5.9.1 Correction of the chromatic shift
Materials:
Image stacks of different color channels comprising ~10 Tetraspeck beads raw Leica file series to be Z-shift corrected
ImageJ, including the plugins Sync Measure 3D and StackGroom / Shiftcorrector Excel
Background:
The chromatic shift gets obvious when overlaying different color channels which contain the same fluorescent object (e.g. a Tetraspeck bead). Calculating the coordinates of the barycenter of the same bead in different channels delivers the geometrical vector of the shift between the channels, with which they can be corrected thereafter. Since confocal images are limited to the dimensions of single voxels, image shift corrections can only be executed with full-voxel step sizes. Since the lateral shifts (x-y) are usually only very small (~50- 150 nm) only Z-shifts are commonly corrected. Z-shift correction can be performed with the plugin Shiftcorrector developed by Dr. B. Joffe, which deletes the first or last slices of the image stacks to yield a best-fitting alignment in Z compensating for the chromatic shift. Shiftcorrector has a defined demand on the file naming and works well with raw Leica file series. Note that every nucleus has to be saved in a subfolder and no additional files are allowed in the main folder as this leads to a crash of the plugin. The plugin reads the information on chromatic shift out of a text-file in which the different channels are sorted according to their emission lights: (b)lue, (g)reen, (o)range, (r)ed and (f)ar red. Since one channel has to act as referee, where all other channels will be aligned to, one should choose the particular channel, where all other channels have a positive shift to. This channel will be given the value 0 (no beginning slice of the stack will be deleted). According to the measured chromatic shifts, a certain number of beginning slices of the stacks of the other channels have to be deleted (e.g. 1= the first; 2= the first and the second or 0= no slice). To equalize the number of slices obtained in the different channels after Z-shift correction, the software automatically deletes slices in the end of the channels till all channels have the same amount of slices
Methods:
- Measure chromatic shift
o Load bead image stacks in ImageJ and analyze chromatic shift with the use of the plugin Sync Measure 3D developed by Dr J. Walter.
o Save chromatic shift vector information for subsequent shift corrections in an Excel file
- Correct chromatic shift
o round Z-shift vectors to whole voxel values according to the voxel-size in the data sets which are to be corrected. Thereby, minimize the left-over shift summed up for all color channels after shift correction (e.g.: Z-voxel-size: 250 nm; Z-shift: 300 nm left-over shift of 50 nm)
o Create a text-file (e.g. actual_shift.txt) matching the demands of the plugin Shiftcorrector
o Start the plugin Shiftcorrector and choose the text-file containing the actual shift
o Define the order in which the different color channels had been recorded (leave out non- recorded channels)
o Choose the main folder in which the raw Leica image series are saved
The plugin will automatically open all stacks consecutively, will delete first or last slices according to the text-file and will save shift corrected files in a new folder
5.9.2 Deconvolution
Materials:
Raw confocal stacks
Deconvolution software Huygens (SVI) Essential 3.5 For all deconvolution parameters to be set see list in 8.5 Fluorescent beads: 175 nm, for each color channel Confocal microscope
Background:
Since deconvolution of confocal images was not commonly applied in the beginning of this thesis, its establishment and standardization was the first aim of this study. The methodological part comprehending deconvolution is therefore kept short since the proof of reliability is described and discussed in detail in the corresponding results chapter (see Results 6.1).
Methods:
- Preparation of 175 nm-beads slide:
o Follow protocol 5.8.3 but substitute the Tetraspeck-beads with the fluorescent 175 nm beads. Since these beads are available with different spectral signatures (blue, green,
orange and red), it is advisable to make separate slides for each signature to facilitate the subsequent recording.
- Measuring the Point-Spread-Function (PSF):
o Record stacks for each laser line with the 175 nm-beads slide best-matching with its spectral signature. Thereby, use an image size of 512x512 pixels and a voxel dimension of 50x50x120 nm.
o Open the recorded image stacks with the Huygens software and distill the corresponding PSFs according to the manual (for detailed description of the parameters see 8.5)
o Save PSF files for subsequent deconvolution of confocal image stacks separately for each spectral signature in the '.ics' and '.ids' file format
- Deconvolution of single stacks:
o To deconvolve a single image stack, open it with the Huygens Essential software
o Additionally open the corresponding PSF file
o Start deconvolution according to the manual (for parameters see 8.5)
o Save deconvolved image stacks
- Deconvolution of multiple stacks (batch mode):
o To deconvolve multiple stacks start the computer in the Linux mode
o Save the raw image stacks, the corresponding PSF files and the three Huygens files ('batchCtl.tcl', 'restOneImage.tcl' and 'doBatch.tcl') in the same folder
o Open the 'batchCtl.tcl' file with Kwrite, adapt all parameters (see 8.5), define the stacks to be deconvolved and save the processed 'batchCtl.tcl' file
o Start deconvolution by executing the 'doBatch.tcl' file
The software will deconvolve all selected image stacks according to the information in the 'batchCtl.tcl' and 'restOneImage.tcl' files and will save them in a subfolder named 'Results'.