Overview
This article describes sample preparation for light-sheet fluorescence microscopy. It also demonstrates a method to mount live zebrafish embryos for long term imaging, which allows recording of long time-lapse movies of live embryos in near-physiological conditions.
Protocol
1. Sample Preparation
- Preparing the Agarose Mix
- 15 min before making of the agarose mix, melt one 1 ml aliquot of 1% low melting point agarose (dissolved in E3 medium) in a heating block set to 70 °C. Once the agarose is completely molten, transfer 600 µl into a fresh 1.5 ml tube, add 250 µl of E3 medium, 50 µl of 0.4% MS-222 and 25 µl of vortexed bead stock solution.
NOTE: This makes 925 µl of the mix, while additional 75 µl is calculated for the liquid added later together with the embryos. - Keep the tube in a second heating block at 38-40 °C or ensure that the agarose is very close to its gelling point before putting sample embryos into it.
- 15 min before making of the agarose mix, melt one 1 ml aliquot of 1% low melting point agarose (dissolved in E3 medium) in a heating block set to 70 °C. Once the agarose is completely molten, transfer 600 µl into a fresh 1.5 ml tube, add 250 µl of E3 medium, 50 µl of 0.4% MS-222 and 25 µl of vortexed bead stock solution.
- Mounting the Embryos
- Take five glass capillaries of 20 µl volume (with a black mark, ~1 mm inner diameter) and insert the matching Teflon tip plungers into them. Push the plunger through the capillary so that the Teflon tip is at the bottom of the capillary.
- Transfer five embryos (a number that can be mounted at once) with a glass or plastic pipette into the tube of vortexed 37 °C warm agarose mix.
NOTE: Try to carry over a minimum volume of liquid together with the embryos. - Insert the capillary into the mix and suck one embryo inside by pulling the plunger up. Ensure that the head of the embryo enters the capillary before the tail. Avoid any air bubbles between the plunger and the sample. There should be ±2 cm of agarose above the embryo and ±1 cm below it. Repeat for the remaining embryos.
- Wait until the agarose solidifies completely, which happens within a few minutes and then store the samples in E3 medium, by sticking them to the wall of a beaker with plasticine or tape. The bottom opening of the capillary should be hanging free in the solution allowing gas exchange to the sample.
NOTE: A similar protocol to the sections 1.1 and 1.2 can be also found on the OpenSPIM wiki page http://openspim.org/Zebrafish_embryo_sample_preparation.
2. Sample Positioning
- Sample Holder Assembly
- Insert 2 plastic sleeves of the right size (black) against each other into the sample holder stem. Their slit sides have to face outwards. Attach the clamp screw loosely by turning it 2-3 rounds. Insert the capillary through the clamp screw and push it through the holder until the black color band becomes visible on the other side. Avoid touching the plunger.
- Tighten the clamp screw. Push the excess 1 cm of agarose below the embryo out of the capillary and cut it. Insert the stem into the sample holder disc.
- Confirm in the software that the microscope stage is in the load position. Use guiding rails to glide the whole holder with the sample vertically downwards into the microscope. Turn it, so that the magnetic holder disc locks into position.
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Materials
| Name | Company | Catalog Number | Comments |
| Lightsheet Z.1 microscope | Carl Zeiss Microscopy | ||
| Low melting point agarose | Roth | 6351.1 | |
| Low melting point agarose | Sigma | A4018 or A9414 | |
| Ethyl 3-aminobenzoate methanesulfonate (MESAB/ MS-222/Tricaine) | Sigma | E10521 | |
| 20 μl (1 mm inner diameter, marked black) capilllaries | Brand | 701904 | sold as spare part for transferpettor |
| Teflon tip plungers for 20 μl capillarie | Brand | 701932 | sold as spare part for transferpettor |
