[Postgrad] FW: Seminar

[Neil Hunt]

Dear All,

Please see below details of a BCP/Physics and Life Sciences seminar that you may be interested in. All welcome.

Best wishes,
Neil


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2pm Friday 10th Oct JA3.27

Robert Pal,
University of Durham.

In the pursuit of higher resolution in optical microscopy

Abstract :
The optical probes and cellular stains commonly used in microscopy are usually fluorescent organic molecules or recombinant proteins which have been used in many areas of cellular biology leading to an enhanced understanding of cellular processes and molecular interactions. However, many of these dyes have inherent drawbacks, such as issues associated with their toxicity, photostability and selectivity.

Over the past few years, emissive lanthanide complexes have been shown to be alternative robust and bright cellular stains. These probes not only stain selected cellular organelles in a wide variety of cell lines, but also possess long lifetimes allowing 'autofluorescence free' time-gated detection to be achieved without perturbation of cellular homeostasis.[1]

Recent years show the emergence of novel optical microscopy techniques to surpass the optical diffraction barrier and visualize the 'living' cell in higher resolution. Governed by Abbe's law, the highest achievable spatial resolution is dictated by the wavelength of excitation light d ~ Lambda(exc.)/2. The invention of confocal microscopy paved the way to the development of new optical (hardware) and software based super-resolution methodologies, such as SIM or STED. Since these techniques are limited by their well-known experimental drawbacks,[2] it is possible to improve lateral resolution using UV light as illumination source (Kohler 1904) with bright non-disruptive molecular probes.

We seek to develop Phase Modulation Nanoscopy (PhMoNa) a novel super-resolution technique using spatially modulated illumination intensity, capable of improving experimental resolution in both lateral and axial domain by a factor of 2. The work initially uses functionalised Ln(III) complexes as probes, synthesized in tandem to instrumental development. The advantageous properties of the lanthanide based probes have been further exploited in Durham in recent years, allowing high resolution visualization (<130 nm at 355 nm excitation) of selected cellular organelles in long term live cell experiments, whilst reporting on the micro-chemical environment. Owing to their beneficial photophysical, brightness and cellular accumulation properties, UV exposure and photo-bleaching were minimized.[3]

The instrumental development involves modification of an existing confocal (LSCM) system integrating a custom spatially (sinusoidal phase) modulated laser to achieve superior resolution of cellular substructures with an 8 fold reduced voxel size. Another important advantage to emphasize is that this approach (PhMoNa) promises to be a facile LSCM based experimental set-up that can be safely used for live-cell imaging. It will be one of the few applicable nanoscopy techniques employing a compact white laser sources for excitation. This is of key importance as the application of white laser sources promotes PhMoNa to be utilized with any currently commercially available cellular stain at any given excitation wavelength. Thus, we seek to develop an attractive alternative instrumental technique to be used by the broad imaging community.

1          C.P. Montgomery, B.S. Murray, E.J. New, R. Pal and D. Parker, Acc. Chem. Res., 2009, 42, 925
2          L. Schermelleh, R. Heintzmann and H. Leonhardt, J.Cell. Biol., 2010, 190, 165
3          J.W. Walton, A. Bourdolle, S.J. Butler, R. Pal and D. Parker, Chem. Comm., 2013, 49, 1600


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