Further, On multi camera systems, like OMX or some HCS system there WILL be a rotation angle difference between the channel images.Moving the images using a whole pixel or z plane shift will not be precise enough for high resolution colocalization analysis.Īt a higher level there may also be a magnification difference between the different colour channels.Or one could imagine a Fourier based method… (using a phase shift?) The Fiji ] plugin contains stuff that might do this but its not exposed in the GUIs… so scripting would be required there?.Use a nice interpolation method to avoid smashing the information in your images - eg quintic B-spline interpolation.Erik M’s TransformJ Translate plugin in Fiji/ImageJ can do a sub pixel resolution shift for for each channel, as we have measured those shifts TransformJ Translate.Now we can use software to fix this systematic error by shifting one image colour channel relative to the other. ![]() Use imageJ to do gaussian fits of the bead images and find the shidsts in x y and z.psfJ from Knop lab can make the measurement.How to measure the colour channel shift systematic error? see the 2 slides titled “Check with multi-colour beads” at Colocalization analysis course notes.BUT, they will never be whole xy pixels or z slice shifts… forget that idea its too crude.These will be between 10-1000 nm in xy and up to 500 or even 2000 micron in z.So if we measure a few 1 or 0.5 or so micron beads (no need to use tiny beads here) near the centre of the field of view (where optics are best) we can calculate or guestimate the center of mass of the bead images in each colour channel, and work out the shift vectors needed.OK, so at the simple level, we can assume that the whole field of view is shifted by some 3D shift per channel compared to the reference, usually green channel (ignoring any geometrical distortions or different magnifications or image rotations for different colours.) There always remain measurable errors (unless you were really really lucky and happen to have a perfect lens, which is a 1 in a 1000 chance at best, plus perfectly aligned fluorescence mirrors/filters).So its best to get is as close as is reasonably possible to do in hardware adjustments, then measure the residual error then correct for it.getting the near UV and IR laser spots to coincide exactly with the visible lines is hard… and so there is a collimation adjustment to make BUT, the 405 lasers come in through a different fibre than the Vis lasers.Nowadays with the Zeiss 7xx, 8xx series and on the Olympus FV1000 and other modern point scanners there is only 1 pinhole for the emission light, so that problem is suppressed.The Zeiss 510 has a different pinhole per channel… and depending on their positions and correct setup, the images of the bead in different channels is also affected by pinhole position settings. ![]() On a single point scanning confocal the matter can be made worse by the more complicated optics. So we are left with a systematic error that can be measured, actually to a precision of 10s of nm by Gaussian fitting bead images, or some other calibration sample (like you do in PALM/STORM type super resolution and single molecule tracking.This is why its not a good idea to screw a Zeiss c-apochromat 100x 1.4 oil into an Olympus stand.Olympus does all the chromatic correction in the lens, but others do some in the tube lens. On the API OMX you can even adjust this… normally its fixed on most systems but might also be tweakable. Also, the alignment of the fluorescence filters in different cubes might bend different channels to slightly different places on the detector.1.4 NA lens on a widefield/confocal/SPIM/super resolution system there will still be a unique colour shift in all three spatial dimensions, xyz, for that particular lens/opics combination, and even for what angle it is screwed into the stand. ![]() When imaging diffraction limited objects with an eg.
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