TimeLapse Microscopy allows a visualization of the processes of development growth, or drift of sample that occurs over this image. Acquisition can result in an inability to follow or track the cell movements, the following protocol. We use open source imaging processing software to correct for three-dimensional drift over time.
Capturing a series of optical sections at different focal planes allows the generation of a three dimensional model of the sample. This can be extended into four dimensions by creating a time-lapse series of 3D data sets. In long-term time lapse experiments, it is common to observe sample movement.
This can be caused by sliding accuracies in the hardware controlling stage, and focal positions, while in other cases drifter as a result of movements induced by sample growth or flexibility within the mounting media methods exist to compensate to limit these movements with many improvements to hardware focusing systems and the increased rigidity of mounting medium. However, these approaches cannot be applied in many cases due to the imaging setup required to provide suitable conditions for the samples maintenance and growth to correct. For sample drift, we have developed a plugin to utilize the open source imaging processing platform of Fiji.
Our correct 3D drift plugin is able to form a phase correlation registration to correct for movement that occurs as a role or sample drift in three dimensional time-lapse experiments in multi-channel experiments. The correct 3D drift plugin utilizes one channel to determine the required correction. This correction is then applied to any additional channels resulting in the registration of The four D dataset.
The open source free Web package. Fiji is a distribution fork, the Image J program, which contains pre-installed plugins to perform numerous processes on data collected from microscopy experiments. Fiji provides an easier plugin architecture and includes in default installation.
A copy of the correct three EG plugin, which we'll Use for this analysis when installation is complete Open Fiji. Fiji supports the import of a vast array proprietary microscopy image formats. This Can be achieved through drag and drop or through the users, the Open microscopy environment.
Bio formats import plugin. If the data set Exceeds the size of memory installed on your system, open the image file as a virtual stack and of a memory Management option. The data set we'll use In this analysis was generated from a three-dimensional time series experiment of the ACTA one, GFP transgenic zebrafish in which the GFP is expressed in the skeletal muscle.
To visualize the nuclei, this transgenic line was injected with the nucleo localizing M cherry to better represent these Fluor floors, we can alter the lookup tables for each of these channels under the color channel tool. Over the course of the time series collected, it appears the zipper fish sample drifted in the field of view. She correct this drift opened the correct 3D drift plugin in the registration sub menu of the plugins installed in Fiji.
Seller Markers that are either widely expressed within a sample or not involved in the marketery events during image acquisition provide the best source for drift correction. In this example, the ACTA one, GFP fluorescence in channel one. If memory is limited on your system, select the virtual stack option and allow the Correct 3D drift plugin to run.
The corrected dataset will have a larger canvas size compared to the original. Accommodating the movement of the sample. Generating maximum Projection images of the optical sections allows the comparison of the drift that has been removed from the corrected sample to Be made.
The maximum intensity Projection removes the dimension from the dataset, but as a useful approach to quickly examine the results of the drift Correction. Here we see the maximum projection images for each tie point in their corrected and uncorrected data sets. When this time-lapse experiment Is projected as a single image, the amount of drift that can occur even in a short time-lapse experiment is apparent.
The corrections made by registering the drift of one channel of your sample are also applied to all other channels contained In this image stack. The procedure for drift Correction if memory is limited on your system is slightly altered. Select The used virtual stack option in both the buy format, import menu, and in the correct 3D drift plugin Popup menu.
When you select This option, you'll be prompted to select a location that a plugin can export individual image files for each channel slice and time point Of your time-lapse experiment. The generated virtual stack contains the same drift correction as our previous example. If we open the selected save location, we'll see the resulting individual image sequence files Generated.
To open this collection of files, use the image sequence import tool. The resulting file will Not separate channels, optical sections or time points. To visualize these features, convert the stack to a hyper stack using the data from the log file, enter in the number of channels, Optical slices and the tie points collected in the, We can again alter the lookup tables to adjust the color displayed of our time-lapse experiment.
This Protocol corrects for drift that occurs between time points drift that occurs within the capture of a single time point cannot be corrected by this protocol. We recommend that the parameters used for image acquisition reduce the time taken for each time point to be captured. The ability to correct for drift in post-processing software allows for cell movements that would otherwise be skills by the drift of the sample to be analyzed and measured.
