Hi, after quite a few years of struggling we have finally finished our homemade OPT-SPIM system. However what would be very handy to check alignment processing etc are a few long-term test samples. Like an Argo-light slide but for light sheet. I make my own for our super-res systems.
Have folk any experience? We thought of an fish embryo and 1um tetraspeck beads in super-glue - so it doesn’t go off and can allow for month to month comparators.
Thanks
Ann
Hi Ann
the easiest would be to use an agarose cylinder with fluorescent beads, which you may use anyway to align your system. I prefer a small mirror or reflective grid that you insert at 45 degrees to the sheet. It gives you a nice overview of your light sheet parameters: waist, length, height, tilt, etc. You can even take a stack and reconstruct the entire light sheet profile, which allows you to characterize the light sheet properties regularly to identify if anything got misaligned. We now make our own little alignment mirrors with different grid patterns. I can send you one if you want.
Best
Jan
How about fluorescein bath? It’s quite handy when aligning a light sheet, checking it’s profile, and making it coplanar with the detection plane.
Thanks guys, we have both of these. What we need is a sample which is not homogenous. What we want to check is how good of a job our stage is doing in moving the samples through the light sheet. We want to do this to determine whether the issue is with the stage, the sample prep or the way we analyse our images. Our systems light sheet itself seems pretty stable.
To determine if it is the stage, I think the service guys have a tiny mirror on a stick–i don’t recall if it had its own stem, if it goes in the T-bar slot or what. Assuming that can be positioned consistently at an angle, taking a z-stack of the reflected light should give you information on step size uniformity.
To test whether the sample is dragging, you can try gluing it to a small piece of coverslip that is attached to the 3D printed adapter from the ExM protocol (scroll to the bottom of the web page for the printing file). Super sturdy, but can’t rotate the sample.
For a more biological type sample, maybe generate a hydrogel with crosslinked fluoropores (similar to CLARITY and ExM) to generate a uniformly florescent barely-solid goo then cut into a shape and embedded in agarose? I have not tried this.
Hi Wendy, I like the creation of Goo option I’d not thought of that. I did try making a blob of fluorescently labelled Gelatin in our early days of building which was surprisingly helpful. I effectively want the equivalent of a ‘convolaria slide’ but for light sheet. Makes you wonder if gluing a convolaria onto a stick mightn’t work. Ann