Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 2 - SAMPLE PREPARATIONLightsheet Z.1 Tips, Troubleshooting and Additional Information Carl Zeiss02/2013 000000-1790-528 374 Tips, Troubleshooting and Additional Information4.1 TipsProtocolsAs a general rule, follow the protocol carefully and try it out well in advance as you may encounterdifficulties and missing parts (chemicals…) that may hinder you to use the sample straight away. Theprotocols presented in this section are not the ultimate solution for every problem. They can be improvedand adapted to your need. If you encounter a problem, check the scientific literature and the otherprotocols presented and see if you can find a tip that you may apply to solve your problem. Be creative.SafetyPlease refer to the safety instructions provided with the Lightsheet Z.1.Setup Requirements and MaintenanceFor optimal performance of the Lightsheet Z.1, please check the Setup Requirements informationdelivered with the system.For information about maintenance and cleaning any part of the Lightsheet Z.1, including the optics,please refer to the according CHAPTER in this manual.Refractive index mismatchLight is refracted when it crosses the interface between two media of differing refractive indices (RI).Mismatching the refractive index of the objective immersion medium and mounting medium is one of themain causes of image degradation in microscopy. Refractive index mismatch results instretching/compression of the z-axis. Also, spherical aberration is worsened by axial spreading of thepoint-spread function (PSF) resulting in reduced axial resolution. This phenomenon is exacerbated withdepth and with a high numerical aperture objective, serious problems arise when imaging deeper than 10μm into an aqueous sample. The mounting medium and the immersion medium should be matched. It isnot a major issue in LSFM but this as to be considered when filling the imaging chamber in regard to thesample preparation technique, especially for embedding as gelling agents are used.4.2 TroubleshootingCommon problemsLSFM is a fluorescence microscopy technique so many troubleshooting guidelines from other microscopytechniques apply here as well. Do not hesitate to ask experts in the field, check the literature, as well asinternet resources that may provide you with a more detailed solution to the problem you haveencountered. Also check with your Lightsheet Z.1 specialist for FAQs and tips for troubleshooting.