[Academic] Seminar this afternoon at 2pm JA3.27

[David McKee]

Reminder for today's seminar. Highly topical subject area!

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*2pm Friday 10^th Oct JA3.27*

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*Robert Pal, *

*University of Durham.*

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*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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