Difference between revisions of "Localization Microscopy"
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==Introduction== | ==Introduction== | ||
This plugin uses Gaussian fitting to localize single fluorescent molecules for tracking or reconstruction of super-resolution images. One window is used to set parameters for fitting and tracking, the second window (which can be opened from the first using the 'Data' button) is used to perform operations (such as rendering, drift correction, etc.) on data-sets of fitting results. | This plugin uses Gaussian fitting to localize single fluorescent molecules for tracking or reconstruction of super-resolution images. One window is used to set parameters for fitting and tracking, the second window (which can be opened from the first using the 'Data' button) is used to perform operations (such as rendering, drift correction, etc.) on data-sets of fitting results. | ||
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==Getting Started== | ==Getting Started== | ||
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[[Image: Main_LMPlugin.PNG]]</td> | [[Image: Main_LMPlugin.PNG]]</td> | ||
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To track a spot of interest in a time-series, draw a box around the spot and press the 'Track' button. After a short while, a row will appear in the '''Gaussian tracking data Window'''. Select the row and press '''Plot''' to visualize movement of the spot in the x direction. | To track a spot of interest in a time-series, draw a box around the spot and press the 'Track' button. After a short while, a row will appear in the '''Gaussian tracking data Window'''. Select the row and press '''Plot''' to visualize movement of the spot in the x direction. | ||
Revision as of 15:15, 17 May 2012
Introduction
This plugin uses Gaussian fitting to localize single fluorescent molecules for tracking or reconstruction of super-resolution images. One window is used to set parameters for fitting and tracking, the second window (which can be opened from the first using the 'Data' button) is used to perform operations (such as rendering, drift correction, etc.) on data-sets of fitting results.
Getting Started
Localization Microscope Window (first window)
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Imaging Parameters: Values entered in this section will be used in subsequent calculations. Press the read button to deduce some of these values from the meta-data contained in Micro-Manager datasets. You will need to measure the Photon-conversion factor (pcf) or use the pcf provided on the camera data-sheet. If you do not use EM gain, enter '1' in the field linear EM gain (to calculate the number of photons, digital numbers will be multiplied by pcf / linear EM gain. The Camera Offset (in digital numbers) is the average of an exposure in which no light can reach the camera. The program uses this to calculate the actual number of photons detected in signal and background. |
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Fitting Parameters
Dimensions: Determines the model used for the Gaussian. 1 - results in symmetric Gaussian in which only the width can vary, 2 - elliptical Gaussian, 3 - elliptical Gaussian in which the angle (theta) of the blob can vary. See Wikipedia article on Gaussian for details. |
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Filtering Data |
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Data Processing |
Data processing window
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General Options |
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2-Color Single Molecule |
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Processing Tracking Data |
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Localization Microscopy |