Multifunctional confocal laser scanning microscope with time resolved and two-photon imaging and fluorescence correlation spectroscopy capabilities
The system will establish a novel research tool – a multifunctional confocal laser scanning microscope with time resolved and two-photon imaging and fluorescence correlation spectroscopy capabilities by combining state-of-the-art technologies in microscopy with the versatility and diagnostic power of traditional and time-resolved spectroscopy. The goal is to provide microscopic information on physical, chemical and biological systems on the micrometre scale. The systems will provide us with completely new techniques of investigating the micro-world not currently available anywhere in Australia.
These techniques will be used to resolve important problems in chemistry, forensic science, geology, biology, physics, biochemistry, physiology and materials science, which cannot presently be addressed using existing infrastructure. Problems to be studied will include the studies of cell signalling, more effective methods for detection of low levels of water borne organisms, the design of better electronic materials and forensic identification, thin film and materials characterisation. We anticipate that this instrumentation, with its broad range of applications, will promote and develop interdisciplinary research on the boundary between physics and other sciences. Being a most recent generation system, it will position Australian scientists on the forefront of the international research effort.
PURPOSE OF EQUIPMENT AND VALUE TO RESEARCH:
The microscopy system is built on a foundation of a modern laser scanning confocal fluorescence microscope, of great scientific utility in its own right. This microscope will be equipped with the Fluorescence Lifetime Imaging accessory (FLIM) and a separate Fluorescence Correlation Accessory (FCS). There are 4 parts in this system:
In confocal microscopy the collected light is reflected or emitted by a single plane of the specimen. This leads to high contrast due to effective suppression of light scattered from outside the focal plane. Moreover, as number of such images generated with the focal length shifted in small steps can be combined in a three dimensional stack, which is accessible to digital processing. Thus such microscopes have the optical sectioning capability – as slices of the specimen can be examined without mechanical cutting and direct specimen preparation. These two important advantages have led to a widespread use of confocal imaging in many areas of science and technology.
4.4.Fluorescence Lifetime Imaging (FLIM)
The powerful technique of time-resolved fluorescence significantly enhances the capabilities of conventional laser scanning microscopy, by offering deeper insights into chemical composition of the examined specimens. This very important capability makes it possible to produce fluorescence signatures that fully characterise the sample in a matter of minutes. Importantly, time-resolved fluorescence imaging is complementary to conventional fluorescence imaging. This facility is of importance in many biological applications, where the objective is frequently to make a fine distinction between two parts of a specimen with broad and very similar fluorescence curves. Applications of time-resolved fluorescence microscopy are as diverse as those of fluorescence and specimens can be examined rapidly and non-destructively.
The fluorescence of molecules is not only characterised by the emission spectrum, it also has a characteristic lifetime. Any energy transfer between an excited molecule and its environment changes this fluorescence lifetime in a predictable way. Since the lifetime does not depend on the concentration of the chromophore, fluorescence lifetime imaging is a direct approach to all effects that involve energy transfer. Typical examples are the mapping of cell parameters such as pH, ion concentrations or oxygen saturation by fluorescence quenching, or FRET between different chromophores in the cell. Furthermore, combined intensity- lifetime imaging is a powerful tool to distinguish between different fluorescence markers in multi-stained samples and between different natural fluorophores themselves. These components often have ill-defined fluorescence spectra but are clearly distinguished by their fluorescence lifetime.
Thanks to these characteristics, the Fluorescence Lifetime Imaging (FLIM) has become a new powerful tool to investigate molecular interactions, reactions and energy transfer including in cells and subcellular structures. These effects cause changes in the fluorescence quantum efficiency and thus in the fluorescence lifetime. Since the fluorescence lifetime does not depend on the unknown dye concentration it is a direct measure for the quantum efficiency. It therefore gives a more direct access to the investigated effects than the fluorescence intensity. Furthermore, the fluorescence lifetime can be used to separate the fluorescence of different luminophores in the cells if the components cannot be distinguished by their fluorescence spectra. Recording time-resolved fluorescence images is achieved by combining the Laser Scanning Microscope with pulsed laser excitation and a new Time-Correlated Single Photon Counting (TCSPC) Imaging technique introduced by Becker and Hickl.
4.4 Fluorescence correlation spectroscopy
In fluorescence correlation spectroscopy (FCS) the fluctuations of fluorescently labelled molecules are used to deduce quantities such as concentrations, interactions, binding strength, diffusions etc. All these quantities are measured by observing molecules in the focal volume of 0.3 x 1.5 um size. (in the volume of less than 1 fl), and has similar dimensions as a single E.coli bacteria. FCS evaluates the temporal behaviour of the fluorescence fluctuations and it evaluates the correlation. The amplitude of the correlation is inversely proportional to the number of particles in the confocal volume. In practice this means the ability to measure minute concentrations of molecules, down to single molecules under physiological conditions. FCS makes it possible to distinguish slowly diffusing from fast diffusing particles. In solutions large molecules move more slowly and molecules of different sizes may be distinguished without any separation procedures. FCS facilitates efficient analysis of molecular interactions, including determinations of binding constants and kinetic examinations. Issues such as binding between ligands and receptors on the living cells, processes on membranes can be addressed as well.
The proposed instrument will thus mark out a new frontier in spectroscopic instrumentation and will represent a significant enhancement of present diagnostic capabilities. In summary, the proposed system will present the latest in the state-of-the-art in microscopy and diagnostics, with a broad range of applications across all sciences.