Introduction
This guide from the Center for Microscopy and Image Analysis will explain how you properly adjust the motorized correction collar of the following objectives available on ZMB instruments:
- 93x / 1.3 GLYC objective mounted at the Leica SP8 inverse STED 3X (Irchel).
- 63x / 1.2 WATER objective mounted at the Leica SP8 inverse (Gloriastrasse).
- 25x/ 1.0 WATER objective available at the Leica SP8 MP DIVE Falcon (Irchel).
Precise adjustments of the motCORR can compensate for deviations in the coverslip thickness, refractive index mismatches/specimen inhomogeneities as well as temperature changes to ensure restoration of optimal resolution, signal intensity and penetration depth.
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Make sure you are in the "Acquire" tab.
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Sample should be focused and lasers set.
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In the XY dialog open the "motCORR Collar Settings" dialog by clicking the arrow symbol.
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Configure your setting by
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adjusting the slider,
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directly entering a value into the input field, or
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alternatively via the control panel (one needs to add the desired setting).
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Follow the next steps for correct adjustment: Steps 2 and 3 applicable for thin samples close to the coverslip, Steps 4 and 5 applicable for any kind of sample.
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Only applicable if your sample is located close to the coverslip. If thick samples are used, the correction ring is best adjusted at the current z-level. Follow Step 4 in this case.
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Activate a laser line (e.g. 550nm). 488 nm recommended.
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Choose "xzy"- Scan Mode.
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Activate a "PMT" and place the detection window underneath the chosen laser line.
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Choose appropriate LUT. "Spectrum" recommended.
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Open the "AOBS Configuration" window and set it to "Reflection".
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Click "Live" to start live acquisition. Adjust "Gain [V]" and laser power appropriately without saturation.
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Adjust the correction ring to minimize reflection signal width corresponding to maximum intensity.
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Not optimized.
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Optimized.
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If the result looks satisfying the adjustment is done.
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Applicable for cells on cover glasses as well as thick specimen like tissue samples.
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Best to work with the HyD detector set to counting mode.
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Focus your sample in the fluorescence mode. Make sure you are in the "xyz"- Scan Mode.
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Activate "Auto-scaling" by clicking the "M" (when activated it displays an "A").
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Carefully adjust the correction collar in one direction. The focus has to be constantly corrected.
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Check whether the intensity and contrast of your specimen are improved - can be easily observed by the photon counts.
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If yes, continue in the same direction and constantly check your image.
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Finally, do this iteratively in both directions and adjust the ring to the maximum intensity and highest contrast.
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Same procedure can also be applied to the profile view of your sample.
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Choose "xzy" scan mode.
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A dotted line appears in the image. Choose the area where you would like to see your cross section (xz view).
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If the correction ring is set poorly your structures show low intensity, and have low resolution (large point spread function (PSF)).
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Adjust the correction ring to minimize the PSF size corresponding to maximum intensity.
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If the correction ring is adjusted optimally the intensity should have increased (can be observed by the photon counts) and the elongation of the PSF decreased.
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![Click "Live" to start live acquisition. Adjust "Gain [V]" and laser power appropriately without saturation.](https://d3t0tbmlie281e.cloudfront.net/igi/zmb/NZb2CQylYawXZmnM.medium)
