Live Cell Imaging with the Zeiss 510 Confocal
A microscope Zeiss XL-3 stage incubation chamber (with temperature and CO2 control) for the Microscopy Core facility's Zeiss 510 MetaHead confocal microscope
Overview
State-of-the-art time-lapse imaging of living cells under the microscope requires the maintenance of the cell culture in suitable conditions for biological processes to continue normally. This is provided by our Zeiss 510 confocal microscope stage incubator that has accurately controlled air temperature and carbon dioxide (media pH) concentrations.
Zeiss/PeCon XL3 Incubator
The Zeiss confocal has a large Zeiss (PeCon) XL3 incubator enclosure which surrounds the microscopes motorised XY stage and objectives. We have added an additional 20x Ph2 (phase-contrast) plan neofluar and a 40x Plan Apochromat Ph3 air objective for live cell and multi-well plate use. The inside of the XL3 enclosure can be thermostatically heated to any temperature from ambient to 37oC (or above) which, with the aid of an air humidifier, can keep cells alive for days. For long term live cell time-lapse, a 5% (variable: user adjustable) carbon dioxide enrichment of the heated air has been provided, via the Zeiss CO2 Controller.
The confocal's Zeiss LSM510 v4.2 operating software includes the 'FRAP', ‘Physiology', and 'Multi-Time Series' modules. The 'Physiology' time-lapse LSM software module is for long-term live cell time-lapse imaging, plus MetaMorph Off-line has various cell tracking applications for quantifying cell movement. In addition the Multi-Time Series module utilises the motorised stage enabling it to run multi-well or multi-position time-lapses, plus it can raster scan entire sections using top-left & bottom-right selection.
The confocal can image multiple fluorescent labels and standard bright-field DIC transmission automatically over the time-lapse period - with full Z focus motor control. The Zeiss Axiovert 200 M is an inverted microscope ideally suited to imaging living cells in culture, e.g. using cover-slip bottomed (Mattek/ibidi) Petri dishes, LabTek/Culture-well slides and multi-well plates. The use of a temperature and CO2 controlled incubator, enclosing the entire stage and objectives, allows the confocal system to be used for stable long-term video imaging of live cells ranging from hours to days.
The live cell imaging upgrade includes a Zeiss XL3 Incubator, a Zeiss heated LabTek/Petri dish Insert P, a Zeiss Universal KM frame for multi-well plates, a Zeiss TempControl 37-2 digital heater Controller, a Zeiss incubator air Heating Unit, and a Zeiss CO2 Controller, plus a 20x phase contrast objective for cell tracking/motility studies. A CO2 supply pipe has been installed into the microscope room from the centre's supply. If you wish to use the confocal with fixed specimens on glass slides the Universal Mouting Frame AK insert is still availabe. Please note that a few of these links to the Zeiss/PeCon manuals only function from within the WTCHG's intra-net.
A photograph of the XL3 incubator, Zeiss TempControl 37-2 Digital and the Zeiss CO2 Controller is shown above. Images of our various stage inserts for live cell imaging and slides are shown below. They can hold LabTek type culture-well slides, standard glass slides, Petri dishes or Mattek sized multi-well plates. For further details of culture vessel options see our ‘live cell' website links.
Before using the live cell system please ask a member of the Core for help and advice on using the system. In particular the switch off procedure for the XL-3 incubator must be followed rigidly to prevent damage to the Heating Unit and TempControl 37-2 controller.
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