Zeiss Deconvolution Microscope: A Quick Guide

Transcription

1 Zeiss Deconvolution Microscope: A Quick Guide Start-up Uncover microscope. Do not put dust cover on the floor. Plug in both cameras. The default camera is the AxioCam HRm (monochrome camera) for fluorescence imaging (left hand side of microscope). The second camera is the AxioCam HRc (colour camera) suitable for transmitted light imaging (top of microscope). Turn on the microscope (green switch on the power supply box; below) and round silver switch on the left hand side of the microscope. Next turn on the UV lamp (if required for fluorescence imaging) and finally the computer (Login Name: User, password: decon). Start the AxioVision Ver 4.8 software (it takes a few minutes to load). Once the AxioVision main menu bar is loaded, select Workarea. 1

2 To set up the microscope for fluorescence imaging Click on the microscope icon in the software or use the touch screen on the microscope to select objectives and filters (for reflective and/or transmitted light microscopy; below). Always start with a low objective ie. X5 and move up and down in order of objectives. The nosepiece that carries the objectives is MOTORIZED so must NOT be moved manually. Alternatively you can use the buttons on the focus wheel (filters on left, objectives on the right wheel) or the TFT touch screen display. Note the RL (reflected light) and TL (transmitted light) controls on the far right of the TFT. Push both pins in all the way to view your sample through the eyepieces. If the top pin is pushed half way in, half the light goes to the eyepieces and half goes to the colour camera. Check that the pin on the left hand side is also out. Open UV light shutter via the software, RL (reflected light) illumination button on right of the TFT display or the RL button on the right hand side of microscope (see pics). Note you can use the attenuator (lower image on right) to cut down UV light reaching specimen if sample very bright or if it is bleaching rapidly. Find specimen and make sure it is in focus. NB. Close shutter when not imaging to reduce sample bleaching. 2

3 Image acquisition Send the light to the correct camera on the microscope. Setup as follows; Monochrome camera setup Bottom pin all the way out. Colour camera setup Bottom pin in and top pin all the way out. Top pin halfway = half to camera and half to the eyepieces. Select camera on software. or Select appropriate frame size. Standard frame size is 1300 x1030 pixels (2.55 MB) for fluorescence imaging. NB. You can crop image but at the expense of resolution. NB. Always check the scalings. Choose the correct filter and open light shutter when ready to image (or as previously). Click on live, select linear display mappings (default) on the live window and find the optimal focus on the monitor. (NB. You can select Best Fit and Min/Max to alter your display settings if required.) 3

4 Set the exposure automatically on live window or manually under camera settings. Use the dynamic range indicator to highlight overexposed regions (indicated by red pixels) Click on Snap to scan a single monochrome image. Multi-dimensional Acquisition. Load experiment settings and/or select C-filter wheel tab multi-dimensional image acquisition for setting up a method for 4

5 Select the appropriate Channels. Right hand click mouse to deactivate and reactivate a channel or simply delete channel completely. Use Duplicate to set up more channels. Select appropriate colour for dye. Alternatively ReUse (see above) a previous zvi file to load settings. Select Measure to set the exposure for EACH channel using range indicator. Select OK. Save Method under experiment as a.zvx file. When ready select Start to image all channels Saving Images Save image as.zvi (AxioVision format) as you go along and batch export as tiff files at the end of the session. Please make sure that you save files to the appropriate folder (D:\users\month\user\). Z-stack acquisition Select Z-stack tab, tick the Z-stack box to enable Z-acquisition and setup the stack to image by centre or start/stop mode. Select z- position using live window and a suitable channel. Select start and stop to select Z-height. Note Z-stack height. 5

6 Click on optimal distance (based on objective NA and λ, requirement for deconvolution) Select all channels per slice (requirement for co-localisation) Click on start Post Acquisition Z-stack observation 2D View. Can scroll through z-series using slider. Or play images as a movie continuously. This file can be saved as a.avi file. Gallery view. Here you can select images within the z-stack. Using the Shift or Ctrl buttons on your keyboard and Extract selection. You can also create an image here. Cut View. Shows a 3-D orthogonal section of the z-stack Note MIP creates a Maximum Intensity Projection (extended focus image) of the z-stack. 6

7 Deconvolution software Deconvolution (removal of out of focus blur) is done using a mathematical algorithm. It gives an image with less noise, better definition and higher resolution. A Z-stack of your sample taken using the optimal distance and with known appropriate scalings is required. Select Deconvolution. Choose appropriate algorithm to use eg. Inverse Filter seems to work well in most instances. Select Apply. The deconvolution process can take time if there are a number of channels and z-slices. Once process complete. Close the deconvolution window by selecting OK. Observe and save the result. Shut down Check microscope bookings. If there is another user coming within 1 hour then leave the system on. If not, shut down completely. If you finish your session earlier than planned please send an to microscopy@lists.bosch.org.au to say the microscope is free OR call the next user. If working out of normal working hours (ie. Mon-Fri 9 am 5 pm), please do not leave the system on for the next user unless you are sure they are coming! Note UV lamp hours. Turn off the Hg lamp and log your usage on the sheet provided. Report any errors on sheet and to Dr Louise Cole directly (in person or by louise.cole@bosch.org.au) Save all images and exit the software. Remove slide from stage. If used an oil objective, please wipe excess oil from objective using lens tissue ONLY 7

8 Leave microscope with a low objective in place and clean for other users. Switch off microscope Unplug both cameras. Shut down the computer. Cover the microscope rubber covers for eyepieces and transmitted light as well as the large blue cover. Leave area clean and tidy. Turn any lights off in room as you leave. 8