downloads:spindlefanalyzer
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+ | ~~DISCUSSION: | ||
+ | ~~NOTOC~~ | ||
+ | [{{ http:// | ||
+ | ===== Spindle Microtubule Frap / Flip Analyzer ===== | ||
+ | |||
+ | ==== Author ==== | ||
+ | Kota Miura\\ | ||
+ | Centre for Molecular and Cellualr Imaging (CMCI), EMBL Heidelberg\\ | ||
+ | miura at embl dot de Tel: +49 6221 387 404\\ \\ | ||
+ | ==== History ==== | ||
+ | 08-08-12? First release\\ \\ | ||
+ | === Source === | ||
+ | Released under the GNU General Public License.\\ \\ | ||
+ | ==== Installation ==== | ||
+ | Download [[http:// | ||
+ | |||
+ | ==== Requires ==== | ||
+ | ImageJ ver 1.34j or higher ([[http:// | ||
+ | ==== 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/ | ||
+ | < | ||
+ | **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; | ||
+ | </ | ||
+ | [{{ 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 | ||
+ | |||
+ | ==== 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 ==== | ||
+ | [{{ http:// | ||
+ | [{{ http:// | ||
+ | **1. Measurment of Profile Dynamics**\\ \\ | ||
+ | **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).? | ||
+ | **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 " | ||
+ | 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.\\ ?\\ | ||
+ | **2. Graphing the Profiles**\\ \\ | ||
+ | [{{ 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).\\ \\ | ||
+ | There are two options. One is 'Plot Increments', | ||
+ | The second option is to avoid plotting the first frame profile, since in many cases the first frame in fluorescence image sequence tends to be dark and cannot be used for the measurement.\\ \\ | ||
+ | After setting these two options, click " | ||
+ | **2.2.** Two graphs will then appear which look like below.\\ \\ | ||
+ | [{{ 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.\\ \\ | ||
+ | [{{ 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.\\ \\ | ||
+ | **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). | ||
+ | [{{ 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). | ||
+ | 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:// |