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Cell Imaging

The Cell Imaging Core Facility allows scientists across the Westmead Research Hub to see their genes and proteins of interest in action in brilliant fluorescent colour. By tagging proteins or cell structures with synthetic dyes or living molecular markers like GFP (green fluorescent protein) and its derivatives, researchers can view still images at set points of time in fixed cells or movies of live cells going through their normal physiological functions. With the current technology available at Westmead we can routinely capture 4-dimensional multicolour movies to explore a wide range of disease models. Opportunities are also now in place to correlate fluorescence images directly with the sub-nanometre detail available in electron microscope images.

Staff

The Westmead Research Hub Cell Imaging Facility is managed by Dr Hong Yu and Dr Laurence Cantrill.

Services

Access to and training in high end light microscopes.
Access to a high pressure freezing service for transmission to electron microscopy.

Technology

Olympus FV 1000 laser scanning confocal microscope is a powerful fluorescence microscopy system combined with the laser scanning method and developed for obtaining high resolution images by optically sectioning samples. It acquires in-focus images from selected depths to eliminate out-of-focus light or glare that otherwise leads to image degradation in specimens and therefore is suitable for thick samples particularly those whose thickness exceeds the immediate plane of focus. The microscope comes with 405, 473, 559, and 633 excitation laser lines and features two free emission settings, rather than fixed filters as in conventional fluorescence microscopy, thereby permitting lower crosstalk and better flexibility with fluorescent probes. Typical applications of the FV1000 confocal microscope include 3D imaging, colocalisation, time course, fluorescence energy transfer (FRET), and photo bleaching recovery experiments (FRAP). In addition, the Olympus confocal is fitted with a heated incubation chamber with 5 % CO2 supply enabling its use for live cell/tissue imaging.

Deltavision restoration deconvolution microscope is a high end wide field epifluorescence microscope featuring restorative deconvolution. It is specially designed for fast, sensitive and high resolution imaging (100nm vs 200-300nm for a confocal system) and therefore is suitable for small/thin and faint fluorescence samples, particularly living cells. Its unique image restoration through deconvolution is a software-based process which reassigns the out-of-focus light to its calculated point of origin rather than simply subtracting from the image, and therefore no data is lost or thrown away. As a result, Deltavision generates an image more closely representing the original sample and also images of better quality than any other de-blurring methods. The system is equipped with an optimised solid-state illumination source with fast excitation switching (microsecond) that allows fast and bright multicolour 3D live cell imaging resulting in reduced specimen fading and photobleaching. Dual cameras (CoolSnap HQ and EMCCD) provide high resolution and high sensitivity options. The popular user applications include Z-stack and 3D imaging, multi-position, panelling and stitching, time course with hardware autofocus. It is fitted with a WeatherStation environmental control chamber and recently updated with an Environmental Chamber Upgrade Kit which provides stable CO2 maintenance allowing for long time live cell imaging experiments. The system is operated by the Linux based DeltaVision Software SoftWorx which enables deconvolution to be performed up to 10 times faster than with other commonly-used deconvolution software.

Leica TCS SP5 laser scanning confocal microscope is a confocal laser scanning microscope designed for optical sectioning of cells and tissues to depths of 200um and resolution of 200-300nm. The system has a tandem scanner with a conventional scanner (fixed cell) for best image definition and a high-speed resonant scanner for live-cell and rapid 3D scanning of thick specimens. The laser lines available are: 405, 452, 476, 488, 496, 514, 561, 633nm allowing a broad spectrum of dyes from blue to far red to be observed. Unlike all other confocal systems, the Leica SP5 does not have dichroic mirrors, using instead a tunable acousto-optical beam splitter that can be tune rapidly and quickly for any wavelength of light. The system has 4 fluorescence PMT detectors for simultaneous 4 colour imaging, 1 hybrid detector for faint fluorescence and photon counting, and 1 transmission PMT detector for brightfield and DIC imaging. There is an environment chamber for live cell imaging. Other capabilities include FRAP, FRET, time course scanning, multipositional scanning, tile scanning, and multispectral scanning.

Nuance multispectral imaging system (Leica upright fluorescence microscope) is a standard wide-field upright epi-fluorescence microscope equipped with a unique Nuance multispectral CCD camera capable of characterising all the spectral components in a sample. The Nuance system allows separation of the background or autofluorescence in order to clearly detect very faint specific labels, and therefore is ideal for samples with low signal-to-noise staining. The ability of this system to eliminate autofluorescence is a major feature, and the camera is much more sensitive than multispectral confocal systems. Nuance enables imaging of multiple markers in tissue sections for both fluorescence and brightfield. Fluorescence filters are suitable for DAPI, FITC, TRITC, and Cy5 channels. To appreciate most of the advantages of its spectral unmixing function, a full set of samples must be prepared including individual single stained slides as well as non-stained specimen as controls. This system is used only for fixed slides and not for live cell imaging experiments.

Zeiss AxioVert 200M live cell imaging system is a fully motorised inverted widefield fluorescence microscope equipped with a temperature controlled CO2 incubator for live cell imaging applications. It comes with standard filters for fluorescence (DAPI, FITC, TRITC, Cy5) and BF/Phase/DIC imaging options. The Mark-and-Find functionality allows multiple time-lapse experiments. The new AxioCam mRm camera (upgraded in 2013) provides a good balance with resolution, sensitivity and imaging speed. This system allows for deconvolution processing of samples using Axiovision software to obtain high resolution images from both fixed and live cells.

PALM/Zeiss Laser Capture Microdissection microscope is for molecular biology. The laser capture microdissection microscope or LCM allows the identification and isolation of cells and tissues for later DNA/RNA or proteomic analysis. Using paraffin or cryostat-cut sections mounted on glass slides, researchers can cut around areas of interest with the microscope’s UV laser and then catapult cell contents into an eppendorf tube for later analysis. The system may also be used for selecting out individual chromosomes from metaphase spreads and clonal selection of cells from live cultures.

Location

The Cell Imaging facility is located on Level 2 of the Westmead Millennium Institute for Medical Research.