Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONCarl Zeiss Sample Mounting for LSFM Lightsheet Z.114 000000-1790-528 02/2013The gel can be cut into various shapes depending on the needs (cylinder, hole). Afterwards the gel withthe specimen is pulled back into the syringe and is covered with more molten agarose. The agarose isallowed to cool and solidify, this time period can be shortened by cooling the whole sample. For examplethe housing of the sample can be rinsed with cold water, although care must be taken to ensure that thepolymer does not come into contact with the water, otherwise the cooling agarose would becomediluted and lose its stability necessary for holding the sample. After polymerization, the sample is readyfor imaging.2.2.2 Hanging SamplesThe Lightsheet Z.1 is optimized for gel embedding samples The sample chamber must be filled witha watery solution (refractive index of 1.33) at all times, to ensure optimal image quality.This mounting technique can also be used, but will require some initial adaptations to the sampleholder.An intuitive way of imaging an object is to simply take it as it is and place it in front of an objective. In anLSFM, this can be done by hanging the object in front of the objective where the axis of rotation andgravity are parallel. This can be achieved using a simple hook made of glass, stainless steel or plastic(Fig. 9/A). This mounting technique can be used for large samples such as organs (for example the brain)or complete organisms (insect, fish). One main drawback is the fact that the hook will partially damagethe object and may also interfere with the field of view.Fig. 8 Aligning an embedded sample.The sample can be aligned in a particular orientation to allow the details of interest to be close to theouter surface of the agarose. The solidified agarose is pushed out the syringe a few millimeters and asmall v-groove is cut into the cylinder to take up the sample (A). The sample is placed into the v-groove(B). The sample on the agarose is pulled back into the syringe and more agarose is added (C). After thecylinder has completely solidified the sample is pushed out of the syringe allowing free sight on to thesample (D). The same approach can be used to carve a central tunnel in the middle of the agarose toalign the sample along the agarose tube axis.