Metamorph users guide

Metamorph Users Guide

Before you begin: The microscope is configured for automated use with Metamorph. If you wish to use Slidebook or ANDOR iQ imaging software, you can do so, but need to manually set the filter sets, and ensure that you are imaging in the correct light path.

Part I: Using Metamorph

1. Turn on microscope control boxes, lamp power sources, and Hamamatsu (ORCA) camera box.
2. Turn on the computer. The ANDOR iXON camera powers up as the computer boots up. The ANDOR camera turns off completely when the computer shuts down. If the computer has been left on and Metamorph does not detect the ANDOR camera, you may want to reboot in order to assure that the software can connect to the ANDOR camera.
3. Click on the “Metamorph Advanced” icon on the desktop. The software will take a minute to load and connect to the microscope.  Error messages might appear regarding connections to the camera or microscope.
4. You should see the following screen after loading. Sometimes the software may stall during acquisition or upon loading. If this stall occurs, close the software, then turn off the control boxes (not the lamp power) and then turn them back on. After the microscope has started, restart Metamorph. Note: If you do not see the task bar, press F4.
5. The “Foundry” task bar (shown on the left) allows you to switch the filter sets, objectives, select camera, or open and close shutters on the microscope. The top bar in each section is the header for the section, denoted with **___**, and does nothing when pressed. The other tabs, when pressed, will either select the filter set indicated or perform the indicated functions. The tabs are separated into groups to indicate the filter sets for each camera/light path. You will need to select the corresponding filter set and camera to image your sample. Filter sets not list can be added manually, but you must see the equipment manager to do so.
6.  The **ND** section will place neutral density filters in the light path of the DSU/ANDOR camera. These filters attenuate the excitation light, allowing the indicated percentage of light to illuminate your sample.
7. The task bar functions can also be found in the pull down menu or icons on top of the Metamorph window. The tool bar is simply a convenient way to access the most useful functions.
8.  IMPORTANT: when you switch between the Hamamatsu (ORCA) and ANDOR cameras, you must click “Select ORCA2” or “Select ANDOR”, respectively, before getting a live image.  This tells the software from which camera it should be receiving an image.
9. After selecting the appropriate filter sets, objective, and selecting the correct camera, you can begin acquiring a live image. Press the Configure Acquisition tab in the task bar, to bring up the “Acquire” window (shown below).
10. The “Acquire” window is used to adjust the camera settings, exposure time, binning, and to take quick snap shots (see image below). Press the “Show Live” button to bring up a live picture from the camera. You can use the live image to focus on your sample.
11. Typically, you will not see your fluorescent sample and will have to adjust the Acquisition settings.  First, you can increase the exposure time to have the camera integrate the signal over that time scale. Doing so increases the sensitivity of the camera and allows you to see your sample better. Some samples may require 1-5 seconds of exposure to get a decent image.
12. Also, note that the “Autoscale” box is checked. This means that the live image will adjust the contrast as fluorophores or other bright objects come into focus. If your signal is low, you may want to de-check the box, and use the Image Scaling bar directly above the “Autoscale” box to adjust the contrast.
13. Finally, you can adjust the binning by increasing the “Live Bin” values. Binning sums the intensity of adjacent pixels (in 2×2, 4×4, or simmply nxn square) and expresses the value as one pixel. Binning will increase the signal to noise of your sample, but at the cost of resolution, so be careful.
14. NOTE: If you image looks different from the last time, try clicking “Reset Display” to reset the default settings. Microscope users may change these settings and Metamorph will not reset the settings each time. You can also save your settings, using your initials, and then load those setting each time you use Metamorph
15. The “Special” tab on the acquire window allows you to change settings for the selected camera. Features such as digitization rate, gains, and sensitivity can be adjusted. For example, when using the ANDOR iXON, in the settings tab you can set the pre-amp gain to 2.4 or 4.9, and adjust a sliding bar to increase the gain on the CCD. These changes make the camera more sensitive to incoming light and can be used to image single fluorophores.
16. Once your image is in focus, and you see a good signal, go back to the “Acquire” window shown on the previous page, and click the “Acquire” button. This will take a snap shot of your live view using the current Acquisition settings. The image will open in a window similar to the one on shown directly to the left. The buttons on the left hand side of the window can be used to adjust the size of the image , color and adjust the brightness and contrast scale (the vertical scale bar at the bottom left). These can also be used in the live image mode and will be carried over when you snap an image. To save the image, click “file/save as” and you can save the individual image as a tiff, jpg, or other image formats. (For a description of the different file types see http://www.martinreddy.net/gfx/).
17. To adjust specific components on the microscope, click the “Device Control” tab in the Foundry taskbar
18. The Device control window can be used for several functions. One, you can monitor the current position of the stage, and z-dimension. You can also set the origin (an arbitrary point on your sample to be the origin), memorize the position, go to a specific position and set the top and bottom for a particular sample for confocal imaging. Second, you can manipulate the individual components of the microscope by going to the “Configure” tab (see image below).
19. The configure tab allows you to change the illumination and magnification settings. Also, you can click “Configure Z” to bring up a Focus window and tweak the adjustment knob such that it can move in specified increments. Lastly, if you click “Component Control”, you can select any component of the microscope (shutters, objective, filter sets, etc.) and control it directly
20. To take a movie of the image, select the Multi Dimensional Acquisition button (the camera with the three axes next to it)  to bring up the Multi Dimensional Acquisition window (below). Here you can select the “Timelapse box” and then click next. This will prompt you for the time, number of frames, etc. When the box is selected, the tabs on the left will change to the input parameters for time-lapse. You can click on these to change any parameter.
21. Once selected, you will bring up a new series of tabs with parameters for the time-lapse (see image below). First, set the directory and file name (“Base name”). Second, determine the length of the movie and frame rate.
22. Third, set the Illumination source/filter sets you want (See image below). Also set the exposure time per frame and gain.  You can also choose to Auto expose, Skip frames, or auto focus. Auto expose should be used when visualizing in bright field (white light) because the lamp takes a few seconds to turn on, but the image will be taken before reaching full voltage. Setting the Auto Expose value to ~3000 should be sufficient to avoid this problem.
23. Finally, the summary Tab will show you all the settings. You can copy paste the text and save is separately for your files. This summary page is save with you movie and can be recalled in Metamorph to see your settings.
24. Also, on the bottom of the window, at any time you can click the camera icon  with the arrow to see a live image using the settings you typed in the “Wavelength “ tab. Once you are ready, click Acquire and your movie should begin recording.
25. After the movie is finished, nothing will pop up. The file will be saved as a tiff stack onto the hard drive. Metamorph saves each frame as a separate file, and then creates a file with the same name as the movie, but with an .ND extension. You will have to load the filename.ND file to be able to view and edit your movie.
26. To open a movie, click on the review multidimensional stack button.  You should see the following window open up.
27. Click on “Select base file” to open a movie file. You should see the window shown above (right image) pop up. The movie file has .nd extension and increments up to the number of images in your movie.  Click on the one or ones you want to open and click “view”. Once the movie opens, you can view each frame, play the movie, color the various frames, add or remove frames and save it as a playable format. Consult the manual for more information. You can combine the frames into one file and play it as movie in other programs.
28. An alternative way to acquire a movie is to use the “Acquire  Timelapse” function under the “Acquire” drop down menu. This function brings up a window in which you simply set the time increment, the length of the movie, and the illumination setting (i.e. filter set). Once you click “OK” the software begins collecting frames using the current settings in the “Configure Acquisition” window. Once it has collected all the frames, Metamorph will display the movie in a new window. You can save this movie as one file using the “save as” command in the “file” pull down menu.
29. Note: This method lets you collect a movie quickly and save it to one file. However, you cannot do multi-channel acquisition or collect a Z-stack.

Part II: Confocal Imaging

1. To view a sample with the confocal disk, first select the appropriate filter set in the DSU/Andor light path and make sure you click the “Select ANDOR” button in the tab. Also open the “Device Control” window to see your stage position and position in the Z-axis
2. Focus your sample in the “Show live” mode of the “Configure Acquisition” window. Adjust the exposure time and binning to get a good signal from your sample.
3. To insert the spinning disc into the light path, press the button on the control pad located near the microscope. This button is the only control for inserting and removing the spinning disk. A green light should appear on the DSU box and the intensity of your live image should decrease. By inserting the disk you eliminate background fluorescence, but also decrease the fluorescence of your sample.
4. Adjust the exposure time in the Acquire window to better see sample. You may have to set the exposure time to 1-5s to see an image.
5. To take a Z-stack of the image, select the Multi Dimensional Acquisition button (the camera with the three axes next to it) . This should bring up the multidimensional acquisition window.
6. Check the box with the name “Z-series” and click next.  Set the desitination folder under the “Saving” tab. Then set the filter set under the “Wavelength tab”. These settings are determined from the “Configure Acquisition” window when you first visualzied your sample.
7. Next, you need the tell the software how many slices you want and the thickness of each slice. Turn the focus knob and pay attention to the position of the stage in the Z-directions using the “Device Control” window as you move in and out of focus. The By moving the focus knob, the position of the objective and thus focus will increase (the focus is moving up) or decrease (focus is moving down). You can find the values of the top and bottom of your image this way.
8. Once you determine the top and bottom values, go to each position and click the “Set top To current” button. This will set your current position to the top of your image. Then do the same at the bottom of your image. Next, you need to set the “Step size”, which is the number of increments or number of slices the software will take. Starting with 1 micron is good, but you will probably want to take pictures with smaller increments to get more detail (0.5, 0.2 microns).
9. Finally, click Acquire, the camera will start at the bottom, move 1 step size up in the Z-direction, take an image, and continue until reaching the top.
10. Alternatively, you can find the center of your sample, and then click “Center Around current” and have the software move some distance above and below the center to take a Z-stack.
11. After the image acquisition is done, you should see an image stack come up. You can press play to view the series of images play as a movie. You can also save this image stack by clicking “file/save as” and selecting the directory and image file type (i.e.  tiff, jpeg, png, etc.
12. To get a 3D reconstruction of your image, right click your image stack and select “view in 4D viewer.” This should open a 3D reconstruction of your image in a new window. The 4D viewer allows you view your image in all directions, zoom in and out, change color, etc.