The Zeiss LSM510 META/FCS is situated in the Center for Cell Signaling, 7th floor, Hospital West.  It is a state-of-the-art confocal microscope that is ideal for multi-color live cell imaging (5D imaging).  The confocal head is attached to an inverted microscope (Axiovert) with an automated XY stage, and an Eppendorf micro-injector.  It has two options for incubating cells at controlled temperatures, one of which uses Bioptech dishes.  The other can accommodate any shape or size of culture system.

The system can perform FRAP and FLIP photobleaching assays, automated long-term time-lapse imaging with auto-focusing, spectral fingerprinting to resolve up to 5 separate fluorophores; high-throughput imaging; single particle detection using the FCS; and routine z-stacks and 3D rendering.

Objectives: It is equipped with an LD Achroplan 20x/0.4 na dry lens for low power routine imaging; a 40x/1.2 na water-immersion lens that is ideal for live-cell imaging; plus Plan Neofluar 40x/1.3 na, and Plan Neofluar 100x/1.3 na oil immersion lenses.

The oil immersion lenses provide high-resolution imaging, ideal for either live cell or fixed samples.

Lasers: The instrument has an argon laser with 458, 477, and 488 nm lines (blue-green); a 543 nm HeNe laser (red); and a 633 nm HeNe laser (far-red).

Automated stage: A big advantage of this instrument is that it has an automated XY stage.  This can be used for tiling – i.e., producing very large, high-resolution images of objects; for multi-location timelapse studies or photobleaching; and for high-throughput imaging.  For example, one can collect images from each well of a 96-well plate, or from multiple locations on a dish of cells.  A macro called ‘Multi-time’ controls the stage to drive it to each location and ensure that each location remains in focus throughout the experiment.  This feature can dramatically increase your rate of data collection.  Click here for additional information on timelapse experiments using the automated stage.

Autofocusing: The autofocusing system uses a linescan to detect the reflection off the coverglass.  The offset from this position to the focus position for the cells is recorded, and the linescan checks the position at set intervals during the timelapse experiment.  Using the Stage control up to 999 locations can be recorded, with distinct focus positions.  We have used this system to maintain the focus of live cells at multiple locations for over 12 hours, using the 100x objective lens.  For additional information on autofocusing in a timelapse experiment click here.

Emission Fingerprinting: The META module uses a diffraction grating and a set of PMTs to collect the emission spectrum (a Lambda stack) consisting of 32 channels, from each point in an image.  It can then deconvolve the spectra to separate the contributions from each fluorophore.  This method is particularly useful for getting rid of autofluorescence during live cell imaging.  See