Imaging Resource
Standard Operating Procedure.
Nikon Timelapse
How to Use the Nikon TE 2000 Timelapse Microscope with PFS and Metamorph
Switch on the Xenon Lamp.
On / off switch for
Xenon lamp. Only
need to switch this
on if you are using
Fl dyes or proteins.
Switch on the Lambda 10-3 Sutter unit.
On / off switch
Switch on the Halogen bulb control unit. The camera power control unit and the camera.
On/off switch at
back of unit.
Switch on the ASI stage control unit.
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Imaging Resource
Standard Operating Procedure.
Switch on the PFS controller for TE2000 microscope systems. See BAIR staff for Ti microscope.
On/off switch on the
side of unit.
Switch on the computer.
Log onto the PC
Double click on the Metamorph software icon to open the software.
On the window 7 systems the software will launch automatically. On the XP systems you may see the tab
below.
Select user name Beatson_Staff and OK
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Imaging Resource
Standard Operating Procedure.
You will be asked to make sure the objective is lowered and the stage will register and home when you select
Continue. This takes at least a 1min to complete the process.
If the taskbar does not appear when the software opens up. Select Journals | Load taskbar and load the
BICR_Tasks taskbar. Again this task bar may look different depending on the system you are using but they all
contain the essential two main tasks.
MDA
Select Multi Dimensional Acquisition (MDA) from the taskbar.
Select the main tab and tick the functions required. What you select will depend on your experiment.
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Standard Operating Procedure.
Select the Saving Tab. Select the directory where your are going to save the timelapse images to. Select a file
base name. Please note it is best to save to the HD of the PC and then transfer your data at the end of the
timelapse run to your own network area. Do not leave your data on the PC as it will be deleted by BAIR facility
staff!
Select the Timelapse tab.
Number of time points = Number of images of each wavelength that you want to capture.
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Standard Operating Procedure.
Time Interval = The time between taking each image.
Duration = Total time the timelapse will run.
Select the Wavelength tab. Select the number of wavelengths you require. eg: two for BF and FL.
This system Has filters for GFP/Cherry , CFP/YFP.
Select the wavelength tab for each channel in turn and set up the exposure required.
Select BF cam for Phase
images. Select live to
see a preview image.
Focus on a cell of
interest. If using BF and
FL channels you may
need to select Bin X2.
Select GFP cam2 for FL
images.
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Alter the exposure until
a bright preview image
is seen. In the live image
make sure Auto scale is
not selected. A 10 bit or
12 bit range is a good
choice. Use colour LUT
to check pixel intensity.
Blue under saturated
Red saturated. Aim for
an exposure that gives
you a few red and blue
pixels.
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Imaging Resource
Standard Operating Procedure.
Alter the exposure until
a bright preview image
is seen. In the live image
make sure Auto scale is
not selected. A 10 bit or
12 bit range is a good
choice. Use colour LUT
to check pixel intensity.
Blue under saturated
Red saturated. Aim for
an exposure that gives
you a few red and blue
pixels.
Select the wavelength you wish to set up with and then select the Stage Tab.
With No PFS
Using the ASI joystick and the live preview find the first cell of interest and add the XYZ position. Repeat this
for all the positions you wish to capture. The software will take the Z position from the Microscope and will not
change the Z if there is a drift due to temperature changes or vibration.
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Imaging Resource
Standard Operating Procedure.
With PSF
Use the ASI joystick to find the first cell of interest focus using the microscope as normal. Once the focus green
light comes on you can then switch on the AF on button on the perfect focus control box and focus using the
control knob. Add positions as above. The Z will be taken form the PFS control box and it will not drift out of
focus due to temperature change or vibration.. Remember the PFS does not work with plastic bottom dishes.
Focus Control Knob
AF on
Select the Display Tab. Select Preview Button.
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Imaging Resource
Standard Operating Procedure.
Sometimes the preview image is scaled differently and your image may look brighter of darker.Set up how you
want the images to be displayed. Make sure autoscale is not selected and the bit range matches the range you set
up with. Select Start to acquire the timelapse.
When the timelapse is acquired select review multi dimentional data
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Imaging Resource
Standard Operating Procedure.
Select | Select Base file.
Select your timelapse name form the list on the left of the window and then select view.
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Standard Operating Procedure.
Select the timelapse
channel you want to
view by ticking the box.
Right hand mouse click
on the number 1 all
frames will be selected
with an X. Select the
position.
Select Load images.
You can now play through the frames of your timelapse.
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Standard Operating Procedure.
From the top file menu select | Stack | Make movie
Select save unselected if you want to make an avi of all the timelapse frames. Select | Selected if you want to
make a timelapse of a range of frames. Select the time you want each frame to be played for and select save.
Give the timelapse a name and save as uncompressed avi. To save the images as tiffs file select File | save as
and save as a metamorph tif. To save a metamorph stack file select file | save as metamorph stk. Metamorph
stacks will open with Image J. Images can also be saved as jpegs and opened in powerpoint.
Files should be transferred to your own network area Do not leave them on this PC or they will be deleted.
NOTE
The AVI movies can be inserted into Powerpoint. If you are intending to insert several large movies into a
powerpoint presentation you may have to reduce the size of the avi moves before inserting them.( See Margaret
or David Strachan).
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Standard Operating Procedure.
Always keep the avi movies you have inserted into your ppt presentation in the same folder as your
presentation.
Always load and play your movies and ppt presentations from the hard drive of the PC/Mac.
Never play your movies and ppt presentations over the network, from a disc or USB stick.
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