Takeoff Guide Tutorial to get started with the AxioVision Imaging System

Takeoff Guide Tutorial to get started with the AxioVision Imaging System
Takeoff Guide
Tutorial to get started with the AxioVision Imaging System
(Based on Release 4.8 June 2009)
This Takeoff Guide has been prepared for Carl Zeiss by
Imaging Associates Ltd.
www.imas.co.uk
Copyright © 2009 by Carl Zeiss
Carl Zeiss MicroImaging GmbH
07740 Jena, Deutschland
BioSciences | Industrial | Standort Göttingen
Postfach 4041, 37030 Göttingen, Deutschland
Telefon: +49 (0) 551 5060 660
Telefax: +49 (0) 551 5060 464
E-Mail:
[email protected]
www.zeiss.de/axiovision
How to Use This Tutorial
This tutorial has been prepared to help you get started with your AxioVision imaging system. You are
invited to work through the topics, step by step. Some of the functionality of AxioVision has been
deliberately left out of the guide; the idea is to explain the main trunk and branches of the program
from an application point of view. You are left to explore the twigs on your own. For a description of
the full functionality please refer to the manual or help file accompanying the system.
AxioVision can support various cameras each with their own characteristics, however in software
versions later than AxioVision 4.6 analog cameras are not supported. In this guide only the AxioCam
interface is described in detail.
Tips, shown in italic text, will give hints and short cuts.
Notes, shown in italic text, will give additional information and explanations.
For further information please contact your local Carl Zeiss representation.
Contents
1
Introduction to the AxioVision Environment ........................................... 1
Image Types ............................................................................................................... 2
2
Image Acquisition using the AxioCam Camera....................................... 3
2.1 Getting Started ........................................................................................................... 3
Exposure ..................................................................................................................... 4
Focus .......................................................................................................................... 4
White Balance (applies only to color cameras)........................................................... 5
Acquiring an Image ..................................................................................................... 6
2.2 AxioCam Features...................................................................................................... 6
Resolution ................................................................................................................... 6
Frame.......................................................................................................................... 6
Monochrome or Color ................................................................................................. 7
Black Reference.......................................................................................................... 7
Shading Correction ..................................................................................................... 7
Gain Factor ................................................................................................................. 8
Sharpening.................................................................................................................. 8
3
Saving and Archiving Images ................................................................... 9
4
Image Display and Adjustments ............................................................. 11
Image Size ................................................................................................................ 11
Image Display Properties.......................................................................................... 11
Image Splitter Display Window ................................................................................. 13
Image Gallery View................................................................................................... 14
5
Image File Types and Exporting and Importing .................................... 15
6
Scaling and Scale Bars............................................................................ 16
Changing the scale bar color .................................................................................... 18
Automatic scaling...................................................................................................... 18
7
Annotations .............................................................................................. 19
8
Workflow Customization & Short-Cuts .................................................. 20
9
Backing up your data............................................................................... 22
10 Automation ............................................................................................... 23
11 Interactive Measurement ......................................................................... 24
12 Multidimensional Acquisition (optional) ................................................ 27
12.1 Multichannel ............................................................................................................. 28
Exposure ................................................................................................................... 28
Hardware Settings .................................................................................................... 28
Channel Actions........................................................................................................ 29
Extended Parameters ............................................................................................... 29
Saving Your Experiment ........................................................................................... 29
Acquiring the Images ................................................................................................ 30
Viewing Complex Images ......................................................................................... 30
12.2 Z-Stack ...................................................................................................................... 30
12.3 Time Lapse ............................................................................................................... 32
12.4 General Remarks...................................................................................................... 32
13 Importing Multidimensional Images from another File Format............ 33
14 Image Editor – Change Image Dimensions............................................ 35
15 Glossary.................................................................................................... 36
1
Introduction to the AxioVision Environment
AxioVision is a powerful and flexible software package for capturing, archiving and preparing
images for publication. Whilst AxioVision can perform many complex tasks it can be customized so
that you only see the information and controls you need. Important information in Chapter 8 will
explain how you can set up your system for easy, efficient work flow.
For the purposes of this tutorial we need to set up a standard configuration.
Start AxioVision by double clicking on the AxioVision icon on your desktop
and compare your screen with the picture below.
Note: If you do not see this icon on the desktop then click on the Microsoft© Windows Start button
and look in the Carl Zeiss Vision program group.
Toolbar
Document area
(Images, Forms, Reports)
Workflow
Workarea
If your AxioVision window does not look similar to the
picture above select Options in the Tools menu and
Reset Window Layout, then restart AxioVision.
1
Microscope Configuration Program (MTB2004): AxioVision allows you to control and
read the settings from motorized ZEISS microscopes. Before you can do this you need to tell
AxioVision about your microscope using MTB2004. This has probably been done for you when the
system was installed and is outside the scope of this guide. If you are not using a motorized or
encoded microscope set the microscope to other.
Note: MTB2004 has a Simulate Hardware check box. You can use this to simulate a motorized
microscope i.e. AxioVision will behave as if a motorized microscope was connected.
Referring to the picture on the previous page, the areas you see in the AxioVision window have the
following functions:
Toolbar: these contain groups of functions belonging together e.g. annotation functions. You can
switch toolbars on and off by right clicking in a toolbar and you can drag them around the screen to
"dock" them at the edges or leave them "floating". To keep things simple we have switched most of
the toolbars off.
Workflow: these are groups of functions put together to perform a particular task e.g. capture,
annotate and save an image. You can make up your own workflows to make repetitive tasks easier
to perform. This is explained in Chapter 8.
Workarea: this is a quick way to access frequently used functions and control panels e.g.
microscope control or scaling.
Document area: this is free space for images and information.
: you can hide or show the Workflow and Workarea using the buttons on the toolbar.
Image Types
AxioVision is a very powerful image acquisition environment. Most of the features will be explained
using standard images, i.e. single color or monochrome images. However AxioVision can acquire
more complex image types as follows:
Multichannel: multiple monochrome images which can be pseudo-colored and superimposed,
usually acquired for various fluorochromes or contrast techniques.
Z-Stack: a series of images acquired at different focus positions.
Time Lapse: a series of images acquired over a period of time.
MosaiX: a series of overlapping tiled images acquired over a defined area.
Mark&Find: s series of images acquired at different predefined positions on a sample.
These image types can also be combined e.g. as a multichannel z-stack. The acquisition of
complex images is covered in Chapter 12.
2
2
Image Acquisition using the AxioCam Camera
The first step is to capture an image from the camera attached to your microscope. If you are
working on an AxioVision system which is‘off-line i.e. not connected to a camera, you may want to
skip this section.ccc
AxioVision can acquire images from a variety of cameras; most of these will use a specific interface
called FireWire. All systems supplied by Carl Zeiss will have appropriate connection for the Carl
Zeiss range of cameras. Some older camera types will require specific capture boards to be
installed in your computer. Some use a TWAIN interface which is a piece of software supplied with
the camera and is used to capture the image. You can start the TWAIN interface inside AxioVision
and import the captured image.
The first thing to do is to check that AxioVision is configured for use with the camera you are using.
In the Acquisition menu select the camera.
The AxioCam camera range are very flexible high performance digital cameras. You can use them
for bright field and low light level fluorescence imaging to acquire images at a range of resolutions.
A detailed explanation of the camera technology is beyond the scope of this guide.
Note: Most modern cameras communicate with the computer via a FireWire cable, however, older
cameras such as the AxioCam HRc and HRm Rev.1 and 2 AxioCams communicate with the
computer via a fiber-optic cable to reduce electromagnetic noise effects and therefore have a
separate power supply. You should take care not to kink this cable sharply. If you need to
disconnect the cable at either end then cover the exposed cable ends and sockets with the caps
provided to keep dust out.
2.1 Getting Started
Set up a sample under the microscope and ensure that the light is directed to the camera.
Activate the camera controls by clicking on the relevant camera button in the acquire menu or by
selecting it from the work area. In this instance we will be using an AxioCam HR.
3
Next click on the live image icon on the toolbar to open the live image window. You may or
may not see an image in the live window. Next we need to set exposure, focus and white balance.
For convenience, many of the routine controls such as measuring the exposure and automatic white
reference are accessible from buttons on the toolbar in the live window or click on the live image
properties icon
to do the camera adjustments.
Exposure
The AxioCam range of cameras can adjust to various illumination
levels by varying the exposure time. Ensure that the Auto Snap
box is not checked and then click on the Measure button to let the
camera find the optimum exposure level. You can further adjust the
exposure manually using the top slider control (the range can vary
from 400 μs to 60 s depending on the type of camera). If you find
that image brightness after using Measure is always too dark or too
bright for your purposes you can adjust the level e.g. set to 90% for
a darker image (underexposed) or 110% to overexpose using the
lower slider control. If you have Auto Snap box checked the
exposure will be adjusted automatically before each single snap.
Checking the Auto Live box enables continuous exposure time
adjustment in the live image.
Note: The region of the image used to measure the exposure is the
same as the focus region (see following section). If the focus
window is inactive then the whole image will be used. Therefore,
you may want to position this over the regions of interest before
clicking the Measure button to set the exposure.
Focus
Focusing is often difficult when using high resolution or low light level cameras. Because of the time
required to transfer high resolution images to the computer, or the long exposure times required for
low light levels, it is not possible to update the live image frequently enough. To overcome this, the
AxioCam range of cameras use a standard resolution and electronic gain (rather than a long
exposure) when in live mode. Thus, the live image you see will be relatively low in resolution and
noisy but will be updated frequently enough to allow focusing and stage movement. The final
acquired image will be at full resolution as set in the Frame tab of the camera driver interface (see
page 6).ccc
You can vary the update speed of the live image by clicking on the button on the toolbar
at the bottom of the live image (fast, medium and slow options). Live image quality will
decrease as update speed increases.
Tip: The toolbar above can be used to adjust the display contrast and brightness, see Chap.4.
4
AxioCams also have a focus meter. Right
click in the live image window to toggle
between spot meter/focus bar on/off.
As you change focus the green bar will
move, the red line shows the peak value
reached. The focus meter measures the
sharpness of edges within the green box. To
get the best results you should position the
box over an area containing some structure
or detail, the focus meter cannot work when
the focus box is positioned over a uniform
background area.
Focus meter
Tip: This region is also used to set the
illumination level for the exposure Measure
function.
White Balance (applies only to color cameras)
The color spectrum of the illuminating microscope lamp varies with voltage and hence the color of
the resulting image will also vary as the lamp intensity is changed. AxioCam can automatically
correct for this as follows.
Before
Move to a region of your sample
which contains an area with no
color i.e. white or light gray. Click
on Interactive. As you move the
mouse pointer over the image it will
change to a dropper. Now click on
the area which should be white (or
light gray).
After
This will adjust the camera color balance to show the true colors of the sample. These
settings will remain in effect for subsequently acquired images however you may need to
repeat the white balance procedure if you change microscope settings. Alternatively click on
Automatic in which case AxioVision will search for an area which is close to white and use it
for correction.
The 3200K button will set the camera color balance for use with illumination of this color
temperature. This can also be considered as a default setting and should be used if it is not
possible to do a white balance e.g. in the case of fluorescence imaging where there will not be a
white area.
Tip: You can manually adjust the color offset by using the slider, thus making the image more
yellow (warm) or blue (cold).
5
Acquiring an Image
Click on the Frame tab on the camera control panel.
Set the resolution to 1300x1030 and click on the
icon. For convenience you can also find this on the toolbar
at the bottom of the live window. You can now close the live
window and you should see the high resolution acquired
image.
Tip: Right click in the live image and select Close after
Snap to automatically close the live window after acquiring
an image.
Now that you have successfully acquired an image we can
take a look at some of the other features of the camera.
2.2 AxioCam Features
Resolution
Click on the Frame tab on the camera control panel.
All AxioCams allow images to be acquired at a variety of resolutions i.e. number of pixels. The
AxioCam HR uses patented technology to achieve high resolution and color quality by scanning the
CCD chip.
Choose a resolution to suit your requirements. You may want to capture a very high resolution
image for publication or you may want to save disk space and processing time by capturing a
medium resolution image. Although very high resolution images can be acquired, many people find
that 1300x1030 pixels are sufficient for "every day" requirements.
If you are working with very low emission level fluorescence samples you may find that exposure
times become unacceptably long and sample bleaching becomes a problem. In this case you can
binning. Binning is a method of altering the camera resolution by combining square groups of pixels
(2x2, 3x3 etc) and outputting them as one pixel in the image. The result is a proportional increase in
sensitivity with an associated reduction in resolution. Binning has the added advantage of
increasing sensitivity without increasing noise.
Frame
You may want to capture a region of interest from the camera rather than the full frame.
Click on Refresh Overview to capture the full image into the window on the right, then click on
Select Frame and select a window size. Now move the window around the full image to an area
suitable for focus fine adjustment. You can interactively resize the region of interest.
6
Monochrome or Color
If you are using a color camera you can switch it to monochrome mode by clicking the B/W button.
This is useful when working with fluorescence samples.
Note: Switching a color camera to monochrome mode does not increase its sensitivity.
Monochrome cameras have several times more inherent sensitivity than color cameras as there is
no color mask in front of the sensor chip.
Black Reference
Click on the General tab on the camera control panel.
Your image can be further improved by correcting for the dark
current i.e. the level of signal when no light is reaching the
camera.
Close the light path to the camera (it is essential that no light
can get to the CCD camera) and click on Black Reference.
Now the camera takes a few seconds to acquire an image at the
maximum resolution which is then used to correct subsequently
acquired images. This is only necessary for exposures greater
than approximately 2 seconds.
Re-open the light path to the camera.
Shading Correction
If you acquire an image from a clear field e.g. a clear glass slide, you may find that the image is not
uniform e.g. it may be slightly brighter in the centre of the field than at the edges. This is due to
optical aberrations and variations in illumination. The Shading Correction can correct for this nonuniformity.
Before
Move to a clear part of your
microscope slide and click on
Shading Correction. You
may want to slightly defocus
your microscope to remove
any small specks. When the
Enable box is checked all
images will be automatically
corrected for shading nonuniformity.
After
7
Note: The Shading Correction is correcting for non-uniformity of illumination (shading) across the
image whereas White Balance is adjusting the color.
Tip: If you do not use a clear field to set the Shading Correction any dark objects or features in the
reference image will be seen as bright areas in all subsequent images as AxioVision will try to
correct the apparent non-uniformity. This can give the impression that there is some dust in the
microscope or on the camera surface.
Gain Factor
If you have low light levels and find that your exposure time is unacceptably long you may use
"binning" as described previously, however this sacrifices resolution. An alternative is to apply gain
to the signal using the controls at the top of the General tab. This will increase the sensitivity of the
camera at all resolutions, but in this case, as previously discussed, you will also increase the noise
in the acquired image.
Sharpening
By checking the Enable box you can apply sharpening to your image automatically upon
acquisition. The slider can be adjusted to determine the strength. You can also choose to use the
same sharpening tool as a post processing function from the Processing menu.
Tip: Sharpening will alter the data contained within your image. If you are subsequently going to
analyze your images, we recommend that you do not use this as a post-acquisition function. The
post processing sharpening may be useful to make images look good for publication purposes.
Note: Upon acquisition sharpening is not available for the AxioCam ICc range of cameras from Carl
Zeiss.
8
3
Saving and Archiving Images
If you have worked through the previous chapters you can now capture some images to use as
examples when working through this section. If not example images are on the DVD delivered with
your system. You can open them using the Open Image option in the File menu. In principle any
image can be used, you will see that all common image file formats are supported.
Capture an image from your camera then click on Info View at the bottom of the image window.
The Info View shows information associated with your image. If you have a motorized or encoded
microscope some of the details will have been automatically entered. You can enter further details
as required including specific comments relating to your experiment for example..
Click on Save or Save As in the File menu. The default folder will be the last folder used to save an
image and is likely to be in the My Documents area of your computer. Create a new folder in the My
Documents area called "AxioVision Images" and save your image here. It will be saved by default in
ZVI format. As well as the optimized ZVI format, AxioVision allows the image to be saved as a
Tagged Image File (*.tif), a Microsoft Windows Bitmap (*.bmp) or as a JPEG compressed file (*.jpg)
– you can choose the level of compression required in the JPEG here. You can also choose to
compress the ZVI and TIFF image formats using the compression factor setting in the Save As
window. Very high values generate very small files. However, in this case image quality may be
significantly reduced. There is no compression available for the BMP file format.
Note: TIFF compression is loss-free and the file size is barely affected.
In the three other file formats (TIFF, Bitmap and JPEG) you can choose to either Burn-in
annotations, Apply display mappings and Convert to 8-bit. This will be discussed further in
Chapter 5.
9
Note: The advantage of using My Documents is that it is specific to the user logged on to the
computer and therefore reduces the risk of confusion between users as you won’t see another
user’s My Documents folder.
Once you have saved your image you can close the image window.
Repeat this to acquire and save several images in the AxioVision images folder. Now we
can use the browser to review these saved images. Click the Browser button and
navigate to AxioVision images.
The AxioVision browser will create small thumbnail images. You can then open any image by
double clicking it or by clicking on the Load button.
Refresh
Load
Change View
You can view the information associated with each image by clicking on one of the Change View
buttons – try all three of these to see what they do, use the arrow buttons to change the image. You
can change the information associated with images here.
Images can be deleted using the red cross.
Tip: If you save new images in this folder the thumbnails will not automatically be updated; use the
Refresh button to update all thumbnails.
You can open several images and view them in separate
windows, alternatively maximize one of the image windows and
use the tabs to switch between images.
Use the Gallery button on the toolbar to open a gallery of open images.
Tip: Hit F6 to swap quickly between open images.
10
4
Image Display and Adjustments
In this section we will look at various image display modes and adjustments.
Image Size
Load an image from your images folder.
Right click the mouse in the image to see the context menu. Try
the various image sizing functions to see the effect:
Zoom In / Zoom Out (also F7 and F8).
Auto Zoom: display the entire image inside the display window.
Normal View: displays the image at its true resolution (no
zoom) i.e. one image pixel per screen pixel.
Full Screen: Press the Esc key to exit this view.
*
Try using the Navigator window. This is particularly useful when viewing large images e.g.
those which do not fit on the display area. You will probably want to drag the Navigator window out
to make it a bit bigger, and then move the red square around inside it.
Image Display Properties
You may want to adjust the contrast and brightness of the displayed image. This is particularly
useful when viewing high bit depth images i.e. images with more gray values, as obtained from the
AxioCam camera range.
11
Right click in the image to see the context menu. Select Properties to open the Properties window
and select the Display tab.
You use this window to modify the way in which your image is displayed. The histogram shows the
number of pixels at each intensity level for each color (RGB). Contrast, brightness and gamma can
be controlled via the sliders or by dragging the curve on the histogram.
Click on Linear for the default display curve.
Try selecting Min/Max to stretch the contrast to the histogram maximum and minimum.
Tip: Best Fit is similar to Min/Max but it will stretch the contrast a little further since it ignores
the extreme tails of the histogram. This is set at 5% by default but can be changed.
Close the Properties window when you have finished adjusting the image.
Note: images acquired with a monochrome camera will only have one histogram line relating to the
grey values as there is no RGB data
If you have modified the display properties of an image you will be asked if you want to save the
changes when you close it, but don’t worry you haven’t really changed the image data. The updated
display properties are saved with the image so that it will be displayed in the same way if it is
reopened, but the image data has not been changed and the display properties can still be put back
to linear.
Image Window Toolbar - The toolbar at the bottom of the image window provides shortcut buttons
for zooming and display adjustment.
Note: You can set the display properties for the live mage window. The acquired image will inherit
these properties.
12
Image Splitter Display Window
You can also compare up to 12 images using the splitter window. The
button is on the
standard toolbar or can be accessed via the View menu. In the View menu select Windows and
Splitter Display. With your images already open, click on the Gallery button to select the image for
each pane in the splitter window. Check Synchronize to adjust all of the images simultaneously.
The down arrow allows the number of images compared to be changed.
By clicking on this button an additional window containing some Extended settings is
opened. From this window it is possible to create images containing the selected images
side by side. If the original images are Z-Stack images then the entire Z-Stacks can be
viewed side by side by selecting Create ZStack image. It is the same for time lapse
images, just select Create Time lapse
image. You can also control the spacing
between the images and the color used to
separate the aligned images.
13
Image Gallery View
The Gallery view enables the individual images in a multidimensional image to be viewed
simultaneously.
In the image below, the individual DAPI, FITC and TRITC channels are viewed as the original
monochrome image – the fourth image is the original images pseudo-colored and merged.
By selecting Channel Color, the
individual channels can be pseudocolored – i.e. the DAPI image will
be displayed as blue.
This example is a three channel ZStack image and by selecting ZStack it is possible to view the
merged images at all z positions. If
the example image were a time
lapse then selecting Time Lapse
would allow all time points to be
viewed simultaneously.
By holding down the Shift button
and scrolling the wheel button of
the mouse all images are zoomed
in/out. If an image is selected it
becomes identified by having a
white spot in the centre of the
image. Once selected you can hold
down Ctrl and scroll the wheel
button of the mouse to zoom in on
each individual image at the same
time.
14
It is possible to use Gallery View to edit the
image dimensions (see Chapter 14). In the
Gallery View, a single image can be selected by
clicking on the image – a white spot is
superimposed on the image. Additional images
can be added to the selection by holding down
the Ctrl button and clicking on the required
images. Complete Z-Stacks can be generated by
clicking on the first z position you want and, whilst
holding down Shift, clicking in the last z position
of interest. In this example below all three
channels in six z positions have been selected.
When Extract Selection is clicked the selected
images will be extracted and inserted into a new
image.
If you select Create Image, a new image of the
current view will be produced.
5
Image File Types and Exporting and Importing
So far we have saved and loaded our images as ZVI files. Now it’s time find out how to export the
images in more common file formats which can be used by other imaging and document processing
packages.
A number of different file types or formats have been developed for storing images to disk,
AxioVision supports all major formats. However, its default native format is a new image format ZVI.
ƒ
Why develop yet another image format?
As imaging technology has advanced so have the requirements of users therefore the current
formats are not able to offer the flexibility required. For example, if you annotate your image with a
scale bar and text you need to burn this information into the image when it is stored as a TIFF file,
thus the annotations can’t be removed or changed at a later date. The new ZVI format keeps all
graphic information separately so that it can always be changed and the underlying image is not
destroyed. The ZVI format can also handle the full range of bit depths, multichannel images and
image sequences.
ƒ
How do I export my images in another image format?
If you just want to export a single image then you can simply use Save As in the File menu and
select the file format. However if you want to export multidimensional images it becomes more
complicated as each image in the ZVI file must be exported as a separate image. In this case you
need to use the function described below.
First ensure that the image to be exported is the active document (i.e. click on the bar at the top)
then use Export in the File menu. Refer to the online help for a detailed description of this function,
we will just cover the main points.
15
Insert a Base name and a Save in location – all images
contained within the ZVI will be exported to a file with a
name starting with the base name. If you click Create
project folder a folder will be created with the Base name
which will contain all exported images.
Mode allows you to export some of the image e.g. a
specified channel, time, z position or all images.
Generate merged images will combine multichannel
images as overlaid color images.
File Type - select the output file format.
Convert to 8 bit (only seen when saving as a *.tif) will
convert a higher bit image down to an 8 bit TIFF.
Gray scale will convert a color image to a black and white
image.
Apply display mappings will retain the current display
property settings (see Chapter 4).
Burn in Annotations will merge any graphics into the image before it is saved.
Click Start to export the images.
Tip: To convert more than one image, select File, Export and click on Batch. Choose Add files
type, select the files to be exported then click Run batch.
ƒ
How do I import an image from another format?
AxioVision will open all of the common image file formats mentioned in Chapter 3 simply by
selecting a file using File and Open Image. You can subsequently save the image in the ZVI
format. If you want to import a sequence of images (such as a Z-Stack) then refer to Chapter 13.
6
Scaling and Scale Bars
There are two ways of creating scale factors in AxioVision. Firstly you can manually define and
store scale factors using images acquired of a stage micrometer. Secondly you can use automatic
scaling and allow AxioVision to calculate the scaling factor for you. Here we will discuss manual
scaling first, followed by automatic scaling.
Select Scaling from the Workarea. Here you can select an existing scale and apply it to the
currently open image or activate it in which case it will be applied to all images subsequently
captured.
16
Before we can make a new scale we must acquire an image
of a micrometer scale using the objective lens we want to
calibrate.
Note: Scaling should be done using the standard camera
resolution (approx. 1300x1000 pixels). If you then switch the
camera to a higher or lower resolution a new corresponding
temporary scale factor will be created.
Click Advanced and then New to start the scaling wizard.
If your scale image was active it will appear in the image
window, otherwise select it from the gallery or load it from a
file. Once you have selected your image, click Next.
Now select the Single distance calibration method.
Enter the calibration distance and units.
Draw a line on the image between two
points corresponding to the calibration
distance entered.
Finally enter a name for your scale and it will be entered in the scale list.
Note: The calibration assigned is stored with the image.
17
: click on the scale bar button on the toolbar and drag a line on the image to draw a scale bar
(any length in x or y).
Use this to draw a scale bar on your image of a scale and check its accuracy (remember to apply
your new scale to the image first).
Try loading another image from your archive, apply this calibration and draw a scale bar.
If you want to make your scales available to all users then copy the files from your user data folder
to the workgroup data folder (see chapter 9 for more information about this).
Note: You can calibrate the system independently in x and y directions although this is often not
necessary since the calibration factors will be similar in both directions. However you should confirm
this for yourself on your imaging system.
Changing the scale bar color
Right-click in the image and select the
Properties option and the Attributes tab. Click
on Scale bar and change the text and line color
attributes using the buttons at the bottom of the
dialog box.
If you have drawn several scale bars or other
graphics you can select any one and change its
attributes independently.
Automatic scaling
The size of the pixels in a digital microscope image is determined by the following microscope
components and camera parameters:
•
Magnification of the objective used.
•
In the case of an upright and inverted microscope: magnification factor of the optovar/tube
lens used, or, if applicable, magnification factor of an optovar cube in the reflector turret.
•
In the case of a stereomicroscope: zoom factor.
•
Magnification factor of the camera adapter(s) used.
•
The physical size of the pixels on the digital microscope camera’s CCD sensor.
•
Binning mode used on the digital microscope camera.
AxioVision and the active MTB2004 configuration allow you to take advantage of the benefits of
automatic scaling.
18
If you are using a motorized or encoded microscope with a correct MTB2004 and camera
configuration, AxioVision will know which components have been used and the automatic scaling
calculates the appropriate scale for you. You do not need to do manual scaling.
Automatic measurements are very convenient and generally sufficient for most applications. Manual
scaling can still be done for all microscope types and, in contrast to the “theoretical” automatic
scaling, a measured scaling takes very small manufacturing tolerances into account. If you require
highly precise measurements we recommend working with measured scalings.
Automatic scaling can be turned on and off by navigating to Measure on the Menu toolbar.
7
Annotations
We have already annotated an image by adding a scale bar. You
may want to add further annotations using the options in the
Annotations menu (or switch on the Annotations toolbar – see
Chapter 8)
Select the drawing tool you require and then draw on the image.
Click the right mouse button to finish drawing a shape. If you want
to change or move the graphics then click simply left click on the
item and move it. The colors and fonts for each graphic object can
be set independently by right clicking in the image and selecting
the Properties option and the Attributes tab (see Chapter 6:
adding a scale bar).
Tip: Use Undo and Redo in the Edit menu to correct mistakes.
Note: If you store the image in ZVI format the graphics are stored
as separate objects with the image i.e. they can be changed at a
later date. For further information on image file types see Chapter
5.
Frequent Annotations: Use this option to add annotations to
image sequences e.g. time lapsed since start of a time sequence.
19
8
Workflow Customization & Short-Cuts
Having mastered many of the AxioVision functions you may want to customize the interface to
make repetitive tasks easier and to help out other users who have well defined requirements. The
best way to do this is to hide all unnecessary toolbars (right click on a toolbar) and make a workflow
to suit your application. It’s very easy to do and may save a lot of time in the long run.
You can make many workflows in AxioVision but note that they are specific to the person who is
logged on to Microsoft Windows. This has the advantage that each user can set up AxioVision to
suit their needs. If you want to make a Workflow for somebody else you will need to use their log in
and then start AxioVision before you make the workflow.
In the Tools menu click on My AxioVision.
Select the Workflow tab.
Click on New Workflow and enter Image Capture as the name.
Click on the Image Capture workflow on the left side of the dialog box.
Now we can add tools to the workflow by selecting them from the right hand side and clicking on
Add.
In the example above we have added:
Live from the Acquisition group
Snap from the Acquisition group
Navigator from the View/Windows group
Scale bar from the Annotations group
Save from the File group
You can change the order of the tools on the Workflow using the Move Up/Move Down buttons.
Close the dialog box and you will see the Image Capture
workflow added to the Workflow area.
Workflows can be exported, see Chapter 9 on backing up your
data.
Tip: Further discussion of customization is beyond the scope of
this guide. However you may like to try assigning a shortcut
key to a function using the Keyboard tab in the My AxioVision
dialog box.
20
There is also a built in short-cut to save an image to disk with
an incremental name.
In the Tools menu select Options and then the Naming tab.
Enter an image name prefix.
Select the number of digits for the incremental number
(e.g. 2 = 01-99)
Choose additional designations from the Format-ID list, e.g.
%U will add the Microsoft Windows user name.
In the Tools menu select Options and then the Storage tab.
The folder for Auto save can be typed in (or selected via
Microsoft Windows tree by clicking on
).
In the Tools menu select Options and then the Acquisition
tab. It is possible to keep the settings for the acquired image
or alter the bit depth.
If Auto save is checked, captured images will automatically be
saved in the place identified in the Storage tab, with the name
identified in the Naming tab. We recommend that you save
the images as Carl Zeiss Vision Image (*.zvi) files but you can
automatically export the images to different file formats.
If you assign a keyboard short-cut to the Snap function you
can acquire and save an image with a single key stroke.
21
9
Backing up your data
As with any computer system you need to make adequate back-up copies of your data. This
consists of settings, data and images. You need to know where these are so that you can copy
them to your back-up media e.g. CD-ROM, ZIP disk or network folder. As long as the images are
stored in ZVI format they will contain all of the additional information and scaling information within
the image file. Backing them up is simply a matter of copying the image files to your back-up media,
then you can either copy them back again to restore the data or use the browser to open an image
directly from the back-up media.
By default AxioVision will store your images, data and settings
in the My Documents area of your computer. This has the
advantage that different users can log on using their own log
on names and work independently. However you may want to
make some files available to everyone e.g. scaling, so
Microsoft Windows provides an additional My Documents area
for All Users which is accessible to everyone. However you
can decide to store your images and other data anywhere on
your computer.
To find out where the default folders are click on Options in
the Tools menu and select the Folder tab. You will see that
folders are defined for user data and templates (usually the
user’s My Documents folder) and also for Workgroup data and
Workgroup templates (usually All Users My Documents).
To change folders or to see the full path, click on
Folder info.
or click on
You need to back-up the files in these folders; they contain
scaling calibration files and microscope setting files.
In addition to backing up your images and calibration files you may want to keep a copy of your
customization features i.e. Workflows, short-cut keys, screen layout. You can do this by exporting
your configuration.
Click on Import/Export Configuration in the
Tools menu.
Select Export in the first screen.
Now check all the boxes except the last and click
Next.
Enter a file name.
To import a set-up use the same function but select
Import on the first screen.
Note: The last option (Save to Zip file) creates a
zipped up configuration file. This is useful if you
need to e-mail your configuration to a colleague or
to support staff.
22
10 Automation
If you have a microscope from Carl Zeiss with some encoded or motorized features you can read or
control the settings from within AxioVision however a detailed discussion of these features is
beyond the scope of this guide. You will need to configure the microscope module using the
Microscope Configuration program before you can use these features, usually this will have been
done by the installation engineer.
To control the microscope you have various options:
Click on Microscope in the workarea and use the control panel.
Use the options in the Microscope menu or Microscope toolbar –
these can be left out on the desktop for easy access.
The potential of microscope remote control is realized when we start to save and recall microscope
configurations. This means that we can then construct image acquisition sequences using different
microscope settings e.g. settings for various fluorochromes.
To save a microscope setting:
Activate the Settings Editor in
the Tools menu.
The example highlights a
setting for FITC.
Click New to make a new
setting.
Select microscope components
from the right side and add
them to the group of selected
settings.
button
Click on the Edit
for each selected component to
set it.
In this case the reflector is set to the TRITC block and the internal reflected light shutter is open.
Tip: Be careful about the components you select e.g. we would not usually want to save the focus
motor position as this would always be used when we activated the setting. Generally, select the
minimum number of settings required.
23
When using fluorescence you may find it useful to make a setting which closes the reflected light
shutter to prevent bleaching e.g. called shutter closed. In this case you only need the internal
shutter (in the closed position) in the group of selected settings.
If you want to check your setting, click the Apply button (or Tools, Settings, User) to activate it.
The microscope will then be adjusted according to the setting.
Microscope settings can be added to the workflow (see Chapter 8) for ease of
use.
11 Interactive Measurement
There are various advanced measurement options for AxioVision. This chapter only describes the
standard interactive measurement functions found in the Measure menu. We will use the Interactive
Measurement Wizard.
Open an image.
Click Interactive Measurement from the Measure menu and
then select Configure Tools...
AxioVision provides you with various tools for defining an area
to be measured whether the radius of a circle, the area of a
circle or the distance between two points for example. The
Configure Tools dialog allows you to select measurement
Features for each individual drawing tool.
You can also choose which measurement values are shown
as a graphic overlay by choosing Draw Features from within
the specific feature. Once the settings have been edited, they
can be saved for either individual users or for the workgroup.
24
To use a configuration, select Measure, Interactive Measurement,
to browse the various saved configurations
Load Tools. Click on
and select the one you want to use. Click OK.
Click on select Measure, Interactive Measurement, Start
Measurement. This opens the active image in the interactive
measurement window, as shown on the following page.
Toolbar AxioVision Basic
25
Select a drawing tool, we have indicated the Outline spline tool here. The features chosen in the
configuration step are now displayed.
Outline spline tool
Toolbar Interactive Measurement module
Draw around the features to be measured.
Click OK to close the measurement window. The desired measurement data is now shown on the
image.
The data can be viewed by right clicking in the image and selecting Properties, and then selecting
the Measurement tab as shown below.
Here you have the option to send the data to the clipboard for subsequent pasting into another
application (such as Microsoft Excel).
Below are some typical region measurement descriptions and their meaning.
Area
Area of region
Center X
Centre of gravity x coordinate
Center Y
Centre of gravity y coordinate
Perim
Perimeter
BoundWidth
Width of rectangle drawn around object
BoundHeight
Height of rectangle drawn around object
Diameter
Diameter of a circle with equivalent area
Radius
Radius of a circle with equivalent area
DensMeanR
Mean intensity of red color channel
DensMeanG
Mean intensity of green color channel
DensMeanB
Mean intensity of blue color channel
DensStrR
Standard deviation of intensity of red color channel
DensStrG
Standard deviation of intensity of green color channel
DensStrB
Standard deviation of intensity of blue color channel
The measurement values and graphics will be saved with the image when you close it. If you reopen the image you can view the measurement data by right clicking in the image and selecting
Properties, and then selecting the Measurement tab.
26
12 Multidimensional Acquisition (optional)
AxioVision is a very powerful image acquisition tool allowing multidimensional images to be
captured. Three optional multidimensional acquisition modules are available:
Multichannel: Up to 32 images can be captured and overlaid with a separate pseudo-color
assigned to each channel. This is generally used to capture and combine images of different
fluorochromes or to combine fluorescence and bright field (typically phase contrast) images.
Z-Stack: Images are acquired for a range of equally spaced focus positions. This allows imaging
through a thick section or of a rough surface. Often these images are further processed using
Deconvolution to improve the resolution prior to 3D reconstruction or using extended focus
techniques to create a single image containing the most ‘in focus’ data from the Z-Stack series.
Note: Deconvolution, 3D Reconstruction (Inside4D) and Extended Focus are optional modules
available for AxioVision and a description of their use is outside the scope of this guide.
Time Lapse: Images are acquired at equally spaced time intervals allowing dynamic processes to
be observed and recorded.
These multidimensional acquisition modes can be combined so that we can acquire image data
sets for several fluorochromes through a focus range and for a series of time intervals. They are
then saved as a single file in ZVI format (see Chapter 5).
Note: There is a further multidimensional acquisition option called Mark&Find. This is used with a
motorized microscope stage to move to a series of pre-defined positions prior to image capture.
However a description of this module is also outside the scope of this guide.
Click on Multidimensional Acquisition in the Workarea; the example
below shows all of the options, however, you may only have some of
them available.
The Experiment tab gives access to the general settings and the C, Z
and T tabs contain settings for Multichannel, Z-Stack and Time Lapse
respectively.
The first thing to do is to enter an image name. In this example the first
image set acquired will be called Experiment_1, a second image will be
called Experiment_2 and so on.
Before we look at the other experiment controls we will see how to set up
each of the Multichannel, Z-Stack and Time Lapse modules.
27
12.1 Multichannel
Even if you are not actually using more than one channel you should still review this
section; you will need to set up a single channel to be able to go on and use the Z-Stack or
Time Lapse modules.
Click on the C (Channel) tab to see the Multichannel settings.
Note: You don’t need any microscope automation to use this option.
If this is the first time the option has been used you will see a single
channel (‘1’).
Settings
In this example we have selected DAPI from the list of dyes. If your dye is
not listed you can type any Dye name in this box.
The blue color is associated with DAPI but you can change it if you wish.
Now we can enter a name, in this case My_DAPI. Very often the name will
be the same as the dye, but we may for example have two DAPI filter sets
and want to call them DAPI_Narrow and DAPI_Wide.
Tip: Make sure your AxioVision window is large enough to show all of the
buttons as shown in the example image.
Exposure
This allows us to set the camera exposure time for this channel. You have three options:
Auto – the exposure is calculated automatically before the image is acquired. This will always give
you a well exposed image but the exposure time may be different for each image and a brief
additional calculation exposure is performed before each acquisition which may increase sample
bleaching.
Note: You should not use this mode for Time Lapse if you want to make relative intensity
measurements. Also you should not use when capturing Z-Stacks if you want to subsequently use
Deconvolution.
Fixed – the value in the Time box is used. To find out what this value should be click on the
measure button.
Camera – the current exposure value set in the camera control dialog (see Chapter 2)
Hardware Settings
This section only applies if you have a motorized microscope. First you must set up microscope
settings for the dyes you want to use (see Chapter 10). Now you can attach a setting to be used for
acquisition to this channel e.g. move filter block to DAPI and open the reflected light shutter, and a
setting to be used after acquisition e.g. close reflected light shutter.
If you have the Autofocus optional module installed you can perform an autofocus before each
acquisition. Otherwise the current focus position will be used.
Tip: Click on the Go buttons to check the hardware setting.
28
Channel Actions
Once you have set the channel up you can save it to the channel pool (Save to pool) and
subsequently load it.
Click on Duplicate to make a copy of My_DAPI. You can change the settings here from now on, for
example to set-up a FITC channel.
Tip: You can temporarily inactivate a channel by right clicking on its colored tab. The tab will gray
out and have a cross through it. To re-activate the channel, right-click on the tab again.
Extended Parameters
We have now set up the basic channel parameters however there are more parameters available.
Click on Extended parameters to open the Extended Channel Parameters window shown below.
Here you can select more settings for the My_DAPI
channel we have just made. For example we have an
additional exposure setting which will start a series
with an automatic exposure and then use it
unchanged for subsequent images.
We might want to use these channels again in another
experiment so before you close this window click on
Save to pool to add each channel to the Channel
pool.
If you now click on Channel pool you will open the
Channel pool and you can add any channel from the
pool into your current experiment by clicking on Add
to Experiment.
Saving Your Experiment
Once you have set up your channels you should save your
experiment settings so that you can always repeat this type of
image capture.
Go back to the Experiment tab.
Click on Save and enter a name for your experiment e.g. DAPIFITC.
Now you can click on the Load button to reload this image
acquisition setup.
Note: ReUse allows you to use the experiment settings from an image captured previously.
29
Acquiring the Images
It seems like we have put a lot of work in so far and still not acquired an image, but it will all be
worth it.
If you have a motorized microscope and have used the Hardware Settings to assign microscope
configurations to each channel then all you have to do is to hit the Start button and your automated
multichannel image acquisition will begin.
If you have a manual microscope then select the first channel (e.g.
My_DAPI), set up your microscope for DAPI and click on the Snap button.
Repeat this for each channel.
Tip: You can repeat and overwrite channels if necessary.
You can continue to acquire images and they will be automatically named for
you.
Viewing Complex Images
If you have acquired multidimensional images you will see additional display controls at the bottom
of the image window.
In this example we have a three channel ZStack image. All three channels are overlaid
and we are showing the first image in the ZStack.
You can view different slices using the Z scroll bar or the player buttons to the right.
To view a selection of the channels click on the corresponding colored button e.g. click on button 3
to switch it off. To change the pseudo color for any channel, right click on the corresponding button
and choose your color.
If you click on this button you can view each channel separately without pseudo-color. You
must switch to this mode before you can adjust the display settings as described in Chapter 4.
12.2 Z-Stack
Click on the Z tab to see the Z-Stack settings.
Note: You must have a motorized focus on your microscope to use this option.
30
There are two ways to define the focus range; in the example shown we
have clicked on the Center mode.
Move the microscope focus to the centre of the focus range; this is a
matter for your judgment.
Click on the Center button to enter the current position.
Now decide how many slices you want to acquire and the spacing
between them and enter the values. In our example we will acquire 15
slices spaced at 0.5 μm. This means that the center slice will be number
8 and we will acquire 7 above and 7 below this position.
You can use the Z-Stack navigation controls at the bottom of the
control panel to step the microscope to each position.
Tip: Click this button to see a live image for focusing.
The alternative way to define the focus range is Start/Stop
mode. In this case you should move the microscope to the top
and bottom of the range and then click on the Start and Stop
buttons. These have deliberately not been called ‘top’ and
‘bottom’ as is doesn’t matter whether you enter the top position
as Start and the bottom position as Stop or vice versa.
Tip: If you are combining multichannel and Z-Stack acquisition, AxioVision will normally acquire all
the z images for channel 1 then all the z images for channel 2 and so on. This gives the fastest
acquisition but may suffer from slight z registration problems. However if you check All Channels
per Slice AxioVision will acquire all channels for the first z position and then move on to the next z
position and so on. This will give exact z registration but usually increases acquisition time.
The Optimal distance button will set the ideal slice spacing to match the z
resolution of the optics you are using. This is also known as the Nyquist criteria as it takes into
account the double over-sampling required by this theorem. It depends on the numerical aperture of
the objective lens and the wavelength of the illumination. Therefore, you must have an encoded or
motorized objective nosepiece which has been properly configured so that AxioVision knows which
objective is being used. You must also have entered all the dyes you are using in the multichannel
setup.
Generally you do not need to use this button as it may result in too many slices being acquired but it
is not worthwhile sampling more finely than this setting. However you should use this setting if you
subsequently want to use the AxioVision Deconvolution option to improve the resolution of your
images since double oversampling is a requirement condition for the algorithm.
See "Saving Your Experiment and Acquiring the Images" in the Multichannel section to capture the
images.
31
12.3 Time Lapse
Click on the T tab to see the Time Lapse settings.
Note: You do not need microscope automation to use this option.
In our example shown here, we have chosen to acquire images from 10
time points, one every 15 seconds. Therefore, the total duration of the
experiment will be 135 seconds (the first time point is at t=0).
Alternatively we could click on Cycles in which case we would enter the
interval and total duration and then the number of cycles would be
automatically calculated.
See "Saving Your Experiment and Acquiring the Images" in the
Multichannel section to capture the images.
Note: The interval is measured from start of image acquisition to the start of acquisition of the
subsequent image. If you are acquiring multichannel, Z-Stack or long exposure images at each
cycle you must ensure that the time required to acquire the images does not exceed the interval
between acquisitions.
12.4 General Remarks
Returning to the Experiment tab there are just a couple more things
worth pointing out.
Saving and reloading your experiments makes it very easy for a less
experienced person to use the system. This is particularly useful for
multichannel acquisition; for Z-Stacks you will probably have to reset the
Center or Start/Stop positions each time.
You can temporarily disable the Z-Stack or Time Lapse elements by unchecking the boxes. This is useful to grab a single image as a preliminary
check.
We described how to attach microscope settings to a channel in the
Multichannel section. You can also attach a microscope setting before
starting the experiment and after e.g. you might use a motorized
binocular head to switch the light to the camera before the experiment
and back to the eyepieces after the experiment.
32
13 Importing Multidimensional Images from another File Format
It may be necessary to import images into AxioVision. If the images are single images in any of the
file formats mentioned in Chapter 5 they can simply be opened using File and Open (or Open
Image).
However you may wish to import more complex Multidimensional images. In the File menu, select
Import to ZVI. This wizard allows you to import various file formats in any combination of
Multichannel Fluorescence, Z-Stack and Time Lapse.
Step 1: Setup Image Dimensions
identify the type of image to be
imported (Multichannel
Fluorescence, Z-Stack or Time
Lapse). For the purposes of this
description, all import steps will be
discussed. In Scale lateral (X) box
the number of μm/pixel can be set.
It may be necessary to create a
different y scaling which can be
achieved by ticking the Different Y
scaling box. If the μm/pixel
relationship is unknown, the scaling
dialog box can be ignored but any
subsequent measurements will be
incorrect. Click Next.
Step 2: Channel Setup allows you to choose up to 8 different
image channels in which you can select the dye, assign a color
and name the channel. Additional channels can be added with
Add channel - click Next when all channels have been identified.
Step 3: Z-Stack parameters allows the number of slices and the
distance between theses slices needs to be entered. Alternatively
the number of slices and the Z-Stack height can be used. It is
possible to add Extended parameters, including the
magnification, numerical aperture of the lens and the immersion
medium. Again if any of these parameters are unknown, the
import process can proceed but any future calculations which use
these values will be affected.
Step 4: T Parameter Setup enables you to enter the number of
cycles and either the time interval between each image acquisition
or the time duration of whole experiment.
33
Step 5: Compose Images allows you select the files to be imported. Locate the position of your
stored files and select the images to import. Once all of the images have been selected, click Get
Selected. The selected images now appear in the bottom half of the panel. Click on Consistency
to check that the images are compatible. At this stage it is possible to check the images that will be
imported by viewing them in Preview.
Select Finish at the bottom to import the images:
Once the images have been imported they can be saved as ZVI files via File, Save (or Save as).
34
14 Image Editor – Change Image Dimensions
This command allows you to edit a Multichannel, Z-Stack or Time Lapse image should some
images not be required (for instance a Z-Stack may have extremities with little or no data). Image
Editor is opened from the Edit menu and Change Image Dimensions or by right clicking on an
image and selecting Change Image Dimensions. It is also possible to edit the image dimensions
from the Image Gallery View (see Chapter 4).
Step 1: Choose action selects whether selected images are to be kept or
removed by ticking the relevant box.
Step 2: Channel Selection allows you to choose which
channels you want to remove or keep.
Step 3: Reduce pixel resolution allows you to dramatically
reduce the volume of data when you are working with very
large images, without necessarily losing information that you
need. From the drop-down list box for X, select the desired
reduction factor.
Step 4: Z-Stack selection allows you to remove selected
images from your Z-Stack. If you wish to skip over this step, you
can deselect the Enable this action check box. To select the
desired z plane images either type in the From and To fields
the positions of the relevant images or to view images click on
Get player position and select image/s using the slider. It is
possible to select every “nth” image in the selected range
(meaning that every nth image will be kept/removed).
Step 5: Time Point Selection allows you to remove certain time points. Again this step can be
skipped by deselecting the Enable this action button. The method of time point selection is similar
to the Z-Stack selection. Select Preview and/or the Finish button.
The images generated can be subsequently saved via File, Save (or Save as).
35
15 Glossary
Analog Camera
Camera which produces an analog video signal.
Annotation
Graphics (e.g. text and arrows) drawn on top of the image.
Binning
Technique used to increase the sensitivity of the camera by
adding together groups of adjacent pixels.
Bit Depth
Number of intensity values resolved e.g. 8 bits = 256 levels, 12
bits = 4096 levels, 14 bits = 16384 levels.
Camera Interface
A board inside the computer which reads the image in from a
digital camera.
CCD
Charge coupled device – solid state technology used for camera
sensors.
Channel
Settings for image capture of a specific fluorochrome or contrast
technique.
Clipboard
A Microsoft Windows facility used by many applications to
exchange data.
Context Sensitive Menu
A menu specific to a particular window, usually activated by
clicking with the right mouse button inside the window.
Co-site Sampling
Technique used to improve color image quality by moving the
CCD chip.
Dark Current
Inherent background signal from camera when no light is
reaching the sensor.
Deconvolution
An image restoration technique in which the blurred information
from planes above and below the plane imaged is removed.
Dialog Box
The window associated with a function used to set up its
parameters.
Digital Camera
Camera which directly produces a digital image.
Drag and Drop
Click on an object with the left mouse button, hold the button
down and move to a new location (drag), then release the button
to drop the object in its new location.
Drop Down Menu
List of options associated with a menu item accessed by clicking
on a down arrow to the right of the entry line.
Encoded
An encoded device (e.g. objective nosepiece) can tell the
computer which position it is in, as distinct from ‘motorized’ in
which case the computer can also move the device.
Extended Focus
Software technique in which the sharpest (most in focus)
features from a Z-Stack are combined into a single image.
Fluorochrome
Marker dye which fluorescence.
Form
Template used to display information from the archive on screen.
Frame grabber
Device, usually a single board, which digitizes a video image and
stores it in computer memory.
Gallery
Collection of miniature images which show the contents of all the
image stores.
36
Gamma
Defines the relationship between the intensity values in the
image and the intensity of the display. If Gamma is set to 1.0
then the relationship is linear. Often a Gamma value less than
1.0 is used e.g. 0.5.
Graphics
Drawings and annotation drawn on top of an image.
Graphics plane
Place where the graphics are drawn.
Histogram
Graph of the number of pixels (y axis) against intensity value (x
axis).
Kohler Illumination
Method for setting up the microscope condenser.
Live Image
Direct display of the image from the camera.
Merge
Copy contents of the graphics plane into an image.
Microscanning
Technique used to acquire very high resolution images by
shifting the camera chip.
Multichannel
Several separate images (usually fluorochromes) captured from
the same field within a sample using different filter sets and
overlaid.
Nyquist Theorem
States that to measure a given frequency you must sample the
waveform at twice that frequency, in terms of imaging it means
the pixel size should be half the size of the theoretical resolution.
Overlay
See graphics plane.
Pixel
Single element of a digital image. The value of a pixel sets the
intensity or color of the point in the image.
Pseudo color
Artificial color applied to monochrome images.
Scroll Bars
Controls at the side of an image or window which change the
window view point.
Shading Correction
Correction of an image for illumination or background nonuniformities.
Sync
Synchronization signal. In the case of a monochrome signal, this
is usually added to the video signal (composite sync.). In the
case of a separated R, G, B color signal the sync signal may be
added to the green channel or it may be separate.
Thumbnail
Small copy of an image used in image galleries etc.
Toolbar
Collection of function buttons.
White Balance
Process by which the image from a color camera is adjusted to
give true colors.
White Reference
Background image used for shading correction.
37
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