Low Power Sub-Wavelength Resolution Fluorescence Imaging

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.

The project seeks to develop a wholly new approach to obtaining super-resolution in fluorescence microscopy. This is distinct from previous approaches in that we use the alteration of the time evolution of a fluorescent image in the presence of a moderate (milliwatt range) continuous wave depletion laser to achieve super resolution. The point spread function (PSF) of a sub-wavelength source evolves (broadens and gains central structure) in the presence of a CW Gaussian depletion field. This evolution can be captured in a fluorescence lifetime imaging microscope (FLIM) and time slices of this evolution can be recombined to yield a sub-diffraction limited PSF. The same approach can be applied to every pixel of the image, leading to a reconstructed image with significant resolution enhancement over that afforded by a confocal imaging system. In order to obtain sub diffraction image resolution a ca. 33-50% reduction in the fluorescence lifetime by stimulated emission is required. This degree of depletion requires significantly lower powers than in 'conventional' STED super-resolution. We will realise the technique by the addition of a depletion laser to a standard fluorescence lifetime imaging microscope and the development of routines 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.

Who will benefit from the research?

Academic Researchers

In the life sciences many bio-molecular (sub-cellular) structures are smaller than the optical limit of confocal microscopy. Our technique affords the possibility of low power non-invasive probing of biological samples below this fundamental limit. This opens up a wide range of research possibilities. The immediate beneficiaries are Biological and Life Scientists who will benefit from access to a technique that affords non-invasive sub wavelength resolution of samples and dynamical processes. Establishing a super-resolution microscopy capacity is a strategic priority for UCL (see letter of support from John Carroll). Our technique (centred in the Department of Physics & Astronomy) has the potential to provide major advantages for this effort.

Medical Researchers

A number of the collaborations have a direct impact on the development of therapies for disease, for example the collaboration with Stephen Moss (UCL Institute of Opthalmology) will investigate the mechanisms responsible for Retinal Cell Ganglion death, this in turn, will contribute to the development of more effective neuroprotective agents in glaucoma.

The future application of super-resolution to questions such as the co-localisation of antibodies to study gap junctions underlying cochlear homeostasis (UCL Ear Institute) would have a considerable benefit to the understanding of the origins of hearing loss.

Are there any beneficiaries within the commercial private sector who will benefit from the research?

The development of commercial super resolution instrumentation is an area of expanding interest. Leica Microscopy SEC 10K filings indicate overall sales in life-science microscopy of $2.5bn [1], at present Leica has a monopoly on the (conventional) STED super resolution technique. The development of an alternative and lower power super resolution imaging system should be of significant interest to Zeiss, Nikon and Olympus as well as manufacturers of precision FLIM instrumentation.

[1] UCL Business preparatory market research (2011).