Super-resolution optical microscopy via nonlinear self-focusing

The main conclusion of modern biomedical science is that the activities of life depend on specific interactions between protein, carbohydrate and lipid molecules. In a single cell there are thousands of different types of molecules, some presented as only a few copies. Unfortunately, the resolving power of a standard optical microscope is approximately 100 times too poor to see individual molecules. Super-resolution is therefore desperately needed in these instruments.Methods for achieving super-resolution have been proposed since the 1990's and have raised hopes. However, the number of super-resolving microscopes in the UK is, so far, probably less than 10, and they have had little impact. The reasons are not only high cost, instrumental complexity and tardy commercialisation: each method has serious practical disadvantages. For example, stimulated emission depletion (STED) microscopy requires the use of special fluorophores and sophisticated multi-wavelength laser sources. Photo-activation microscopy (PALM) needs the specimen to be frozen through many cycles, each cycle consisting of activation and then imaging to the full bleaching of a subset of photo-protein molecules. Stochastic methods such as STORM and structured illumination techniques are slow and computationally intensive and do not provide as large an improvement in resolution as the previous methods, at least with the available linear optics. A simple and inexpensive method to increase the resolution in nonlinear optical microscopy would be a boon to every biomedical researcher.This proposal concerns just such an approach, using nonlinear optical self-focusing. Self-focusing is a nonlinear effect caused by the propagation of a high-power laser source in a medium with a positive Kerr nonlinearity. The high-magnitude optical power of the light source along the propagation axis causes an effective increase in the higher order refractive index. This modified refractive index distribution then acts like a focusing lens and the net result is self-focusing of the input beam within the transparent material. Self-focusing is well recognised in photonics and is employed to great effect in Kerr lens mode-locking to develop ultra-short pulsed laser sources such as those used in nonlinear optical microscopy. Crucially, we have recently demonstrated this as a means for producing better-resolved images in an optical microscope. Common immersion media (air, water, oil) have very low positive Kerr nonlinearity and considering the laser parameters typically employed in nonlinear optical microscopy, the self-focusing threshold condition is not met. However, calculations and preliminary experimental studies show that certain organic water-soluble compounds may have sufficiently high Kerr nonlinearity to support self-focusing of the excitation beam. The successful implementation of this method could easily bring about a revolutionary improvement in spatial resolution of pre-existing microscope instrumentation. These are of the type known as multi-photon laser scanning microscopes, and are already widespread in the UK and overseas, in spite of their high cost. Consequently, this research could lead to major biomedical discoveries and add vastly enhanced value to the existing equipment stock of laboratories, not only in the UK.

-Who will benefit from this research? The entire community will benefit from this research through improvements in healthcare which may be expected to be made as a result of the novel method for basic research envisaged here. -How will they benefit? At present, molecular information is revolutionising medicine. Identification of proteins presently involves elaborate fine-structure methods such as X-ray crystallography and high-resolution electron microscopy (EM). Unfortunately the light microscope is a hundred times too poor to resolve even the outline of protein molecules: identifying which protein to study often involves genetic inference, which is impossible in many diseases. If the proposed research is successful, the new method is likely to be adopted immediately by all biomedical researchers who are users of multi-photon microscopes (approximately 100 systems in the UK at present, representing a huge investment of capital equipment funding over the last 20 years, since each system costs around 0.5 million). It will become possible to see individual macro-molecular assemblies & protein molecules that are currently invisible in living cells. For example, molecular signs of disease such as prion particles and individual viruses may become visible in the live cell. Direct and short-term beneficiaries (<2 years) Super-resolution has been reported for over ten years, but in this time it has been realised convincingly in only a few cases and with some doubt over the performance of all commercial offerings in this field. The new approach offered here may provide super-resolution with existing multi-photon microscope systems, thus removing the need for substantial funding to purchase the new and relatively untried special-purpose instruments. The first beneficiaries will be the biophotonics community at Strathclyde, who will apply the method to research on cell signalling, receptor function, novel drug targets such as ion channels for cardiovascular therapy and other current research fields, but the method is likely to be adopted quickly world-wide. Longer term (5 years plus) If even a modest success is achieved, it should produce an avalanche of research world-wide, with particular relevance to healthcare. This research will probably involve a search for more water-soluble compounds that support self-focusing but which have the required spectral properties for use in conjunction with current fluorophores, lasers and microscopes. Concomitant laser development may also be required. Since it is a basic method, it may well also find application in materials science and micro-fabrication. It is likely that an optical method such as this, which can be applied to hydrated samples including biopsies and which permits the examination of far larger volumes than by EM, may create totally new methods for the diagnosis of disease by observation of bacteria, virus particles and abnormal details of cell morphology. -What will be done to ensure they have the opportunity to benefit from this research? The new method proposed may be so useful that it will be self-propagating. Preliminary experiments (as explained in the Case for Support) demonstrate the efficacy of beta-carotene in increasing resolution in an oil medium. The aqueous extension of the work will be applied to well-known biological subjects such as microtubule arrays & actin filament bundles in standard immune-fluorescence preparations. This work will be published in leading journals in the field of biomedical microscopy. As well as having a wide circle of international contacts in microscopy, the PI is active in the Scottish Universities Physics Alliance (SUPA), as a conference organiser for ELMI (the European Light Microscope Initiative) and the EMBO Practical Course on Advanced Optical Microscopy (Plymouth) and engages in scientific outreach through a host of organisations, including SETPoint and National Science Week.