Light microscopy core facility
The light microscopy facility provides training and support to scientists in a wide-range of imaging acquisition and analysis techniques. Instruments in the Light Microscopy Core range from wide-field imaging systems to non-linear multi-photon and lifetime imaging systems for cell and intra-vital tissue studies.
The facility is specialised in the following areas of expertise:
- Advanced live-cell imaging using wide-field and spinning disc imaging systems
- High-quality confocal scanning light microscopy
- Non-linear imaging techniques such as multi-photon and second harmonic imaging
- Fluorescence life-time imaging (FLIM)
- Live-animal imaging at high resolution
- Quantitative high throughput image acquisition and analysis (e.g. iCys)
Figure 1: Left panel: Cell tracking for Mitotic Index Analysis (with C-B. Schoenlieb, DAMTP). Middle panel: Image Analysis of mitotic spindle components in 3D (with F. Gergley). Right panel: High content Analysis with CompuCyte iCys iGen software.
The facility has two Nikon and two Leica confocal laser-scanning systems for high-quality fluorescence imaging. The Leica systems have additional fast resonance scanning modes, whilst the Nikon systems have the advanced spectral detection option of imaging 32-channels simultaneously. Three of the confocal systems have large area scans or multiple time-point experiments with CO2 and temperature-controlled incubation chambers.
Wide-field imaging systems
Time-lapse observation systems are available for studies of apoptosis and growth development (>24 hours). These microscopes have CCD or EMCCD cameras, high-quality DIC optic or phase contrast, perfect focus, and multi-position stages. Solent or Tokai-hit portable chambers for CO2 humidity and temperature control and an Eppendorf microinjector are available.
Spinning disc system
A parallel-scanning spinning-disc system (Improvision) is available with a fast piezo-drive for imaging of cells in XYZ at full frame resolution at 30fps acquisition speed.
Multi-photon imaging is the state-of-the-art technique for the observation of living mammalian cells and special physiological probes within tissues. It is highly recommended for monitoring thick biopsies, as the depth penetration of IR is greater than for shorter visible wavelengths.
The facility has two multi-photon systems: (1) the Leica SP5, equipped with a pulsed Coherent Chameleon Ultra II laser infra-red laser. (2) The LaVision BioTec ‘TriMscope’, with a 4W Coherent Ultra II (690 – 1080nm) and an additional APE OPO, extending the excitation wavelength from 1050 to 1580nm. This allows for background-free deep tissue imaging of fluorescence ranging from UV to IR-probes and second harmonic imaging e.g. of collagen.
Fluorescence life-time (FLIM)
The Becker & Hickl FLIM module is part of the Leica SP5 MP system. Lifetime measurements can yield information on the molecular micro-environment of a fluorescent molecule, independent of the concentration of the fluorophore. Factors such as ionic strength, hydrophobicity, oxygen concentration, binding to macromolecules and the proximity of molecules, can be studied using ion indicator probes for e.g. FRET studies of intermolecular distances.
High throughput image acquisition and analysis – Laser Scanning Cytometry
Laser scanning cytometry (LSC) with the CompuCyte iCys system for quantitative high-throughput image analysis combines the advantages of flow cytometry with those of image analysis systems (Zecchini et al., 2012). The iCys allows rapid scanning and automated analysis of solid phase samples including fixed cells, live adherent cells, tissue sections and microarrays. The iCys inverted format supports a range of carrier types from single slides and Petri dishes to 96-well micro-titre plates, facilitating measurement of any sample size. Current applications include studies of DNA damage in cancer cells, apoptosis assays, tumour vasculature and drug distribution and ligand uptake.