C2D2 research 2a - Exploring novel multi-modal photonic techniques for live imaging of synaptic activity

FINDINGS/RESULTS
The major aims of the project were:
1. Construct a microscope that can simultaneously image a) fluorescence and b) neurotransmitter secretion and changes in synaptic plasticity via refractive index in live cells.
2. Optimise the functionalization of photonic crystals with antibodies to detect secretion from cells.
3. Measure endogenous glutamate release from cultured neurons during stimuli that mimic learning.

Excellent progress has been made in aim 1, where we have designed and constructed a novel microscope that can visualise cellular fluorescence and detect cell features due to fluctuations in refractive index (see attached document outlining preliminary data).

To address aims 2 and 3, considerable effort has been spent i) optimising the design and fabrication of silicon crystals that resonate and can detecting changes in refractive index and ii) optimising the culture of neuronal cells on photonic crystals. Issues with the fabrication process and cell culture media degrading the crystal have slowed progress.  However, we have recently fabricated resonant crystals that can detect cultured cells.  Using a simpler 'ring' resonator, we have also successfully detected antibody-antigen interactions on a crystal surface.

Thus the components of the proposed system to measure neurotransmitter release have been developed independently and through our joint PhD student, collaborative work between our labs will continue to combine these technologies to image neurotransmission and synaptic plasticity label-free and in real time.

COLLABORATIONS
The Krauss-Evans collaboration was further strengthened by Evans becoming a joint supervisor of Graham Triggs, an EPSRC PhD student whose project focuses on biosensor development.  This C2D2 project stimulated further discussions between the Krauss lab and other biologists to develop specific biosensors.  A collaboration was hence formed between the Krauss lab and Mark Coles (HYMS, CII) for detecting chemokines.   Krauss, Evans and Coles and Steve Johnston (Electronics) also submitted an application for further C2D2 funding for a discipline-hopping intern to develop novel biosensors.

STAFF
Matthias Fischer was recruited in June 2013 to work on the project (focusing on the development of the fluorescence/resonance microscope) and has now been employed on a 3 yr contract in the Krauss lab.
A PhD student,  Graham Triggs (2012-2015, jointly supervised by Krauss and Evans), contributed to the project by developing the photonic crystal technology.

COMMERCIALISATION/TRANSLATION

The biosensors we have designed could be amenable to commercialisation but have not yet reached a sufficiently advanced stage of development.

APPLICATIONS SUBMITTED
Further funding to study mechanisms of synaptic plasticity will be sought by Evans and Krauss once more robust preliminary data in neurons has been obtained.

On the basis of the biosensor development conducted in our project, Krauss, Coles and Johnson submitted a successful application for a Tools and Resources grant from the BBSRC.
Label-free, Real-time, Spatial-resolution (LRS) immunoassay: 2D mapping of extracellular signalling molecules
PI T. Krauss, Co-I Steve Johnson, Mark Coles
BBSRC Tools and Resources Development Fund (BB/L018160/1)
£181,954.14
01/04/2014 - 01/08/2016

ARTICLES SUBMITTED
Two articles are planned in the short term:
i) A photonics study demonstrating how the grating configuration of silicon crystals determines the resolution of detection.
ii) A microscopy study showing the first demonstration of simultaneous fluorescence and resonance measurements from cells.