Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONLightsheet Z.1 Sample Mounting for LSFM Carl Zeiss02/2013 000000-1790-528 13There are two principal methods of embedding an object. The first is to directly mix the object with theagarose then pump it into the sample embedding cylinder. This is a convenient way of embedding verysmall objects such as pollen grains (Swoger et al., 2007); yeast (Taxis et al., 2006) or cell clusters(Pampaloni et al., 2007) or even large objects like fish embryos. The action of pumping in the samplewith the agarose results in a self-alignment of the specimen within the tube (Fig. 7/A, E and F).The second method is to fill the sample embedding container with the gelling agent, then to place theobject within the gel using a needle or forceps (Fig. 7/A, B, C and D). This approach is more suitable forthose samples that cannot be easily aligned using the first technique.In some cases, it may still be challenging to align the specimen in the most suitable way for imaging. Theorientation of the sample must then be optimized, so that interesting details are facing the surface of theagarose cylinder with as little material as possible in the optical path. One solution is to fill a syringe withagarose and allow it to cool until it solidifies. The agarose is then pushed out of the syringe(Fig. 8/A and B). A small V-shaped groove can be cut into the gel and the sample then positioned in theV-groove.Fig. 7 Basic principles of sample embedding.A cylinder with a suitable plunger is used as a mounting device (A). The 1 % low melting-pointagarose is melted, then brought to 37 °C, then pumped into the cylinder. (B) The object is thenintroduced to the agarose with a needle or forceps. (C) Once solidified, the embedded sample can bepushed out and imaged (D). Alternatively, the object, devoid of water, or other solution, is added to asolution of 1 % low melting-point agarose at just above gelling temperature (typically 40 °C) andsucked into the cylinder (E) and then allowed to polymerize. The embedded sample can then bepushed out and imaged (F).