Update generate-and-exploit-kymographs.md

_pages/tutorials/generate-and-exploit-kymographs.md

@@ -12,17 +12,17 @@ Examples of usage can be found in the references listed at the end of this page.
 
 ## Context
 
-This is a movie made in the lab of Ewa Paluch, MPI-CBG, featuring a floating L929 cell (mouse fibroblast), stained for myosin (RLC-GFP) being ablated in the cortex. The transmitted signal is overlayed with a confocal image of the GFP channel in various ways. The acto-myosin cortex is the part of the cytoskeletton that lies just below the membrane This cortex is active and generate a contractile force that presses on the cell volume. At rest, this tension gives the floating cell this spherical shape.
+This is a movie made in the lab of Ewa Paluch, MPI-CBG, featuring a floating L929 cell (mouse fibroblast), stained for myosin (RLC-GFP) being ablated in the cortex. The transmitted signal is overlayed with a confocal image of the GFP channel in various ways. The acto-myosin cortex is the part of the cytoskeleton that lies just below the membrane. This cortex is active and generates a contractile force that presses on the cell volume. At rest, this tension gives the floating cell this spherical shape.
 
-A picosecond pulsed laser allowed us to make a micro ablation of the cortex, without disrupting the membrane. The ablation laser power saturates the transmitted detector, which results in white frames in the movie.
+A picosecond pulsed laser allowed us to make a micro ablation of the cortex without disrupting the membrane. The ablation laser power saturates the transmitted detector, which results in white frames in the movie.
 
-When ablated at the left spot, the constraint on volume is relaxed, and the cortex can now contracts, expulsing some cytoplasm through the hole, like a relaxing ball that would be squeezed by your hands. This leads to the formation of a bleb, a woughly spherical bulge of membrane, initially devoid of cortex. After a while, a new cortex is polymerized and the bleb retracts. Goal of this study is to get the dynamics of the bleb growth and retraction.
+When ablated at the left spot, the constraint on volume is relaxed, and the cortex can now contract, expulsing some cytoplasm through the hole, like a relaxing ball that would be squeezed by your hands. This leads to the formation of a bleb, a roughly spherical bulge of membrane, initially devoid of cortex. After a while, a new cortex is polymerized and the bleb retracts. The goal of this study is to get the dynamics of the bleb growth and retraction.
 
 ## Bleb growth dynamics
 
 First, get the raw transmitted movie [here (22MB)](https://fiji.sc/tinevez/web/Bleb_growth_transmitted.tif).
 
-We would like to get the speed at which the front of the bleb grows. The simplest way to do so is to use kymographs. The idea is to draw a line from the center of the cell that passes through the bleb. To asjust the line position, select a frame where the bleb is still small, and draw a line ROI from the center through the bleb.
+We would like to get the speed at which the front of the bleb grows. The simplest way to do so is to use kymographs. The idea is to draw a line from the center of the cell that passes through the bleb. To adjust the line position, select a frame where the bleb is still small, and draw a line ROI from the center through the bleb.
 
 ![](/media/tutorials/generate-and-exploit-kymographs-1.jpg)
 
@@ -32,17 +32,17 @@ Check the feature using a profile plot (shortkey {% include key key='K' %} or {%
 
 The edge feature is not that clear on this picture. At about 7 um, one can see that the dark pixels and the bright halo around the bleb front generated some kind of ridge.
 
-The kymograph consists in plotting this profile, encoding the intensity in grayscale level (or color for color images) for all slices. Rather than creating a plugin for this, we can use a function that was initially made for Z-stacks: the {% include bc path='Image | Stacks | Reslice'%} command (shortcut "/"). An option dialog appears; the defaults are fine for us, just press OK. We then get the kymograph:
+The kymograph consists of plotting this profile, encoding the intensity in grayscale level (or color for color images) for all slices. Rather than creating a plugin for this, we can use a function that was initially made for Z-stacks: the {% include bc path='Image | Stacks | Reslice'%} command (shortcut "/"). An option dialog appears; the defaults are fine for us, just press OK. We then get the kymograph:
 
 ![A kymograph](/media/tutorials/generate-and-exploit-kymographs-3bis.png)
 
 On this image, space is encoded along the x axis, following the line ROI from left to right. Since we drawn the ROI from the middle of the cell to the exterior of the cell, intensities measured within the cell are reported to the left of the image. At almost the extreme right, we get the cell's border and the bleb front.
 
 Time is encoded along the y direction. The first frame measurements is reported on the top line, and the bottom line reports the last frame. The white band correspond to the ablation, between frame 11 and 16, and appears between the 11 and 16 pixels of the kymograph.
 
-ImageJ can't deal with a calibration unit that is different in x and y axes, which is the case here (space and time). So for further analysis, we will have to put it right ourselves using the pixel size for the x calibration and the frame interval for the y calibration. I found it safer to remove totally any calibration from the images, even from the source stack. This way no confusion can be arise from a delay that would be measured in µm.
+ImageJ can't deal with a calibration unit that is different in x and y axes, which is the case here (space and time). So for further analysis, we will have to adjust it ourselves using the pixel size for the x calibration and the frame interval for the y calibration. I found it safer to completely remove any calibration from the images, even from the source stack. This way there is no confusion from a delay that would be measured in µm.
 
-The front of the growing bleb can be seen below this white band, as a dark edge at the right of the image. The bleb growth appears here as a rather steep curve below the white band. Its retraction takes much longer, which results in having a drak edge slowly moving towards the left part of the image as we screoll down the kymograph. Other features within the cell can also be seen within its volume. In the case of this transmitted image, they are various granules movements which we do study here.
+The front of the growing bleb can be seen below this white band, as a dark edge at the right of the image. The bleb growth appears here as a rather steep curve below the white band. Its retraction takes much longer, which results in having a drak edge slowly moving towards the left part of the image as we scroll down the kymograph. Other features within the cell can also be seen within its volume. In the case of this transmitted image, they are various granules movements which we do study here.
 
 To get the bleb front dynamics, one can directly plot a multi- line ROI and get its coordinates for further analysis. We could also try to segment it a bit automatically.
 
@@ -66,13 +66,13 @@ Next step is to get numerical values and model for this growth. We leave it to a
 
 ## Myosin dynamics at bleb front
 
-Now download [here (22 MB)](https://fiji.sc/tinevez/Bleb_growth_myosin.tif) the movie for the same experiment, but measured in the fluorescence channel. It pictures myosin dynamics at the cortex. Our goal is to measure the myosin recovery rate at the bleb front during its growth. This would typically be complicated: we are trying to measure the intensity of something that moves over time. A sophisticated solution would be to segment the bleb front as an object and then measure the intensity given the objects. We propose here a simple solution involving a bit of manual interaction.
+Now download [here (22 MB)](https://fiji.sc/tinevez/Bleb_growth_myosin.tif) the movie for the same experiment, but measured in the fluorescence channel. It pictures myosin dynamics at the cortex. Our goal is to measure the myosin recovery rate at the bleb front during its growth. This would typically be complicated: we are trying to measure the intensity of something that moves over time. A sophisticated solution would be to segment the bleb front as an object and then measure the intensity given the objects. Here, we propose a simple solution involving a bit of manual interaction.
 
-First,change a bit the contrast so that we can see the cortex well, so as to place correctly our line ROI. To do so, use {% include bc path='Image | Adjust | Brightness/Contrast'%} and play with the *Maximum* slider. Do not press *Apply*: we just want to improve what we see on the screen, not the actual intensities stored in the stack. Then draw a line ROI that goes - as before - from the center of the cell to the exterior, crossing the bleb grow spot.
+First, adjust the contrast such that the cortex is clearly visible, so as to correctly place our line ROI. To do so, use {% include bc path='Image | Adjust | Brightness/Contrast'%} and play with the *Maximum* slider. Do not press *Apply*: we just want to improve what we see on the screen, not the actual intensities stored in the stack. Then draw a line ROI that goes - as before - from the center of the cell to the exterior, crossing the bleb grow spot. If needed, adjust the calibration before reslicing.
 
 ![](/media/tutorials/generate-and-exploit-kymographs-7.png)
 
-Get the kymograph from this movie using the *Reslice* command. Thrash the calibration before doing it if you feel the use for it. You can also use the dynamic version of *Reslice*, shipped with Fijim and that can be found in {% include bc path='Image | Stacks | Dynamic reslice'%}. The advantage is that the kymograph is updated live when you move the ROI. After improving the contrast, it should look like this:
+Get the kymograph from this movie using the *Reslice* command. You can also use the dynamic version of *Reslice*, shipped with Fijim and that can be found in {% include bc path='Image | Stacks | Dynamic reslice'%}. The advantage is that the kymograph is updated live when you move the ROI. After improving the contrast, it should look like this:
 
 ![A kymograph for fluorescence image](/media/tutorials/generate-and-exploit-kymographs-8.png)
 
@@ -86,7 +86,7 @@ $$s=\sqrt{(x_2-x_1)^2+(y_2-y_1)^2}$$
 
 but since on our kymograph the x coordinates report the space and the y coordinates report the time, the x axis of the profile has no meaning, and can't be exploited as is.
 
-<u>Exercise:</u> Think of a way to put this right, that is to get a profile where the x axis would be the correct delay measured from the ablation offset ([please discuss](/discuss/#ways-to-get-help)).
+<u>Exercise:</u> Think of a way to get a profile where the x axis would be the correct delay measured from the ablation offset ([please discuss](/discuss/#ways-to-get-help)).
 
 ## Examples and references