====== Stripper ====== STRIPPER: fa**ST** **R**obust f**I**lament **P**icking **P**roc**E**du**R**e Helps you to strip your filaments out of the micrograph automatically :-) {{:actin.png?direct&300 |}}{{ :actin_processed.png?direct&300 |}} ===== Overview ===== {{::screen_shot_2017-12-08_at_12.43.57.png?direct&200 |}} Stripper is the filament particle picking procedure used in our lab. ===== Installation ===== Activate the "biomedgroup" update site and add [[https://imagej.net/Following_an_update_site|a new update site]]: http://sites.imagej.net/Mpi-do-stripper/ ===== Get your data into ImageJ ==== === Load the raw data === - File -> Import -> Image Sequence - Select the folder of your images - Type the file ending into "File name contains:" (e.g .mrc, .tif) - Check "Use virtual stack" - Press OK Now you have read in your data as an image stack. === Reduce the size === For the sake of memory consumption and speed your should reduce the size / image depth of your image data. - Image -> Adjust -> Size - Set the width to 1024. The height is calculated automatically. - Press OK. - Image -> Type -> 8 Bit Wait until the processing is finished. === Save the file for later use === File -> Save As -> Tiff... ===== Parameters ===== ==== Detection ==== As there is no easy way to guess the lower and upper threshold, it is recommended to use the detection assistant (File -> Detection assistant). **Filament width:** The width of your filaments in pixel. **Mask width (Advanced option):** As the response along the filament randomly fluctuates and sometimes disappear, it is important to average along the filament. **Lower threshold:** Filter responses lower than that value will be considered as background and filament tracing stops here.Higher values lead to more segmented lines, as low contrast regions of a filament will be considered as background the line is splitted at this position. **Upper threshold:** Filter responens higher than that will be considered as valid filament starting point.With higher values the contrast of the filaments have to higher be to be picked. **Equalize:** After enhancement of the filaments, all images are adjusted that filamants have a more similar absolute response. Especially when the image contains high contrast contaminations, this option has a positive effect. By defaut this option is activated. ==== Filtering ==== **Min number of boxes:** The minimum number of boxes that have to be placed per line. ---- === Response filter === **Sigma min. response:** The mean response M and the standard deviation S are estimated over all lines per image. If a line has a signficant number of line points __below__ the threshold T = M-F*S it is considered as false-positiv (background) and is removed, whereas F is value to specifiy.By default, this filter is deactivated (set to 0). A resonable value lies typically between 2 - 3. **Sigma max. response:** The mean response M and the standard deviation S are estimated over all lines per image. If a line has a signficant number of line points __above__ the threshold T = M+F*S it is considered as false-positiv (ice,overlapping filament) and is removed, whereas F is value to specifiy.By default, this filter is deactivated (set to 0). A resonable value lies typically between 2 - 3. **Sensitivity:** Threshold value for minimum straightness. Line segments (see window size) with a straightness below that threshold will be removed and the line is splitted. Higher values means that the filament have to be more straight, where 1 means a perfect straight line. ---- === Straightness filter === **Min. straightness:** Threshold value for minimum straightness. Line segments (see window size) with a straightness below that threshold will be removed and the line is splitted. Higher values means that the filament have to be more straight, where 1 means a perfect straight line. **Window size:** The number of line points which are used to estimate the straightness. Larger values lead to more robustness against noisebut decrease to ability to detect small curves. ---- ** Allowed box overlap: ** Relative amount to what extend two boxes auf different filaments are allowed to overlap. The default value 0.5 which is the maximum value that ensures that a box contains only one filament ==== General ==== ===== Step-by-Step Tutorial =====