Our institute, National Institute of Genetics, has just bought a Zeiss lightsheet microscope. So, I wanted to see how it woks in calcium imaging. Sample preparation and setting up seems a bit comlicated for the first time and here is a record of the procedue.
Embedding zebrafish larva in agarose
One percent low melting point agarose (LMA) was too soft and could not be used. So, I used 2% LMA. To orient the larva in the solidifying agarose, I anesthetized the larva in tricaine. Our lab has 10xconcentrated tricaine.
What you see in the picture is, a bottle of 2% LMA agarose stock, larvae in 10mL dish, a big drop of system water (1 drop of 10x tricaine + 9 drops of system water) and a big drop of 2% LMA that contains tricaine (1 drop of 10x tricaine + 9 drops of microwaved-2% LMA solution) in 10mL dish, and a big drop of 2% LMA that contains tricaine uder a stereo microscope.
- Pick a larva and transfer it into a system water that contains tricaine (1 drop of 10xtricane + 9drops of system water).
- Move the larva into the 2% LMA that contains tricaine. (This step is not to bring the water into the final LMA.)
- Move it again into 2% LMA(+tricaine).
Use a needle to orient the larva.
Somehow keep the larva a bit above the bottom of the dish and keep the side up. Keep orienting the larvae with the needle until you see the agarose starts to solidify.
I usually wait for 5 min after I thought it got solidified and pour system water over the agarose. Then, using a fine knife,
make cuts as seen in the picture below. Here I plan to shine the light sheet from the front and observe from the dorsal side. So, I tried the agarose outside be as thin as possible.
Zeiss Lightsheet microscope
The lighsheet microscope is seein as a white box on the table in the picture below.
Choosing the objective lens and the lenses for the lightsheet illimination
We have a water-immersion 20x lens and 5x lens. Choose the one for your experimental purpose. Here I will use the 20x lens.
Choose the adaptor
To observe a sample in the water with the water immersion 20xlens, choose the adaptor labeled “n=1.33 2064-450”.
and attached it to the lens as in the picture below.
Choose the right lenses for the lightsheet illumination compatible with the objective lens that you chose for the observation. In this case, the left ones are used.
First, set the objective lens in the microscope on the side. I didn’t know until the microscope was delivered that observation is made from the side!!! This looks very weired to me as I want to use a living animal that can sense gravity.
(to be continued)