LIMS Bioimaging Facility

Light microscopy

The Bioimaging facility offers researchers access to a range of high performance confocal and widefield fluorescence microscopes. Capabilities include live imaging of cell dynamics and multichannel imaging of stained cells, 3D cultures, tissues and small organisms. Support in advanced fluorescence techniques such as FLIM, FRET, FRAP and FCS is also available for quantitative analysis of interactions and mobility of molecules in cells.

Electron microscopy

Core resources for transmission electron microscopy (TEM), scanning electron microscopy (SEM) and EM sample preparation are now available to researchers through the Bioimaging facility. Advanced TEM techniques available include tomography, scanning TEM (STEM) and cryo-TEM.

Instrumentation

Confocal imaging

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 LSM 510 laser scanning confocal
The 510 is equipped with 5 lasers, high resolution objectives (20x - 100x) and has 3 conventional PMT detectors, 2 high sensitivity APD detectors and an incubator. Use the 510 for 3-channel fluorescence imaging of live or fixed cells. The APD detectors can be employed for imaging of weaker fluorescence signals or combined with low laser power to reduce sample fading. Enquire about fluorescence correlation spectroscopy (FCS), available only on this instrument, to measure diffusion and interactions of fluorescent molecules in living cells or solution with single photon sensitivity.

Leica TCS SP2 laser scanning confocal
The SP2 is a 6-laser confocal with choice of high quality objectives in the 10x – 100x range and adjustable bandwidth PMT detectors. It is suitable for 3-channel fluorescence imaging of specimens on slides. Acquire lambda (wavelength) stacks to record the emission profiles of fluorophores of interest. Equipped for FRAP experiments.

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.

Widefield imaging

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.

Lifetime imaging

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.

Image analysis

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.

Transmission electron microscopy

Jeol JEM-2100 TEM

The Jeol JEM-2100 is a 200 kV transmission electron microscope equipped with two Gatan digital cameras for widefield or high resolution imaging. Advanced options available on this microscope include tomography and scanning TEM (STEM).  Cryo-TEM, for imaging of frozen hydrated specimens is also available. Recent projects: imaging of protein nanocrystals, complexes and fibrils, virus particles, extracellular vesicles. Resolution in the micron to sub nanometer range

Cryo-TEM equipment

An FEI Vitrobot is available for semi-automated preparation of frozen hydrated specimens for cryo-TEM. A Gatan 626 cryo specimen holder and SmartSet workstation enable transfer of frozen samples into the TEM.

Scanning electron microscopy

Hitachi TM3030Plus Tabletop SEM

The TM3030Plus is a compact variable pressure benchtop SEM with optional 5 or 15 kV accelerating voltages and BSE and SE detectors for imaging. The instrument is additionally fitted with a Bruker elemental dispersive X ray spectroscopy (EDS) detector and Quantax software for element mapping. Can be used effectively with many fresh samples (e.g. pollen, seeds, insects).

Jeol JSM-6340 FESEM

The Jeol JSM-6340 field emission scanning electron microscope is available for high magnification, high resolution imaging projects.

Support

We support researchers with equipment training and can assist with project planning, basic and advanced microscopy techniques and digital image analysis. Contact us and arrange for a tour to see what facilities are available or schedule a meeting to discuss your project.

Training and equipment bookings

To use the equipment researchers should arrange for instrument-specific training with the facility manager. Training is designed to enable users to operate the instruments skilfully and obtain publication quality data.

Trained users can book equipment and view availability online using Outlook (La Trobe students and staff).

Fees

Annual subscription plans are available to La Trobe research groups to access the confocal and electron microscopy equipment. Operator training is included at subscription plan rates.

Fees for external users including staff-assisted services are available on request.

TRANSMISSION AND SCANNING ELECTRON MICROSCOPY

Subscription Plan La Trobe research group
Up to 25 h $1500
Up to 50 h $2750
Up to 100 h $5000
Up to 200 h $9000

CONFOCAL IMAGING

Subscription Plan La Trobe research group
Up to 30 h $1100
Up to 100 h $2600
Up to 200 h $4000
Uncapped $6000

Contact

For more information about accessing the LIMS Bioimaging Facility, please contact:
Dr Peter Lock
Manager
LIMS Bioimaging Facility
T: +61 3 9479 3349
E: P.Lock@latrobe.edu.au