17
Tilt
User’s Guide
Tips for Mounting Samples
Sample prep with the Tilt is straightforward, and indeed is similar to how you would nor-
mally mount a sample for imaging on any inverted microscope. That said, there are a few
key considerations to keep in mind when mounting samples for the Tilt.
2.1 Mount as you normally would
As a first pass, mount your samples as you would normally mount them for imaging on an
inverted microscope. For instance, if you immobilize your sample in 0.5% agarose, then do
that as well for imaging with the Tilt; if you do some sort of trick to bring your sample as
close to the cover slip as possible, such as glue, then use that as well with the Tilt; etc.
2.2 Tilt-specific considerations
The goal here is to maintain an unobstructed lightsheet all the way to your sample of
interest. Anything that gets in the way of your lightsheet (meniscus of the medium, a dirty
side wall of the sample chamber, other specimens, etc.) can create shadows and/or act as
a lens
(“Anything curved can act as a lens”)
and lead to a deterioration of the lightsheet.
Here are several tips for minimizing obstructions:
• Keep the sides of your sample chamber clear of fingerprints, oil, grease, etc.!
• Make sure there is adequate medium in your sample chamber. The meniscus can have a
lensing effect and distort the incoming lightsheet.
• If you use agarose or similar mounting gel, make sure you fill the entire well - either with
the gel or a medium that matches its refractive index (i.e. water for agarose). Also make
sure the gelling agent is well dissolved/incorporated. Undissolved particulate can cause
shadowing. Boiling for a longer period of time or filtering will help with this.
• If your specimen has features that obstruct the path of the lightsheet — such as a large
yolk sac — orient the sample with your region of interest facing the incoming lightsheet,
such that the obstructing element is not in the way.
• Mount your samples at low density. If plating cells, plate at low density as the “shad-
ows” from a nearby cell may in some cases disrupt the lightsheet. The same principle
holds for larger specimens.
• Sample multiple specimens per well, and multiple wells. Illumination artifacts sometimes
vary between individual specimens within a single well. Mount a sufficient number of
specimens to allow adequate sampling.