Fluorescence lifetime imaging in the study of the cellular disease state
Photodynamic therapy (PDT) continues to be a technique that uses tissue friendly red and near infra-red light (NIR) to activate light-sensitive drugs (photosensitisers) for the treatment of cancer and other diseases in a non-surgical and non invasive way. Understanding the mode of action of the photosensitiser drugs is vital to their usage in patients and the development of more efficacious molecular systems. The efficacy of most photosensitisers is dependent on their quantum yield of singlet oxygen (1O2) generation within the cellular localisation. Hence knowledge of how the photosensitisers distribute themselves in cells and tissues is vital to understanding their action. Furthermore, the activity of these drugs can be environmentally dependent and therefore within the many types of conditions found in a cell (pH, [Ca2+] etc.) intra cellular studies are vital in this area of research.
Research within the STFC at the LSF Laser Microscope laboratory at RAL began such work nearly 10 years ago and its efforts have been instrumental in discovering the cellular organelle involved in some of the first generation photosensitisers such as the metal phthalocyanines. In these studies by using a steady state line scanning confocal microscopy it was demonstrated that during photoexcitation of the sensitised cells, a redistribution of the fluorescence is accompanied by an increase in the emission intensity which may be a result of monomrisation or diffusion from concentrated sites to areas where less quenching, and hence more drug activity is possible.
Since steady state fluorescence spectroscopy and imaging cannot account for these effects, time resolved techniques make an ideal tool to compliment these studies. During mammalian cell and tissue studies, the lifetimes of these excited drugs was found to be vary from 4.2-6.6 ns, depending on the particular location within the cell. This programme of work is ongoing and is stimulated by new advances in using multiphoton excitation which offers benefits over the one photon process for both the PDT treatment and the understanding of the biophysics of the process.
The measurements will feed directly back to the drug manufactures and enable them to design more optimum drugs capable of targeting particular environments found within cancer cells. The proposed Network will provide direct interaction with the medical community not only for the field of PDT but also to bring an awareness of time-resolved fluorescence and multi-photon imaging techniques for drug targeting within cells.
Current network members:
- Stan Botchway (CLF)
- Marisa Martin-Fernandez (SRD)
- Stephen Webb (SRD)
- George Santis (Consultant Physician, Guys Hospital)
- Prof Douglas Mitchell (Consultant Physician and Director of Research, Royal Preston Hospital)
Systematic studies on single cells
The ability of individual cells (mammalian, bacterial or virus) to change from normal behaviour to a rogue or disease state is the key behind unraveling health and well-being. For example the process of programmed individual cell death (apoptosis) is paramount to the survival of the organism. The 2002 Nobel prize in Physiology and Medicine was awarded to scientists investigating the behaviour of individual cells in an organism revealing the a key role of apoptotic cells (This process has been linked to neurodegeneration, autoimmune disease and cancer).
Currently laser optical tweezers provide the only clean tool available to the life-sciences to study these medically important processes at the individual cellular level. The optical tweezers apparatus based at the CLF Laser Microscope Laboratory at RAL has been used to capture, manipulate and measure forces on a wide range of particles, mammalian cells, bacteria, spores and virus size objects. Recently, a number of new experimental studies have been reported where optical tweezers have been combined with a range of spectroscopic techniques such as infra-red absorption and confocal fluorescence microscopy.
Of particular interest is the combination of optical tweezers with Raman spectroscopy to characterise the chemical species within single optically trapped microdroplets (for drug delivery and physiological interaction) and biological cells. With further developments, arising from collaborations within the proposed network, the above techniques have the potential to create a powerful spectroscopic imaging tool-set for use by biologists and biochemists.
Such techniques, will in the future provide the basis to extend current studies in key areas such as cell death, intra-cellular communication and drug delivery mechanisms. For example, they will enable the identification of chemical changes and abnormalities that occur in situ within the natural life cycle of an individual cell as well as when in close proximity with other cells. In addition, previous work on cellular form and function has typically used data averaged from populations of thousands of cells. It has therefore been difficult to obtain information on the triggering events within a population that leads to catastrophic malfunction of cells and tissue.
Although methods exist that are capable of imaging whole tissue cultures, they cannot probe the initiation events that occur during physiological changes of single cells. Optical tweezing is an ideal technique to allow such studies, such as the transformation of individual stem cells.
The proposed network will provide the platform and forum for leading scientists in the above field to meet and accelerate the use of manipulation of individually trapped cells (Mammalian, bacteria or virus) as well as spectroscopic imaging of optically trapped cells.
Current network members:
- Andy Ward (CLF)
- Stan Botchway (CLF)
- Tim Humphry (MRC, Harwell)
Signal transduction across cell membranes
The aim of this research is to characterise the conformational dynamics governing signal transduction by cell-surface receptors across the plasma membrane. The importance of receptor conformational changes in signal transduction has been thoroughly documented. For cell-surface receptors where crystallographic structures have been resolved activation has been shown to be critically dependent on the relative orientation of the extra cellular and intracellular domains in a dimer.
Understanding how signals are transduced by growth factor receptors is a central goal in Cell Biology, as these receptors play a key role in the regulation of embryonic development, metabolism, immune system function, development of the vascular system, angiogenesis, mitogenesis, cytoskeleton restructuring, and transformation
Interrogating how individual membrane proteins interact and/or how the associated changes in conformation regulate cell processes is, however, a challenging task. Multidimensional single-molecule fluorescence imaging is a method being developed at the Daresbury Laboratory to do just that, by combining total internal reflection illumination and light detection optics to allow the recording of the x-y distribution of the fluorescence from single fluorophores discriminated in colour and polarisation.
Single pair FRET (SpFRET) can be measured in this fashion, and the distance between two sites in a bio-molecule, or between two interacting molecules, to be determined in the range between 1-10 nm, a length-scale encompassing the typical diameter of membrane proteins. The rate of energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor fluorophores, so time-dependent spFRET can report on very small fluctuations in the distance distribution of donor-acceptor pairs by ratioing the anticorrelated two-colour time-traces for donor and acceptor. In turn, single molecule fluorescence polarisation (smFP) can determine the mean orientation of a single fluorophore relative to any convenient system of coordinates and is determined from the intensity values of the anticorrelated parallel and perpendicular emission traces as a function of excitation polarisation.
Current Network members:
- Marisa Martin-Fernandez (SRS)
- Stan Botchway (CLF)
- Clive Bagshaw (Leicester University)
