Related Manuals for Zeiss LSM 710 SIM
Summary of Contents for Zeiss LSM 710 SIM
- Page 1 ZEISS LSM 710 SIM Superresolution microscope Manual/Quick guide confocal image SIM image Matyas Molnar, Biovis 2016...
- Page 2 Starting the microscope No need to touch… Condenser, tiltable Laser safety box; make sure all openings are closed before acquisition is Microscope stand started Real time PC, open front and reset if ZEN cannot start Computer (left side) Main switch Fluorescent lamp Argon confocal laser Confocal and SIM laser boxes...
- Page 3 Check the microscope if everything looks clean and normal. If not, report it in the logbook. Start the system with the Main switch (on the microscope panel, located on the laser box) Switch the Systems/PC ON (on the microscope panel, located on the laser box) Switch the Components ON (on the microscope panel, located on the...
- Page 4 b. Fluorescent lamp: switch on with the button on its control box (white box located on the laser box, marked with X-cite sign)
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Page 5: Locate Tab
Using the ZEN software Locate tab Use the locate tab to focus onto the sample and to get a first overview with the eyepiece. Turning ON/OFF the transmitted (brightfield) lamp Changing the intensity of the transmitted (brightfield) lamp Changing objectives Switching between fluorescence channels (OBS! The only available filter is the FSet77 HE,... - Page 6 Acquisition tab, confocal imaging This is the tab where image acquisition can be started and change the properties of the imaging. Always check “show all tools” here and for all the blue menus in the view menu (show all global). Smart setup: quick setup for channels Starting/Stopping...
- Page 7 Smart setup (only for the confocal mode) Smart setup is a quick and easy way to create the settings for fluorophores and channels. Unexperienced users should start with the smart setup. Please note that smart setup might not working with the multiphoton laser so multiphoton channels should be setup manually. •...
- Page 8 Experiments A possibility to select advanced imaging methods such as: • 3D imaging (Z-Stack) • Time lapse imaging • Bleaching experiments • Tile scan (no stitching possibility exist for this instrument) • Imaging with specific regions Laser menu SIM lasers (Elyra) can be turned on here. All the other lasers are either always on (HeNe lasers, 405 diode) or turned on manually on a laser box (like the Argon laser).
- Page 9 Imaging setup A tool to manually setup channels for fluorophores either is for simultaneous (two detector is activated for one or more track) or for sequential imaging (all fluorophores are in different tracks). Adding/removing tracks. Changing between WF (Widefield), SIM, LASER WF, LSM (confocal), or lambda mode (using the instrument as a spectrometer)
- Page 10 Channels Here the intensity of the signal for the different channels can be adjusted. When adjusting the intensity for a channel be sure that the desired channel is highlighted and activated (checked) otherwise settings cannot be modified, or settings are modified for a different channel. Existing tracks and channels.
- Page 11 quality and time/bleaching! Don’t use a default laser or gain settings, always change them freely to get the best image without ruining the sample. Note that if you change the pinhole, the signal is collected in a different Z range (intensity is changed), therefore new intensity adjustment is needed.
- Page 12 Acquisition mode In the acquisition mode the quality of the imaging acquisition can be changed. Selected objective Scan mode: spot, line or frame X-Y resolution: adjusting the X-Y resolution. If you are unsure, use the optimal button. For 3D imaging use 512x512 to save time/avoid bleaching.
- Page 13 The image window Split channels Gallery images (stacks) for Z-stack or time lapse imaging Orthogonal projection for 3D 3D rendering of a Z-stack Range indicator: to see overexposure Info and setting of the acquisition Histogram: use it for contrasting the image or check the intensity Reuse: Reusing the acquisition settings of an...
- Page 14 Experiments – Z-stack (3D imaging) With Z-stack it is possible to acquire 3D dataset. There are two ways of doing Z-stacks, “first/last” or “center”. Diagram showing the stacks and first/center/last positions First/last: 1. Go to Live mode, use the focus knob to find the end of the sample and push Set Last to select the end position of the...
- Page 15 Diagram showing the stacks and first/center/last positions Center: 1. Go to Live mode, use the focus knob to find the center of the sample and push Center to select the center position of the sample 2. Select Slice and give the slice number for the total range of the Z-stack.
- Page 16 Experiments - Tiles It is possible to acquire tiles to get a bigger field of view of the sample. Note that no stitching module is purchased; therefore stitching of the tiles is not possible. However the tiles can be exported as a single .tiff file using the Contents of the image window – single plane of the export menu (for exporting see next chapter).
- Page 17 Saving/exporting Data is not automatically saved after acquisition, it is only stored in the memory. It is recommended to save always after every acquisition. If an unsaved data is visible in the image window and Live/Snap/Start experiment is pushed, the unsaved data will be overwritten. Check BioVis guidelines where to save/export your data.
- Page 18 Acquisition tab, SIM imaging Here is only the SIM imaging showed, for a detailed description of the ZEN Acquisition tab, please check first the “Acquisition tab, confocal” part. Resolution, quality and cropping of the imaging, make no change here Grid rotation for SIM imaging, use 5 rotation for the best result Select SIM imaging mode...
- Page 19 Adjusting the correct intensity for SIM imaging Since we use a 16-bit camera for SIM imaging, the histogram should be monitored continuously to get an overview about the correct intensity. Use “Auto” contrast settings (Min/Max or Best Fit) to see your image as during SIM imaging we are working in the darker region of the histogram.
- Page 20 SIM processing After the SIM acquisition is done, save the file as .czi before processing. Always start with automatic processing then if needed fine tune the data with manual processing. Automatic processing Go to Processing tab Select Structured Illumination from the list Select the desired dataset (image) to process Select the output as SR-SIM.
- Page 21 Manual processing If SIM artifacts and aberrations are visible in the processed image, fine tuning with manual processing can help to eliminate the problems. First check what parameters were used for automatic processing under the info tab. Noise filter has the biggest effect in changing the resolution and removing artifacts...
- Page 22 Shutting down the microscope When you are finished with your study do the following: Transfer your data files to the “DATASHARE” computer (In the workstation room). From this computer you can remove your files with USB connection or upload it to the server.