Differences
This shows you the differences between two versions of the page.
| Both sides previous revision Previous revision Next revision | Previous revision | ||
|
picker:tilt_pair_picking [2017/06/12 13:22] kbertra2 |
picker:tilt_pair_picking [2017/06/20 17:17] (current) kbertra2 |
||
|---|---|---|---|
| Line 1: | Line 1: | ||
| ===== Random Conical Tilt (RCT) ===== | ===== Random Conical Tilt (RCT) ===== | ||
| - | The CowSuite offers the option to analyze random conical tilt (RCT) data. The following tutorial provides a step-by-step guide of how to use the **Tilt Mode** function. | + | The CowSuite offers the option to analyze random conical tilt (RCT) data. The following tutorial provides a step-by-step guide of how to use the **Tilt Mode** function. Be sure to check out the [[picker:shortcuts|]] for easier navigation in the [[:picker|]]. |
| === Tilt Pair Selection === | === Tilt Pair Selection === | ||
| - Open the [[:picker|]]. Load micrographs by clicking on the folder icon in the top-left corner of the GUI. Select "Tilt mode" and then either "Folder" or "Files" (depending on how you've stored your data) when prompted. Following the prompts, first select untilted data and second select tilted data. Make sure that the files are in .tif format before proceeding. | - Open the [[:picker|]]. Load micrographs by clicking on the folder icon in the top-left corner of the GUI. Select "Tilt mode" and then either "Folder" or "Files" (depending on how you've stored your data) when prompted. Following the prompts, first select untilted data and second select tilted data. Make sure that the files are in .tif format before proceeding. | ||
| - | - Open "Settings." If working with negative stain samples, make sure this box is checked. Click on one of the micrographs to make a test selection, then adjust "particle size" in "settings" until the circular selection is the same size as your particle. (To delete a selection, right click on it.) Ensure that the settings for "particle size" of the tilted and untilted micrographs are identical, as below: {{ :picker:selection_012.png |}} | + | - Open "Settings." If working with negative stain samples, make sure this box is checked. Click on one of the micrographs to make a test selection, then adjust "particle size" in "Settings" until the circular selection is the same size as your particle. (To delete a selection, right click on it.) Ensure that the settings for "particle size" of the tilted and untilted micrographs are identical, as below: {{ :picker:selection_012.png |}} |
| - | - To make a tilt pair selection, select a particle in one of the micrographs and then find the corresponding particle in the remaining micrograph. Zoom by holding down Ctrl and then moving the mouse wheel. To move the micrographs independently, click on the icon immediately to the left of the red X in the toolbar (Ctrl+L). {{ :picker:selection_013.png |}} | + | - To make a tilt pair selection, select a particle in one of the micrographs and then find the corresponding particle in the remaining micrograph. Zoom by holding down Ctrl and then moving the mouse wheel. To move the micrographs independently, click on the icon immediately to the left of the red X in the toolbar (shortcut: Ctrl+L). {{ :picker:selection_013.png |}} |
| - Save your progress periodically by clicking on the floppy disk icon in the toolbar. Select whether you would first like to save tilted or untilted data. Select the destination where your data will be stored and create a unique filename for both the tilted and untilted data, since the data will be saved as a separate .plt file for each dataset. | - Save your progress periodically by clicking on the floppy disk icon in the toolbar. Select whether you would first like to save tilted or untilted data. Select the destination where your data will be stored and create a unique filename for both the tilted and untilted data, since the data will be saved as a separate .plt file for each dataset. | ||
| - Once all of your tilted pairs are saved as .plt files, you are ready to preprocess them for import into CowEyes. | - Once all of your tilted pairs are saved as .plt files, you are ready to preprocess them for import into CowEyes. | ||
| Line 25: | Line 25: | ||
| - If working with more than one set of tilted pair files of the same sample, you may want to combine your data. To do so, find the [[eyes:logics:Append|]] logic and drag it into your workspace. Connect this logic with your imported data. Continue this process until all of your data for untilted or tilted particles is combined into a single [[eyes:logics:Append|]] logic (i.e. at the end you should have two [[eyes:logics:Append|]] logics; one for tilted data and one for untilted data). At this point, your logics should be connected as shown by the following: {{ :picker:selection_006.png |}} | - If working with more than one set of tilted pair files of the same sample, you may want to combine your data. To do so, find the [[eyes:logics:Append|]] logic and drag it into your workspace. Connect this logic with your imported data. Continue this process until all of your data for untilted or tilted particles is combined into a single [[eyes:logics:Append|]] logic (i.e. at the end you should have two [[eyes:logics:Append|]] logics; one for tilted data and one for untilted data). At this point, your logics should be connected as shown by the following: {{ :picker:selection_006.png |}} | ||
| - | == Tilted Data == | + | **Tilted Data** |
| - From this point, work with //tilted data// until otherwise noted. You will deal with the tilted and untilted data separately. Once the data is appended, it needs to be filtered. Find the [[eyes:logics:Filtering|]] logic and connect it with the [[eyes:logics:Append|]] logic. Click on the [[eyes:logics:Filtering|]] logic and apply the following settings: Processor = Gauss bandpass, Upper frequency = 0.6, Lower frequency = 0.04, Show filter function = unchecked, Transmission = 0. Click the "Play" icon to run the logic. | - From this point, work with //tilted data// until otherwise noted. You will deal with the tilted and untilted data separately. Once the data is appended, it needs to be filtered. Find the [[eyes:logics:Filtering|]] logic and connect it with the [[eyes:logics:Append|]] logic. Click on the [[eyes:logics:Filtering|]] logic and apply the following settings: Processor = Gauss bandpass, Upper frequency = 0.6, Lower frequency = 0.04, Show filter function = unchecked, Transmission = 0. Click the "Play" icon to run the logic. | ||
| - Next select the [[eyes:logics:Normalization|]] logic and connect it to the [[eyes:logics:Filtering|]] logic of the previous step. Click on the [[eyes:logics:Normalization|]] logic and apply the following settings: Processor = Normalize against background, Invert = (select yes if micrographs were acquired in negative stain). | - Next select the [[eyes:logics:Normalization|]] logic and connect it to the [[eyes:logics:Filtering|]] logic of the previous step. Click on the [[eyes:logics:Normalization|]] logic and apply the following settings: Processor = Normalize against background, Invert = (select yes if micrographs were acquired in negative stain). | ||
| - | - Find the [[eyes:logics:Rct2|]] (random conical tilt) logic and connect it to the output of [[eyes:logics:Normalization|]] to "io:tilted" input of the [[eyes:logics:Rct2|]] logic. Before running the [[eyes:logics:Rct2|]] logic, click on it and adjust the settings as shown below (note: pixel size varies): | + | - Find the [[eyes:logics:rct|]] (random conical tilt) logic and connect the output of [[eyes:logics:Normalization|]] to "io:tilted" input of the [[eyes:logics:rct|]] logic. Before running the [[eyes:logics:rct|]] logic, click on it and adjust the settings as shown below (note: pixel size varies): |
| {{ :picker:selection_008.png |}} At this point, your logics should be set up in the following way: {{ :picker:selection_009.png |}} | {{ :picker:selection_008.png |}} At this point, your logics should be set up in the following way: {{ :picker:selection_009.png |}} | ||
| Note that the input labeled "io:untilted" has not been connected. Your //untilted// data must now be processed. | Note that the input labeled "io:untilted" has not been connected. Your //untilted// data must now be processed. | ||
| - | == Untilted Data == | + | **Untilted Data** |
| - Repeat steps 1-2 from the instructions for Tilted Data. | - Repeat steps 1-2 from the instructions for Tilted Data. | ||
| - | - Perform 2D operations as listed in the [[:tutorial|]]. Once you have a 2D classification, connect the "output" of your [[eyes:logics:Classification|]] to the "io:untilted" input of the [[eyes:logics:Rct2|]] logic. Click the "Play" icon to run the logic. | + | - Perform 2D operations as listed in the [[:tutorial|]]. Once you have a 2D classification, connect the "output" of your [[eyes:logics:Classification|]] to the "io:untilted" input of the [[eyes:logics:rct|]] logic. Click the "Play" icon to run the logic. |