Thick Specimen Sample Prep Recommendations for the Tilt

Thick specimen sample prep for the Tilt light sheet is straightforward. Follow these sample prep recommendations to learn how to prepare thick specimens (things like embryos, tissue sections, whole mounts, etc) for light sheet imaging with the Tilt.

Transgenic tadpoles to be prepared for imaging
Photo of transgenic tadpoles swimming in a dish. Follow the sample prep recommendations in this post for thick specimens like this.

Thick Specimen Sample Prep: Something to Keep in Mind

When preparing your thick specimens for imaging on the Tilt, keep in mind that the excitation light enters the sample from the side. This is different than what you are used to when imaging on traditional inverted widefield, TIRF or confocal microscopes. On inverted scopes, the excitation light enters your sample from below and then the emitted light is also collected from below. On the Tilt, the 4 light beams that make the light sheet are launched from the side (Figure 1) and then, like conventional imaging on an inverted microscope, the emission is collected from below. This distinction means thinking about thick specimen sample prep a little differently.

For more information on how the Tilt works, click here for more information.

Light approaches sample from the right on the Tilt.

Figure 1: On the Tilt, the light beams to create a light sheet at the sample approach from the side. This is important to keep in mind when planning your thick specimen sample prep. 

Recommended Sample Holders: Ibidi u-slides or Mizar Imaging Chambers

Because the light beams creating the light sheet approach the specimen from the side, it is important to ensure that the path the light travels is clear. Therefore, the sides of the sample holder used to mount your specimen are also important. The sides of your sample holder must be optically clear (no fingerprints please) and flat (no round sample dishes allowed).

Mizar recommends using the u-slide 4 well or 8 well sample chambers from Ibidi (cat no 80426 or 80826). Similar looking 4 well or 8 well sample holders can work but beware of a big lip around the bottom edge (Figure 2). This lip can be so large that it can block some or all of the light beams from getting to your sample! Remember that if any of the light beams pass through a different amount of air, glass, plastic or media this will affect your light sheet.

 

Example of 4 well sample holder with lip
Figure 2: Be careful of 4 or 8 well sample holders that have a large lip around the bottom edge (red arrows). These can interfere with the 4 light beams making the light sheet on the Tilt.

Another option is the Mizar Imaging Chambers (Figure 3). These are custom made glass cuvettes that can be affixed to a #1.5 coverslip with vacuum grease. These are easy to use and the best part is that they are reusable! Reach out to us if you want to get some of your own.

 

Use #1.5 Coverslips

Objective lenses that we use daily and often take for granted are amazingly sophisticated. The fancier the objective the more corrections it has. When manufacturers of objectives are making lenses and correcting for common defects, they must make an assumption on the thickness of the coverslip that will be used. Most often they assume you are using a #1.5 coverslip. This means a coverslip that is ~170 microns thick. Using a #1.5 coverslip is especially important for high numerical aperture objectives. For the best image quality always use #1.5s. Please don’t mess around here and take care to ensure that your coverslip is indeed a #1.5 or ~170 microns thick (Figure 4).

Screenshot of Ibidi u-slide 4 well #1.5 coverslips
Figure 4: Screenshot of Ibidi u-slide 4 well sample holder with selection for #1.5 coverslip highlighted in the red box. When purchasing sample holders for imaging on the Tilt, ensure you are selecting the product with #1.5 coverslips. Screenshot captured here: https://ibidi.com/chambered-coverslips/37–slide-4-well.html

Mount Directly on the #1.5 Coverslip

Arguably the coolest thing about the Tilt is the ability to use high numerical aperture (NA) objectives. High NA objectives mean high resolution and short working distances. This means getting your thick specimen as close as possible to the surface of the #1.5 coverslip (Figure 5). Preferably directly on it. 

Has this ever happened to you: You’re on the microscope with your awesome sample. You can see the sample but it’s out of focus and no matter what you do, you cannot get it in focus. Often times this means your sample is outside of the working distance of the objective and you will need either 1) fix your sample prep (get it on the #1.5 coverslip) or 2) use a different objective with a longer working distance.

 

Embryo mounted on coverslip in Mizar Imaging cuvette.
Figure 5: Photo of an embryo mounted on the surface of a #1.5 coverslip in a Mizar Imaging chambers. For imaging with high numerical aperture objective, be sure to get specimen directly on the #1.5 coverslip.

Pro tip: transfer your thick specimen to the coverslip surface with as little liquid as possible. Remove excess liquid. If there is a lot of liquid it is more likely that the sample will float. Then use your favorite manipulation tool to gently push the specimen down onto the coverslip. You can also try coating the coverslip with poly-L-Lysine to help it stick. For some sample types, this helps adhere the specimen to the coverslip.

Immobilize Thick Specimen

If your sample is floating, wiggling, swimming, beating, rolling, etc this is going to make imaging hard. You have quite a few options here to get your sample to hold still (Figure 6) and will need to figure out what works best for your science. Here are some options you may consider:

1) 1% low melt agarose with 20% Optiprep (more on Optiprep later, keep reading). Very common and will do the job, but some specimens get very upset in agarose.

2) Hydrogel. This recent paper by Burnett et al., shows that organisms stay happier longer in hydrogel along with several other advantages.

3) Some specimen types stick to coverslips better with a coating like poly-L-Lysine, fibronectin or ’embryo-glue’ (double sided sticky tape dissolved in heptane, section 8 in this paper) for example. If you are against low melt agarose or other encapsulation methods, trying out different coatings may be a good option for you.

4) High doses of anesthetic. Also known as putting to death instead of putting to sleep. This may mess with the physiological process you are trying to study and may not be an option for you.

This step can take some optimization, but is totally worth it! You don’t want to find yourself in a situation where you have the most perfect sample ever but can’t image it because it moves too much.

 

Cartoon of immobilized embryo.
Figure 6: Cartoon of immobilized embryo mounted on #1.5 coverslip. For imaging on the Tilt, ensure that your sample does not move around.

 

Low Plating Density & Orientation

Remember, the light beams on the Tilt enter your sample from the side. And that you want to ensure that nothing is obstructing the path of the light, including other thick specimens. Otherwise, this can lead to stripe artifacts in your image. You can certainly mount multiple thick specimens in one well or on one coverslip, but make sure they are oriented perpendicular to the light beams. Check out Figure 7:

 

Cartoon illustrating low plating density and sample orientation of thick specimens.
Figure 7: Cartoon illustrating low plating density and sample orientation of thick specimens for imaging on the Tilt. Because the light beams approach from the side the specimens should be oriented perpendicular to the light (orientation displayed on the right). This avoids having to image through another specimen (as would happen with orientation on the left).

More on Thick Specimen Orientation

If possible, orient the structure you want to image so that it is facing the light beams (i.e. facing the side the light will be coming from, Figure 8). For the best image quality, avoid imaging through the rest of an embryo or yolk. This will also help minimize the striping artifact.

Cartoon illustrating how the structure to be imaged orientation.
Figure 8: Cartoon illustrating how the structure to be imaged should be imaged relative to the incoming light (it should be facing the direction of the light).

Thick Specimen Sample Prep: Key Take Homes

1) Use appropriate sample holders. We recommend Ibidi u-slides or the Mizar Imaging cuvettes.

2) Mount directly on #1.5 coverslips.

3) Immobilize your thick specimen.

4) Plate few specimens per coverslip and orient perpendicular to the light source.

5) Orient structure to be imaged towards the side the light will be coming from.

 

Download an infographic for large sample mounting  here.

 

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