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software:mipav_generatefusion [2016/02/05 05:34]
Jon Daniels 		software:mipav_generatefusion [2016/06/23 20:40]
Jon Daniels [Tutorial] added dropbox link to sample data from Hari
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 MIPAV is a framework not unlike Fiji, and the generatefusion plugin was developed in collaboration with the Shroff lab.  It does intensity-based registration and joint deconvolution.  You can download MIPAV at [[http://mipav.cit.nih.gov/]] and then install the plugin. MIPAV is a framework not unlike Fiji, and the generatefusion plugin was developed in collaboration with the Shroff lab.  It does intensity-based registration and joint deconvolution.  You can download MIPAV at [[http://mipav.cit.nih.gov/]] and then install the plugin.
  
-In general this software is much slower and more computationally intensive that Fiji MVR but crucially it has image-based registration that can handle registration of datasets without distinct interest points.  (Image-based registration is in the future plans for MVR per [[http://imagej.1557.x6.nabble.com/3D-affine-transformation-Deskewing-with-anisotropic-voxels-td5013781.html|this thread]] but it is not implemented as of Jan 2016).+In general this software is much slower and more computationally intensive that Fiji MVR.  However, it uses image-based registration so it can register datasets without distinct interest points.  (Image-based registration is in the future plans for MVR per [[http://imagej.1557.x6.nabble.com/3D-affine-transformation-Deskewing-with-anisotropic-voxels-td5013781.html|this thread]] but it is not implemented as of Jan 2016).
  
 MIPAV generatefusion is referenced in the 2014 Nature Protocols paper, though some things have changed since.   This is the place to look for updated information. MIPAV generatefusion is referenced in the 2014 Nature Protocols paper, though some things have changed since.   This is the place to look for updated information.
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 diSPIM image processing involves several steps, including background subtraction, cropping, transformation, fusion, and deconvolution. The first two steps in this process, background subtraction and cropping, are carried out in ImageJ or FIJI, while the remaining steps are carried out in MIPAV. diSPIM image processing involves several steps, including background subtraction, cropping, transformation, fusion, and deconvolution. The first two steps in this process, background subtraction and cropping, are carried out in ImageJ or FIJI, while the remaining steps are carried out in MIPAV.
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 +Sample data for this tutorial is at https://www.dropbox.com/sh/qua01k7v8bi9sge/AAB7ZLN2DN19GPL2lGQFh0nza?dl=0.
  
 ==== File Organization ==== ==== File Organization ====
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   - Import the background image. Go to File -> Import -> Image Sequence, navigate to the folder where the background image sequence is stored, and import it. The settings for the import image sequence dialog box are as follows:   - Import the background image. Go to File -> Import -> Image Sequence, navigate to the folder where the background image sequence is stored, and import it. The settings for the import image sequence dialog box are as follows:
   - Go to Image -> Stacks -> Z Project and set Projection type to Average intensity. Click on the OK button. Then close the original bg0 window, leaving only AVG_bg0.   - Go to Image -> Stacks -> Z Project and set Projection type to Average intensity. Click on the OK button. Then close the original bg0 window, leaving only AVG_bg0.
-   - Go to Plugins -> Macros -> Record. The screen should look like this: {{:software:mipav_fig1.png?1000|}} +   - Go to Plugins -> Macros -> Record. The screen should look like this: \\ {{:software:mipav_fig1.png?1000|}} \\ 
-   - Next, open the first image sequence for the diSPIM images. To do this, go to File -> Import -> Image Sequence, navigate to the folder containing the diSPIM image sequence, and open it {{:software:mipav_fig2.png?1000|}} +   - Next, open the first image sequence for the diSPIM images. To do this, go to File -> Import -> Image Sequence, navigate to the folder containing the diSPIM image sequence, and open it \\ {{:software:mipav_fig2.png?1000|}} 
-   - Subtract the image background by going to Process -> Image Calculator. Set Image1 to the image you are working with (in this case bv0), Operation to Subtract, and Image2 to AVG_bg0. Press OK. Press Yes when the Process Stack dialog box appears. The result should appear as: {{ :software:mipav_fig3.png?1000|}}+   - Subtract the image background by going to Process -> Image Calculator. Set Image1 to the image you are working with (in this case bv0), Operation to Subtract, and Image2 to AVG_bg0. Press OK. Press Yes when the Process Stack dialog box appears. The result should appear as: \\ {{ :software:mipav_fig3.png?1000|}} \\
   - Use the Polygon select tool to draw a box around the sample. This box will be used to crop the image. While size will vary depending upon the sample, make sure that room is left (perhaps 25 pixels minimum) surrounding the sample in the cropped image in all directions. In this demo I have used a crop size of 300 x 400 pixels.   - Use the Polygon select tool to draw a box around the sample. This box will be used to crop the image. While size will vary depending upon the sample, make sure that room is left (perhaps 25 pixels minimum) surrounding the sample in the cropped image in all directions. In this demo I have used a crop size of 300 x 400 pixels.
-  - Go to Image -> Crop to crop the image. {{ :software:mipav_fig4.png?1000|}}+  - Go to Image -> Crop to crop the image. \\ {{ :software:mipav_fig4.png?1000|}} \\ 
   - Go to File -> Save As -> Tiff… to save the image. It should be saved in a new folder; conventionally I name this new folder SPIMA or SPIMB depending on which arm the images come from. The image itself is named SPIMA_XX, where XX is the number of the image in the image sequence.   - Go to File -> Save As -> Tiff… to save the image. It should be saved in a new folder; conventionally I name this new folder SPIMA or SPIMB depending on which arm the images come from. The image itself is named SPIMA_XX, where XX is the number of the image in the image sequence.
   - Close the SPIMA_20 and bv20 images.   - Close the SPIMA_20 and bv20 images.
-  - Edit the macro to run through as many images as required. An example of a finished macro can be seen below. We also attach a sample version of the macro edited to run through the sample data.  {{ :software:mipav_fig5.png?1000|}}+  - Edit the macro to run through as many images as required. An example of a finished macro can be seen below. We also attach a sample version of the macro edited to run through the sample data. \\ {{ :software:mipav_fig5.png?1000|}} \\ 
   - Repeat the process for the SPIMB images. Make sure that the size of the cropped images is the same between SPIMA and SPIMB images.   - Repeat the process for the SPIMB images. Make sure that the size of the cropped images is the same between SPIMA and SPIMB images.
  

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