User manual | AxioCam HR Installation + Reference

AxioCam HR
Installation + Reference - 10.2003
000000-1246-946
Number of this manual: B 48-0020 e
Date of issue: 03.2003
Date of the last revision/update: 10.2003
Carl Zeiss Vision draws the User's attention to the fact that the information and
references contained in these documents may be subject to technical modifications, in
particular due to the continuous further development of Carl Zeiss Vision's products.
The documents enclosed do not contain any warranty by Carl Zeiss Vision with regard
to the technical processes described in the documentation or to certain reproduced
product characteristics. Furthermore, Carl Zeiss Vision shall not be held liable for any
possible printing errors or other inaccuracies in this documentation, unless proof can
be furnished that any such errors or inaccuracies are already known by Carl Zeiss Vision
or that these are not known to Carl Zeiss Vision due to gross negligence and that
furthermore Carl Zeiss Vision has for these reasons refrained from eliminating these
errors or inaccuracies appropriately. Carl Zeiss Vision hereby explicitly draws the User's
attention to the fact that this manual only contains a general description of the
technical processes and information, the implementation of which in any individual
case may not be appropriate in the form described here. In cases of doubt, we
recommend the User to consult Carl Zeiss Vision.
This manual is protected by copyright. Carl Zeiss Vision has reserved all rights to this
documentation. It is prohibited to make copies, partial copies, or to translate this
manual into any other language. Carl Zeiss Vision explicitly draws attention to the fact
that the information contained in this manual will be updated regularly in compliance
with the technical modifications and supplements carried out in the products and
furthermore that this manual only reflects the technical status of Carl Zeiss Vision's
products at the time of printing.
© Copyright 2003 by Carl Zeiss Vision GmbH
Reproductions, copies, and even excerpts may only be made with explicit approval
from
Carl Zeiss Vision GmbH
Zeppelinstraße 4
85399 München-Hallbergmoos
Germany
http://www.zeiss.de
[email protected]
References
Microsoft, Windows, Windows 2000 Professional and Windows XP Professional are
registered trademarks or trademarks of Microsoft Corporation in the U.S. and/or other
countries.
Safety
Refer to the safety notes and instructions in the manuals of all
necessary devices (e.g. microscope peripherals, cameras,
computers, computer additionals, etc.) before installing and
using the software.
Revision / Update Information
27.10.2003
Installation adapted from Product CD 03.2003 to the installation of
the product CD number 15 of October 2003.
Table of Contents
Table of Contents
1 Installation of the AxioCam HR ............................................................ 1
1.1 Safety Regulations....................................................................... 1
General Notes.......................................................................... 3
Backup Copies ......................................................................... 4
1.2 System Requirements / Contents of Delivery ................................ 4
Minimum Hardware and Software Requirements ...................... 4
Recommended Hardware and Software.................................... 5
Contents of Delivery................................................................. 5
1.3 Setting up the Camera ................................................................ 6
Installing the PCI Interface Board .............................................. 6
Connecting the Camera ........................................................... 7
Mounting the camera on the microscope ............................ 8
Connecting camera to PCI interface board........................... 9
Connecting camera to power supply ................................... 9
Power control ................................................................... 10
Extension connector.......................................................... 10
Checking the Setup................................................................ 10
1.4 Installing the Camera Driver ...................................................... 11
General.................................................................................. 11
Installing the Camera Driver with the Hardware Wizard .......... 12
Installing the Camera Driver with the Setup Program .............. 15
How to correct the driver entry in the device manager ....... 18
1.5 Notes on Maintenance .............................................................. 24
Camera Electronic .................................................................. 24
Optical System ....................................................................... 24
Cleaning the Infra-red Barrier Filter ......................................... 24
Important Note on the Use of C-Mount Objectives ................. 24
2 Image Acquisition with AxioCam HR ................................................. 25
2.1 General..................................................................................... 25
2.2 Quick Guide to the First Image with AxioVision.......................... 26
The Workflow Standard ......................................................... 27
Step by Step to the First Image ............................................... 28
2.3 Adjust Property Page................................................................. 33
Exposure................................................................................ 34
Automatic exposure .......................................................... 35
White Balance........................................................................ 36
AxioCam HR Installation + Reference
i
Table of Contents
Color Adjustment................................................................... 37
Histogram.............................................................................. 37
2.4 Frame Property Page ................................................................. 39
Camera Mode........................................................................ 39
Resolution......................................................................... 40
Binning............................................................................. 42
Size of the resulting files ................................................... 43
2.5
2.6
2.7
2.8
Frame .................................................................................... 43
General Property Page............................................................... 45
Digital Gain............................................................................ 45
Image Orientation .................................................................. 46
Black Reference...................................................................... 46
White Reference .................................................................... 47
Shutter Control ...................................................................... 48
Filter Operations..................................................................... 49
Practical Notes on Operation in AxioVision................................. 50
Speed of the Live Image ......................................................... 50
Notes on Optimum Color Reproduction in AxioVision ............. 51
Information for Displaying 14 Bit Images in AxioVision ............... 52
Recommended Camera Settings ............................................. 54
Recommended Monitor Settings ............................................ 54
Background Information............................................................ 55
Gamma Setting...................................................................... 55
Linearity................................................................................. 55
Exposure Times for Microscanning Resolutions........................ 55
Fluorescence Images .............................................................. 57
Configurable Toolbars for Operating the Camera.................... 57
3 Trouble Shooting ................................................................................ 58
3.1 Software................................................................................... 58
3.2 Image Quality ........................................................................... 59
4 Technical Data ..................................................................................... 60
4.1 AxioCam HRc (color-version) ..................................................... 60
4.2 AxioCam HRm, Vers. 2.0 and Later (black/white-version)............ 62
4.3 Spectral Sensitivity..................................................................... 64
ii
B 48-0020 e 10.2003
Installation of the AxioCam HR
1
Installation of the AxioCam HR
The AxioCam HR is a professional highly-resolution digital camera with
additional integrated scanner technology for universal application in the field
of light microscopy.
There are two versions available: AxioCam HRc (color camera) and AxioCam
HRm (b/w camera).
To make it easier for you to set up the camera, please follow the instructions
in these chapters step by step.
If the power supply is connected to the mains and to the camera the LED on
the top of the camera is redn when the computer is started. After the first
initialization (starting the software) the color of the LED changes from red to
green. During continuous image acquisition the color of the LED changes
between red (exposure) and green (ready).
NOTE:
In the description that follows the term AxioCam HR is used for both the
color camera and the black/white camera. A distinction is only made
where this is necessary for technical reasons.
1.1
Safety Regulations
Please read this chapter carefully and observe the regulations in
order to ensure your safety and the intended operation of the
system. Please observe the warnings and notes printed in this
manual and on the unit.
The AxioCam HR CCD camera has been manufactured and tested by Carl
Zeiss according to the regulations specified in TÜV, GS and cUL and has left
the manufacturer’s premises in perfect working order.
In order to ensure that this condition is maintained and to avoid any risks
when operating the system, the user must comply with any notes and
warnings contained in this manual.
AxioCam HR Installation + Reference
1
Installation of the AxioCam HR
Exemption from statutory liability for accidents
The manufacturer shall be exempt from statutory liability for accidents should
the operator fail to observe the safety regulations.
Limitation of liability
No warranty shall be assumed by Carl Zeiss during the warranty period if the
equipment is operated without observing the safety regulations. In any such
case, Carl Zeiss shall be exempt from statutory liability for accidents resulting
from such operation.
Exemption from warranty
Carl Zeiss shall be exempt from any warranty obligations should the user fail
to observe the safety regulations. Carl Zeiss only guarantees the safety,
reliability, and performance of the system if the following safety regulations
are closely observed.
The electrical installations of the room where the system is to be set up
must conform to IEC requirements.
Warning
Any interruption of the PE conductor, either internally or externally, or
removal of the earthed conductor will make the system unsafe to use.
Any intentional interruption of the earthed conductor is illegal.
Attention
To interrupt the power supply simply disconnect from the mains.
Setup, expansions, re-adjustments, alterations, and repairs must only be
carried out by persons who have been authorized by Carl Zeiss.
Use only those cables supplied by Carl Zeiss.
Do not allow any cables, particularly power cords, to trail across the floor,
where they can be snagged by people walking past.
2
B 48-0020 e 10.2003
Installation of the AxioCam HR
General Notes
Please ensure the notes described below are adhered to when setting up and
operating the AxioCam HR camera.
All connectors must be firmly and securely attached.
Please protect all camera cables and the fiber optic cable from excessive
heat (e.g. halogen lamps, microscope fluorescence illumination).
Please prevent the fiber optic cable from being bent in a radius lower
than 50 mm.
Only use the camera in a clean and dry location. If voltage is applied in an
environment with over 70% humidity, the Piezo actuator insulating
resistance may be reduced.
The camera and its power supply must be protected against mechanical
impact. External damage may affect the operation of inner components.
Keep chemicals and fluids away from the camera. To avoid the risk of fire,
do not use near inflammable liquids or gases.
Make sure there is sufficient ventilation of the camera head and its power
supply. Avoid direct exposure to sunlight and locations near to heat
sources (radiators, stoves). Overheating can cause noisy images.
The camera housing can be cleaned using normal microscope cleaning
material, but never use compressed air.
If a repair is necessary send the camera including the power supply with a
detailed description of the fault to:
Carl Zeiss
Service Mikroskopie
Abt. MS-LO2
Königsallee 9-21
D-37081 Göttingen
AxioCam HR Installation + Reference
Tel.: (++49) 551 5060 428
Fax: (++49) 551 5060 325
eMail: [email protected]
3
Installation of the AxioCam HR
Backup Copies
We strongly recommend that all users save the data they create, such as
images, measurement data, archives, reports, forms and documents, at
regular intervals on an external medium. Otherwise it cannot be excluded that
access to this data may be lost as a result of operational errors or hardware
defects. Carl Zeiss accepts no liability for consequential damage resulting from
insufficient data protection.
1.2
System Requirements / Contents of
Delivery
Minimum Hardware and Software Requirements
Pentium III processor 600 MHz or faster
256 MB RAM
20 GB hard disk (minimum)
1280x1024 bit graphics adapter for real color
CD-ROM drive
One free PCI slot (PCI Local Bus Specification Rev. 2.1 or higher)
17" Multisync monitor
Windows 2000 Professional SP3 or Windows XP Professional SP1
AxioVision software release 4.1 (or higher)
AxioCam HR camera driver
Microscope adapter 1x or 0.63x
4
B 48-0020 e 10.2003
Installation of the AxioCam HR
Recommended Hardware and Software
Xeon workstation 2 GHz or faster
1 GB RAM
120 GB hard disk
1280x1024 bit graphics adapter for real color
CD-ROM drive
One free PCI slot (PCI Local Bus Specification Rev. 2.1 or higher)
17" Multisync monitor
Windows 2000 Professional SP3 or Windows XP Professional SP1
AxioVision software release 4.1 (or higher)
AxioCam HR camera driver
Microscope adapter 1x or 0.63x
Contents of Delivery
AxioCam HR camera head
PCI interface board
Fiber optic cable (data transfer between camera and PCI interface board)
Power supply
Power cord for mains connection
CD-ROM with device driver for AxioVision
This Installation Guide
AxioCam HR Installation + Reference
5
Installation of the AxioCam HR
Camera, PCI interface board, fiber optic cable, power supply, power cord
1.3
Setting up the Camera
Installing the PCI Interface Board
Static electricity may damage electronic components. Make sure
you discharge any static electricity by grounding yourself to the
chassis of the device before touching electronic components.
Never touch the contacts of electronic components. In addition, it
is advisable only to work on antistatic sheets.
PCI interface board
6
B 48-0020 e 10.2003
Installation of the AxioCam HR
The PCI interface board requires the space and power of a single 32 Bit PCI
slot. PCI interface boards do not require any configuration.
For special instructions on how to open your computer and
information about the configuration of the slots, please refer to
your computer documentation.
Connecting the Camera
After closing the computer please connect the AxioCam HR's fiber optic cable
to the PCI interface board.
Fiber optic cable, PCI interface board, protection caps
The AxioCam HR consists of these components:
Camera head with C-Mount thread for microscope or objective
Power supply
PCI interface board
In order to operate the camera all the interfaces above must be connected.
AxioCam HR Installation + Reference
7
Installation of the AxioCam HR
Connection of the AxioCam HR's components
Mounting the camera on the microscope
Please use a 1.0x or a 0.63x C-Mount adapter to mount the camera on the
microscope's TV port.
Possible adapters (example):
Port 44
Port 60
1.0 x
No. 452995 (44 C2/3" 1.0x)
No. 456105 (60 C2/3" 1.0x)
0.63 x
No. 452997 (44 C2/3" 0.63x)
No. 1069-414 (60 C2/3" 0.63x)
For the Axiovert 25 please use adapter 451268.
The adapters are not supplied with the camera.
For mounting on the microscope please remove the protection cap from the
camera's C-Mount interface. Screw in the adapter until you reach the end of
the thread. Then put the camera on the microscope's TV port. Please ensure
that no dust drops into the camera's opening.
Never touch the surface of the IR barrier filter or the CCD sensor
with your fingers. If you remove the camera from the microscope
please close the C-Mount opening immediately using the
protection cap supplied in order to avoid dust contamination.
8
B 48-0020 e 10.2003
Installation of the AxioCam HR
Connecting camera to PCI interface board
When plugging and unplugging please hold the fiber optic cable's
connector by the connector case only, in order to avoid damage.
Please remove the protection caps from the camera's fiber optic socket
and from one end of the fiber optic cable. Plug in the fiber optic cable
until you hear an audible click. Please note that the fiber optic cable's
connector has a groove preventing the wrong orientation of the
connector.
Protect all cables from the microscope's hot light sources.
Please keep these protection caps in a safe place for later
transportation or storage of the camera in order to prevent
damage or dust contamination of the fiber optic transmission line.
Connecting camera to power supply
Connect the cable of the power supply to the camera.
The camera and power supply are designed for continuous operation. The
warming-up of the camera body during operation is harmless and does
not in any way affect the function of the camera.
The power supply is automatically adapted to the local main voltage in a
range from 110V/AC to 240V/AC.
The camera and power supply do not have an on/off switch. We
recommend that you use a switchable mains connector or disconnect the
camera cable at the back of the camera.
The mains socket must be close to the camera's power supply and easily
accessible. The mains plug may only be inserted into an earthed socket. The
earthed conductor must not be interrupted through the use of an extension
cord.
AxioCam HR Installation + Reference
9
Installation of the AxioCam HR
Power control
If the power supply is connected to the mains and to the camera the LED on
the top of the camera is red. After the first initialization the color of the LED
changes from red to green.
During continuous image acquisition the color of the LED changes between
red (exposure) and green (ready).
Extension connector
The camera has a fibre optic cable connector and also another wrep
connection. An external shutter can also be opened and closed by trigger
output signals.
Checking the Setup
Please check the following connections:
The camera is mounted on the microscope and sufficiently illuminated.
The PCI interface board is installed in the computer.
The camera is connected to the PCI interface board via the fiber optic
cable.
The 12V output of the power supply is connected to the camera.
The power supply is connected to the mains.
If the LED on the top of the camera is not now red, please check
all the points above.
Please start your computer now.
10
B 48-0020 e 10.2003
Installation of the AxioCam HR
1.4
Installing the Camera Driver
In order to operate the AxioCam HR the AxioVision software is needed.
First insert the PCI interface board into your computer and then install
the driver software.
General
Firstly we would like to emphasize a number of important points:
Allow sufficient time for installation. As a rule this will take just over five
minutes, although in exceptional circumstances considerably more time
may be necessary. The actual time you need will depend on the current
status of your computer's Windows operating system.
Carl Zeiss's image processing products have been developed using the
most up-to-date software tools and methods. This means that you must
first update your computer's operating system to this same standard to
ensure that things will run smoothly. The files to be installed are
contained in the enclosed installation program and are standard Microsoft
products or updates.
For some of the Microsoft update components the installation instructions
are often only available in English. If you are installing a Carl Zeiss product
in English then this presents no problems. If, however, you are installing a
Carl Zeiss product in German or another language, then the language will
change during installation if, for example, a Microsoft update component
is installed.
During installation you will have to answer a sequence of questions with
YES or NO. We have kept such questions to a minimum. Take sufficient
time to read through the questions carefully and only then decide which
answer applies.
When installing the components it is possible that some of the files to be
installed may already exist. You are informed if this is the case. You are
then asked if you want to retain or overwrite the existing files. Always
select the answers suggested by the installation program.
AxioCam HR Installation + Reference
11
Installation of the AxioCam HR
Installing the Camera Driver with the Hardware
Wizard
NOTE:
For installation on a computer running under the operating system
Microsoft Windows 2000 Professional and Windows XP Professional, you
need administrator rights. Enter the system using a user name with
administrator rights, or consult your system administrator.
The installation procedure is the same for Windows 2000 Professional and
Windows XP Professional, but is displayed in this Installation Guide with
screenshots of Windows 2000 Professional.
Read this chapter when you have just entered a new PCI interface board in
your computer. If you want to update the camera installation or if you have
interrupted the driver wizard, follow the instructions in chapter "Installing the
camera driver with the setup program".
After inserting the PCI interface board and restarting your PC, the following
dialog box Found New Hardware Wizard will be displayed. Make sure that
during all the steps described below, the CD-ROM containing the AxioCam
HR driver remains inserted in the CD-ROM drive of your computer.
The wizard detects new hardware in the system and searches for new drivers.
Click Next.
12
B 48-0020 e 10.2003
Installation of the AxioCam HR
Select the option Search for a suitable driver for my device
(recommended). Click Next.
Activate the check box CD-ROM drives. Click Next.
The following dialog window shows the actual path of the AxioCam HR
driver. Click Next.
AxioCam HR Installation + Reference
13
Installation of the AxioCam HR
Under Windows 2000 Professional this dialog window is displayed.
Click Yes to continue.
Under Windows XP Professional the Hardware Installation dialog window is
displayed. In this window click Continue Installation.
The last dialog window of the hardware wizard is displayed.
Click Finish to complete the installation.
14
B 48-0020 e 10.2003
Installation of the AxioCam HR
Installing the Camera Driver with the Setup
Program
NOTE:
For installation on a computer running under the operating system
Microsoft Windows 2000 Professional and Windows XP Professional, you
need administrator rights. Enter the system using a user name with
administrator rights, or consult your system administrator.
In this chapter the correction of the driver entry is described with
Windows 2000 Professional screen shots, but the procedure is similar
under Windows 2000 Professional and Windows XP Professional.
After you have inserted the CD into the CD drive installation starts
automatically.
If the automatic start function has been switched off on your system
(Autorun) you must start the installation program manually in the following
way:
Double-click on the desktop icon My Computer
Double-click on the CD-ROM Drive icon
Double-click on the entry setup.exe
After the start procedure (automatically resp. manually) this dialog window
will be displayed on your monitor:
AxioCam HR Installation + Reference
15
Installation of the AxioCam HR
Select the language you want to install (English or German) and click OK.
Follow the instructions of the setup program until the window is displayed,
where you can customize your setup. In this window you can also choose to
read the ReadMe files.
It is recommended that you carefully read the ReadMe file. This file provides
complementary or up-to-the-minute information concerning AxioCam HR
hard- and software. The text window can be closed by selecting this
Window's
symbol.
Set the entry "Cameras and Framegrabbers" - "Carl Zeiss AxioCam" to This
feature will be installed on local hard drive. Check the other settings and
continue by clicking Next.
Under Windows 2000 Professional this dialog window will be displayed during
installation of the AxioCam HR.
Click Yes to continue.
Under Windows XP Professional the Hardware Installation dialog window is
displayed. In this window click Continue Installation.
16
B 48-0020 e 10.2003
Installation of the AxioCam HR
The system prompts for a restart, depending on the used operating system.
After the restart the installed driver will be activated.
Start the systems Device Manager to check the installation:
Click Start
Settings
Control Panel.
Double-click the System icon. The System Properties dialog will be
displayed.
Select the property page Hardware and here the Device Manager
button. Now double-click the entry Imaging devices to check the entry
Carl Zeiss AxioCam HR.
AxioCam HR Installation + Reference
17
Installation of the AxioCam HR
How to correct the driver entry in the
device manager
NOTE:
In this chapter the correction of the driver entry is described with
Windows 2000 Professional screen shots, but the procedure is similar
under Windows 2000 Professional and Windows XP Professional.
If the entry in the Device Manager must be corrected, i.e. if the PCI interface
board was entered as Multimedia Video Controller, proceed as follows.
Please make sure that during all the steps described below, the CD-ROM
containing the AxioCam HR driver remains inserted in the CD-ROM drive of
your computer.
Start the systems Device Manager:
Click Start ⇒ Settings ⇒ Control Panel.
Double-click the System icon. The System Properties dialog will be
displayed. Select the property page Hardware and click the Device
Manager button.
18
B 48-0020 e 10.2003
Installation of the AxioCam HR
Double-click on Other devices and select the entry Multimedia Video
Controller.
The property page General opens.
Click the Reinstall Driver... button.
AxioCam HR Installation + Reference
19
Installation of the AxioCam HR
The Upgrade Device Driver Wizard starts.
Click Next.
Select the option Search for a suitable driver for my device
(recommended). Click Next.
20
B 48-0020 e 10.2003
Installation of the AxioCam HR
Activate the check box CD-ROM drives. Click Next.
The following dialog window shows the actual path of the AxioCam HR
driver. Click Next.
Under Windows 2000 Professional this dialog window is displayed.
Answer the question by clicking Yes.
AxioCam HR Installation + Reference
21
Installation of the AxioCam HR
Under Windows XP Professional the Hardware Installation dialog window is
displayed. In this window click on Continue Installation.
The last dialog of the upgrade procedure will be displayed.
Click Finish to complete the installation.
22
B 48-0020 e 10.2003
Installation of the AxioCam HR
The last dialog of the installation will be displayed.
Click Close to complete the installation.
The entry in the Device Manager is now Imaging devices
AxioCam HR.
Carl Zeiss
The system will be restarted and the installed driver for the AxioCam HR will
be activated.
AxioCam HR Installation + Reference
23
Installation of the AxioCam HR
1.5
Notes on Maintenance
Camera Electronic
Camera, interface and power supply are maintenance free.
Optical System
The internal optical components of the camera should always be protected. If
no lens or TV adapter incorporating optics is screwed into the camera's CMount thread, the camera's sensor and IR-filter must be protected by
screwing the protective cap into the camera's C-Mount thread.
Cleaning the Infra-red Barrier Filter
Contamination of the infrared filter has an adverse effect on the quality of the
resulting image (dark points, cloudy structures in the image). If there is dry
dust on the front side of the infrared filter, you can remove it with a soft
brush or with cotton (wool) after unscrewing the lens or TV adapter.
Important Note on the Use of C-Mount
Objectives
The camera can be mounted onto microscopes with standard TV-adapters
with C-mount connections. Carl Zeiss supplies the camera complete with a
pre-mounted infrared barrier filter, which offers the optical advantage for use
on microscopes of a lower sensitivity to dust.
The IR barrier filter is situated 5 mm behind the outer edge of the C-mount
opening in the camera.
Due to this pre-mounted IR barrier filter, C-mount objectives that
screw more than 5 mm into the thread cannot be screwed into the
camera.
24
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
2
2.1
Image Acquisition with AxioCam HR
General
The AxioCam HR is a professional highly-resolution digital camera with
additional integrated scanner technology for universal application in the field
of light microscopy.
Two versions are available: The AxioCam HRc (color camera) and the
AxioCam HRm (black/white camera).
The color camera is suitable for all situations where stained or naturally
colored samples need to be acquired with very high levels of quality.
The black/white version of the camera has been optimized in particular for
acquisition when light intensities are low, e.g. for fluorescence images. In
these cases it is important for the widest possible spectral response and the
highest possible sensitivity to be achieved. Due to the elimination of the color
filter mask, there is no need for the IR filter usually required with color
cameras.
The integrated, high-precision scanner technology gives you the option of
increasing the spatial resolution of the AxioCam HRc in several steps up to the
limits of the optical capabilities: from the sensor’s basic resolution of 1300 x
1030 pixels up to 3900 x 3090 pixels per color channel (with the HRm up to
4164 x 3120 pixels can be achieved).
The 14-bit digitization of the sensor signals ensures an optimum combination
of spatial definition and outstanding resolution of intensity levels.
The camera’s very low level of background noise (supported by active Peltier
cooling of the sensor), and possible exposure times ranging from 1 ms up to
600 sec, make it suitable for an extremely wide range of applications in the
field of microscopy.
AxioCam HR Installation + Reference
25
Image Acquisition with AxioCam HR
2.2
Quick Guide to the First Image with
AxioVision and AxioCam HR
If you are using several cameras on your system you must always make the
AxioCam HR camera the active camera before proceeding.
Start AxioVision by double-clicking on the corresponding symbol.
Select from the Acquisition menu the Select AxioCam HR function.
The image acquisition can also be easily done via the toolbar. Open the
corresponding menu by clicking on
.
The text below describes how to achieve your first image in AxioVision with
just a few clicks of the mouse.
The Standard workflow is the easiest way to control image acquisition. If the
workflows are not displayed, in the View menu select the Windows
function, and there select the Workflow command.
NOTE:
The workbar may in fact be open but covered by the work area. If that is
the case, simply click on the Workflow tab at the bottom edge of the
work area
26
.
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
The Workflow Standard
Live starts and closes the live window.
Show live window properties opens and
closes the window for adjusting the display
characteristic curve and for controlling the
camera (exposure time, white balance etc.).
Snap acquires an image using the active
camera.
Show Properties adapts the image display
using a characteristic curve and shows the
parameters of the image.
Scale bar inserts a scale bar.
Annotations inserts a caption and scale bar
into the acquired image.
Navigator opens the Navigator window for
zooming.
Save saves the image.
As the icons are identical on all menus, the following description applies both
to operation via the toolbar and to the workflow.
AxioCam HR Installation + Reference
27
Image Acquisition with AxioCam HR
Step by Step to the First Image
Select the camera you want to use (if
only one camera is installed, this step
is not necessary).
Set the light path to the camera.
Then click on the live image icon.
The live window is opened to display
the camera image.
This function can also be called up via
the Acquisition ⇒ Live menu.
Now focus the camera image and
select the frame you want to acquire.
In the footer you can select a suitable
scaling for the objective you are using
from the List of available scalings.
Clicking on the gamma icon in the
bottom line of the live image
accomplishs the optimum color
reproduction.
Click on the icon Show live
window properties to open the live
properties.
28
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
Here you can see the settings for the display of the image like Brightness,
Contrast and Gamma. Settings made here are passed on the acquired image.
NOTE:
Only the display is adapted, the camera data remain unchanged.
AxioCam HR Installation + Reference
29
Image Acquisition with AxioCam HR
Make changes for the camera settings on the following controls:
The central element is the setting for
the exposure time on a digital
camera. The Measure button can be
used to determine an optimum
exposure time. Check the quality of
the exposure in the live image.
The arrow keys and sliders can be
used for fine adjustment of the
exposure time.
If you are using a color camera, you
need to perform a white balance for
the image. If you select the automatic
balance (Automatic button), the
camera tries to determine an
optimum value itself.
Enter further settings for the selected
camera on the Adjust, Frame and
General property pages.
Click on the camera icon to acquire
an individual image, or in the
Acquisition menu click on Snap.
Click on the Show Properties icon
and activate the Display tab. Any
changes you make here are displayed
in real time in the image window.
30
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
Move the sliders
Gamma
Contrast
Brightness
up or down to adjust the image.
Good color reproduction on a
monitor is achieved with a gamma
value of around 0.45.
To find the optimum setting for
contrast and brightness, click on
Min/Max.
To undo all your changes, click on
Linear.
You can use this icon to insert a scale
bar into your image. The appropriate
scaling must have been generated
and selected in advance.
This icon is used to open the window
for generating annotations.
Using the Navigator you can magnify
or reduce the image and position the
displayed image area in the overview
window.
AxioCam HR Installation + Reference
31
Image Acquisition with AxioCam HR
Captions and markings can be
inserted using the Annotations
window.
Click on this icon to save the image
you have generated to the
computer’s hard drive.
NOTES:
Once you have found a good camera setting, you can save it with the
Settings Editor in the Tool menu and load it again automatically in the
future. This allows you, for example, to acquire particular samples under
the same conditions every time.
To allow correct scalings to be selected in the footer of the live image
during acquisition, you need to have generated these before you start
your work. If you are using a motorized or encoded microscope, the
scalings can be allocated automatically. For further information see
Chapt. 9 "Configuration".
32
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
2.3
Adjust Property Page
This property page contains a summary of the most important camera
settings, which are constantly used during operation.
The property page is splitted in the fields Exposure, White Balance (with
color settings) and Histogram (for the display of an image histogram).
AxioCam HR Installation + Reference
33
Image Acquisition with AxioCam HR
Exposure
The camera’s exposure time can be adjusted to the lighting conditions on the
microscope using a slider or by entering an exposure time directly into the
input field. When entering the exposure time manually you can state the value
range directly using the abbreviations "ms" for milliseconds and "s" for
seconds.
Exposure time measurement
The program supports you in calculating an optimum exposure time, at which
the camera will be maximally saturated without being overexposed. To
perform this calculation, click on the
button.
The exposure time calculated is available for subsequent images. It can be
changed at any time by moving the slider or by entering a new exposure time
manually.
This measurement function uses all the image information from the camera
sensor for the exposure measurement as standard (integral measurement).
NOTE:
More detailed information to use the live image you can find in Chapt.
2.6 "Practical Notes on Operation in AxioVision" - "Speed of the live
image".
Weighting of exposure time measurement
During an exposure time measurement an attempt is made to expose the
camera in such a way that the sensor is not overexposed in any area of the
image. The value calculated therefore corresponds to 100% of the camera’s
maximum exposure range.
The exposure time measurement can also be adjusted to particular lighting
conditions. If under certain acquisition conditions the exposure time
measurement does not achieve satisfactory exposure of the areas of the
image in which you are interested, it is possible to influence the result of this
measurement by adjusting the weighting.
34
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
A setting of 200% means that 200% of the exposure time set in the display
window or measured by pressing the appropriate button is used for actual
acquisition.
A setting of 50% means that 50% of the exposure time set in the display
window or measured by pressing the appropriate button is used for actual
acquisition.
The setting range is between 5% to 500%.
EXAMPLE:
A thick sample is acquired in bright field. The empty image background is
much brighter than the darker sample. The measurement produced an
exposure time of 50 ms, at which the background would not be overexposed
during acquisition. Setting a correction factor of 200% extends the effective
exposure time to 100 ms, which ensures that the darker area of the sample is
exposed more brightly. The brightest area of the image, which is not of
interest, is overexposed here by 200%.
Automatic exposure
The software sets the exposure automatically for each individual shot when
the check box Automatic is activated. This makes it possible to generate
acquisitions with the correct exposure without having to enter manual
presettings. For each individual acquisition the correct exposure time is
calculated and set in advance.
can be pressed at any
The exposure measurement button
time. This is useful if you want to update the exposure time for a current live
image. If you deactivate Automatic, the program will continue to use the last
setting calculated.
NOTE:
In this mode it is not possible to edit the exposure times in the
multichannel mode for the individual channels.
AxioCam HR Installation + Reference
35
Image Acquisition with AxioCam HR
White Balance
In order to achieve a color display which is neutral and close to reality it is
necessary to balance the individual color channels against one another. For
this, you need to use the pointer to click in the live image on an area of the
microscope’s specimen, which is white, neutral and illuminated as evenly as
possible. The scene must not be overexposed.
Clicking on White Balance automatically starts up a live image. It is
important to set the exposure correctly before carrying out the white balance.
This can be carried out via automatic exposure measurement, for example.
If the measurement cannot be performed because of unsuitable lighting
conditions an error message is issued. In this case the amount of light must be
reduced or increased accordingly.
icon allows you to switch back to the basic white
The
balance setting. This setting is optimized for a halogen light temperature at
3200 K.
NOTE:
Setting the white balance to 3200 K generally leads to image
reproduction that is closest to the impression of the image in the
microscope’s eyepiece. Note that it is recommended to set the gamma
curve for the image display on a value of 0,45 (please find more
information in Chapter 2.6 "Practical Notes on Operation in AxioVision" "Display characteristic curve").
Clicking on the
button starts an attempt to automatically
adjust the white balance. This requires the presence of sufficient neutral-tint
areas in the image.
NOTE:
With fluorescence samples or image areas that are completely filled, this
automatic method can lead to color distortion. In this case use the 3200 K
standard setting or the manual color adjustment function.
The color setting is only available for the color camera AxioCam HRc and
not for the AxioCam HRm.
36
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
Color Adjustment
Clicking on the buttons
and
allows you to change the weighting
of the individual color channels manually. Using this method a white balance
you have generated can be subsequently changed. Each click on the arrows
shifts the weighting of the colors in the direction indicated. These changes
can be observed immediately in the live image and in the histogram display.
NOTE:
Further information on setting optimum color reproduction in AxioVision
can be found in Chapter 2.6 "Practical Notes on Operation in AxioVision"
- "Notes on optimum color reproduction in AxioVision".
A setting found using this method can be saved as a setting with a name of
your choice and subsequently reloaded. Activate the corresponding
parameters (WhitebalanceWhiteRed, WhiteBalanceWhiteGreen,
WhiteBalanceWhiteBlue) in the menu Tools - Settings Editor.
Histogram
A histogram indicates the intensity distribution of the image signal in the live
image and the acquired image. It provides a clear representation of the
brightness distribution in the current image. The horizontal axis corresponds
to the scale of possible intensity values from dark to light and the vertical axis
shows the number of pixels calculated from the current image which possess
this intensity value on the x axis.
With the color camera the value distribution is displayed separately for each
color channel in three lines (red, green and blue).
If an image is overexposed, at least one curve on the histogram is cut off at
the right-hand edge of the display area, where it forms a maximum. Reduce
the exposure time or insert gray density filters into the microscope’s beam
path to reduce the amount of light. It is also possible to reduce the lamp
voltage of the microscope illumination. However, this changes the basic color
of the illumination (color temperature) and requires a new white balance.
AxioCam HR Installation + Reference
37
Image Acquisition with AxioCam HR
If an image is underexposed, the histogram does not reach the right-hand
edge of the display area. As a result optimum use is not made of the camera’s
resolvable gray value range. You should therefore increase the exposure time
or the amount of light in the microscope.
Log: By clicking on this button it is possible to influence the histogram display.
You can toggle between a linear scaling and a logarithmic display of the
signals represented in the histogram.
The logarithmic display makes it possible to view small amplitudes and high
peaks at the same time in a single overview. Otherwise, because of the
automatic scaling of the display, small signal values are often not visible in the
diagram.
Skip: This function can be used to influence the automatic scaling of the
histogram. If an image contains an extremely bright glare or extremely dark
features the result will be overexposure or underexposure of the image in
these areas. All of these pixels possess the maximum / minimum numerical
value which can be displayed (e.g. in an 8-bit image 255 for overexposure or
0 for underexposure). Consequently, for the gray value 255 the histogram has
to display an extremely high number of pixels with the same value. As the
scaling of the histogram curves is automatically adjusted to this high value,
smaller values can no longer be resolved in the display.
By using the Skip function the maximum values at the upper and lower end
of the histogram are omitted during the scaling of the display.
38
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
2.4
Frame Property Page
The Frame property page can be used to select a frame for the image
acquisition and is splitted in the fields Camera Mode and Frame.
Camera Mode
RGB and B/W: Two types of AxioCam HR are produced; one with a CCD
sensor with a color mask on the chip (AxioCam HRc), and the other with a
black and white sensor (AxioCam HRm).
AxioCam HR Installation + Reference
39
Image Acquisition with AxioCam HR
With the color variant (AxioCam HRc), you can use these setting options (RGB
or B/W) to determine whether the camera should supply a color or a black
and white image. This may be desirable for multichannel fluorescence images,
or to reduce the amount of data. The camera will always acquire a color
image, which is then converted to a black and white image.
In RGB mode only odd Binning modes, in B/W mode only even Binning
modes are available. This is caused by the structure of the color filters on the
pixels.
AxioCam HRm works always in the b/w mode. All Binning modes are always
available.
Resolution
Optical adaption of the camera
The possibility of selecting the camera resolution is an important feature of
the AxioCam HR. It allows the camera’s image resolution to be adapted to suit
the possibilities of the microscope's optical system.
One of the camera’s special features is increased resolution with microscanning allowing high color quality to be retained. This means that the loss
of resolution necessitated by the color mask on the CCD sensor can be fully
compensated for.
The camera has a 2/3" CCD sensor with dimensions of 8.8 mm x 6.6 mm.
This corresponds to an image diagonal of approx. 11 mm.
To optimize the size of the camera’s field of view a camera adapter with a
magnification factor of 0.63x is recommended. In this case approx. 42% of
the intermediate image is acquired with a microscopical field of view of
20 mm in diameter. The optical resolution of the microscope objective is
optimally suited to the resolution of the camera.
The color quality is (with concurrent drastical increased resolution),
comparable with that of 3-chip CCD video cameras, without the need for an
expensive beam splitter.
40
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
In the color mode of the AxioCam HRc the following resolutions are available:
260 x 206
432 x 342
1300 x 1030
1300 x 1030
2600 x 2060
3900 x 3090
3900 x 3090
Binning mode 5x5
Binning mode 3x3
Single image, color interpolation
Microscanning
Microscanning
Microscanning
Fast Scan
The "1300 x 1030" resolution is available twice. The first option is generated
via a single image with subsequent color interpolation. This method is useful if
recording conditions are changeable, e.g. if the sample bleaches out with
fluorescence or if specimens are moving. The second option with microscanning generates significantly higher image quality, which is comparable to
that of a camera with three corresponding sensors. The three color channels
will be captured exactly on each image area.
In opposite to the above mentioned microscanning resolution the "3900 x
3090 Fast Scan" resolution is a compromise between the scan position and
the acquisition speed: a quadruple accelerated acquisition speed will be
reached by a reduction of the scan position from 36 to 9 positions, which
means to abandon on the accurate color scan (color co-site-sampling).
In the black and white mode of the AxioCam HRc only the following image
resolutions are available:
1300 x 1030
650 x 514
324 x 256
Interpolated image
Binning mode 2x2
Binning mode 4x4
For the AxioCam HRm (black and white camera) the following image
resolutions are available (from camera version 2.0)
260 x 208
364 x 260
462 x 346
694 x 520
1388 x 1040
2776 x 2080
4164 x 3120
Binning mode 5x5
Binning mode 4x4
Binning mode 3x3
Binning mode 2x2
Single image
Microscanning
Microscanning
AxioCam HR Installation + Reference
41
Image Acquisition with AxioCam HR
NOTE:
With the AxioCam HRm the omission of the color filter mask on the
CCD sensor and the associated generation of monochrome images offers
the following advantages:
•
The acquisition speed in scanning modes is increased by a factor of
four, as fewer positions have to be located.
•
The light sensitivity is increased.
•
The visible spectrum for the camera expands, because no IR barrier
filter is required.
•
Even single-frame exposures produce images with the full basic
resolution of the CCD sensor.
•
The quantity of image data is reduced by a factor of 3 with the same
spatial resolution, as no RGB color channels are acquired.
Binning
The process of Binning ensures that the information in neighboring pixels, in
both a horizontal and vertical direction, is summarized on the camera's
sensor. This increases image brightness in accordance with the number of
pixels summarized.
However, this process also reduces the sensor's spatial resolution accordingly.
Since the amount of data to be transferred is reduced, the potential image
rate is increased.
EXAMPLE:
In the case of 2x2 binning, out of 1400 x1000 pixels 700 x 500 pixels remain.
42
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
Size of the resulting files
The size of the resulting files depends on the choice of resolution and gray
value scaling.
The following table gives an overview:
Image size
1300 x 1030
2600 x 2060
3900 x 3090
3 x 14 bit
8 MB
32 MB
71 MB
NOTE:
The images of the AxioCam HRm are reduced in size by a factor of three,
as only a monochrome channel is present.
In AxioVision, images in 14 bits per pixel or in 3 x 14 bits per pixel mode can
be saved in ZVI image format. When exporting the images to other image
formats, (TIF, BMP, JPG etc.) this option is not available. With these image
formats the image data are converted from 14 bit to 8 bit data in accordance
with the graduated display characteristic curve.
Frame
In this field you can specify whether the frame or the entire area of the sensor
is used for the acquisition.
Refresh overview: This button makes it possible to insert an overview image
into the area where the frame can be positioned. This makes it extremely easy
to target your specific area of interest.
In the displayed overview image a frame can be positioned by using the
mouse, which corresponds to the selected frame of the acquired image. The
shape and size of the frame can be changed by clicking on and dragging the
edges of the frame while holding down the left-hand mouse button. It is also
possible to move the frame by clicking inside it and keeping the button
pressed as you change its position.
AxioCam HR Installation + Reference
43
Image Acquisition with AxioCam HR
Center: By clicking on Center you can automatically center the selected
frame. This can be useful as interesting parts of the sample are usually moved
to the center of the view area. In addition the center of the view area is the
place of the highest optical quality.
Framestart / Framesize: The start point and the selected frame size will be
displayed in these input fields. Alternatively you can enter the values manually.
It is also possible to select default frame sizes from the drop-down list box and
position it in the center of the image by using the Center button.
Memory used: Memory used shows the memory size of the acquired image,
which depends on the camera resolution, the image size of the selected
image frame and of the selected gray value scaling.
44
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
2.5
General Property Page
On this property page you enter the basic settings for the camera.
Digital Gain
Using the slider it is possible to change the interpretation of the brightness of
the image data received. This allows you to display dark images as very bright
images, which means you can acquire images with very high contrast using
short exposure times.
AxioCam HR Installation + Reference
45
Image Acquisition with AxioCam HR
The disadvantage of this technique, however, is that noise is greatly
intensified, and the potential dynamic range of the corresponding image data
is reduced. You should therefore carefully weigh up the benefits of using
digital signal gain before actually employing the technique. For this reason the
recommended value is gain by a factor of 1.
From a technical viewpoint, this technique involves multiplying the data by a
corresponding factor. The maximum numerical pixel value generated is always
limited to the maximum value of the analog/digital converter you are using
(14 bits per pixel per color channel in the case of the AxioCam HR).
NOTE:
You can also achieve a bright image display with short exposure times by
making the display characteristic curve steeper.
Image Orientation
An AxioCam HR image is adjusted in such a way that the image orientation
corresponds to the image in the eyepiece when mounted on an upright
microscope with the front of the camera facing you.
This starting position can be adapted via this menu to suit your requirements.
If, for example, the camera’s cable routing needs to be changed on an inverse
microscope, the camera can be mounted at a different angle, without the
correspondence between the image orientation and the eyepiece being lost.
Black Reference
Setting a black reference ensures that the image background retains a
uniform darkness, even with exposure times in excess of several seconds. This
setting is therefore particularly recommended for the acquisition of weak
fluorescence or other images with weak light intensity and consequently long
exposure times (starting from approx. 3 seconds).
NOTE:
Perform this black-value calibration approx. 15 minutes after switching on
the camera, as the camera will then be in a thermally balanced condition.
46
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
For this calibration process make sure that the camera is viewing a completely
black image. For example, close the light path to the camera on the
microscope. In exceptional cases it may be necessary to remove the camera
from the microscope for this calibration process and to cover the sensor with
the cap provided.
Calibration takes approx. 20 seconds. At the end of this period it will continue
to be available even if the software is restarted. Under certain circumstances it
may be necessary to repeat this calibration process from time to time.
NOTE:
The black reference is needed to correct deviations in the dark current for
individual pixels. This measure is additional to the camera’s available
Peltier cooling, and allows a uniformly dark and even image background
to be achieved, even with long integration times.
Activating the black reference can reduce the maximum possible speed of
the live image.
White Reference
Activate this setting to compensate a decrease of image brightness to the
borders of the view area.
For this calibration process ensure that the camera is exposed to a completely
empty image. To do so place the sample to a completely empty position of
the slide.
NOTE:
Use a clean slide, a clean objective and defocus your microscope in order
to avoid the footprint of dust particles. Ensure that the microscope is
properly adjusted (Köhler illumination).
After changing the objective a white reference must be done.
Activating the white reference can reduce the maximal possible speed of
the live image.
AxioCam HR Installation + Reference
47
Image Acquisition with AxioCam HR
Shutter Control
Via a supplementary cable the AxioCam HR offers a TTL control pulse, which
can be used, for example, to control a mechanical shutter (e.g. "Uniblitz") in
synchronism with the camera’s exposure time.
As soon as this output is activated you can select the polarity that you want to
set for this signal:
High active means that the Control Signal jumps from 0V to 5V when the
camera’s exposure begins and returns to 0V when exposure is complete.
Low active means that the Control Signal jumps from 5V to 0V when the
camera’s exposure begins and returns to 5V when exposure is complete.
Please carry out configuration for this procedure in accordance with the
requirements of the device you have connected.
NOTE:
Simultaneous control of the external Uniblitz shutter by AxioVision via a
serial interface and by the shutter control of the camera is resulting in the
following behavior:
•
Shutter closed via AxioVision → than AxioCam HR shutter control.
•
Shutter open via AxioVision → than AxioCam HR shutter control does
not work.
Acquisition Delay: Due to the inertia of the masses being moved, a
mechanical shutter requires a certain amount of time to change from the
closed to the open position after the control signal has been initialized. To
make sure that when the sensor is exposed it does not record this transition
state and is not thereby exposed unevenly it is possible to delay the start of
actual acquisition. Please set a value here in accordance with the data for the
device you have connected.
48
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
Filter Operations
NIR-Mode: The designation "NIR" stands for "Near Infrared". If you activate
this mode, it is possible to achieve a further increase of up to 20% in the light
sensitivity of the sensor in the range of red and near infrared wavelengths
(from approx. 650 nm). However, this makes the CCD sensors more
susceptible to overexposure (so-called "blooming"). We recommend that you
use this mode for particularly weak fluorescence samples in this wavelength
range, to reduce the exposure times required.
NOTE:
This mode is only available for the black/white camera AxioCam HRm
from product version 2.0 onwards.
Enhance Color: Activating this check box enables you to switch the color
optimization algorithm on or off. Under normal circumstances this check box
should always be activated in order to obtain vivid color reproduction during
image acquisition.
If it is deactivated the noise in the image is reduced and the speed of the live
image increased.
NOTE:
For the Gamma curve a value of approx. 0,45 is recommended as a
setting for the image display (please read also the Chapt. 2.6 "Practical
Notes on Operation in AxioVision" – "Display characteristic curve").
AxioCam HR Installation + Reference
49
Image Acquisition with AxioCam HR
2.6
Practical Notes on Operation in AxioVision
Below you will find a few practical notes on the speed of the live image, color
adjustment, working with exposure times, fluorescence images and
configurable toolbars.
Speed of the Live Image
There are three speed modes available for the AxioCam HR live image. If you
use the pointer to click on the
icon at the bottom edge of the live
image, a pop-up menu with the possible setting options appears. The current
setting is displayed at the bottom edge of the live image.
Each mode has its own particular compromises.
Live mode
Description
Fast
Greatly reduced resolution, fast frame rate
Medium
Medium resolution, moderate frame rate
Slow
High resolution, slow frame rate
The image rate can be further increased by deactivating certain AxioVision
features:
Deactivating the focus bar.
Deactivating the live image scaling (1:1 representation of the image).
Activating a frame for the live image (Size) on the camera property page
Frame.
Set image orientation to original on the General property page (no
rotation).
NOTE:
Note that the above mentioned notes are only valid for short exposure
times, as for very long exposure times the speed of the acquisition is
mainly dominated by the exposure time.
50
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
For very long exposure times, the live image will appear grainy, as
automatic amplification is active. However, this will not influence the
quality of the final image.
Notes on Optimum Color Reproduction in
AxioVision
Below you will find a detailed description of how best to set up AxioVision to
achieve optimum color reproduction with the AxioCam HR.
NOTE:
For an optimum color reproduction the monitor first has to be adjusted.
To do so please use a well-suited image.
Adjusting the display characteristic curve in AxioVision
In order to achieve optimum reproduction of color images of the AxioCam
HRc data on a monitor, the display characteristic curve must be used to set
gamma compensation. The advantage here is that only the display is
influenced and the actual data remain unchanged. Further details on this can
be found below. The settings can be done as follows:
With the mouse click on the icon
in the footer of the live image.
This adjusts the display characteristic curve automatically for an optimum
color reproduction of the monitor (gamma = 0.45).
In the Properties dialog window, set the gamma characteristic curve in
the histogram image on the Display property page.
NOTE:
This also influences the reproduction of the color saturation.
For fluorescence images, background correction can also be carried out
simply by shifting the offset of the characteristic curve.
Clicking on this icon
returns to a linear display of the data.
AxioCam HR Installation + Reference
51
Image Acquisition with AxioCam HR
2.7
Information for Displaying 14 Bit AxioCam
HR Images in AxioVision
A PC graphics card cannot display more than 3 x 8 bits per pixel. However, as
we have already mentioned, the AxioCam HR is able to generate 3 x 14 bits
per pixel. So that intelligent use can be made of this feature, the display's
characteristic curve can be altered in AxioVision.
To do this, right-click in the image acquired and select from the context menu
the Properties function. The dialog window Properties is displayed on the
screen:
52
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
You can now select the area to be displayed by dragging the black rectangles
along the characteristic curve or carry out automatic optimization by clicking
on the corresponding buttons.
The Best Fit, Min/Max and Linear buttons can be used to quickly adjust the
histogram display. You can influence the display by clicking on the Log or
Skip buttons.
Best Fit calculates in the current histogram the values which exclude 1‰ of
the pixels contained in the image. The color/color range calculated in this way
is displayed on the screen. (You can adjust the value of 1‰ to suit your
requirements).
Min/Max displays on the screen the smallest and largest color or color value
present in the image.
Linear displays on the screen the total possible value range.
Log displays the histogram in a logarithmic scaling, allowing you to effectively
evaluate histograms of images with high dynamics.
Skip: By using the Skip function the maximum values at the upper and lower
end of the histogram are omitted during the scaling of the display.
The
,
and
scrollbars can be used to change the
brightness, contrast and gamma value of the display. This can also be
achieved by dragging the individual "handles" of the characteristic curve in
the histogram. You can adjust the brightness and contrast by changing the
left or right-hand end points of the characteristic curve individually or by
shifting the entire line using the center "handle". The gamma value can be
changed by shifting both "handles".
Click the Save button to store the current settings. You can use these settings
when displaying another image by clicking the Restore button. This enables
you to compare different images.
If you activate the Apply to all T check box the settings in all the acquired
instants of exposure are applied to the relevant fluorescence channel.
AxioCam HR Installation + Reference
53
Image Acquisition with AxioCam HR
NOTE:
You can get the best display of colored objects, if the gamma setting is in
the range of approx. 0,45.
Recommended Camera Settings
Activate the Enhance Color check box on the General property page to
improve the color reproduction quality (this slows down the live image rate
slightly as additional calculations are required).
The standard setting installed for 3200 K (3200 K button on the Adjust
property page) often offers the camera the most natural reproduction of the
image situation on the microscope. Here the image background is reproduced
as it appears on the microscope. This setting is also useful in acquisition
situations where no neutral-tint area is available to carry out a balance
manually, e.g.: fluorescence.
If the image background in the acquired image is to be displayed with a
neutral tint (gray), a manual white balance is required. Click on the White
Balance button; then click on an area in the image that has a neutral tint and
is not overexposed.
If the lamp voltage selected on the microscope is too low, the color of the
image background may have been shifted a long way into the red. If a white
balance is carried out in this event, the result may be visible deviations in the
display of the color of the current sample. Correct this situation by increasing
the lamp voltage to achieve a more neutral background, and insert gray filters
into the microscope’s illumination beam path to adjust the intensity.
Recommended Monitor Settings
The output medium has a significant influence on color perception!
Use a familiar specimen to adjust the brightness and contrast rendering. If
possible also set the color temperature of the monitor to approx. 5000 K or
6500 K.
54
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
2.8
Background Information
Gamma Setting
This setting is required to compensate for the inherently non-linear behavior
of the image tube ("gamma"). It also influences the quality of the color
display. You can get the best display of colored objects, if the gamma setting
is in the range of approx. 0,45.
Many video cameras have built-in gamma compensation, as they are intended
for use on video monitors.
Linearity
All versions of AxioCam HR have been designed to determine linear,
quantitative measurement data, as such data is frequently used for tasks in
the field of image analysis. Consequently, internal gamma compensation has
been deliberately omitted from the AxioCam HR.
Exposure Times for Microscanning Resolutions
Images that are generated with micro-scanning resolutions are composed of
several component images upon acquiring. This process allows for an increase
in resolution and color quality. The number of component images needed for
the final image varies according to the resolution you have selected. The
exposure time is multiplied in accordance with this number.
260 x 206
Single image with no microscanning, color
interpolation, binning mode 5x5.
324 x 256
Single image with no microscanning, black
and white image, binning mode 4x4.
432 x 342
Single image with no microscanning, color
interpolation, binning mode 3x3.
650 x 514
Single image with no microscanning, black
and white image, binning mode 2x2.
AxioCam HR Installation + Reference
55
Image Acquisition with AxioCam HR
1300 x 1030 standard
Single image with no microscanning, color
interpolation.
1300 x 1030 scanned
Microscanning with 4 positions, maximum
color quality.
2600 x 2060 scanned
Microscanning with 16 positions, maximum
color quality and double resolution.
3900 x 3090 scanned
Microscanning with 36 positions, maximum
color quality and triple resolution.
3900 x 3090 fast scan
Microscanning with 9 positions.
EXAMPLE:
With an exposure time of 100 ms and 150 ms readout time for a component
image, a scan with a 3900 x 3090-pixel resolution takes 250 ms x 36 positions
= 9 seconds. This is a fact, which must be taken into account with long
exposure times in particular.
NOTE:
The AxioCam HR has two terms: "exposure time" and "acquisition time",
which have to be distinguished.
"Exposure time" is the period of time for which the sensor is exposed to
the light. This period can be optimized automatically by starting the
exposure measurement icon
.
"Acquisition time" is the period of time, which is used for the microscanning acquisition method. Depending from the used resolution in the
microscanning mode a different amount of partial acquisitions are done,
which were united to a high resolution image. With a constant exposure
time the whole acquisition time can vary when the camera resolution will
be switched.
56
B 48-0020 e 10.2003
Image Acquisition with AxioCam HR
Fluorescence Images
Several shots are needed for increased resolution. As fluorescence specimens
can be subject to bleaching out during exposure, there is the risk that the
image intensity for micro-scanning shots may diminish from component
image to component image. This makes artifacts occur in the final image.
Check whether your fluorescence specimen bleaches out within the necessary
exposure time. If this is the case, you must revert to the camera resolution
with 1300 x 1030 pixels. For cameras with black and white sensors, this
corresponds to the full basic resolution of the CCD sensor; for a color camera,
color interpolation is used.
With the color camera AxioCam HRc, it is usually possible to use a 1300 x
1030 pixel resolution with 4 component images when bleaching out occurs to
a lesser extent. One further possibility for reducing the effects of bleaching
out is to reduce the camera’s necessary exposure time by increasing the
amplification.
For the acquisition of fluorescence images a black/white camera is the
optimum choice.
Configurable Toolbars for Operating the Camera
The AxioVision software allows you to configure your own toolbars. This
means that the procedure can be tailored to your own requirements, and thus
optimized. The size of the toolbar buttons can also be adjusted, helping you
to work quickly with the mouse. A detailed description of how to create
toolbars can be found in Chapt. 9 "Configuration" - "Adapting Toolbars" in
the AxioVision Manual.
AxioCam HR Installation + Reference
57
Trouble Shooting
3
Trouble Shooting
If errors occur, check first the setup instructions of the camera.
3.1
Software
Please also pay attention to all the comments in the file ReadMe.TXT, which
is displayed during the driver installation.
AxioCam HR does not appear in the menu of selectable cameras
Make sure that the AxioCam HR files have been installed in
accordance with the installation of the software.
For Windows 2000 Professional and XP Professional: Make sure that
the camera drivers have been installed with administrator rights.
No camera image on your screen
Check the light setting of the microscope.
Check the cable connections between camera and computer.
Is the green control light on the camera on? If not, check the cable
connections between camera and computer.
Execute an automatic exposure measurement (Autoexposure).
Check display adjustments for the live image
.
Check the aperture diaphragm of the microscope.
Check the position of the beam splitter between the ocular and the
TV port.
The color of the image is green and not realistic coloring
Check white balance and, if necessary, repeat white balance.
58
B 48-0020 e 10.2003
Trouble Shooting
3.2
Image Quality
Sudden jolt, vibration or moving objects
If the microscope is jolted whilst you are taking a shot, the image will
show periodic interference.
Repeat the shot and take care to ensure the conditions remain calm.
Do not use resolutions with micro-scanning for moving objects.
An example of a shaky shot
Compare: An error-free image
Fluctuations in illumination
If the light source has a fluctuating quality of light intensity, stripes or
blotches will show up in the image.
This is particularly true for fluorescence shots where a sample, which
has bleached out to a great extent, is recorded using a microscanning resolution.
In general, the 1300 x 1030 Co-Site Sampling resolution should be
the maximum used for fluorescence shots.
AxioCam HR Installation + Reference
59
Technical Data
4
Technical Data
4.1
AxioCam HRc (color-version)
CCD Basic resolution
1300 x 1030 = 1,3 mega pixels
Pixel size
6,7 µm x 6,7 µm
Sensor size
8,7 mm x 6,9 mm, equivalent 2/3"
Spectral range
Approx. 400 nm - 700 nm with BG 40 IR barrier filter,
Bayer color filter mask
Max. charge / pixel
Full Well ca. 25.000 e
Selectable resolution
Horizontal x Vertical
260 x 206
(Binning 5x5, RGB)
324 x 256
(Binning 4x4, b/w)
432 x 342
(Binning 3x3, RGB)
650 x 512
(Binning 2x2, b/w)
1300 x 1030
(Single Shot)
1300 x 1030
(Co-Site-Sampling)
2600 x 2060
(Co-Site-Sampling)
3900 x 3090
(Co-Site-Sampling)
3900 x 3090
(Fast Scan)
Live image frame rates
(Binning in color mode)
Max. frame
(@20 ms)
Maximal values when using 5 images / s
13 images / s
optimal hardware
20 images / s
Binning factor
/ Mode
1 / slow
3 / middle
5 / fast
Horizontal x Vertical
1300 x 1030
432 x 342
260 x 206
Readout of sub frames
Random definition of regions of interest on the sensor
("ROI")
Digitization
14 bit / 10 Mhz pixel clock
Dynamic range
Typical >2000 : 1 at <9 e readout noise
Range of integration time
1 ms to several minutes
Cooling
Single stage Peltier cooling
Signal output connector
TTL signal output for control of external shutters
Interface
PCI interface (32 bit / 5 V) card with thin fiber optic
60
B 48-0020 e 10.2003
Technical Data
cable for data and control lines at 200 Mbit / s
Optical interface
C-Mount (17,5 mm)
Max. file size per image
Approx. 71 MB @ 3900 x 3090 @ 3 x 14 bit (color)
Operating systems
Windows 2000 Professional or Windows XP
Professional
Size / weight
ca. 11 x 8 x 6,5 cm / 0,5 Kg (camera head)
Housing
Aluminium - with fins - ¼" connection for tripod
Registration
GS, CE, cUL
Power input
12V DC, 1A
Power supply
Please refer to the mains voltage specification, given
on the used power supply. The power supply must be
tested according to IEC/EN 60950 standard (Europe).
The power output has to comply with the conditions
for SELV and limited power. The power supply
provided with the camera complies with these
conditions.
Ambient condition
(operation)
0° ... +35° Celsius
10% ... 80% relative humidity, no condensation, free
air circulation required
Ambient condition
(storage)
-15° ... +60° Celsius
90% relative humidity at 40° Celsius
80% relative humidity at +20° Celsius
no condensation
Above frame rates are supported by the camera electronics at 20 ms
exposure time. Computer hardware, operating system and application
software may decrease the frame rates. Selecting a part of the sensor
area can increase the frame rate. All specifications are subject to
change without notice.
AxioCam HR Installation + Reference
61
Technical Data
4.2
AxioCam HRm, Vers. 2.0 and Later
(black/white-version)
CCD Basic resolution
1388 x 1040 = 1,4 mega pixels
Pixel size
6,45 µm x 6,45 µm
Sensor size
8,9 mm x 6,7 mm, equivalent 2/3"
Spectral range
Approx. 350 nm - 1000 nm with BK-7 protection
glass without IR filter (BG40 can be inserted if
necessary)
NIR Mode
Mode for higher sensitivity in near IR
Max. charge / pixel
Full Well approx. 17.000 e
Selectable resolution
Horizontal x Vertical
276 x 208
(Binning 5x5)
346 x 260
(Binning 4x4)
462 x 346
(Binning 3x3)
694 x 520
(Binning 2x2)
1388 x 1040
Single Shot
2776 x 2080
Microscanning Mode
4164 x 3120
Microscanning Mode
Live image frame rates
(Binning in color mode)
Max. frame
(@20 ms)
Maximal values when using 5 images / s
13 images / s
optimal hardware
20 image / s
Binning factor
/ Mode
1 / slow
2 / middle
3 / fast
Horizontal x Vertical
1388 x 1040
694 x 520
462 x 346
Readout of sub frames
Random definition of regions of interest on the sensor
("ROI")
Digitization
14 bit / 10 Mhz pixel clock
Dynamic range
Typical >2000 : 1 at <8 e readout noise
Range of integration time
1 ms up to several minutes
Cooling
Single stage Peltier cooling
Signal output connector
TTL signal output for control of external shutters
Interface
PCI interface card (32 bit / 5 V) with thin fiber optic
cable for data and control lines at 200 Mbit / s
62
B 48-0020 e 10.2003
Technical Data
Optical interface
C-Mount (17,5 mm)
Max. file size per image
Approx. 25,9 MB @ 4164 x 3120 @ 14 bit
Operating systems
Windows 2000 Professional or Professional XP
Professional
Size / weight
Approx. 11 x 8 x 6,5 cm / 0,5 Kg (camera head)
Housing
Aluminium - with fins - ¼" connection for tripod
Registration
GS, CE, cUL
Power input
12 V DC, 1 A
Power supply
Please refer to the mains voltage specification, given
on the used power supply. The power supply must be
tested according to IEC/EN 60950 standard (Europe).
The power output has to comply with the conditions
for SELV and limited power. The power supply
provided with the camera complies with these
conditions.
Ambient condition
(operation)
0° ... +35° Celsius
10% ... 80% relative humidity, no condensation, free
air circulation required
Ambient condition
(storage)
-15° ... +60° Celsius
90% relative humidity at 40° Celsius
80% relative humidity at +20° Celsius
no condensation
Above frame rates are supported by the camera electronics at 20 ms
exposure time. Computer hardware, operating system and application
software may decrease the frame rates. Selecting a part of the sensor
area can increase the frame rate. All specifications are subject to
change without notice.
AxioCam HR Installation + Reference
63
Technical Data
4.3
Spectral Sensitivity
Normalized to maximum amplitude
The use of the AxioCam HRc (color version) without infrared barrier filter is
not recommended as color is only defined for visual spectral range.
The AxioCam HRm (black/white version) is used without infrared barrier filter.
If necessary, an additional infrared barrier filter can be inserted between the
microscope adapter and the camera.
64
B 48-0020 e 10.2003
Index
Index
1
14-bit images 52
A
acquisition delay 48
Adjust property page 33
automatic exposure 35
automaticalle adjust white balance 36
AxioCam HRc 25
AxioCam HRm 25
B
b/w sensor 39
backup copies 4
basic settings 45
binning 42
black reference 46
black/white camera 41
black/white mode 41
brightness, change 53
color mode 41
color value, change 53
contents of delivery 4
contrast, change 53
control signal 48
D
delivery contents 5
digital gain 45
display characteristic curve 51
E
enhance color 49
ensure the notes 3
exposure 34, 56
exposure time measurement 34
extension connector 10
F
file size 43
filter operations 49
fluorescence images 57
Frame property page 39
frame selection 39
C
camera connection 7
camera electronic 24
camera mode 39
camera on microscope 8
camera settings 54
camera setup 6
checking 10
camera to PCI interface board 9
camera to power supply 9
center frame 44
checking the setup 10
C-mount objectives 24
color adjustment 37, 49
color channel balance 36
AxioCam HR Installation + Reference
G
gamma settings 55
general notes 3
General Property Page 45
H
hardware requirements 4
histogogram, influence 38
histogram 37, 38
I
image acquisition 25
I
Index
image frame 43
image orientation 46
image properties 52
image quality 59
infra-red barrier filter 24
installation 1
installing software 11
L
limitation of liability 2
linearity 55
live image speed 50
logarithmic display 38
M
memory size for image 44
microscanning 41
microscanning resolution 55
N
notes on maintenance 24
resolution 40, 41
S
safety regulations 1
scaling histogram 38
seting up the camera 3
settings 33
shutter control 48
software problems? 58
software requirements 4
spectral sensitivity 64
speed modes 50
starting AxioVision 26
statutory liability for accidents 2
step by step to the first image 28
system requirements 4
T
technical data 60
toolbars 57
trouble shooting 58
U
O
operating the camera 3
optical adaption 40
optical system 24
optimum color reproduction 51
overexposure 38
P
PCI interface board 6
power control 10
practical notes on operation 50
underexposure 38
W
warranty 2
weighting of exposure time
measurement 34
white balance 36
white reference 47
Workflow 27
R
repair is necessary
address 3
II
B 48-0020 e 10.2003

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project