Article Title: Direct contact of umbilical cord blood endothelial progenitors with living cardiac tissue is a requirement for vascular tube-like structures formation. Figure Lengend Snippet: Immunohistochemical analyses for human vWF and human mitochondria expression, showing modest integration of EPCs into the ischaemic embryonic murine ventricular tissue and lack of vascular tube-like structures formation — day 6 of coculture A—D ; polyconal rabbit anti-human vWF-Alexa Fluor green , monoclonal IgG1mouse anti-human mitochondria-Alexa Fluor red , and Hoechst nuclei, blue ; negative controls-secondary antibodies only E and murine ventricular slice only F.
Article Snippet: Cell and tissue preparations were examined under an inverted epifluorescence microscope with an incorporated digital camera system for imaging and a module for optical sectioning Axiovert M ApoTome, Carl Zeiss MicroImaging GmbH ; picture acquisition was performed with the AxioVision 4.
Figure Lengend Snippet: Immunohistochemical analyses showing EPCs integration and vascular tube-like structures formation in living murine embryonic ventricular slice preparations. Human vWF and human nuclei expression — day 6 A—C , day 10 D—F , and day 14 G of coculture; polyclonal rabbit anti-human vWF-Alexa Fluor green , monoclonal IgG1 mouse anti-human nuclei-Alexa Fluor red , and Hoechst nuclei, blue ; negative controls-secondary antibodies only J and murine ventricular slice only K.
Nuclear telomere distribution patterns define TAs indicated with an arrowin the three categories. Article Snippet: AxioVision 4. Nuclei DAPI, blue. F-actin fluorescent phalloidin, green and nuclei DAPI, blue were also stained. Article Title: Disturbed distribution of proliferative brain cells during lupus-like disease.
B Higher magnification confirmed the selective. Buy from Supplier. Structured Review. However you should confirm this for yourself on your imaging system. Cha nging t he sc a le ba r c olor 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.
AxioVision and the active MTB configuration allow you to take advantage of the benefits of automatic scaling. You do not need to do manual scaling. Automatic measurements are very convenient and generally sufficient for most applications. 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. 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. For further information on image file types see Chapter 5. Frequent Annotations: Use this option to add annotations to image sequences e. 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.
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. 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.
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. 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.
If you assign a keyboard short-cut to the Snap function you can acquire and save an image with a single key stroke. 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. 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. 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.
To change folders or to see the full path, click on or click on Folder info. 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. Workflows, short-cut keys, screen layout. You can do this by exporting your configuration.
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.
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. 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.
Click on the Edit button 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. Generally, select the minimum number of settings required. 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. This chapter only describes the standard interactive measurement functions found in the Measure menu. We will use the Interactive Measurement Wizard. Open an image. 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. Click on to browse the various saved configurations and select the one you want to use. Click OK. This opens the active image in the interactive measurement window, as shown on the following page. The features chosen in the configuration step are now displayed. Toolbar Interactive Measurement module Outline spline tool 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. If you re- open the image you can view the measurement data by right clicking in the image and selecting Properties, and then selecting the Measurement tab. 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. 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. 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 first thing to do is to enter an image name. 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. Click on the C Channel tab to see the Multichannel settings.
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. Tip: Make sure your AxioVision window is large enough to show all of the buttons as shown in the example image. Ex posure 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 H a rdw a re Se t t ings 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 Now you can attach a setting to be used for acquisition to this channel e. 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. 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.
Ex t e nde d Pa ra m e t e rs 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. 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. Sa ving Y our Ex pe rim e nt 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. 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. 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. 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. V ie w ing Com ple x I m a ge s 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 Z- Stack image. All three channels are overlaid and we are showing the first image in the Z- Stack.
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.
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. Note: You must have a motorized focus on your microscope to use this option.
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 slices spaced at 0. This means that the center slice will be number between them and enter the values.
In our example we will acquire 15 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. 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.
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. 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. 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. 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.
Saving and reloading your experiments makes it very easy for a less experienced person to use the system. You can temporarily disable the Z-Stack or Time Lapse elements by un- checking 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. 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. This wizard allows you to import various file formats in any combination of Multichannel Fluorescence, Z-Stack and Time Lapse.
In Scale lateral X box It may be necessary to create a different y scaling which can be scaling box. 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. 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. 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. 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. The images generated can be subsequently saved via File, Save or Save as.
Annotation Graphics e. 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. 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. 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. 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. If Gamma is set to 1. Often a Gamma value less than 1. 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 non- uniformities.
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.
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