Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONLightsheet Z.1 Specific Examples of Sample Preparation Carl Zeiss02/2013 000000-1790-528 298. Suck in the agarose/beads by pulling the opposite end of the wire/plunger.9. Let the gel polymerize (approx. 5 minutes) before imaging.10. Make sure that only a very short part of the agarose block is pulled out of the glass capillary duringimage acquisition.11. When multiple views are recorded, it is best to image from the centre of the agarose block.Beads should be fluorescent in the part of the spectrum you would like to analyze for 1 channelsystems. With the Lightsheet Z.1, a two channel system, one can use one channel for the beads(e.g., red) and one channel for the specimen label (e.g, GFP.) Fluorescent beads covering wholevisible spectrum are nowadays easily available from various different suppliers (e.g. Polysciences,Invitrogen, Estapor / Merck etc.). In our case, the density of the fluorescent beads is chosen to endup with several hundred beads in the imaged volume. For example, for a 40x magnification lens thevolume of interest is around (200 μm) 3 = 8*10-6 ml. If the fluorescentbeads are shipped as a solution of 5*1013 particles/ml , you have to dilutethem 1:106 in agarose to have approximately 400 particles in the volume of interest. Havingtoo few of them (less than 100) in the three-dimensional image will give you no or poor processingresults, while too many of them (more than 1000) might considerably increase processing timewithout a significantly improving the final results.Moreover, a gel with sufficient stiffness but minimal impact on the image has to be used to immobilizethe beads. 1 % low-melting agarose (Sigma, Type VII) is being routinely applied for lenses with numericalapertures up to 0.8 NA. For 1.0 NA objective lenses and above, a more diluted (e.g. 0.5 %) gel must beused to minimize gel-caused image aberration.3.2 Preparation of a Medaka Fish Embryo (Oryza latypes)Embryos have been extensively used for many decades to study developmental mechanisms as well asdiseases. They can range from micrometers to centimeters depending on the species used (frog, fish, fly,worm, etc.). This protocol applies to most embryos. The important point is the temperature. The embryomust not be damaged by temperature shock during embedding. Moreover, the embryo should not beconstraint by the stiffness of the gel. This may impair its normal development.Equipment and reagents− 1.5 % Low Melting Point (LMP) Agarose in E3 (Fish buffer)− Mesab/Tricaine 0.4 % stock (3-Aminobenzoic Acid Ethyl Ester)− Capillary (Size 4, Blue, #701910, BRAND GmbH)− Electrical thread (1,6 mm) or plunger− Heating block