- Incubation system allowing multi-day timelapse
- Fluorescence and transmitted light DIC imaging
- Up to 8 samples can be imaged per timelapse
- Multiposition timelapse
- Liquid handling - change media or add drugs
- If you will be using fluorescence, turn on the X-Cite lamp (#1), otherwise leave it off
- Turn on the HAMAMATSU camera controller (#2) by pushing and holding the power button for 2 seconds
- Start up computer (#3), start VivaView FL (MetaMorph)
The actual incubator stays on all the time.
To load your samples
- Open the incubator doors
- Swing the transmitted light illumination arm to the right
- Unscrew the specimen tray and take the tray out of the incubator
- (The following steps can be done in the hood)
- Step 1: Replace the original dish lid with the special Olympus dish lid. Remember the identity of each dish if you have multiple specimen dishes! Writting on the side of the dish isn't a bad idea.
- Step 2: Load the dish (with the Olympus lid on) on the specimen tray. Make sure to push it all the way to the back. There are marks #1 through #8 for each position on the tray. Remember the position of each dish.
- Return the specimen tray to the incubator and tighten the screws.
- Return the transmitted light illumination arm and close the doors of the incubator
Possible modes: timelapse, multi-wavelength, multi-position, and z-stacks acquisition
Open the dialog boxes for "LCV Control Panel" and "Multi Dimensional Acquisition"
n "LCV Control Panel"
- Choose "Magnification"
- Click to choose the dish you want to observe. The dish number will be shaded.
In "Multi Dimensional Acquisition" tab, under "Wavelengths"
- Decide how many channels needed for the experiment
- Choose an "illumination" method from the dropdown list (e.g. DIC, GFP 25%, RFP 12%) for each selected channel, start with DIC
- Press the full chip button once before you start to make sure the entire camera chip is used
- Use the "Live" button to focus with transmitted light (the same button is used to stop "Live".) In "LCV Control Panel", use the arrow heads to control the position in X-Y direction; use the Z handle to focus on the cells (coarse or fine focusing is selectable). When the cells are in focus with the 20X objective, the Z position should be at about 3200. (0 is the lowest position for the objective, 3368 the highest)
Go through the tabs in "Multi Dimensional Acquisition" dialog window and set up other conditions for acquisition.
Main - Select the things you want to do and then go through each tab:
- Choose directory of "C:\Data " to save your images. If needed, make a new folder for the experiment under your lab folder
- Choose a base file name (all the images will be named basename-t1z1 etc)
- Check the box for "Break up into multiple files based on sample loader position " if you wish to group the images from different positions in each sample dish.
- (The hard drive on the VivaView is not for permenant storage of your data. You are expected to move your data as soon as you can. Per LMCF policy, any data can be deleted without prior notice if space is required for new experiments.)
- Choose number of time points (or duration) and interval (one will always be a consequence of the other two)
Stage - For assignment of multiple positions
- Choose a "Sample" (dish) number;
- Choose a "Position Label"
- Choose illumination and start the "Live" mode.
- You can add a position ("+") to the list or delete a position ( "X ")
- To revisit a memorized position, highlight that positon and click "Move to Position". Any change in X-Y or Z of a revisited position can be saved by clicking the modify button and overriding the old X-Y or Z information.
- In addition to setting up the exposure time for each channel (see above), you may allow the microscope to "Auto Focus" in one channel (e. g. DIC) every "n" acquisitions to correct possible focus drift (this is generally not necessary). Let the microscope update the new Z position for all other channels.
Display - Default is okay for most things, for high frame rates they should be minimized to not slow down the computer
Summary - It is an overview of the settings for your experiment
Press the green "Acquire" button to start your multidimensional acquisition. The most recent image/stack of each channel will be displayed as you acquire. An acquisition status dialog box appears.
The acquisition can be paused for modification of positions (X-Y and/or Z), adjustment of time interval, or for the addition of treatment reagents
For adjustment of positions:
- Click "Pause" button.
- Choose a "Position Item" and click "Go to"
- Choose a "Wavelength " and start "Live"
- Adjust X-Y and/or focus of the position, "Set to current" to update the new information
- Repeat for other positions as needed
- "Resume" to continue the experiment
For addition of reagents to the medium
- Click "Injection" button and the "LCV Injection" dialog box appears
- Choose a sample (dish)
- Click "Start " and watch in wonder as that dish moves into place and the lid opens!
- Open the incubator door and load the reagent through the window on the inner glass door
- "Finish" when it is done
- "Resume" acquisition
Review Multidimensional Data
Open the "Review Multi Dimensional Data" window. Tell MetaMorph which images you want to build.
Select the wavelengths, stage positions, Z-slices and timepoints you want to view.
- Select the "Stage Position" (one at a time here) and channels you want (probably all of them)
- Right clicking in the boxes selects that image. Right click on the row/column heading selects the entire row/column. Right clicking again unselect the image.
- You can choose multi-color overlay (under "Display"), "Z projection", "Select Best Focus", or range of time points to be reviewed if you want.
- Use the play and reverse arrows to view the time course etc.
- "Load Images " loads the select images to a single file which can be saved as one or exported to "avi " or "quicktime " (Stack/Make Movie from the top menu).
Make Movie from MetaMorph Stack File
- Load image stacks (see above)
- To stamp "scale bar" on the images, you need first to make sure the image size is calibrated. In menu, "Measure "--> "Calibrate Distance", pick the magnification and click "Apply"
- Open "Calibration Bar" dialog box from "Display " --> Graphics --> "Calibration Bar "
- Find a place on the image for the future bar. Choose bar size, bar and label intensities (usually choose the absolute white point intensity of the image)
- "Stamp" the scale bar on the image.
- Similarly, you can stamp "Date and Time " on the images. Choose "All Planes " to display "date and time " on all image stacks
- "Stack " -->"Make Movie ". Those steps in there are self-explanatory.
Finishing and shutting down
If there is somebody using the scope after you within 1 hour, close MetaMorph, move your data, record your use of the system using the CoreResearch booking system, log-off the computer (If there are updates please install and shut down the computer so it is ready for the next person)
If the person after you is definitely not going to use the Xcite for fluorescence imaging, it can be turned off even if the rest of the system is on.
If there is nobody booked after you within 1 hour, record your use of the system using the CoreResearch booking system then shutdown the system in the reverse order of startup
- Close Metamorph, move data, shutdown the computer (#3)
- Turn off the camera by pressing and holding #2 for 2 seconds
- Turn off the fluorescence lamp (#1)
If you have used the tissue culture hood, please close the sash and unplug the power from the wall socket.