Low Power Sub-Wavelength Resolution Fluorescence Imaging

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Super resolution refers to the ability to resolve objects and/or observe contrast on a distance scale below that afforded by conventional optical imaging (e.g. the confocal microscope) which is restricted to approximately half the wavelength of the illuminating light. In the visible region of the spectrum this is on the order of a quarter to a third of a micron (250- 330nm). Non-invasive sub-cellular observations below this length scale are impossible using conventional optical microscopy . There has been considerable academic and commercial activity aimed at developing techniques that reveal structure on the 100nm length scale and below. These fall into four categories [1] Stochastic Reconstruction Techniques (PALM & STORM) [2] Structured Illumination (SI) [3] Stimulated Emission Depletion (STED) Techniques [4] Ground State Depletion (GSD) Microscopy All have serious drawbacks. PALM and STORM (photo-activated localization microscopy and stochastic optical reconstruction microscopy respectively) require the use of specialised (photo-activatable) fluorescent probes and often long (several hours) data collection. SI uses structured illumination of the sample with patterned light (complex optical input) and detailed computer analysis of the resulting fringe structure to provide increased resolution, yet increases in resolution above a factor of two require high input powers with an increased risk of sample damage. STED creates a sub-micron fluorescent spot by the overlap of the initial exciting beam (PUMP) with a depletion (DUMP) laser (pulsed or continuous wave) which is 'shaped' to provide a 'doughnut' intensity profile. The drawbacks of STED are [a] the expense and complexity of the DUMP beam-shaping optics and [b] the on sample DUMP powers that are required to obtain high resolution. These correspond to intensities where the onset of photochemical damage and sample heating becomes a significant risk. GSD microscopy is a two laser technique and is similar to STED in that resolution is intensity dependent. A spatially offset second laser is used to (strongly) drive molecules into long lived non-fluorescing triplet states resulting in a reduced fluorescent spot. GSD resolution is degraded by triplet lifetime shortening due to quenching (collisions with oxygen) requiring the development of customised fluorescent probes and/or the removal of oxygen by specialised mounting media.Our new technique for super-resolution breaks the diffraction limit through imaging the modifications to the time and spatial dependence of fluorescent probe emission following pulsed excitation using a moderate power (0.1W) continuous wave depletion laser. Time slices of the fluorescent image can be recombined to yield an image which reveals contrast and structure below the conventional diffraction limit of a confocal microscope. The technique does not require sophisticated laser beam shaping as in SI and STED. Also, in contrast to STED spatial resolution is not critically determined by the degree of depletion and on-sample powers will at the very least be an order of magnitude below that of the typical STED doughnut. We will realise the technique by the addition of a depletion laser to a conventional fluorescence lifetime imaging microscope and the development of software to analyse and reconstruct the (modified) information provided by the intensity-space-time data that is routinely collected in FLIM systems. The apparatus will be used firstly to obtain super-resolution in test structures (20-100nm fluorescent nanoparticles) and biological structures in fixed cells. The final phase of the project will involve the application of the technique to the study of biological processes in live cells involving collaborations with UCL groups in Cell & Developmental Biology, The UCL Institute of Opthalmology and the UCL Ear Institute.