downloads:spindlefanalyzer
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- | ~~DISCUSSION~~ | + | ~~DISCUSSION:closed~~ |
~~NOTOC~~ | ~~NOTOC~~ | ||
- | [{{ http:// | + | [{{ http:// |
===== Spindle Microtubule Frap / Flip Analyzer ===== | ===== Spindle Microtubule Frap / Flip Analyzer ===== | ||
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miura at embl dot de Tel: +49 6221 387 404\\ \\ | miura at embl dot de Tel: +49 6221 387 404\\ \\ | ||
==== History ==== | ==== History ==== | ||
- | 08-08-12 First release\\ \\ | + | 08-08-12? First release\\ \\ |
=== Source === | === Source === | ||
Released under the GNU General Public License.\\ \\ | Released under the GNU General Public License.\\ \\ | ||
==== Installation ==== | ==== Installation ==== | ||
- | Download [[http:// | + | Download [[http:// |
==== Requires ==== | ==== Requires ==== | ||
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==== Description ==== | ==== Description ==== | ||
The specific purpose of this macro was to follow the intensity dynamics occurring within double labeled microtubule spindle. Intensity changes that are occurring at stable position is easy, but this macro enables measurement of intensity changes that accompanies movement of the bleached/ | The specific purpose of this macro was to follow the intensity dynamics occurring within double labeled microtubule spindle. Intensity changes that are occurring at stable position is easy, but this macro enables measurement of intensity changes that accompanies movement of the bleached/ | ||
- | <quote> | + | <blockquote> |
**Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.**\\ | **Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.**\\ | ||
Uteng, M., Hentrich, C., Miura, K., Bieling, P. & Surrey, T.\\ J Cell Biol. 2008 Aug 25; | Uteng, M., Hentrich, C., Miura, K., Bieling, P. & Surrey, T.\\ J Cell Biol. 2008 Aug 25; | ||
- | </quote>\\ | + | </blockquote>\\ |
- | [{{ http:// | + | [{{ http:// |
- | [{{ http:// | + | [{{ http:// |
- | [{{ http:// | + | [{{ http:// |
- | Using a pair of two-channel image stacks, this macro measures dynamics of integrated intensity profile along x-axis of a channel masked by a mask created using the other channel. For example, we take red channle and green channel | + | Using a pair of two-channel image stacks, this macro measures dynamics of integrated intensity profile along x-axis of a channel masked by a mask created using the other channel. For example, we take red channle and green channel |
- | + | ||
+ | ==== Other papers using this macro ==== | ||
+ | |||
+ | - Fu, J., Bian, M., Xin, G., Deng, Z., Luo, J., Guo, X., Chen, H., Wang, Y., Jiang, Q., Zhang, C., 2015. TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux. J. Cell Biol. 210, 373–83. | ||
==== Work Flow ==== | ==== Work Flow ==== | ||
- | [{{ http:// | + | [{{ http:// |
- | [{{ http:// | + | [{{ http:// |
**1. Measurment of Profile Dynamics**\\ \\ | **1. Measurment of Profile Dynamics**\\ \\ | ||
**1.1.** Install the macro by **[Plugins -> Macros -> Install...]**.\\ \\ | **1.1.** Install the macro by **[Plugins -> Macros -> Install...]**.\\ \\ | ||
- | **1.2.** Open two stacks, one containing a movie for masking (c.f. Tubulin) and other for measurements (c.f. MAP). Threshold the tubulin window by **[Image -> Adjust -> Threshold...]** (do not actully convert the image to black and white, meaning don't click the apply button in the threshold panel) and by this determine a lower threshold value that highlights the tubulin successfully. This value will be used in the following step. One could also store the lower threshold value in memory by [Plugins -> Macros -> get Threshold Lower] while the thresholded area is highlighted.\\ \\ | + | **1.2.** Open two stacks, one containing a movie for masking (c.f. Tubulin) and other for measurements (c.f. MAP).?Threshold the tubulin window by **[Image -> Adjust -> Threshold...]** (do not actully convert the image to black and white, meaning don't click the apply button in the threshold panel) and by this determine a lower threshold value that highlights the tubulin successfully. This value will be used in the following step. One could also store the lower threshold value in memory by [Plugins -> Macros -> get Threshold Lower] while the thresholded area is highlighted.\\ \\ |
**1.3.** Do **[Plugins -> Macros --> Y projection profile two channels]**. A dialogue window pops up (Fig 4.). Choose appropriate window names using the pull-down menu, and also input the threshold value determined in **1.2**. Uncheck "Noise spot Removal" | **1.3.** Do **[Plugins -> Macros --> Y projection profile two channels]**. A dialogue window pops up (Fig 4.). Choose appropriate window names using the pull-down menu, and also input the threshold value determined in **1.2**. Uncheck "Noise spot Removal" | ||
- | **1.4.** Two new image stacks will be created, one called " | + | **1.4.**? Two new image stacks will be created, one called " |
Column header explanation: | Column header explanation: | ||
* " | * " | ||
* " | * " | ||
* " | * " | ||
- | These results could be saved as a .xls file (excel format) by **[File -> Save As...]** from the menu bar of the results window.\\ | + | These results could be saved as a .xls file (excel format) by **[File -> Save As...]** from the menu bar of the results window.\\ |
**2. Graphing the Profiles**\\ \\ | **2. Graphing the Profiles**\\ \\ | ||
- | [{{ http:// | + | [{{ http:// |
**2.1.** While the above Results window is still opened (plotting loads data from Results window), do **[Plugins -> Macros -> Profile Plotter]**. If Results window is absent and you know that you have saved the file somewhere, you can re-open it by [File -> Import -> Results]. Data will be loaded into the Results window. Then a dialogue window pops up (Fig.6).\\ \\ | **2.1.** While the above Results window is still opened (plotting loads data from Results window), do **[Plugins -> Macros -> Profile Plotter]**. If Results window is absent and you know that you have saved the file somewhere, you can re-open it by [File -> Import -> Results]. Data will be loaded into the Results window. Then a dialogue window pops up (Fig.6).\\ \\ | ||
There are two options. One is 'Plot Increments', | There are two options. One is 'Plot Increments', | ||
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After setting these two options, click " | After setting these two options, click " | ||
**2.2.** Two graphs will then appear which look like below.\\ \\ | **2.2.** Two graphs will then appear which look like below.\\ \\ | ||
- | [{{ http:// | + | [{{ http:// |
The first graph (Fig. 7) shows integrated intensity (sum of pixel values) vs x-position. Each color corresponds to different time point, the legend of which is printed in the Log window.\\ \\ | The first graph (Fig. 7) shows integrated intensity (sum of pixel values) vs x-position. Each color corresponds to different time point, the legend of which is printed in the Log window.\\ \\ | ||
- | [{{ http:// | + | [{{ http:// |
Second graph (Fig. 8) plots the height of mask in each x-position. If you divide the y value in the first graph by the y values in the second graph, you will get the average intensity for each position at each time point.\\ \\ | Second graph (Fig. 8) plots the height of mask in each x-position. If you divide the y value in the first graph by the y values in the second graph, you will get the average intensity for each position at each time point.\\ \\ | ||
**3. Optional function: realignment of spindle**\\ \\ | **3. Optional function: realignment of spindle**\\ \\ | ||
**3.1.** Realign spindle when spindle is tilted against x-axis. For example, here is a spindle that is tilted (Fig. 9). | **3.1.** Realign spindle when spindle is tilted against x-axis. For example, here is a spindle that is tilted (Fig. 9). | ||
- | [{{ http:// | + | [{{ http:// |
- | [{{ http:// | + | [{{ http:// |
To realign these spindles, draw a Line ROI from one spindle pole to the other pole in the tubulin channel like the example above. Line ROI should start from left side of the spindle (this is because of calculation purpose, starting point should always be left). Then do **[Plugins -> Macros -> Rotate Sync 2ch to Horizontal]**. A dialogue wiindow pops up (Fig. 10). | To realign these spindles, draw a Line ROI from one spindle pole to the other pole in the tubulin channel like the example above. Line ROI should start from left side of the spindle (this is because of calculation purpose, starting point should always be left). Then do **[Plugins -> Macros -> Rotate Sync 2ch to Horizontal]**. A dialogue wiindow pops up (Fig. 10). | ||
- | Choose the appropriate windows. Other options do not affect the rotation operation. Then click OK. After the processing, the resulted windows should look like below.\\ \\ [{{ http:// | + | Choose the appropriate windows. Other options do not affect the rotation operation. Then click OK. After the processing, the resulted windows should look like below.\\ \\ [{{ http:// |
downloads/spindlefanalyzer.1267647287.txt.gz · Last modified: 2016/05/24 12:46 (external edit)