Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONLightsheet Z.1 Sample Mounting for LSFM Carl Zeiss02/2013 000000-1790-528 7Fig. 1 Relations between the sample and the objective in microscopy.Samples are traditionally isolated from the objective by a glass coverslip (A and B) limiting access toone side only. (C). In LSFM, the illumination is positioned at 90° compared to the detection axis andcan be set up in a sideways geometry ("horizontal microscope").In LSFM, the sample is usually imaged in a water-based buffer. Generally, it can be kept dry and imagedin air but this has extensive limitations like diffraction due to the significant jump in refractive index fromair to the sample material. This has several consequences for sample preparation. First, the refractiveindex of the mounting medium should be close to that of the sample buffer. The mounting mediumshould not scatter the illumination or the detection light. Second, the mounting medium should notdissolve in water. Third, its diffusive properties should be close to those of water/medium. Fourth, themedium should be non-toxic for live samples. Fifth, the medium should be flexible to allow the sample todevelop. Finally, it should not change its mechanical properties during a period of observation (72 hrs andmore).The following part of this section will deal with sample as a general term but we have devised them infour main classes (Fig. 2) and you can check their size relationship (Fig. 2) and keep that in mind asdifferent samples of different sizes will mean different sample preparation approaches and handling.