IMAGEJ THRESHOLD SELECTION DIALOG REGISTRATION
IMAGEJ THRESHOLD SELECTION DIALOG SERIES
IMAGEJ THRESHOLD SELECTION DIALOG WINDOWS

We can examine the performance of a particular threshold by browsing through the stack and zooming in and out using the Fiji toolbar tools. The goal here is to find a threshold where most beads are detected - ideally only once. The lower the threshold the more 'beads' are segmented. Typically we only need to play with the Threshold. The parameters can be interactively changed in the Adjust Difference-of-Mean Values window. In the browsable stack window the view selected in the previous step is shown and beads segmented using the current parameters are highlighted with small green circles.

IMAGEJ THRESHOLD SELECTION DIALOG WINDOWS

That's it, we can for now safely ignore the remaining options of the dialog and proceed by clicking OK.ĭesktop showing windows opened during interactive segmentation.Īfter a short delay two windows will pop-up. We will leave Affine as a useful and, in fact, necessary default for OpenSPIM data. Affine - includes scaling and shearing (necessary for OpenSPIM data due to aberrations introduced by diffraction index mismatch between water and agarose).Translation - uses transformation model that takes into account only translation between views (nto particularly useful for multi-view OpenSPIM data).We will need it during all subsequent steps.įinally we will examine the pull down menu Transformation model that offer three options It will also be stored as the value in the *.registration files as discussed below. The 9.0323 or z-scaling is an important number to remember and write down. We could also express the ratio as xy = 1 and z = 9.3023. In the case of OpenSPIM sample data it is 6/0.645 = 9.3023. Note that the important number here is the ration between the z and xy resolutions. z resolution (un/px) = 6 - the step size of the motors on OpenSPIM are 1.5 um and we sued 4 z steps between planes when acquiring the sample data, i.e.xy resolution (um/px) = 0.645 - this is dictated by the optics of the detection path of OpenSPIM and the size of the pixels on the CCD camera.For the sample OpenSPIM data discussed here the following parameters work. We will select 3-dimensional quadratic fit for our sample OpenSPIM data.įor OpenSPIM data it is very important to select the Specify calibration manually checkbox and enter the xy and z resolution. Gauss-fit (all detections) - the slowest option where Gauss fitting is applied to all bead detections which can be many and it can take a while.Gauss-fit (true correspondences) - more precise but slower, limited only to true correspondences for performance reasons.3-dimensional quadratic fit (all detections) - fastest, but least precise option, sufficient for OpenSPIM data.The Subpixel localization is a pull down menu offering three option for more-or-less precisely localizing the beads In our initial run we will select the option Interactive to play around with the threshold that is best suited for our data. Interactive - the threshold can be manually adjusted based on visualization of bead detection on a user selected time point.Advanced - the threshold for bead detection can be set manually in the following dialog.Strong - the threshold for bead detection set to 0.25.Comparable to sample - the threshold for bead detection set to 0.075 (typically when using beads emitting in the same wavelength as the sample, i.e.Weak - the threshold for bead detection set to 0.02.red green beads for red fluorescent sample)

Very weak - the threshold for bead detection set to 0.0025 (typically when the beads emit in a different wavelength compared to sample, i.e.

Bead Brightness is a pull down menu offering 6 options. In the second section of the dialog we need to initially pay attention only to the parameters affecting the initial segmentation of the bead. Any comma separated list will do, for instance 60,180, 235.

IMAGEJ THRESHOLD SELECTION DIALOG SERIES

Discontinuous series is also possible, for example 1,5,10.įinally we specify the Angles to process, in our case 0-4 (0.1.2.3.4 = 5 angles). The range does not need to start with 0 or 1. To register all time-points in the time series we would enter 0-10.

IMAGEJ THRESHOLD SELECTION DIALOG REGISTRATION

We will initially optimize the registration using time point 5. lsm - a format used to store data from the Zeiss SPIM demonstrator. This is a common mistake at the default extension is for historical reason. Importantly, do not forget to adjust the extension to. If we have a three digit series (000 - 100) we use in case of our sample data that range from 0 to 10 we need tt as time point placeholder. tif files generated in the previous step of the pipeline.Įnter the pattern of the files, where tt is a zero padded place holder for time points and a a zero padded placeholder for angles/views. Screenshot of the second dialog of the bead based registration pluginĬlick Browse to locate the directory with the.

Imagej threshold selection dialog