Fluorescence microscopy is a technique that enables the molecular composition of the structures being observed to be identified through the use of fluorescently-labelled probes of high chemical specificity, such as antibodies. Immunofluorescence is perhaps the most widely used microscopy technique today, mainly because it can provide high detection sensitivity, there is a high signal-to-background ratio, and a huge range of highly-specific fluorescent probes are available.
There are three major areas of improvement in the field of fluorescence microscopy. The first is that its use is mainly confined to studies of fixed specimens because of the difficulties of introducing antibody complexes into living specimens. For proteins that can be extracted and purified in reasonable abundance, these difficulties can be circumvented by directly conjugating a fluorophore to a protein and introducing it back into a cell. It is assumed that the fluorescent analogue behaves like the native protein and can therefore reveal the distribution and behavior of this protein in the cell.
Additionally, simple fluorescence microscopy only works well with very thin specimens or when a thick specimen is cut into sections. This is because structures above and below the plane of focus create interference in the form of out-of-focus flare. Optical sectioning techniques can minimize interference; part of LOCI's mission is to develop these techniques to increase the amount of information they can produce.
Another serious issue with fluorescence microscopy is phototoxicity. When a fluorophore (endogenous or exogenous) is excited there is a probability that, instead of decaying to a singlet state and emitting a fluorescence photon, intersystem crossing will occur to a triplet state. These long-lived states are very reactive and can damage living cells and bleach the fluorophore. One of the most significant damage mechanisms is the generation of highly reactive singlet oxygen from triplet state. When a specimen is being observed in a fluorescence microscope, the fluorophore is excited throughout the bulk of the sample, even though only one focal plane is being observed at any time. Most of the phototoxic load in a live specimen therefore comes from regions away from the thin focal plane being observed.
- Development of new optical instrumentation to facilitate studies of the dynamics of living specimens. We are focusing our efforts on improved in vivo imaging techniques that provide significantly more information about a specimen than can currently be obtained.
- Development of software for the capture and visualization of dynamic, three-dimensional cellular, or sub-cellular events.
- Development of optical methods for imaging deeper in live specimens
- Development and application of methods for fluorescence lifetime imaging
- Development of spectral and polarization based methods