A confocal microscope for multidisciplinary dynamic studies of complex biological systems

Fluorescence microscopy is a key technique for probing the organisation of living cells and tissues. It can be used to observe and quantify dynamic changes in live cells, for example the complex processes in involved in cell division, the development of animal embryos or the perception of chemical signals. Techniques developed over the last decades have hugely increased the potential of fluorescence microscopy by allowing the specific labelling of cell components such a specific proteins, cell membranes or particular DNA or RNA sequences. Many variants of fluorescence microscopy are available, all with their own advantages and drawbacks. The crucial parameters when judging the usefulness of a particular fluorescence microscopic method for tackling a particular problem include the spatial resolution (which determines how much fine detail can be observed), the time resolution (which determines the ability to observe rapid processes in action), the spectral resolution (which determines how many distinct cell components can be labelled and observed simultaneously), the working distance (which the determines the thickness of the sample that can be imaged), and the detector sensitivity (which determines how long a sample can be observed before it starts to be damaged by exposure the light used to excite fluorescence). Different forms of fluorescence microscopy have different trade-offs between these parameters. Confocal microscopy is a versatile technique in which the sample is imaged by rapidly scanning a highly-focused laser spot across it, in 1-3 dimensions. It lacks the extremely high spatial resolution of some recently-developed forms of fluorescence microscopy, but it provides particularly good spectral resolution with an ability to resolve fine details within thicker samples and excellent time resolution. Sample damage due to exposure to high-intensity lasers was a significant issue in older confocal microscopes, but this problem is greatly decreased in the latest generation of confocal microscopes by the use of higher-sensitivity detectors. Our proposal is to purchase a very versatile and state-of-the-art confocal microscope that will be made available to users from across Queen Mary and also to outside academic and industrial users. It will complement the other fluorescence and electron microscopic techniques available here to give us access to a suite of techniques that will help us to understand the function of living systems on scales from molecules up to cells and tissues. We will apply it to a huge range of biological problems, including the function of bacterial cells, the trapping of sunlight by plants, the perception of chemical signals by cells, the re-organisation of chromosomes as a cell divides and the development of animal embryos.

Laser-scanning confocal microscopy is a versatile tool for probing structure, dynamics and molecular interactions in living systems. It can be combined with a wealth of specific fluorescent labelling techniques including fluorescent protein tagging and immunofluorescence to highlight specific proteins, Fluorescent in situ Hybridisation (FISH) to highlight specific DNA and RNA sequences and a whole range of fluorescent indicators to report on the local chemical environment. The use of laser lines to excite the sample and monochromators to define the emission bands allows versatile spectral resolution and is optimal for visualising multiple fluorophores simultaneously. With pulsed laser excitation, confocal systems can be used for Fluorescence Lifetime Imaging Microscopy (FLIM), which provides a superb way to distinguish fluorescent labels from background autofluorescence and is optimal for probing intermolecular interactions using Förster Resonance Energy Transfer (FRET) measurements. Confocal microscopy is also optimal for measurements of fluidity and dynamics using Fluorescence Recovery after Photobleaching (FRAP) and related techniques. As compared to super-resolution microscopy techniques, confocal microscopy has lower spatial resolution but offers greater spectral resolution, faster kinetics and the ability to probe structure and dynamics in thicker samples, such as tissue sections, Drosophila oocytes and zebrafish embryos. Our proposal is to purchase an exceptionally versatile state-of-the-art instrument with high-sensitivity detectors that will allow dynamic imaging of living systems with minimal photobleaching, combined with capabilities for FLIM, real-time deconvolution for super-resolution, and light-sheet microscopy for three-dimensional dynamic imaging of thicker samples. The instrument will be run as a well-maintained and well-supported facility available to users across Queen Mary and to external academic and industrial users.