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Calibration SCAPE IV

I follow the protocol in the SCAPE manual to perform an XYZ calibration of the setup. The protocol described is very generic, and not optimized. In the following, I will describe a point-by-point protocol:

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2020/10/22 05:29 · lalvarez

Revealing pumping with different microscopes

Jun and I acquired several movies to show pumping in C elegans. The following are examples of the quality obtained with different microscopes/settings.

In terms of Ergonomy and image quality, the Olympus inverted microscope is the worse to image in agar plates. Only long working distance objectives can be used and the quality of the image is not good. I have no examples of images, but they are not as good as the ones shown below.

Axio Illumination settings 1. Magnification 5.59x.

Axio illumination settings 2. Magnification 5.59x

Axio Illumination settings 3. 5.59x magnification

Olympus upright. 10x magnification

2020/10/08 02:18 · lalvarez

SCAPE - removing bead artifacts

Week 21-25 Sept 2020

I investigated the source of some aberration observed in images of beads.

To correct for this, I went through the setup and slightly adjusted the position of the individual components, to identify where might be a critical point determining the aberration. This was not very successful, as I ended up with a totally misaligned setup.

For this reason, I went through the alignment sequence again, having a critical look at the centering of the red and blue lasers, and trying to improve overall their optical path. While doing this, I corrected some small offsets in the center of the beam that appear as we introduce some of the lenses. I did not pay too much attention to it in the past, as I thought that this should be within the tolerance, but I was more strict now. It turned out not to be critical, but on the way to do this, I dismantled the Ploss lenses because the laser beam was slightly out of the center and the shape of the beam was not ideal after the lens. I found out that one of the lenses was very tight within the tube lens. The tesa film had many air bubbles, and I was afraid that as tight as it was, it might stress the material. I improved the Tesa film layer, cleaned the two lenses, and reassembled this component. The centering was slightly better, but the beam shape defect was still present.

I scrutinized the beam before the lens, and I found that this strange laser shape was already present before the ploss lenses. It was just more clearly visible after the ploss lenses because these lenses enlarged the beam a bit. I traced back this defect up to the laser itself. The following shows a picture of a similar profile from another laser on the internet. A tail in one axis (here the vertical) is apparent.

lasershape.jpg

I cleaned the laser profile using a diaphragm. This turned out, at a later point to be irrelevant. Once I found the problem generating the aberration of the beads, I tested with the diaphragm fully opened, and the images are identical. I removed the diaphragm.

After careful alignment of the setup, I managed to have decent images of beads in the absence of these aberrations. I prepared a solution 1:200 of the beads from Johannes. I used 40 µl in my old 150 µm height chambers. I wanted images of beads that are not located at the surface. Just in case the aberration is partially due to surface effects.

The aberration similar to the comma is disappearing for those beads in focus. The image is now almost a point. Out-of-focus beads have a characteristic pattern with a star-like shape at the center.

While performing the alignment of the camera again, I noticed that the aberration was partially back. I also noticed that the changes in the focal plane imaged changing the height of the Z stage could be partially compensated by changing the focal position of the third objective, but the overall quality of the image was strongly dependent on this setting. Coming to think about it makes sense because by changing the last, we do not change the position of the light sheet, which will result in a widening of the illumination, and a less localized image. Furthermore, the image plane of the telescope is not overlapping with the focal plane of the third objective. This will have more critical effects if there is a mismatch between the orientation of the third objective and the 135° positioned image. Finally, we are likely losing part of the light and reducing the overall NA as the image is acquired on the last step from a defocused image. I have to find a way to optimize this by using a single plane Tesa film.

The following is an image of the same bead sample above after detuning the focus of the third objective and refocusing using the Z-stage. The aberration is clearly visible again:

I also took a picture of the Rainbow 5 µm beads that are used for the XY calibration. The bead shape is fairly good.

I also aimed to correct a bending on the borders of the Ghosts plane. This was easily corrected by adjusting the two cylindrical lenses making the sheet. The light sheet was not perfectly vertical.

After a decent adjustment of the settings, I also played around with the offset of the sliding element in the setup. It is clear that making the angle more shallow (more parallel to the plane of the sample), we obtain a better resolution in Z. The spacing between planes becomes larger. We can use this also to increase the acquisition frequency at the cost of losing Z resolution.

The following is using a shallow angle. The slight bending of the upper plane at the leftmost part is still visible here because I played still with the settings after correcting it. I need to adjust it again.

The following uses a more steep gradient:

2020/09/25 04:56 · lalvarez
wiki/documentation/luisalvarez.txt · Last modified: 2020/08/13 03:46 by mscholz

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