Zeiss Cell Observer Live Cell Imaging System Imperial College

Zeiss Cell Observer Live Cell Imaging System Imperial College
Imperial College London
QUICKSTART GUIDE:
Facility for Imaging
WIDEFIELD WF3
by Light Microscopy
Zeiss Cell Observer
Live Cell Imaging System
(SAF,
ROOM 409)
Observing Life As It Happens
Startup procedure ................................................................................................................ 2
Viewing your sample ................................................................................................................................. 2
Adjusting brightfield Köhler illumination .................................................................................................... 3
Setting user-defined pre-sets .................................................................................................................... 3
Image Acquisition ................................................................................................................ 3
Saving .................................................................................................................................. 4
Additional Methods .............................................................................................................. 4
Software Autofocus ................................................................................................................................... 4
Z stack ....................................................................................................................................................... 5
Tile and Positions ...................................................................................................................................... 5
Time series................................................................................................................................................ 5
Heating & CO2 units ............................................................................................................. 6
Shutdown procedure ............................................................................................................ 6
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Startup procedure
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All switches on wall to on
Check camera is on (left hand side)
Start PC and login
Run Zen blue and click on “Zen Pro”
If you are the first user after switching on – the stage will want to
calibrate. Select the 5x objective on the TFT controller and calibrate
TFT touchscreen control
Can be used to:
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Select objective
Switch on/off Transmitted light
Switch on/off Reflected light
Select reflector cube
Control heating and CO2
Additional focussing control
Other microscope controls
Viewing your sample
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In the software select locate tab
Select BF (brightfield)
Turn on Transmitted light either in software or using the TFT touchscreen
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Adjusting brightfield Köhler illumination
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Select 10x objective either with the TFT touch screen or
software
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you can move your specimen in xy-direction with the
joystick, holding the button on top of the joystick makes
the stage moving faster
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focus on your sample
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adjust brightness with large black wheel on front / bottom
of microscope)
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make sure condenser iris is not completely closed (two
buttons on left top of condenser turret)
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completely close field iris (top black wheel) (if the image
turns completely black, reopen until you see some light,
proceed to next step and close again after you have
focussed the iris)
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focus iris (black condenser focus wheel)
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centre iris (2 silver Köhler screws)
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reopen field iris until the edge is just not visible anymore
Image Acquisition
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Select the Acquisition tab (make sure “show all tools” checkbox is ticked)
Either open and image and “Re-Use” or load one of the saved experiment setups (just below the Acquisition tab
– Experiment Manager)
o WF3 Basic 5 channel (Dapi, GFP,TRITC/Texas Red, CY5, Brightfield)
o WF3 Quad (Fast acquisition - Brightfield, Dapi, GFP,TRITC/Texas Red, CY5)
o WF3 Colour Camera
o WF3 Flash BF (Flash camera brightfield only – live cell imaging)
o WF3 CFP/FRET/YFP (request filter set)
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To adjust Channels use the “Channels” tab
Click “Live”
Select each Channel in turn – adjusting LED power (using the Lumencor button) and exposure time (range
indicator button is below image window)
Additional camera setting are in Camera tab
To capture image click Snap
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Settings can be saved for future use
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Lumencor LED’s
Click the button above the image window and this will open the LED intensity controller
These settings are not stored with the image so need to be remembered (use the windows snipping tool to
capture and save the settings
Saving
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Images are stored in your temporary folder until you “save” them
To save the image – select it and press the save icon.
Select location and name
Additional Methods
Select check box for Z stack, Tile and Time Series. This will activate the respective tabs
To capture images using these methods use the Start Experiment button
Software Autofocus
Select Focus Strategy tab
Options will depend on what other functions are selected eg Z stack and Tile
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Absolute fixed Z position
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Software autofocus
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Definite focus
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Software autofocus as a reference for Definite focus
Definite focus as a reference for Software autofocus
Local focus
Local focus for software autofocus
Z stack
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Set First and Last positions
Set interval
Start experiment
Tile and Positions
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Setup tile or positions
Start experiment
(ask for more instruction)
Time series
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Setup duration and cycles
Start experiment
For very fast acquisition single channel
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Load WF3 Flash BF protocol
o Select fluorochrome
o Select “Speed”
Set the camera settings as required
Go “Live” and set exposure and focus
Setup duration under “Time Series” and tick “use Camera streaming if possible”
Run “Start experiment”
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Heating & CO2 units
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Found to the right of the microscope
Switch on heatingat least 1 hour before required
For CO2 open cylinder in room 408
Unclip hoseclips and adjust flow
Shutdown procedure
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check if anyone is booked after you within 2 hours
If nobody is booked within two hours:
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remove specimen
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clean oil objective lenses with lens tissue
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Important: close incubation chamber completely (
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update time in Sharepoint
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secure your data (e.g. copy them to the server)
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shut down hardware in reverse order from startup
protects from dust)
If someone’s booked within two hours:
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update usage in Sharepoint
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remove your samples
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clean objective lenses with fresh lens tissue and close incubation chamber
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clear up the desk
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secure your data (e.g. copy them to the server)
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log off
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