3. TCS - SP5 Off-Line Confocal Work Stations

Transcription

1 3. TCS - SP5 Off-Line Confocal Work Stations 1 Processing image data for 3D rotations and movies takes substantial time on our confocal microscope work station. Clients are asked to transfer data to our off-line work station (Confocal Simulator) so that the microscope can be available for other users. Two LAS software versions are available: Modified home version No rotation capability single monitor Room 249 West Full processing version Hardware key required 3D ROTATION MOVIE.avi Z- STACK (SERIES) MOVIE.avi Please ask for the LAS AF Lite application if you would like to install it on your PC computer. (Windows only)

2 TCS - SP5 Off-Line Confocal Work Stations Table of Contents 2 TITLE PAGE 1 TABLE OF CONTENTS 2 The LEICA Application Suite Advanced Fluorescence Lite (Confocal Simulator Lite) Application 3-6 ADJUSTING IMAGES 7 MORE IMAGE TOOLS 8 INSERTING A SCALE BAR 9 CREATING ORTHOGONAL VIEWS 10 ELIMINATING UNWANTED LABELS 11 SAVING YOUR NEW DATA 12 STAMPING AVI MOVIES WITH TIME AND DIMENSIONAL DATA STREAMS The LEICA Application Suite Advanced Fluorescence (Confocal Simulator Full Version) Application CROPPING [SEPARATION OF] A SERIES CREATING A 3-D ROTATED PROJECTED STACK AND.AVI MOVIE CREATING MEAN INTENSITY EMMISIONS STACK PROFILE CHARTS Donald W. Pottle

3 DWP The LEICA Application Suite Advanced Fluorescence Lite (Confocal Simulator Lite) Application This application is available free of charge and is included in the CD-ROM that comes with this set of User Guides. It cannot process image data as extensively as the full version of this software (e.g. no projected z-stack rotations). This manual considers only selected elements of the software but please explore it for additional processes that may be of use to you. 1. On the desktop, double click on the LAS AF Lite icon. 2. Click on <OK> build 1240 The application initializes and, in about 20 seconds, the user interface appears. Note: Clicking on an.lei (TCS-SP2) or.lif (TCS-SP5) file can automatically load this application. DWP Multiscan x291

4 DWP Multiscan x291 New Experiment ctrl + N Open Experiment ctrl + O Print Print Setup Print Preview (List of experiments on file) Exit 3. Click on <File> and <Open Experiment> and select an.lei or.lif image file from your image data folder. 4

5 IMAGE TEMPLATE WINDOW Click on an image series title (e.g., Series030 (7.9mb,xyz)......and a set of images, editing and annotation icons as well as a movie player will appear in the image template window Click on the overlay and maximum projection to merge the image slices. Descriptions of the image template icons follows.

6 Look Up Table Color Selection Slider right click for color choices or move the slider to produce a gray scale. 6. slider 6 6. Clicking on the <Show Gallery> icon produces image slices and Z-data. The series can be moved by the slider or by the movie player icon.

7 7. ADJUSTING IMAGES This image was enlarged using the magnify tool 8. Channel Selection dropdown menu Contrast Brightness Gamma 7. Click on <Process> to adjust Contrast, Brightness, and Gamma for all or separate channels. 8. Click <Apply> to save the adjusted image or <Reset> to start over. 8. Apply 7

8 8 Click on <Quantify> and select from among these tools Click for Annotation tools Select single Region Of Interest Select All Regions Of Interest Clear selected Regions Of Interest Draw Line MORE IMAGE TOOLS Lines, inscribed Regions Of Interest, Rectangles, Arrows,Text and Numbers can be embedded into your images. Create a Group Ungroup Draw Rectangle Draw an Elipse Draw an Arrow Draw a Polyline Draw Text Draw a Scale Bar Draw Counter (e.g. 1, 2, 3) To change the colors or other characteristics, right click on the line and adjust the properties menus. Draw a Polygon Draw a Range

9 INSERTING A SCALE BAR To insert a scale bar, click on the scale bar icon and draw an estimated scale bar in any area and direction on the image. Click on the line and a pair of white dots will appear. Right click on one of the dots and select White Dot <Properties>. Type in the desired length of the scale bar in the Scale Bar Properties window. To change the color of the scale bar, Click on <Color...> and select from the color menu. Click <OK> in both boxes. To re-do or discard the scale bar, right click on it and click on the delete (Trash Can) icon. 9

10 X Convallaria 400x with 3.4 zoom Y Lines can be deleted by right-clicking and selecting <Hide Crosshair> CREATING ORTHOGONAL VIEWS 10 This orthogonal (X and Y) edge views of vertical planes through a Z-Series was constructed through the Orthogonal View icon. Clicking-and-dragging lines will present edge views in real-time.

11 ELIMINATING UNWANTED LABELS Sometimes unwanted labels automatically appear in inscribed regions of interest. To eliminate the (e.g. ROI# ) text in an inscribed area, right click on the outline and select <Properties>. Highlight the name in the Name box and Click <OK>.

12 SAVING YOUR NEW DATA Clicking on <SaveAll> will save the additions or changes that you have made to your images. Right clicking on any Image title will offer The options to Rename, Delete, or Export a file As Tiff As JPEG AS AVI NOTE: The TCS SP5 LAS AF Lite software will allow the showing, saving and export of movies from previously saved.lif or.lei files. 12

13 STAMPING AVI MOVIES WITH TIME AND DIMENSIONAL DATA STREAMS 13 Series030(7.9MB,xyz) EXPORT As Tiff As JPEG AS AVI Right-clicking on an image series and exporting to an AVI movie file presents windows that allow the user to stamp the movie with time and dimension data. Such data can be positioned (be careful not to superimpose) as desired along the top and bottom margins of the movie and in a variety of colors. Three (3.0) frames and no compression produces high quality movies that present in a comfortable viewing speed.

14 AVI movie files can be inserted, edited and played through MS PowerPoint but confocal movie files are sometimes quite large (300MB). 14 These confocal Images and movies of retinal tissue were created by Bertin Tchedre (D.F. Chen lab) Z- Series Triple labeled March 2009 WINDOWS MOVIE PLAYER Windows Movie Maker and many other movie editing applications can convert AVI files to high quality movie files (.wma) that are much smaller in size; (e.g. 300MB 3.0MB).

15 The LEICA Application Suite Advanced Fluorescence (Confocal Simulator Full - Version) Application This full version application offers the same image data editing options as does the Lite version and several more. The remainder of this manual will consider; 1. Cropping a series (formerly known as separation ) 2. Creating rotated 3D projected stacks (e.g. lower left frame) 3. Creating Mean Intensity Emissions Stack Profile Charts 3D ROTATION MOVIE.avi 15 Z- STACK (SERIES) MOVIE.avi Please ask for the LAS AF Lite software if you would like to install it on your PC computer. (Windows only)

16 Room 249 West Full processing version Hardware key required The LEICA Application Suite Advanced Fluorescence (Confocal Simulator Full - Version) Application 16 Mouse Kidney Glomerulus (Molecular Probes ) Confocal Sequential Scanning 400x with 2.77 zoom DWP 09 Software Key (Dongle) Clicking a Leica.lif file can automatically open the Leica Confocal Simulator if the application is installed on a PC and if the software key is inserted into a USB port.

17 CROPPING [SEPARATION OF] A SERIES Sometimes a collected z-stack series contains slices at the beginning and/or end that contain little or no image value. These slices can be cropped [separated] from the rest through the use of the Crop tool. Select an image series from the <Experiments> menu and click on the <Process> and <Tools> tabs. Click on <Crop> and your selected series (e.g. Series 005) will appear in the image template window. 17

18 CROPPING [SEPARATION OF] A SERIES Moving the SLIDER up and down will help you to select the range of slices to be eliminated. The Slice number(s) appear in a box just below the SLIDER. Enter the desired slice range in the Z stack selection From and To boxes ( 2 and 44 in this example). Click on <Set Start> and <Set End> and <Apply> and the new cropped image series file will appear in your experiments list. To recreate a newer series click <Reset> SLIDER 2

19 19 CREATING A 3-D ROTATED STACK AND.AVI MOVIE Open a desired experiment as you normally would through the file tab. Click on a series title and click on the overlay and maximum projection icons in the tool tray at the right of the image set. When the new merged image appears. determine whether or not it is worthwhile making a 3-D rotation projection and movie. Very thin stacks generally do not yield a great amount of 3D detail, although for this example the specimen happens to be only about 8µm thick.

20 20 CREATING A 3-D ROTATED STACK AND.AVI MOVIE Click on the <Process > tab and then click on the <Tools> tab. Click on <3D Projection> beneath the Visualization header. A 3D Projection window will appear with a gray scale graphic of your image series. Click in the <Create Movie> box.

21 CREATING A 3-D ROTATED STACK AND.AVI MOVIE 21 When this panel appears, click and drag a corner of the graphic until the outline becomes a reasonable start position of your 3D movie. Alternatively, You can type in values for the Start and End Rotation boxes.

22 22 CREATING A 3-D ROTATED STACK AND.AVI MOVIE When you release the moved corner, a progress bar will automatically begin and a color rendition of your choice will appear.

23 23 CREATING A 3-D ROTATED STACK AND.AVI MOVIE If you approve of this view, click on <Set Start> and the rotation settings (degrees) will appear in X, Y, and Z. If you wish to try another Start Rotation setting, click <Reset> or click and drag another corner of the color graphic, and try again

24 24 CREATING A 3-D ROTATED STACK AND.AVI MOVIE Click and drag another corner of the graphic until the outline becomes a reasonable end position of your 3D movie. This may take some practice, but by trial and error appropriate Start and End frame positions can be assigned to the graphic

25 25 CREATING A 3-D ROTATED STACK AND.AVI MOVIE When you release the moved corner, another progress bar will automatically begin and a second color image will soon appear, representing your choice of the last frame position of your 3D stack Projection. If you approve of this view, click on <Set End> and the rotation settings (degrees) will appear in X, Y, and Z Type in the desired number of frames. (40 through 70 works well.)

26 26 CREATING A 3-D ROTATED STACK AND.AVI MOVIE NOTE: <Stereo On> creates a 3D anaglyph movie that you can view using red green 3D glasses. Click <Apply> to create the final 3D Projected Rotation and soon the new file will appear in your Experiments list Use the <Preview> command to watch the projection before you save it. Use the <Reset> command to re-do this procedure. 22%

27 27 CREATING MEAN INTENSITY EMMISIONS STACK PROFILE CHARTS Stack Profile Chart DAPI ALL IN ONE FITC FITC TRITC DAPI TRITC To create a graph (Stack Profile Chart) illustrating Mean Emission Intensity Values vs. multiple fluorophore z-stack slices, select a series and click the <Quantify> tab.

28 28 DAPI ALL IN ONE FITC FITC DAPI TRITC TRITC SLIDER Click on the <Graphs> tab and a Stack Profile Chart appears with images of each fluorophore and their corresponding Mean Intensity values. Raising or lowering the SLIDER moves the Viewer Position indicator along the X axis (depth in microns [µm]) and a corresponding slice number appears at the bottom of the slider.

29 29 CREATING MEAN INTENSITY EMMISIONS STACK PROFILE CHARTS DAPI FITC TRITC To create a graph (Stack Profile Chart) illustrating the Mean Emission Intensity Values vs. SINGLE fluorophore z-stack slices, one must first create a series containing ONLY one fluorophore. ( Use the Crop [separation] tool - described earlier - and delete the extra channels) Select a series, click the <Quantify> and <Graphs> tabs.