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Low Light Tracking Tool

58 bytes added, 12:23, 8 October 2015
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{{Infobox Plugin
| name = Blob Tracking
| software = Fiji
| author = [[User:{{Person|Alex-krull | Alexander Krull]]}}, [[User:damienrj {{Person| Damien Ramunno-Johnson]]Damienrj}}| maintainer = [[User:{{Person|Alex-krull|Alexander Krull]]}}, [[User:damienrj {{Person| Damien Ramunno-Johnson]]Damienrj}}
| source =
| released = May 22<sup>nd</sup>, 2013
"A Divide and Conquer Strategy for the Maximum Likelihood localization of Low Intensity Objects" by Alexander Krull et al, 2013 Optics Express, Vol. 22, Issue 1, pp. 210-228 (under preparation2014).
=Installation of the plugin=
=The User Interface=
==The different windows==
When the plugin starts on a 2D image stack you have three windows and the control panel. The main data window lets you move through the slices like normal. On the sides you see the two kymographs, which make is makes it easier to see movement over time.
The control window will help you to organize your tracking activity.
If you would like the windows to become larger or smaller, change the size of the main data window and then select '''Arrange Windows''', from the '''View''' menu. Alternatively you can use the [[Low_Light_Tracking_Tool#Hot-Keys | hot-key]] '''{{key|Ctrl-}}+{{key|W''' }} (when the focus is on the control panel) or '''{{key|W''' }} (when the focus is on one of the other windows). This will also organize the windows if they have become scattered.
If you close some of the windows you can bring them back using the '''Windows''' menu in the control panel.
* You can use ImageJ/Fiji slider controls below the main window.
* You can set frame number, slice number and channel number in the control window.
* You can use your middle mouse button to click directly click into one of the kymographs to set the frame number. If you are working with 3D data you can click into one of the side projections with your middle mouse button to set the slice number.
=Basic tracking=
==Create Sessionsession==
[[Image:LLTT ChooseSession.jpg]]
Click on the '''New Session ''' button, or select from the menu. The '''New session ''' window opens. Here you can pick a name for the session as well as the channel you want to track on. Finally you can choose your tracking method.
==Choose a tracking method==
There are currently two tracking algorithms available:
This algorithm implements the [http[wikipedia:// Maximum likelihood estimator|maximum likelihood estimator]] based on a [http[wikipedia:// Shot noise|shot noise]] model. This means it considers the statistics of photon distribution in the image generation process to find the optimal location estimate. In our publication this is referred to as the inner loop.
This algorithm implements the maximum likelihood estimator based on a more sophisticated model which in addition includes the stochastic amplification process in an [http[wikipedia:// multiplying CCD|EMCCD]] camera. We have shown, that it yields more accurate results in situations with low light levels. In our publication this algorithm is referred to as the '''Nested Maximum Likelihood Algorithm'''.
==Pick session options==
After clicking '''OkOK''', a new window will open. In this window you can choose a '''σ''' value, which denotes the standard deviation (in pixels) of the Gaussian used to approximate the [http[wikipedia:// Point spread function|Point Spread Function]] (PSF) of the tracked objects. If you select '''Automatic σ''', this value is used only as initialization. In this case '''Min''' and '''Max''' are used as bounds for the estimation. These values will be the default values for all new traces you make in this session.
Fixed '''σ ''' can be more stable and also faster, this is more so for 3D data.
All settings can later be altered by clicking on '''Session Options''', under '''Edit'''.
==Adding objects to track==
==Edit individual objects==
The default settings made in the session options dialog will only effect only new traces. If at this point you find that the parameters you picked were less ideal you can change the parameters with the '''Edit Object''' selection from the '''Edit''' menu.
==Deleting and manipulating traces and sessions==
Using the '''Merge''', '''Split''', '''Trim''', and '''Delete''' buttons allows you to edit your traces by combining or deleting them. Sessions can be deleted using the ''''Delete sessionSession'''' button.
==3D data==
The plugin can be used to track objects in 3D volumes, as well. When working with 3D data you will be asked upon startup to provide the '''ΔZ ratio''' in addition to the '''Intensity Offset''', '''EMCCD gain'''and '''Electrons per A/D count'''.
The '''ΔZ ratio''', is the slice thickness, divided by the pixel width. This value is only used for 3D data.Example: My pixel size for my data is 100 nm, and my slice thickness is 200 nm. I would then enter a value of 2.
Otherwise, tracking function is the same. It is best to optimize the σvalue σ-value for 3D tracking, and then use a fixed value to increase speed.
==Using multiscale tracking for fast moving objects==
If you are tying trying to track fast moving objects that move over a big distance between two frames, the software might loose the target at some point. To avoid this you can use multiscale tracking, which uses iterative smoothing and down-sampling to find the target in each frame. To start tracking in multiscale mode hit the '''Multiscale'''.
You can change the default behavior in the edit menu.
The algorithm builds a [http[wikipedia:// %28image_processing28image processing%29 |pyramid]] by repetitively smothing the image with a Gaussian kernel with the standard deviation given in the '''Smoothing σ''' field and than down-sampling it by a factor set in the '''Downscaling''' field. The pyramid's hight (i.e. the number of repetitions) can be set in the '''iterations''' field.Localization is than first performed on the top level of the pyramid, and repeated until its bottom, which is the original image.
* If you want to be able to see the original image data and find your tracking results get in the way, you use the hot-key '''{{key|P''' }} or '''{{key|Ctrl-}}+{{key|P''' }} respectively to hide all overlays (all circles lines and cross-hairs drawn on top of the image).
* If some of the windows appear to bright or to dark use '''View->Adjust Brightness/Contrast''' to correct them.
* You can use '''File->Export images''' to export a sequence of image with the tracking results drawn on top of them. You can use this to create a movie in order to demonstrate your tracking results. In your working directory a new folder named '''movieMain''' will be created and filled with images.
* If you have multichannel data, each channel needs its own session. You can select which sessions are visible by checking their boxes in the '''Visible Sessions ''' list. You can use the current session drop down menu to switch between sessions.* You can zoom into the kymographs by moving the cursor over the kymograph window and holding down '''{{key|Shift'''}}, while using the scroll wheel on the mouse.
* You can use the '''optimize''' button. This will perform the tracking of the selected objects on the single frame you are looking at. You can use this for example with '''Automatic σ''' turned on to determine the size of the target's PSF on a single frame.
=HotkeysHot-keys=There are several hot-keys you can use. You can find the key to press in the menus of the control panel. When the focus is on the control panel you have to press '''{{key|Ctrl''' }} to access hotkeys. When the focus is on one of the other windows this is not necessary.
=File format=
'''<name of image file>_<name of session>_<label of trace>_<id of trace>.trcT'''
The files are created in the working directory. Each file begins with some lines starting with '#'. These lines store information for the tracking program they will be ignored by most programs like matlab [[MATLAB]] or gnuplot potentially used to further analyze the data.
The columns have the following meaning:
* column 16 the total estimated flux in the considered window
= Example Data =
This link contains the data used to generate this tutorial, and a two channel 3D data set to try.
= License =
Packages hep.aida.*
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