Light Microscopy

Zeiss LSM 800 Airyscan laser scanning confocal (New)

The Zeiss 800 Airyscan confocal is equipped with 4 lasers (405, 488, 563, 633 nm), five high resolution objective lenses (10x, 20x, 25x multi immersion, 40x and 63x oil), sensitive detectors, an auto-focus drive and an incubation system with CO₂ and temperature control. The 800 is used for multi-channel fluorescence imaging of fixed cells and tissues as well as stable long-term live-cell imaging work. Enhanced resolution below the diffraction limit (to about 140 nm) is possible in SR mode, by utilising the 32 channel Airyscan detector (1.7-fold resolution improvement). A high resolution monochrome camera fitted to this microscope also facilitates rapid widefield imaging and navigation of large areas of interest.

Users of the Zeiss 800 may take advantage of a CellASIC ONIX device to record high resolution movies of live cells and their response to treatments under physiological-like conditions. The ONIX couples with a range of dedicated microfluidic plates suitable for mammalian, yeast or bacterial cells to enable constant perfusion of media under defined flow rates and also facilitate the timed delivery of agents of interest (agonists, inhibitors, concentration gradients, washing steps, flow conditions) during time-course experiments.

Zeiss LSM 780 laser scanning confocal

The 780 has seven laser lines for sample excitation and features a 32-channel spectral detector, a choice of five high resolution objectives (10-100x) and incubator. Use it for 4-channel fluorescence imaging of cells (live or fixed), Z stack 3-D reconstructions and extended focus images, for imaging of 3-D cultures and for creation of mosaic "overview" images.

Several advanced capabilities are also available on the 780. Employ spectral imaging and unmixing to discriminate multiple overlapping fluorescence emission profiles at once. Access the 780 to perform FRAP experiments to measure fluorescent molecule translocation in living cells. Use the integrated PicoQuant FLIM system for fluorescence lifetime applications such as FLIM-based FRET to visualise inter and intramolecular interactions in cells.

Zeiss Cell Observer spinning disk confocal

The Cell Observer is a hybrid instrument with 5-laser lines for sample excitation, 63x objective, Yokogawa CSU-X1 spinning disk, dual Photometrics EMCCD cameras and incubator. Designed for speed, the spinning disk employs multi-point rather than single point scanning to illuminate the sample. This allows for 4D imaging experiments where 3-dimensional Z stacks can be captured at every time point (rather than a single focal plane) to gain greater information. Simultaneous 2-channel imaging (e,g, GFP + mCherry) is possible using synchronised cameras. 3-channel fluorescence imaging and DIC imaging is only available sequentially in one camera mode.

Zeiss AxioObserver Z1

The AxioObserver is an inverted microscope with Axio Cam MRm monochrome camera, five objectives (5x to 100x) and DAPI, GFP, TRITC and Cy5 filter blocks. Use the AxioObserver for automated 4-channel fluorescence imaging and phase contrast imaging. Mainly used for imaging slides and cultured cells in dishes or 96 well plates, this instrument can also be used for live cell imaging experiments in combination with a CellASIC ONIX microfluidic system. With this device cells are grown under perfusion conditions in sealed, incubated microfluidic plates. The CellASIC can be used for timed delivery of drugs or other agents of choice and continuous imaging of cellular responses.

Nikon Ti Eclipse

Versatile semi-motorised fluorescence microscope with 7 objectives (2-100x) and two cameras. Use the DS-Qi1Mc monochrome camera for 4 channel fluorescence imaging (DAPI, FITC, TRITC and Cy5 filters) or alternatively utilise the DS-Fi1 high res colour camera for imaging of stained samples (e.g. H&E tissue sections). Also use this instrument for phase contrast imaging of cells in dishes. A motorised Z-drive is available for obtaining Z-stacks and NIS software can be used to perform deconvolution and produce confocal-like 3D images and extended focus images.

Olympus IX81

The IX81 is a semi motorised inverted microscope with 20x 40x, 60x and 100x objectives and filters for imaging DAPI, FITC, TRITC and Cy5. It is fitted with an F-view digital monochrome camera running on SIS software. Use the IX81 for fluorescence of fixed specimens mounted on slides. DIC capability.

Olympus BX50

The BX50 is a manual upright fluorescence microscope with filters for DAPI, FITC and TRITC imaging and objectives ranging from 20x - 100x. Use this instrument for three colour fluorescence imaging of cells and tissues on slides.

Fluorescence lifetime imaging microscopy (FLIM) is an advanced imaging technology to measure fluorescence decay rates of molecular probes in cells or tissues in nanosecond timescales. Data is displayed in colour coded images which map lifetime changes at high spatial resolution. The power of FLIM is in its ability to detect small changes in decay rates of fluorophores due to local temperature, pH and salt concentration changes. FLIM is also particularly sensitive to binding interactions in cells (e.g. interaction of GFP and Cherry fusion proteins).

Specialist instrumentation for time-resolved FLIM experiments is available through the facility. A PicoQuant FLIM workstation is operated in combination with our Zeiss 780 confocal microscope. Instrumentation includes a single 485 nm pulsed laser for excitation of fluorophores such as GFP, single photon detectors, and a device for timing laser pulses and arrival of fluorescent photons.

Use the PicoQuant system to image intermolecular binding of GFP- and mCherry-tagged binding partners or to detect intramolecular conformational changes of GFP - mCherry double labelled biosensors.

A workstation with Imaris image analysis software is available to facility users for processing and analysis of 3D and 4D datasets (e.g. Z stacks, times series, Z-stack over time). Step-by-step tutorials cover many applications and can be accessed via the software. Use Imaris to render confocal Z stacks in 3-D and produce dynamic movies for Powerpoint presentations. Measure volumes and surface areas of objects in 3D images. Track paths, velocities and displacements of moving objects in time series experiments. Trace the paths of filament-like objects (e.g. neuronal networks) and sort and plot objects graphically on the basis of size, shape and other parameters. ZEN and ImageJ/Fiji image software is also provided on the workstation. These programs and Imaris all offer colocalisation analysis software. Enquire about deconvolution processing of 3-dimensional image datasets to increase resolution and contrast.