International Collaboration in Chemistry: Spatially resolved measurements of cellular releasates

Lead Research Organisation: University of Dundee
Department Name: Electronic Engineering and Physics

Abstract

Our understanding of the cell and of cellular interactions is greatly advanced by the development of high-resolutionimaging techniques, from bright-field optical and confocal fluorescence microscopy to electron microscopy and scanningprobe microscopy. Optical microscopy provided the first visualization of a single cell and the organization of multiple cellsto form complex cellular networks we call tissues. In combination with immunostaining, electron microscopy offered keyinsight into the localization of proteins and the organization of subcellular architectures with unprecedented spatialresolution. Fluorescence microscopy has provided striking visualizations of the dynamic environment inside a cell, suchas protein trafficking visualized using green fluorescent proteins.Despite these advances, the visualization of cellular releasates - chemicals released to initiate cell signalling response -has been much more challenging. For certain small molecules, such as calcium and zinc, it is possible to usefluorescence indicators to monitor their spatiotemporal changes. For most other cellular releasates, such as cytokines,metabolites, and peptides, it has been much more difficult to map from where and when they are released. Yet, the abilityto follow the such release pattens is critical in deciphering the complex chemical communications between cells. In thenervous system, for example, the pattern of neurotransmitters and peptide neuromodulators released directly dictatespatterns of activity of the entire network. In the immune system, formation of the immunological synapse results inclustering of cytokine receptors and causes T cells to secrete their vesicular contents..Several techniques have been developed towards addressing this need to monitor cellular releasates. For small electroactivemolecules, Wightman and co-workers have pioneered the use of microelectrodes for the electrochemical detectionof quantal releases from cells, which has been applied towards understanding drug addiction and the brain rewardsystem. Sweedler and co-workers have demonstrated the use of single-bead solid-phase extraction and massspectrometry for measuring various secreted peptides, and Kennedy and co-workers have studied the secretion fromsingle islets of Langerhans using electrophoresis-based immunoassay. Here, we propose to develop a newmethodology, based on holographic optical trapping and ultrasensitive single-molecule bead-based assay, for monitoringthe spatiotemporal dynamics of cellular releasates. We believe this method will offer significant improvements in spatialresolution and sensitivity over current techniques.This project is joint with Prof. Daniel Chiu's group at the University of Washington in Seattle. The Chiu group are leadingexperts in microfluidic design with particular applications in biosensing. The two groups will combine their microfluidicsand optical knowledge to try and create new techniques and devices for biochemical sensing.
 
Description The key findings in this grant were focused around the development of approaches to investigate cellular processes within microfluidic systems and in optical trapping geometries. Beyond this optical tweezers control mechanisms and work moving towards new forms of detection of chemical release were developed.



The specific key outcomes were as follows:



1. We demonstrated that optical manipulation and a new form of microfluidics, called 'rails and anchors', which moves away from conventional microfluidic 'channels' could be used for droplet manipulation and the control over chemical reactions within droplets. A future goal is to develop this further to look at cells contained within the droplets.



2. We explored the binding properties of T cells under flow, and showed that a specific kind of molecule, called an integrin, leads to adhesion strengthening under such conditions. This behaviour is a key part of the immune system response, and the quantification of such results will lead to a better understanding of cellular mechanics. We are continuing to explore this process using optical tweezers and microfluidic techniques.



3. We have shown that cell chemotaxis (the migration of cells in chemical gradients) can be studied in confined microfluidic geometries, and in doing so this significantly simplifies the analysis of the cell behaviour. Specifically we now have preliminary results showing motion of the cells through the generation of blebs in advance of the production of actin at the bleb site.



4. We have developed aerosol lasers that can be trapped and excited using optical tweezers. These lasers are only a few millions of a metre in diameter and offer new possibilities for sensing of aerosol behaviour. We now also have unpublished data that shows that cells can be trapped within such droplets and we can produce a laser from the chemicals that they release opening up a whole new area of study for the sensitive measurement of cellular chemical release.



5. We have developed techniques that allow the study of ultrasound cavitation in a more controlled way than is currently possible, by combining lasers with ultrasound - this is of interest in developing ways to treat tumours with ultrasound, and also for new methods of drug delivery.



As a package of results these are very much waypoints on the journey to understand chemical release from cells, and improvements in the detection of the small volumes involved. Our work will take all of the above forward in future as we move to single molecule measurements in new grants won on the back of the results above, and to explore further mechanobiological processes within cells using optical tweezers techniques.
Exploitation Route The major exploitation routes of the work carried out in this project are clearly interdisciplinary and offer great promise for better understanding cellular processes in future.



The major non-academic context for this work is in the medical arena - the work on laser induced ultrasound might pave the way for new types of medical treatment and drug delivery, but this is still some way off. The work on cell adhesion could (in the very long term) lead to better understanding of how some diseases modify the adhesion properties of cells and hence could lead to treatments downstream.



The other possible avenue is that the new microfluidic techniques developed coupled with our work on aerosols could be exploited to form new sensing technology, a sort of microfluidics for airborne particles. This has great potential in environmental sensing applications - and with more development could be ready for commercial exploitation.
The work we have carried out can be put to use in a range of areas - the ultrasound work is likely to lead to a better understanding of how cavitation works, and how it can be controlled to make it a robust and safe technique with which to try and kill tumours, and locally deliver drugs to an area within the body. The work has led to the major fellowship award to one of those working on the project, and the goal is to apply this basic science to controllably deliver ultrasound therapies into the brain. The work will be carried out at Ninewells hospital in Dundee, and the goal is real translational research.



Additionally the work looking at cell chemotaxis and new microfluidic techniques will be applied to interdisciplinary research within life sciences looking at range of cell migration studies, trying to elucidate better the processes that occur as a cell travels in a chemical gradient - very fundamental work on model organisms.



The optical tweezers work will continue (it has not quite come to fruition just yet) to explore the properties of cells that bind underflow, and again this work will be carried out in collaboration with life scientists - the ideas developed have also led to new applications in developing magnetic tweezers and are also part of the work which led to the major award of an EU ITN programmes, in which we will extend these techniques to single molecule studies within cells.



Finally, the aerosol laser work is going to lead to new types of sensors for the study of chemical release form cells - by placing the cells within an optical cavity (formed by the aerosol) we open up new ultra-sensitive detection systems, whereby the output of the laser will be altered by the changing chemical environment produced by the cell.



The major exploitation routes of the work carried out in this project are clearly interdisciplinary and offer great promise for better understanding cellular processes in future.
Sectors Environment

Healthcare

Other
 
Description Collaboration with Koc University, group of Prof. Alper Kiraz 
Organisation Koc University
Country Turkey 
Sector Academic/University 
PI Contribution This grant allowed new work to be established with the group on Prof. Alper Kiraz on droplet lasers. The work between our groups led to an experiment and publication being carried out in Koc on droplet lasers, this subsequently led to a collaborative visit (funded through a COST action) for one of my group and work on as yet unpublished experiments on this area.
Start Year 2013
 
Description Kinect Controlled Optical Tweezers 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Work was on the development of a Kinect controlled holographic optical tweezers system.

Led to students coming to the University to undertake advanced higher projects.
Year(s) Of Engagement Activity 2012
URL http://www.bbc.co.uk/programmes/p0104kvj