the computer and scope loop through the focus zone that you
have set, select the LOOP radio button and press the START button. The
software will drive the scope from the bottom to the top of the focus zone pausing
the user selected time at each image. After the top is reached, the software sends
the stage directly back to the bottom. This loop will continue until you press the
STOP button.
16) Once the software has captured your images in Image-Pro Plus, you should see
something like the following:
In the example above, we have an image frame titled UNTITLED000 which contains
7 images. Currently the 7th of 7 images is being displayed. Play through these
images using the controls in the SEQUENCE TOOLBAR. Extra options are
“behind” the OPTIONS button in this dialog box.
17) To store these image on your disk, select FILE + SAVE or FILE + SAVE AS
When the dialog box appears, direct the operating system to store the file in your
desired location with your desired name. In this example “MyExample.TIF” will be
stored in “My Documents”.
18) For Image-Pro Plus to store all of the separate images (7 in this example) in one
file, the APPLY TO setting has to be ALL (see below).
If you attempt to save a multiple image window without this setting in the ALL
position, Image-Pro will prompt you.
19) If you want to combine your multiple images using Image-Pro Plus’ EXTENDED
DEPTH OF FIELD tool, do the following:
- Make sure the image you want to process is open
- Select the image you want to process on the left side of the IMAGE LIST tab and
then press ADD to put it on the SELECTED FRAMES list
- Change to the FOCUS SETTINGS tab
- Select the settings as they are shown above and press the CREATE button.
- The resulting image will be displayed and will be given a name based on your
original image’s name and the EDOF algorithm used to create the image.
This image has not been saved to the disk yet, so please do so.
20) The size of these images on disk can be very large so please take this into
Note that the file named “MyExample Local Contrast Composite.tif” contains the
composite image of all the “in-focus” regions of all the contributing images through
the depth of focus. The really large file named “MyExample.tif” is a file containing
each of the individual images making up the composite.
This document has outlined the procedure required to utilize the Extended Depth
of Focus feature which automatically acquires the composite images of a substrate having
topology deeper than the depth of focus of the objective lens. Note that the software
cannot know the critical z-stage position of a collision, so care must be taken in setting
the z-stage limits; i.e. carefully follow the procedures outlined in this document.
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