Usage
At the moment the plugin only supports 16-bit grayscale images from 3D localization microscopy experiment. In order to start an analysis run, drag the localization microscopy image stack into ImageJ. Run the LookUpSTORM plugin from the ImageJ menu Plugins -> LookUpSTORM -> Analyse Experiment. The first step is to create the lookup table (LUT) for analysis.
During generation, the LUT is populated with template images of a selected PSF model at discrete lateral/axial positions. The PSF model's x- and y-positions are varied over a defined range at defined steps (see Lateral Range and Lateral Delta). In addition to the variation in x- and y-position, z-positions are also varied over a defined range at a defined steps (see Axial Range and Axial Delta), which influences the shape of the PSF model (based on the used model, see Source). Each variantion of xyz is used to generate a 4-dimensional (PSF model and their derivatives in xyz) template image within the LUT.
The field Source is used to select the LUT generation method/model. Currently supported sources are:
-
Astigmatism: Generate LUT from a astigmatsim calibration file using 2D elliptical Gaussian model (see Create Calibration using ThunderSTORM) -
Astigmatism Integrated Gauss: Generate LUT from a astigmatsim calibration file using 2D elliptical integrated Gaussian model (see Create Calibration using ThunderSTORM) -
From Binary File: Load an already created LUT as binary (see LUT Binary File Format)
The window size in pixels describes the size of the to generate LUT template image width and height. This value is also used for fitting (window size around an emitter canidate).
This value sets the step size at which PSF models are positioned around the center of the window size in both x- and y-direction within a template image. (default is 0.1 pixels)
This value sets the maximum range at which PSF models are positioned around the center of the window size in both x- and y-direction within a template image. (default is 4 pixels)
This value sets the step size at which PSF models are varied in axial direction. (default is 25 nm)
This value sets the range at which PSF models are varied in axial direction. (default is 1000 nm)
File path to either the calibration file (YAML file format) or binary LUT file.
This field shows the estimated RAM usage for LUT depended on lateral/axial range and step size as well as the window size.
Press this button to start generating the LUT.
LookUpSTORM calculates the photon count and Cramer-Rao lower bound (CRLB) of fitted single emitter signals. Therefore, the camera pixel values are required. LookUpSTORM can only manage EM-CCD cameras at the moment, however the EM-Gain can set to be 1 if only a CCD camera is used.
This field is used to set the image pixel size of the to be analysed localization microscopy image stack in nm.
This field is used to set the pixel photon conversion factor, which is camera specific in photons per AD counts.
This field is used to set EM Gain of the EM-CCD camera used during the image acquisition of the experiment. If not used set this value to 1.
This field is used to set camera baseline in AD counts. Typically, camera add some counts to each pixel. If not used set this value to 0.
In this tab general settings are selected for fitting and post processing (save rendered image/localizations). The directory in which the rendered image and/or the resulting localization table (as CSV) can be selected by the field Output Path.
This field is used to set for finding single emitter signal candidates in AD counts. Signals (local background is subtracted) with values below this threshold are rejected. This field can be changed during analysis. Threshold is a unsigned short value (0-65535).
This field set the magnification from the original localization microscopy stack image size used for rendering. If this value is too high (scale > 1/lateral-delta) a LookUpSTORM warns the used (there could be artefacts in the rendering image since localizations are discrete in xyz).
This field is used to sets the maximum iterations for the Gauss-Newton algorithm. (default is 5)
This field is used to sets the residual squared difference stop value used by the Gauss-Newton fitting algorithm. (default is 1E-2)
This field is used to select an output path for the results of the analysis.