User Manual

LCmicro
IMAGE ANALYSIS SOFTWARE
 Contents
1. About your software..........................................................................................3
1.1. Overview - LCmicro ................................................................................................................. 3
2. Working with LCmicro ......................................................................................4
2.1. Overview - User interface ........................................................................................................ 4
2.2. Working with documents ......................................................................................................... 5
3. Acquiring images ..............................................................................................7
3.1. Snapshot ................................................................................................................................. 7
3.2. Camera Control ....................................................................................................................... 8
3.2.1. Switching on the automatic exposure time .............................................................. 12
3.2.2. Switching on the manual exposure ......................................................................... 14
3.2.3. Setting the camera resolution .................................................................................. 16
3.2.4. Toggle Subarray ...................................................................................................... 18
3.3. Acquisition Settings ............................................................................................................... 20
3.3.1. Acquisition Settings - Acquisition - General ............................................................ 20
3.3.2. Acquisition Settings - Camera - General ................................................................. 22
3.3.3. Acquisition Settings - Camera - Adjustment ............................................................ 23
3.3.4. Acquisition Settings - Camera - Color ..................................................................... 24
3.3.5. Acquisition Settings - Camera - Information ............................................................ 25
4. Working with images.......................................................................................26
4.1. Changing the way an image is displayed .............................................................................. 26
4.2. Processing images ................................................................................................................ 27
4.3. Performing a white balance on an image .............................................................................. 28
4.4. Working with drawing objects ................................................................................................ 29
5. Measuring images ...........................................................................................31
5.1. Overview................................................................................................................................ 31
5.2. Measuring images ................................................................................................................. 32
6. Configuring the system ..................................................................................34
6.1. Overview................................................................................................................................ 34
6.2. Configuring the system.......................................................................................................... 35
6.3. Carrying out a shading correction ......................................................................................... 36
6.3.1. Overview .................................................................................................................. 36
6.3.2. Carrying out a shading correction ........................................................................... 37
6.4. Manual Magnification Calibration .......................................................................................... 37
 About your software

1. About your software

1.1. Overview - LCmicro
Acquiring images
Your system enables you to acquire high resolution images of a sample in just a
few steps. Your system is comprised of your software, a camera, and a
microscope (or, alternatively, a macro stand). With the image acquisition
process, data is read out from the camera that is attached to the microscope and
is displayed on the computer's monitor.
You can first observe the live-image and make good settings for it. The live-
image is constantly refreshed. This means that if you move the stage, the live-
image changes correspondingly. You can switch the live-image on and off and
acquire an image of the parts of the sample that interest you. When you do this,
you create a digital image that you can save and measure.

Processing images
You can process the images you acquired and optimize the image quality for
your purposes. You can, for example, adjust the intensity or optimize the
contrast.

Measuring images
You can make various measurements on images. You can measure the length of
a line for example. The measurement objects are displayed in the image's
measurement layer. All of the image information is therefore retained. The results
are saved together with the image.
00017 20112015

3
 Working with LCmicro

2. Working with LCmicro

2.1. Overview - User interface
The graphical user interface determines your software's appearance. It
determines which menus are shown, how the individual functions are called up,
how and where images are displayed, and much more.
In the following, the basic elements of the user interface are described.
Appearance of the user
interface

The illustration shows the schematic user interface with its basic elements.
(1) Menu bar
(2) Document group
(3) Toolbars
(4) Tool windows
(5) Status bar

(1) Menu bar You can call up many commands by using the corresponding menu. You can find
a variety of commands in the View menu with which you can change the
appearance of the user interface.
(2) Document group The document group contains all of the loaded images.
When you start your software, the document group is empty. While you use your
software it gets filled - e.g., when you load or acquire images, or perform various
image processing operations to change the source image and create a new one.
(3) Toolbars Commands you use frequently are linked to a button providing you with quick
and easy access to these functions. Please note, there are many functions which
are only accessible via a toolbar, the drawing functions required for annotating an
image for example.
(4) Tool windows Tool windows combine functions into groups. These may be very different
functions. For example, in the Properties tool window you will find all the
information available on the active image.
In contrast to dialog boxes, tool windows remain visible on the user interface as
long as they are switched on. That gives you access to the settings in the tool
windows at all times.
(5) Status bar The status bar contains information, a brief description of each function for
example. Position the mouse pointer on the name of a command or on a button
to display a brief description of a function.
00108 14092015

4
 Working with LCmicro

2.2. Working with documents

There are a number of ways to open, activate, save, or close images.

Saving images
You should always save important images immediately following their acquisition.
You can recognize images that have not been saved by the star icon after the
image's name.
There are a number of ways in which you can save images.
1. To save an individual image, activate it in the document group. Then use the
File > Save As... command, or the [Ctrl + S] keyboard shortcut.
2. Use the Documents tool window.
Select the desired document and use the Save command in the context
menu. For the selection of documents, the standard MS-Windows
conventions for multiple selection are valid.
3. Use the Gallery tool window.
Select the desired document and use the Save command in the context
menu. For the selection of documents, the standard MS-Windows
conventions for multiple selection are valid.

Automatic save 1. When you exit your software, all of the images that have not yet been saved
will be listed in the Unsaved Documents dialog box. This gives you the
chance to decide which images you still want to save.
2. You can also configure your software in such a way that all images are saved
automatically after image acquisition. To do so, use the Acquisition Settings
> Saving dialog box. Here, you can also configure your software in such a
way that all images are automatically saved in a database after the image
acquisition.

Closing images
There are a number of ways in which you can close images in your software.
1. Use the Documents tool window.
Select the desired images and use the Close command in the context menu.
2. To close a single image, activate the image in the document group and use
the File > Close command. Alternatively, you can click the button with the
cross [ x ]. You can find this button at the top right of the image's tab's
header next to the name of the image.
3. Use the Gallery tool window.
Select the desired images and use the Close command in the context menu.

Closing all images To close all of the images that are loaded, use the Close All command or the [Ctrl
+ Alt + W] keyboard shortcut. You can find this command in the File menu, and in
both the Documents and the Gallery tool windows' context menu.
Closing an image To close an image immediately without being prompted to save it, press the
immediately
[Shift] key while closing it. Data you have not saved will be lost.

5
 Working with LCmicro

Opening images
There are a number of ways in which you can open or load images.
1. Use the File > Open... command.
2. Drag the images you want directly out of the MS-Windows Explorer into your
software's document group.
Note: At the same time, up to 150 documents can be loaded in the document
group.
Generating a test If you want to get used to your software, then sometimes any image suffices to
image
try out a function.
Press [Ctrl + Shift + Alt + T] to generate a color test image.
With the [Ctrl + Alt + T] shortcut, you can generate a test image that is made up
of 256 gray values.

Activating images in the document group

There are several ways to activate one of the images in the document group and
display it on your monitor.
1. Use the Documents tool window. Click the desired image there.
2. Use the Gallery tool window. Click the desired image there.
3. Click the title of the desired image in the document group.

4. Click the small arrow at the top right of the document group to open a list
of all of the loaded images. Left click the image that you want to have
displayed on your monitor.
00143 20112015

6
 Acquiring images

3. Acquiring images
3.1. Snapshot
You can use your software to acquire high resolution images of a sample in just a
few steps. For your first acquisition you should carry out these instructions step
for step. Then, when you later make other acquisitions, you will notice that for
similar types of sample many of the settings you made for the first acquisition can
be adopted without change.
1. Use the View > Layout > Reset Current Layout command.
 You can find the Microscope Control (1) toolbar at the upper edge of the
user interface, right below the menu bar.
To the right of the document group, you can find the Camera Control (2)
tool window.

Setting the 2. On the Microscope Control toolbar, click the button with the objective that
magnification
you use for the image acquisition.
Switching on the live- 3. In the Camera Control tool window, click the Live button.
image
 The live-image (3) will now be shown in the document group. A new
image document will automatically be created for the live-image.
4. Go to the position on the sample you are interested in.
Settings in the Camera Use the functions of the Camera Control tool window to set the image quality.
Control
5. Bring the sample into focus.
The Focus Indicator toolbar is there for you to use when you are focusing on
your sample.
6. Check the color reproduction. If necessary, carry out a white balance.
7. Check the exposure time. You can either use the automatic exposure time
function, or enter the exposure time manually.
8. Select the resolution you want.
9. Carry out a Gamma correction.
To do so, click the Acquisition Settings button in the Camera Control tool
window's toolbar. Select the Camera > Adjustment entry in the tree view.
Move the Gamma slide control to increase the image contrast either in bright
image areas or only in dark image areas.

7
 Acquiring images

Acquiring and saving 10. In the Camera Control tool window, click the Snap button.
an image
 The acquired image is shown in the document group.
11. Use the File > Save As... command to save the image. Use the
recommended TIF or VSI file format.
Note: The VSI file format is an image file format exclusive for your software. For
this reason, an image file in the VSI format can't be opened with any other
program.
In contrast to TIF images, the file size of VSI images is not restricted to 2
gigabytes per image.
00027 20112015

3.2. Camera Control

Use the Camera Control tool window to acquire images. This tool window is
displayed on the right side of the user interface by default. Should this tool
window not be visible, use the View > Tool Windows > Camera Control
command to show it.
Note: Some of the settings in the Camera Control tool window are camera
specific (e.g., the available resolutions). This is why the Camera Control tool
window looks slightly differently depending on the camera model you are using.
The tool window is made up of several parts.

8
 Acquiring images

Schematic illustration of
the Camera Control tool
window.

(1) Toolbar of the tool window

(2) Acquiring images
(3) Adjusting the exposure time
(4) Setting the resolution
(5) Entering an image comment

9
 Acquiring images

(1) Toolbar of the tool window

In the Camera Control tool window, you will find the following buttons. You can
find these buttons in a toolbar, at the top of the tool window.

Perform a white balance on the live-image. You can

White Balance on ROI only perform a white balance when you are working in
your camera's color mode.

To do so, click this button to have a toolbar displayed

Focus Indicator
that supports you while focusing your sample.

Toggle You can select whether you acquire true-color images

RGB/Grayscale mode or gray-value images with your camera.

Clicking this button will enable you to change a variety

Acquisition Settings
of settings for the image acquisition.

Click this button to have its corresponding tool window

Adjust Display displayed. This tool window offers you
possibilities for changing the appearance of the
Adjust Display current image.
When the tool window is on display, this button
appears clicked. You can recognize this status by the
button's background color.

Click this button to see only the live-image on

your monitor. The user interface will be
completely faded out.
Maximize to Screen
To switch off the mode and return to the normal
user interface, press the [Esc] key or click the
Exit Maximized Screen button.

Toggle RGB/Grayscale Some cameras can also be used to acquire gray-value images.
mode
The Toggle RGB/Grayscale mode button looks like this when your camera is in
RGB mode and you are acquiring true-color images. Release this button to
acquire gray-value images with your camera.
The Toggle RGB/Grayscale mode button looks like this when your camera is in
grayscale mode and you are acquiring gray-value images. Click the button to
acquire color images with your camera.

(2) Acquiring images

Acquiring images Click the Live button to switch your camera to the live mode. The live-image will
then be displayed in the image window. The button will change its appearance.
This enables you to immediately recognize that you are in the live mode.
When you are in the live mode, click the Live button once more to end this mode
without acquiring an image. You can change this behavior. To do so, use the
Acquisition Settings > Acquisition > General dialog box.

10
 Acquiring images

Click the Snapshot button to acquire a snapshot. The live mode will then, as a
rule, be exited. The acquired image will be shown in the document group.
Image acquisition - You have several ways of switching to the live mode to acquire snapshots:
several ways
 Use the Camera Control tool window.
 Use either the Acquire > Live or the Acquire > Snap menu commands.
 Use the [F7] key to switch to the live mode.
Use the [F8] key to acquire an image.

(3) Adjusting the exposure time

Use the Exposure group, to set the exposure time.
You can find more The functions in the exposure time control depend on whether you choose to use
information on the
exposure time on page
the automatic exposure time option or to set the exposure time manually. The
12. Automatic option is always set by default.
The exposure time is adopted in the Properties tool window after every image
acquisition. This information will be retained if the image is saved in the TIF or
VSI file format.

(4) Setting the resolution

You can find more Use the Resolution group to set the camera resolution. You can set different
information on the
resolutions for the live-image and the acquired image.
resolution on page 16.
Another way of reducing your camera's resolution, is to reduce the size of the
image area by using a subarray.

(5) Entering an image comment

In the Camera Control tool window, you can enter text in the Note field. This text
will then be saved as a comment along with every image that is acquired.
Note: The text in the Note field isn't saved when you save the current camera
settings. When you close your software, the text in the Note field is deleted.

Viewing a comment After the image has been acquired, you can view and edit the image comment in
the Properties tool window's Document > Note field. To do so, load the image,
and use, e.g., the View > Tool Windows > Properties command to make the tool
window appear.
Displaying the You can have the image comment displayed in the image window, together with
comment in the image
the info stamp.
window
Deleting or editing 1. Go to the Documents group, located in the Properties tool window.
comments for individual
images  You will see that the comment you entered in the Camera Control tool
window has been adopted in the Note field.
2. Click in the Note field.
 When the Note field is active, a small button with three dots will appear.
3. Click this button.
4. Delete or change the comment in the Note dialog box.
6103 20112015

11
 Acquiring images

3.2.1. Switching on the automatic exposure time

Some of the exposure time correction settings depend on the model of camera
that you are using. For this reason, the Exposure group might have a somewhat
different appearance for your camera.
Switching on the The functions for setting the exposure time can be found in the Camera Control
automatic exposure
tool window.
time
Select the Automatic option located in the Exposure group to choose an
automatic exposure time. In the automatic exposure time mode, your system will
continuously calculate the optimum exposure time for the live-image. The
snapshot acquisition always uses the last exposure time that was calculated in
the automatic exposure time mode.
The Exposure group in the Camera Control tool window offers options for
effecting the functioning of the automatic exposure time mode.
Schematic view of the
Exposure group when
the Automatic option is
selected.

(1) Displaying the exposure time

(2) Slow down Auto Exposure
(3) Lock current exposure time
(4) Setting the region
(5) Setting the gain
(6) Setting the exposure compensation

12
 Acquiring images

(1) Displaying the exposure time

The current exposure time is displayed in the Camera Control tool window and
on the Camera Control toolbar.
Visual exposure The visual exposure indicator has the following statuses:
indicator

1) If the left arrow is green, the image is too dark.

2) If the right arrow is green, the image is too bright.
3) If the point in the center is green, the image is optimally exposed.
4) When the left arrow is red, the image cannot be optimally exposed because
even with the greatest exposure time possible it will remain underexposed.
5) When the right arrow is red, the image cannot be optimally exposed because
even with the smallest exposure time possible it will remain overexposed.

(2) Slow down Auto Exposure

Moving the sample can lead to a frequent changing of the brightness in the live-
image, since the automatic exposure time calculation constantly readjusts the
exposure time.
To prevent this from happening, click the Slow down Auto Exposure button. In
this mode, the automatic exposure time will be readjusted more slowly.

(3) Locking the current exposure time

Click the Lock Current Exposure Time button to have the currently calculated
exposure time used for the complete duration of the live-acquisition.
In this way you will switch off the automatic adjustment for the duration of the
acquisition. That means that even when you move to a completely different place
on your sample, the exposure time will not be adjusted accordingly.
The Lock Current Exposure Time button remains active (also for later live-
images) until you switch it off again.

(4) Setting the region

The exposure time is always calculated on the basis of the histogram, that is, the
intensity distribution in the image. From the Region list, select the image segment
that is to be used for the calculation of the histogram and thus of the automatic
exposure time.
Full Image Select the Full Image option so that the histogram of the entire image goes into
the calculation of the exposure time. This method is recommended if the
structures to be observed are equally dispersed throughout the entire image.
Spot Select the Spot option to have the histogram of only a rectangular image
segment calculated. The sizes shown in the Region list depend on the camera
model being used. As soon as you switch to the live-image you will see a white
dashed rectangle that identifies the relevant segment of the image.
With some cameras you can move the rectangle to any position in the image by
dragging it there with your left mouse button pressed, and in this way you will
then influence the exposure time. You can use this method if relevant details are
only visible in a part of the image.

13
 Acquiring images

The appearance can thus be optimized for precisely this segment of the image.
You can also change the image segment you've selected, however you want to,
at a later date.
Click the Center Spot on Image button to have the image segment that is
relevant for the exposure time returned to the center of the image.

(5) Setting the gain

Note: The Gain slide control is not displayed for every model of camera.
Use the Gain slide control when you want to decrease the exposure time without
making the image darker. The more you increase the gain, the shorter the
exposure time becomes. This increases the image noise, however. So, only use
the Gain slide control if it's especially important to have a short exposure time.

(6) Setting the exposure compensation

You can correct the automatic exposure time that has been calculated by your
software. To correct the automatic exposure time, select a value from the
Exposure compensation list.
Should your images be overexposed or appear to be too bright, when you use
the automatic exposure time, select a negative correction value. Should your
images be underexposed when you use the automatic exposure time, select a
positive correction value. Test the effect of the exposure time correction in the
live-image. Should you not want a correction of the automatic exposure time,
select the value 0.
00127 04112015

3.2.2. Switching on the manual exposure

Switching on the The functions for setting the exposure time can be found in the Camera Control
manual exposure
tool window. Select the Manual option. Then you can freely set the exposure
time.
Schematic view of the
Exposure group when
the Manual option is
selected.

(1) Setting and displaying the exposure time

(2) One Time Auto Exposure
(3) Setting the gain

14
 Acquiring images

(1) Setting and displaying the exposure time

Adjusting the exposure There are several different ways of manually setting the exposure time.
time
 To do this, move the slide control.

 Click the [-] and [+] buttons to alter the exposure time in small steps.
 Enter an exposure time in the edit field, then press the [Enter] key.
Displaying the The current exposure time is displayed in the Camera Control tool window and
exposure time
on the Camera Control toolbar.
Visual exposure The visual exposure indicator has the following statuses:
indicator

1) If the left arrow is green, the image is too dark.

2) If the right arrow is green, the image is too bright.
3) If the point in the center is green, the image is optimally exposed.
4) When the left arrow is red, the image cannot be optimally exposed because
even with the greatest exposure time possible it will remain underexposed.
5) When the right arrow is red, the image cannot be optimally exposed because
even with the smallest exposure time possible it will remain overexposed.

(2) One Time Auto Exposure

Click the One Time Auto Exposure button to have the current automatic
exposure time calculated and set for a single exposure while you are in the
manual exposure time mode.
This method of operation makes sense when the image has been absolutely
wrongly exposed and you need an orientation value that you can change at will.

(3) Setting the gain

Use the Gain slide control when you want to increase the sensitivity without
changing the microscope's exposure time or illumination intensity. The more you
increase the gain, the brighter the image becomes.
00128 04112015

15
 Acquiring images

3.2.3. Setting the camera resolution

In the Camera Control tool window, you can use the Resolution group to set the
camera's resolution.
Note: Most cameras can acquire images in various resolutions. Which
resolutions are possible, depends on the camera being used.
Schematic view of the
Resolution group

(1) Setting the resolution for a live image

(2) Setting the resolution for a snapshot
(3) Using the same resolution for live and snapshot
(4) Toggle Subarray

(1) Setting the resolution for a live image

Select the value you want for the live-image's resolution, in the Live list. A
resolution of 1920x1080 means, for example, that the image you acquire will be
made up of 1920x1080=2073600 pixels.
With most cameras you can reduce the resolution in the live-image. The method
will be specified in brackets behind the resolution. The smaller the resolution you
select is, the quicker the live-image will react. Inversely, the spatial resolution in
the image will improve with the increase in resolution.
Binning With binning, pixels on the camera will be combined when they are read out.
Then less data will be transferred from the camera into the software, and the live-
image will be updated more quickly. At the same time, the camera's sensitivity
will increase, and the exposure time will become shorter.
When you select a resolution using the binning mode the term 'binning' will be
added in brackets after the resolution.
Toggle Subarray When you reduce the size of the image area the speed in the live-image will be
increased, just as it is with binning, and you reduce the size of the file. In contrast
to binning, however, when you reduce the image area, the image segment
displayed in the live-image will be displayed in the same spacial resolution, that's
to say, without loss of quality.

When does which resolution make sense?

High resolution In the highest resolution even sample structures that are very near to each other
are still shown separately in an image. This image contains the most information
on the sample. The disadvantage is, that a high resolution reduces the speed at
which the live-image is acquired.
Low resolution for live- If you want to increase the live-image's reaction speed, it can make sense to set
image
a lower resolution. Then less data will be transferred from the camera into the
software, and the live-image will be updated more quickly. Admittedly, a
reduction of the resolution will also always result in less image information being
acquired.
Different resolution for Note: You can set a different (lower) resolution for the live-image than that which
live-image and
you use for the snapshot. That makes sense, for example, when you wish to
snapshot

16
 Acquiring images

have a live-image that reacts quickly, but do not want to accept a loss of image
quality when you acquire a snapshot.

(2) Setting the resolution for a snapshot

Select the value you want for the snapshot's resolution, in the Snapshot list.
If you acquire images with a lower resolution, it will provide you with files that are
smaller. When you use a camera resolution of 1920x1080 an image with the
highest resolution will need approx. 6000 KB of storage capacity, and the same
image with the lowest resolution around 500 KB.
After the image has been saved, its size will be shown in the Size on Disk field in
the Documents group in the Properties tool window.
Display of the The current resolution for the image acquisition is also shown in the function
resolution
group's header in the Camera Control tool window. This ensures that the
resolution is always displayed, even if you minimize the function group and only
the header remains visible.

(3) Using the same resolution for live and snapshot

To use the same resolution for the live-image and the snapshot acquisition, click
the button with the chain icon. Then you only need to set another resolution in
one place.
From that moment on, the Live list will be grayed out. Then you can't set anything
there. The value shown will, however, be updated if you set another value in the
Snapshot field.

(4) Toggle Subarray

Trim the image that your camera acquires (see chapter 3.2.4).
00129 05112015

17
 Acquiring images

3.2.4. Toggle Subarray

The Toggle Subarray button is located in the Camera Control tool window. You
can find the button in the Resolution group. With it you trim the image that your
camera acquires.

Why use the "Toggle Subarray" mode?

You can use the Toggle Subarray mode for a variety of purposes.
Quick live-image With cameras that support this function, the Toggle Subarray mode will lead to a
quicker live-image acquisition. You define a rectangular image segment and the
readout for the live acquisition limits itself to this area. A higher frame rate is
attained because less data needs to be transferred. Please note that you have to
select the Apply to Live check box before you can use the Toggle Subarray mode
on the live-image.
Toggle Subarray - Like with the Toggle Subarray mode, binning (decreasing the resolution)
Binning
increases the speed of the live-image.
As opposed to binning, with the Toggle Subarray mode you will in fact see only a
small sample segment, this sample segment can, however, be displayed with the
greatest possible camera resolution. Use the Toggle Subarray mode when you
need an active-image that reacts quickly for focusing, and you only want to
reproduce very small sample structures.
The binning functions can also be found in the Resolution group.
You can also combine binning with the Toggle Subarray mode. In this way you
will increase the speed in the live-image still further, and also reduce the size of
your files even more.
Trimming the image Sometimes it can be useful to trim an image you have acquired. By using the
Toggle Subarray mode, you can already trim uninteresting borders from your
images when you acquire them, and in this way considerably reduce the size of
your files.
Should you not have set an area for the Toggle Subarray mode before you
acquired an image, but are only interested in a part of this image, you can trim it
at any time by using the Image > Crop command.

Description of the function

Switching "Toggle Switch to the live mode. Click the Toggle Subarray button to switch on the Toggle
Subarray" mode on or
Subarray mode. The button will retain its clicked appearance. You can then
off
immediately see whether the Toggle Subarray mode is switched on or off.
Note: You can only switch the Toggle Subarray mode on and off in the live-
image.
Using the "Toggle The Toggle Subarray mode will, in every case, have an effect on the snapshot
Subarray" mode for the
acquisition. Should you also want to use the Toggle Subarray mode with the live-
live-image too
image, select the Apply to live check box.
Setting the image When the Toggle Subarray mode is switched on, a blue frame in the image
segment for the
window will show the active image segment.
"Toggle Subarray"
mode You can at any time change the image segment that has been selected in the
live-image with the help of your mouse.
Note: The image segment for the Toggle Subarray mode can only be changed
when the Apply to live check box has not been selected.
This is because, when the Toggle Subarray mode has been activated, only a part
of the image will be on display, which means it would only be possible to define a
new image segment within the segment on display, but not outside this segment.

18
 Acquiring images

Specifying the image segment for the "Toggle

Subarray" mode
1. Make the Camera Control tool window appear. You can use the View > Tool
Windows > Camera Control command to do this.
2. Switch to the live mode. To do this, click the Live button.
3. If necessary, clear the Apply to Live check box in the Resolution group.
4. Switch on the Toggle Subarray mode. To do so, click the Toggle Subarray
button.
 In the image window a blue frame will then identify the active image
segment for the Toggle Subarray mode. If you can't see the frame, click
the Toggle Subarray button once more.
5. To change its position, while pressing the left mouse button, take the frame
and drag it into the position you want. Position the image segment for the
Toggle Subarray method over an important image detail.
To change its size, move your mouse pointer over one of its handles. By
dragging the marker, while pressing the mouse button, you can adjust the
frame's size as wished.
6. Click the Snap button to stop the live mode and thus acquire a new image.
 The acquired image will now only show the image segment you have
chosen.
00130 20112015

19
 Acquiring images

3.3. Acquisition Settings

Use the Acquisition Settings dialog box to change a variety of settings for the
image acquisition.
Opening the dialog box To open this dialog box, click the Acquisition Settings button. You can find this
button on the Camera Control toolbar and on the Camera Control tool window's
toolbar.
The dialog box's tree view offers you access to dialog boxes with a variety of
setting options. Select an entry in the tree view to display the respective dialog
box.
Note: The exact functionality in the camera settings depends on the camera
model used. For this reason, the functionality in your software may differ from
that described here.
3987 20112015

3.3.1. Acquisition Settings - Acquisition - General

This dialog box offers you several general options for working with live images.

Live
The live-image will be allocated its own window in the document group. This
window's title will be Live (active). The behavior of this live window depends on
the settings in the Live group.
Closing the live window Select the Close document when Live is stopped option to have the live window
when stopping the live-
image
closed every time you exit the live mode.
When you exit the live mode by clicking the Live button, in this case, no image
will be acquired.
To acquire an image, exit the live mode by clicking the Snap button.
Keeping the live Select the Keep document when Live is stopped option to keep the live-image
window when stopping
when you exit the live mode.
the live-image
When you exit the live mode by clicking the Snap button, the live-image will be
stopped and additionally an image acquired. You will find the stopped live -image
in the image window with the title Live (stopped). You can save the stopped live-
image just as you can every other image.
Note: A stopped live-image is not the same image as the acquired snapshot. This
means that you can set different resolutions for the live-image and the snapshot
acquisition. In that case, a stopped live-image and an acquired individual image
each have different resolutions.
When you exit the live mode by clicking the Live button, the behavior in this
mode depends on the status of the Create new document when Live is started
check box.
Creating a new The Create new document when Live is started check box is only relevant when
document when the live
you've selected the Keep document when Live is stopped option.
mode is started
Clear the Create new document when Live is started check box to have only one
single live window.
When you exit the live mode by clicking the Live button, you will find the stopped
live-image in the Live (stopped) live window. The next time you start the live
mode, the contents of the live window will be overwritten.
Select the Create new document when Live is started check box, if you want to
have a new window opened every time you start the live mode. In this way you'll
prevent the overwriting of the last live window's contents.

20
 Acquiring images

If you finish the live mode now, the stopped live-image is kept in all cases. A new
image document will be created for the stopped live-image.
Use this setting (Keep document when Live is stopped together with Create new
document when live is started) when you want to make a measurement in the
live-image, for example. When you have finished making your measurements,
stop the live-image. You can then save the image you have measured along with
the measurements. When you start the next live-image, you can perform a new
measurement.
Continuing the live Select the Continue live after acquisition check box to only make a pause in the
mode after a snapshot
live mode while you acquire a snapshot. A snapshot acquisition will then create a
new image window, but the window for the live-image will remain active and will
immediately switch back into the live mode.
To exit the live mode, click the Live button, located in the Camera Control tool
window.

Calibration
Choosing the basic unit You can set the basic unit for the X/Y-calibration that is to be used for the image
acquisition. To do so, select the unit you want to use from the Basic Unit list. As
basic units Meters [m] and Inches [in] are available.
When you select another basic unit, all of the images that you from that moment
on acquire will be automatically calibrated in this new basic unit. Now, all values
that apply to the X/Y-calibration will be specified in this new basic unit. These
could be:
 the labeling of the scale bar
 the calibration data in the Properties tool window
 the measurement results when you make measurements on an image
 the labels of the rulers that you can display in the image window. Use the
Options > Images > View dialog box to show or hide rulers. The rulers'
labels only change after you restart your software.

Note: The basic unit for the X/Y-calibration of images you have already acquired
will not be changed, when you alter the basic unit. If you have acquired an image
with the basic unit Meters it will remain calibrated in meters or in a unit derived
therefrom, such as mm or µm. If you wish to measure this image in another basic
unit, use the Image > Calibrate Image... command to alter the basic unit.

Confirm after acquisition

Select the Confirm magnification after acquisition check box to have the Calibrate
Image dialog box automatically opened after every image acquisition.
Use this feature whenever you want to have the possibility to still change the
objective magnification for the image acquisition after switching to the live-image.
In the Calibrate Image > XY-Calibration dialog box, select the Magnification
option. Now, you can choose the objective magnification you have acquired the
image with from the Set magnification > Objective list. This ensures that the
image is properly calibrated.
When you clear the check box you have to select the current objective
magnification, e.g., by clicking the corresponding button on the Microscope
Control toolbar, before you start the image acquisition. Only then is the image
you acquire correctly calibrated.
3029 05112015

21
 Acquiring images

3.3.2. Acquisition Settings - Camera - General

Use this dialog box to make a variety of general camera settings. For example,
you can mirror the acquired image, use a color camera in black & white mode, or
select a color table.

Image type
Bit depth Some cameras can acquire images of different image types. From the Bit depth
list, select the type of image that you want to acquire with your camera. The
image types differ basically in their depth of bit, i.e., the amount of intensity
values that each pixel can adopt.
Which entries are available in the Bit depth list depends on your camera.
Select the 8-bit RGB color entry to acquire 24-bit true-color images.
You can use some cameras in a black & white mode. Select the 8-bit grayscale
entry to acquire 8-bit gray-value images.
Switching between the If you have to frequently switch between the color and the grayscale mode, use
color and grayscale
the Toggle RGB/Gray Scale Mode button. You can find this button on the
modes
Camera Control tool window's toolbar. You can then switch from one mode to the
other with a single mouse click.

Mirror
Horizontal Select the Horizontal check box to mirror the image horizontally during
acquisition. The illustration shows left the source image and right the horizontally
mirrored image.

Vertical Select the Vertical check box to mirror the image vertically during acquisition.
The illustration shows left the source image and right the vertically mirrored
image.

Horizontal and Vertical The two forms of mirroring can be combined with each other. The illustration
shows left the source image and right the horizontally and vertically mirrored
image.

22
 Acquiring images

Pseudo Color
Prerequisite: The Pseudo color group is only available if you selected the 8-bit
grayscale entry in the Bit depth list.
You can use color tables to have images displayed in color on your monitor.
Then every intensity value in an image will be allotted a hue.
Using no pseudo colors By default, when you acquire images, no pseudo color tables will be used. In this
case, the Pseudo Color > Off option will be selected.
Using the saturation Select the Use saturation indicator option to have all of the pixels with a low
indicator
intensity value displayed in blue, and all of those with a high intensity value
displayed in red. This color table being used now only applies to the live-image.
When you acquire this image, it will show all of the pixels back in their actual
color, in the document group.
With this pseudo color table you can immediately see if the image is well
illuminated. When there are both blue and red pixels in the image, the camera's
complete dynamic range will be in use. If too many blue pixels can be seen, the
image is underexposed. If too many red pixels can be seen, the image is
overexposed.
Using a pseudo color You can apply a color table at as early as the image acquisition. To do this,
table for gray-value select the Use pseudo color table for grayscale images option.
images
You can color only the live-image, or only the acquired image, or both, with the
pseudo color table you choose. To do so, select the one of the following entries
For live only, For snapshot only or For live and snapshot from the list.
Selecting the pseudo Click the Pseudo color... button to change the pseudo color table you have
color table
chosen, or to define a new one.
3002 06112015

3.3.3. Acquisition Settings - Camera - Adjustment

Use this dialog box to manually change settings on the camera that affect the
image quality.
Note: Some of the settings are camera specific. For this reason, your camera's
functionality can differ from what is described here.
Changing the image Before you change the settings, you should switch to the live-image, so that you
contrast in the live-
can immediately see how it changes. To do this, use the Acquire > Live
image
command.

Enhancement
Move the Gamma slide control to increase the image contrast either in bright
image areas or only in dark image areas.
When each image acquisition is made, the Gamma value will be adopted in the
Properties tool window. This information will be retained if the image is saved in
the TIF or VSI file format.
You will change the parameters for all color channels simultaneously. If you want
to accentuate or tone down one particular color, use the functions in the
Acquisition Settings > Camera > Color dialog box.

Correction
With the shading correction you can correct the effect of an inhomogeneous
illumination. You can find more information on the shading correction on page 36.

23
 Acquiring images

Select the Off entry in the Shading list, to switch off the shading correction. The
Shading list is only active when correction images are available.
Select the Flatfield entry to switch the shading correction on. With the flatfield
correction you will automatically also activate the dark correction.
It is also possible to switch on the dark image correction only. To do so, select
the Dark current entry.
3001 06112015

3.3.4. Acquisition Settings - Camera - Color

Use this dialog box to change the camera's color settings manually.
Note: Some of the settings are camera specific. For this reason, your camera's
functionality can differ from what is described here.
Changing the color Before you change the color settings, you should switch to the live-image, so that
settings in the live-
you can immediately see how it changes. To do this, use the Acquire > Live
image
command.

White Balance
Prerequisite: You can only change the color settings when you are working in
your camera's color mode.
With the gain value, you can weight the individual color channels separately.
Increase, e.g. the R value, to increase the amount of red in the image.
You shouldn't usually change the gain value manually. Instead, use your
camera's white balance functions. The result of the white balance will be
automatically adopted in the fields of this dialog box.
You can change the gain individually for each color channel. To change a
parameter, move the slide control. R, G and B stand for the Red, Green and Blue
color channels.
Gain value in the image The gain values being used are transferred to the Properties tool window with
properties
every image acquisition. You will find the Gain (White Balance) entry in the
Camera group. This information will be retained if the image is saved in the TIF
or VSI file format.

Device dependent white balance

This setting is only relevant when you want to switch between existing hardware
configurations.
Select the Device dependent white balance check box to automatically save
each white balance performed. When you acquire images later on using a
hardware configuration with a valid white balance, this white balance will
automatically be loaded and applied.
If you have cleared the Device dependent white balance check box, the latest
white balance will be used, regardless of the current hardware configuration.

24
 Acquiring images

Saturation
Note: The Saturation slide control is not available for all cameras.
Note: Activate the live-image before changing these acquisition settings. You can
then watch the changes directly in the live image.
Increase the Saturation value when there is a preponderance of one color in your
images. In this way you will attain a better reproduction of the other colors in your
image. To change the parameter, move the slide control. A change made in the
Saturation will have an effect on all of the color channels.
3003 20112015

3.3.5. Acquisition Settings - Camera - Information

Use this dialog box to look at data pertaining to your camera.
3014 06112015

25
 Working with images

4. Working with images

4.1. Changing the way an image is displayed
An image can be displayed in very different ways in your software's image
window. There are several ways in which you can change the way your image is
displayed.

Enlarging or reducing the size of the image in the

image window
There are several different ways to change your image's zoom factor in the
image window. The current zoom factor is displayed on the bottom right in the
status bar.

On the left, the whole image is displayed in the image window. On the right, the
zoom factor has enlarged the image segment so that it can be viewed in higher
resolution. The scale bar relates to the magnification of the image in the image
window and is adjusted accordingly.
The different zoom options are described in the text that follows.
1. Use the buttons on the Zoom toolbar.
2. Use the Zoom Tool button on the Toolbox toolbar.
3. If you are not in zoom mode: Right click on an image window. In the context
menu you can find several commands with which you can alter the image's
zoom factor.
4. Rotate the mouse wheel to change the zoom factor.
00270 28092015

26
 Working with images

4.2. Processing images

The Process menu offers several image processing functions, with which you
can change an acquired image (for example, increase the image contrast).
1. Load the image you want to process, or activate the image in the document
group.
 Please note that the Process menu will only be visible when an image is
loaded and active in the document group.
2. Use one of the commands in the Process menu, e.g., Process >
Enhancements > Adjust Intensity... .
 The image processing dialog box opens. The image processing
operation that is active will be shown in the dialog boxes header.
3. Click the small arrow next to the Preview button to open a list of all of the
preview functions. Select the Original and Preview entry.
 This preview function displays the same image segment twice in the
dialog box. The first one shown is the source image. The second is the
image that results when the current parameters are used.
 Most of the image processing operations need one or two of the
parameters that are shown in the Settings group.
4. Change the image processing operation's parameters. Decrease the gamma
value and increase the brightness, for example.
 After every change that is made in a parameter, the operation will be
immediately applied to the source image, and the resulting image will be
shown in the preview window.
5. Click the Default button, to readopt the preset parameters in the Settings
group, when the current parameter doesn't make sense to you.
6. When you have found the optimal parameters, click the OK button to have
the active image processing operation applied to the image with the active
parameters.
 The image processing dialog box will closed.
 Please note that the image processing operation changes the source
image. No new image document will be created.
You can, however use the Edit > Undo command to restore the source
image.
 The new image is not automatically saved. The fact that a change hasn't
been saved is indicated by an asterisk shown behind the image name in
the document group.

The source image (left) has low contrast. Adjust the intensity to get a resulting
image (right) with significantly better contrast.
00175 29092015

27
 Working with images

4.3. Performing a white balance on an image

Should an area in your image that ought to appear white look reddish, or is
another color, this means your image has a tinge. In such cases, use the
Process > Enhancement > White Balance... command to perform a white
balance.
1. Load the image you want to process, or activate the image in the document
group.
2. Use the Process > Enhancements > White Balance... command.
 The Filter: White Balance dialog box opens.
3. For the start, define a white point in the image. This should be a point or
surface that is actually white, but isn't displayed in white in the image.
4. For example, click the Select white point using the pipette button.
 The dialog box disappears. The mouse pointer has turned into a pipette.
 The size of the pipette's point is predefined at 3x3 pixels. Alternatively,
you can also use the ellipse or the rectangle to define the white point,
and by doing so, define an arbitrarily large area as the white point.
5. Click a position in the image that you want to define as the white point.
 The position is then marked with a red cross. When that has occurred,
you can still move the point. To do this, move your mouse over the red
cross so that it takes on this shape. Then, while pressing the left mouse
button, you can move the white point wherever you want to.
6. Confirm your choice by clicking your right mouse button, then using the
Confirm Input command in the context menu.
 The Filter: White Balance dialog box reopens.
 In the Interactive white-point definition group, the color values of the
three colors for the defined white point are displayed.
 In the White-point correction factors group, the correction values
determined for each color, are displayed.
 In the dialog box's preview image, you can see what the corrected image
will look like, should you adopt these values.
 If necessary, you can still manually change the white point correction
factors that have been determined. The preview window will then be
immediately, correspondingly updated.
7. When you are satisfied with the preview image, click the Apply button, then
click OK.
 The white balance will be performed immediately. This means that the
colors in the image are adjusted to make the selected white point and all
of the areas with the same color values appear as white. You can see
the results immediately in the image.
8. If you are not satisfied with the results: Please note that you can undo
the white balance by using the Edit > Undo command.
00391 29092015

28
 Working with images

4.4. Working with drawing objects

Various drawing functions are available that you can label your images with.

Inserting drawing objects

Example of use You want to insert an arrow into an image and label it.
1. Load the image that you want to label.
2. Set the zoom factor of the image window to display the image in a size that
makes the label is easy to read. You can rotate the mouse wheel to change
the zoom factor in the image window.
3. Use the View > Toolbars > Drawing command to have the Drawing toolbar
displayed.
Inserting an arrow 4. Click the Arrow button in the Drawing toolbar.
 There is a button for each drawing object on the Drawing toolbar. Click
the button for the drawing object that you want to insert and then define
the drawing object on the image.
5. Keep the left mouse button pressed to draw the arrow.
6. Double click the arrow to open the Drawing Object Properties dialog box.
Make the settings for the arrow's color and line width here. The size of the
head of the arrow is automatically adjusted to the width of the line.
Inserting a text object 7. Click the Text button on the Drawing toolbar.
8. Keep the left mouse button pressed and draw a rectangle. Enter the text you
want.
9. Click once outside the text object to leave the text-entry mode. If required,
select the text object and change the font size or font color.
Showing and hiding 10. You can display the image and the drawing layer together, on your monitor,
drawing objects
or alternatively the image without the drawing layer.
Open the Layers tool window and click the eye icon in front of the Drawing
layer, to hide all drawing objects. Then click the now empty cell again, to
make the Drawing layer reappear.

Two drawing objects have been defined on the image: an arrow and a label for
the arrow.

Selecting drawing objects

Selecting several For certain actions it is useful to select more than one drawing object. This
objects
enables you to copy several drawing objects from one image to another at the
same time, or to format several drawing objects simultaneously.
1. Use the buttons in the Drawing toolbar to create several drawing objects.
2. If necessary, switch to the edit-object mode. To do so, click the Select
Drawing Objects button on the Drawing toolbar. The button will appear
clicked when the edit-object mode is active. You can recognize this status by
the button's colored background.
When the button already appears clicked, you will already be working in the
edit-object mode.

29
 Working with images

3. Use one of the following methods to select a collection of drawing objects.

 Left click an object.
While pressing the [Shift] key, click other objects you want to edit.
Objects you have selected will be identified by white or gray markers.
 While pressing the left mouse button, drag a rectangle around several
objects.
All objects that are completely inside this rectangle will be selected. All of
the other existing selections will be deleted.
 To select all of the drawing objects on the image, use the [Ctrl + A]
shortcut.
Clearing the selection 1. If necessary, switch to the edit-object mode. To do so, click the Select
Drawing Objects button on the Drawing toolbar.
If the button already appears clicked, this means that you are already in the
edit-object mode.
2. There are several ways of clearing a selection of drawing objects:
 Use the Edit > Deselect command.
 Use the [Ctrl + D] keyboard shortcut.
 Click any place in the background of a document with your left mouse
button. The complete current selection will be cleared.
 To deselect individual objects, click an object that has already been
selected.

Determining the default formatting for drawing

objects
1. Insert a drawing object and assign the properties to it that you want to use as
default settings. For example, change the line color from black to red if you
want the lines in your images to be red by default.
2. If necessary, select the drawing object that you want to use as a template for
the formatting.
3. Use the Draw > Set As Default For Drawings command. You can find the
Draw menu on the Drawing toolbar.
 The drawing object that has been selected is now used as a template for
all new drawing objects.
4. Insert another drawing object, e.g., an ellipse.
 The new drawing object will then be formatted in exactly the way you
determined that the first drawing object should be.
00079 20112015

30
 Measuring images

5. Measuring images
5.1. Overview
You can measure distances and angles with your software. All of the results are
saved together with the image.
Prerequisite For making measurements, correctly calibrated images are an essential
prerequisite.
Images that you have acquired with your software will have been automatically
correctly calibrated when you have specified the objective you used.
Should the image not yet have been calibrated, use the Image > Calibrate
Image... command to carry out a calibration.

Starting a measurement
Begin a measurement by selecting the measurement function you want. You can
find the measurement functions in the Measurement toolbar or in the Measure
menu.
As soon as you have clicked a measurement function, your software will
automatically switch to measurement mode. In measurement mode, your mouse
pointer becomes a cross. The button that has been selected in the Measurement
toolbar remains active to identify the current measurement function.
In measurement mode, you can make further measurements with this
measurement function. The measurement mode is valid for all loaded images.
You can, therefore, easily measure numerous images one after the other.
You will remain in this measurement mode until you explicitly switch it off. To do
so, click the Select Measurement Objects button on the Measurement toolbar.

Displaying and saving measurement results

The measurement results are displayed in the image.
Saving the The measurements will be saved along with the image, if you save the image in
measurement results
the TIF or VSI file format. You can edit existing measurement objects at any time.

Editing measurements
Selecting measurement Before you can edit measurement objects, you have to select them. To do so,
objects
click the Select Measurement Objects button, and then select the
measurement object(s). You can find the button on the Measurement toolbar.
Moving measurement You can move a whole measurement object while keeping the left mouse button
objects
pressed.

Increasing/decreasing You can also change the size of a measurement object. Move the pointer onto a
the size of
marker. By dragging the marker while keeping the mouse button pressed, you
measurement objects
can adjust the frame's size as wished.
Deleting measurement Click the [Del] key on your keyboard in order to delete the selected measurement
objects
object or click the Delete Measurement Object button.

31
 Measuring images

Measuring in the live mode

The measurement functions are also available in the live-image. You can
therefore, e.g., quickly measure a segment in the live-image.
Note: The measurements that you perform in the live-image aren't transferred to
the image when it is acquired.
00150 14102015

5.2. Measuring images

You can measure distances and angles with your software.

Measuring image objects interactively

Example of use You want to measure the filaments in a superconductor.
To do this, load a suitable image, or acquire one. Measure the diameter of some
of the hexagonal filaments, between opposite corners.
1. If the Measurement toolbar is not displayed: Use the View > Toolbars >
Measurement command.
2. Acquire an image or load one.

Setting the labeling The measurement results will be written into the image according to the default
color
settings, in a red font and without a background. This can make the
measurement results hard to read on some images. Change the labeling
settings.
3. Use the Tools > Options... command.
4. Click the Measurement > Measurement Display entry in the tree view.
5. Click in the Background Color field and choose a color, black for example.
6. Select the Text color > Fixed colors option and select a suitable color from
the palette. Select the color white to display the measurements in white and
the labels in white on a black background.
7. Close the dialog box with OK.
Measuring lengths 8. Click the Arbitrary Line button. You can find the button on the Measurement
toolbar.
9. Click with your left mouse button at the starting point and end point of the
reference distance.
10. Repeat the measurement several times.
11. Take a look at the results in the image. If the display of the results has been
deactivated, click the Measurement Options button on the Measurement
toolbar and select the Show value check box in the Options dialog box.

32
 Measuring images

 The illustration shows the image with three executed measurements.

The measurement 2 has been selected.

Deleting measurements 13. Press the [Del] key.

 The selected measurement is deleted.
 When a measurement has been deleted, the image contains one
measurement less. The IDs of the remaining measurements won't be
changed by the deletion of a measurement.

Closing the image 14. Click the button with the cross [ x ] to the right of the image name in the
document group.
 You have changed the image because you've added interactive
measurements. For this reason, you'll receive a query whether you wish
to save the image or not.
15. Save the image in the TIF or VSI file format. The measurements will then
also be saved in the image file. They can at any time, be edited deleted or
augmented.
00154 16102015

33
 Configuring the system

6. Configuring the system

6.1. Overview
You should configure and calibrate your system when you start the computer
after installing the software for the first time. Only when you have done this will
you have made the preparations that are necessary to ensure that you will be
able to acquire high quality images that are correctly calibrated.
Process flow of the To set up your system, the following steps are necessary:
configuration
Specifying which hardware is available

Configure the hardware

Calibrating the system

Specifying which Your software has to know which hardware components your microscope is
hardware is available
equipped with. Only these hardware components can be configured and
subsequently controlled by the software. In the Acquire > Devices > Device List
dialog box, you select the hardware components that are available on your
microscope.
Configure the hardware Every microscope is equipped with a different objective. In addition, your
microscope can also have a magnification changer or a camera adapter with a
particular magnification. Use the Acquire > Devices > Device Settings dialog box
to enter your microscope components.
You can also find all of the camera settings in the Device Settings dialog box.
Calibrating the system When you have entered the objectives being used and, if necessary, the camera
adapter's and the magnification changer's magnifications, all of the images that
you acquire with your software are automatically X/Y-calibrated. Use the Manual
Magnification Calibration calibration process if this calibration is not precise
enough.

About the system configuration

When do you have to You should configure and calibrate your system when you start the computer
configure the system?
after installing the software for the first time. If you later change the way your
microscope is equipped, you will need to change the configuration of certain
hardware components, and possibly also recalibrate them.
00159 10082015

34
 Configuring the system

6.2. Configuring the system

Your software requires information about your camera, the objectives, and the
magnification of the microscope's camera adapter in order to be able to acquire
correctly calibrated images. That's why you need to configure the system.
Preconditions Your software has been installed and the camera is connected to the computer.
The camera driver is installed in MS-Windows.

Specifying which hardware is available

1. Use the Acquire > Devices > Device List... command.
2. Select the corresponding entry in the Microscope Frame list.
If you use the camera together with a microscope, select the Manual
Microscope entry. Alternatively, if you use a stereo microscope, select the
Manual Stereo Microscope entry.
If you use the camera together with a macro stand, select the No Microscope
entry.
3. Then, on the Camera tab, select the camera you use.
4. Finally, check if the default settings on the other tabs of the Device List dialog
box are correct for your system or not. If necessary, make the required
changes.
5. Close the Device List dialog box with OK.
 The Device Settings dialog box opens.

Configuring the specified hardware

1. If the Device Settings dialog box is not open, use the Acquire > Devices >
Device Settings... command.
2. Select the Lightpath entry in the Sort by list.
Configuring the camera 3. In the tree view on the left-hand side, expand the Camera > <camera name>
entry (e.g. LC30).
4. Select the Camera Adapter entry.
5. Select your camera adapter's magnification in the Magnification list on the
right. The magnification is imprinted on your camera adapter. Typical values
would be 1.00 or 0.63.
Configuring the 6. Select the General > Manual Nosepiece entry in the tree view.
objective nosepiece
 The current configuration of the nosepiece is shown on the right side of
the dialog box.
7. Check the preset objectives and adjust the values if necessary.
8. Select the objectives with which your nosepiece is currently equipped from
the Magnification lists on the right. Start with the smallest magnification, then
a higher magnification, and so on. The magnification is printed on the
objective itself.
9. Select the corresponding objective in the Objective Type lists. The type is
printed on the objective itself. If your objective type doesn't appear in the list,
select the None entry.
 In the Description field, a description of the objective will be suggested.
You may change the description of the objective in the Description field,
if you wish.

35
 Configuring the system

10. If the objectives don't use air as their refraction medium, select the immersion
medium from the Refraction Index list. In this case, you find an appropriate
label on the objective.
Configuring the Manual 11. Select the General > Manual Zoom / Magnification Changer entry in the tree
Zoom / Magnification
view.
Changer
(if available) 12. Enter one of your magnification changer's magnifications in the Magnification
field and click the Add button.
 All of the magnifications that have been entered will be available on the
Microscope Control toolbar.
Finishing the 12. Close the Device Settings dialog box with OK.
configuration of the
system  You may get a message asking you to check the calibrations. You can
perform the calibration either now or later.
13. Use the View > Toolbars > Microscope Control command to display the
toolbar.
 The Microscope Control toolbar contains buttons with all of your
objectives, correctly color coded.
00156 20112015

6.3. Carrying out a shading correction

6.3.1. Overview
What is a shading Where every optical system with camera and microscope is concerned, the
correction?
sample will, as a rule, not be homogeneously illuminated, even when the
complete system has been carefully set up. This type of non-homogeneous
illumination leads to image flaws, that are called shading. When a shading
correction is employed, these faults in the image will be determined and
immediately corrected in the live-image.
How does the shading For the shading correction you need two correction images, the dark current
correction function?
correction image and the flatfield correction image. Before you can use the
shading correction, you have to acquire these correction images.
What is a dark current The dark current correction image is an acquisition during which no light falls on
correction?
the camera. Here flaws resulting from noise or defective camera pixels can be
corrected by the correction image. The dark correction image is characteristic for
the camera and need only be acquired once. It is valid for all objectives.
What is the flatfield The illumination of the complete optical system without a sample (or with a
correction?
reference sample in the reflected light mode), will be shown on the flatfield
correction image. In addition to the camera characteristics, the microscope's
optical characteristics, especially the objective being used, are incorporated in
the flatfield correction images. Correspondingly, an individual correction image
must be made for every objective and for every resolution setting of your camera.
00187 05012011

36
 Configuring the system

6.3.2. Carrying out a shading correction

Take great care when you acquire correction images for the shading correction
since incorrect correction images can produce artifacts in the resulting images.
The process flow for the actual acquisition will be controlled by a wizard. This
wizard will guide you through the procedure step by step.
1. Use the Acquire > Calibrations... command.
2. Select the Shading Correction calibration process in the Calibrations dialog
box and click the Calibrate... button.
3. Then follow the instructions given you by the software wizard.
00187 10032015
Switching the shading The shading correction should, as a rule, always be switched on. However, you
correction off
can switch off the shading correction when you require a quick live-image to
focus on. The frame rate in the live-image increases when the calculations
required for the shading correction don't need to be made.
In the Camera Control tool window, click the Acquisition Settings button in the
tool window's toolbar.
Select the Camera > Adjustment entry in the tree view.
In the Correction group, select the Off entry in the Shading list, to switch off the
flatfield correction.

6.4. Manual Magnification Calibration

All images you acquire using your software are automatically X/Y-calibrated. Use
the Manual Magnification Calibration calibration process if this calibration is not
precise enough. You need a calibration standard to perform the calibration
process.
How is the preset The X/Y-calibration of an image you have acquired with your software is
magnification
calculated from the size of a pixel and from the total magnification. The total
calibration determined?
magnification at the time of acquisition is usually a combination of the objective
magnification and the camera adapter's magnification. If you are using a
magnification changer, this will also have an effect on the total magnification.
The preset magnification calibration uses the hardware components from the
Device Settings dialog box and the camera's pixel size, which can be read out
from the camera driver.
Starting the Manual Use the Acquire > Calibrations... command. Select the Manual Magnification
Magnification
Calibration entry in the Calibrations dialog box. Then click the Calibrate... button.
Calibration
Returning to the preset When you have manually calibrated your objectives, you can at any time return
magnification
to the preset values. Select the Manual Magnification Calibration calibration
calibration
process in the Calibrations dialog box. Then click the Edit... button. Clear the Use
manual calibration check box in the Manual Objective Calibration dialog box.

37
 Configuring the system

Calibrating the objective manually

1. Place a calibration standard under your light microscope.

2. If you are using a magnification changer, set the magnification changer to a

magnification of 1 and select the magnification 1x on the Microscope Control
toolbar.
3. Start the Manual Magnification Calibration calibration process.
 In the Calibrate objective list all of the objectives that you have registered
with your software are listed.
 All of the objectives that have already once been manually calibrated are
identified by a check-mark .
4. Select the check box in front of each objective that you want to calibrate.
5. Click the Next > button.
 Your software will automatically switch to the live mode.
6. Bring the objective you want to calibrate into the light path. The dialog box
shows the name of the objective.
7. Focus on the calibration standard.
8. Click the Next > button.
9. Click the Set Reference Distance button.
 The image is acquired and live mode is closed.
 The mouse pointer appears in the image window.
10. Determine the starting and end point of the calibration reference distance
with your mouse pointer, for example, a distance of 800 µm. You define
these two points by clicking with your left mouse button.

11. Click the right mouse button and select the Confirm Input command in the
context menu.

38
 Configuring the system

 The Set Reference Distance dialog box opens.

12. First select an appropriate unit .

If, for example, you are using a calibration standard with a length of 1 mm
and 100 scale units, select the unit µm.
 Note: If you select the unit in here, this image will be calibrated in inches.
That doesn't mean, though, that all of the images will now be acquired
with in as the basic unit. You specify the basic unit for the image
acquisition in the camera settings, in the Acquisition Settings >
Acquisition > General dialog box.
13. In the Length field, you enter the length of the calibration standard that you
want to use for the calibration. With a calibration standard that is 1 mm long
for example, you can enter 800 in this field if you have defined the reference
distance between the scale divisions 10 and 90.
14. Click the OK button to confirm the calibration.
15. Click the Next > button if you want to calibrate further objectives, or the
Finish button when you don't have any more objectives to calibrate.
16. Repeat the manual calibration for the other objectives that you've chosen.
 When the last magnification calibration has been carried out, the Manual
Objective Calibration dialog box opens.
17. Check the results and close the Manual Objective Calibration dialog box with
OK.
 In the Calibrations dialog box, the Manual Magnification Calibration
calibration process will then have the Calibrated status.
18. Close the Calibrations dialog box.
 When you then acquire an image, the image's X/Y-calibration will be
calculated with the new magnification values. Please note that before
you make the image acquisition, you will have to select the objective you
want to use, in the Microscope Control toolbar. If you are using a
magnification changer, you will also have to select the magnification level
used.
19. You can view the actual objective magnifications resulting from the manual
magnification calibration at any time.
Select the Manual Magnification Calibration calibration process in the
Calibrations dialog box. Then click the Edit... button.
2134 25092015

39
OLYMPUS SOFT IMAGING SOLUTIONS GMBH
Johann-Krane-Weg 39
48149 Münster, Germany
Phone: +49 (251) 798 00-0
Fax: +49 (251) 798 00-6060
info.osis@olympus-sis.com

510_UMA_LCmicro22_Shinano_en_00_21Sept2017