Photothermal imaging of biomimetic nanoparticles to investigate the real-time dynamics of transcription at the single molecule level in living cells

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences


One of the greatest challenges in biology is to be able to measure biological reactions as they happen in single cells. One important process is transcription, the reaction in which the DNA sequence of a gene is read by a polymerase to produce an RNA which is then translated to produce a protein. Transcription has been monitored indirectly in single cells through imaging of the synthesis of the firefly luciferase reporter protein in cells since it makes living cells glow and can therefore be measured using a camera that counts photons. Biological processes in living cells can also be monitored by microscopy using naturally fluorescent proteins which are genetically synthesised in the cell fused to proteins of interest. Over the past 10 years this has led to a revolution in cell biology because for the first time single biological processes such as protein movement can be watched as they happen in single cells. The Centre for Cell Imaging in Liverpool has been a leading centre in the development and application of these technologies. Using these approaches it is not generally possible to watch the movement of single molecules in cells. This would allow a new level of understanding of many biological processes. The ability to study in a single cell exactly when transcription is switched on by activating proteins (called transcription factors) is an important objective. It is becoming clear that these events may often be governed by probability and that averaging such processes or measuring them indirectly misses important information. I pioneered the development of biomimetic gold nanoparticles which can be easily coupled to proteins. Due to the small size of the nanoparticles the resulting molecules can behave in the same way as the normal protein. I will build the world's second photothermal microscope, which will be the first to be specifically designed to study living cells (with Lounis, Bordeaux, and White, Liverpool). This microscope allows the easy visualisation of gold and silver nanoparticles in optically complex environments. This has many applications in the important emerging field of nanobiotechnology. Using this new microscope we will be able to see single nanoparticles within living cells for long periods of time without any loss of signal and without damaging the cell. I will continue to develop biomimetic nanoparticles and optimise their ability to couple to functional proteins and other molecules that can be used to label biological processes (with Brust, Liverpool, and Desbat, Bordeaux). At present it would be necessary to bind proteins to the nanoparticles in the test tube outside the cell and then to introduce the resulting conjugate into the cell. This has the disadvantage that it takes time to purify the protein and has the risk that the protein may not be fully functional. I will therefore develop a new methodology for binding nanoparticles to proteins within cells (with Johnsson, Lausanne). The nanoparticles will be introduced into the cell where they can specifically couple with the protein of interest. I will use this technology to study transcription at single genes in living cells. I will use a combination of gold and silver nanoparticles which can be distinguished from each other to allow different processes to be watched at the same time. The first aim will be to use triple helix forming oligos (which form stable and specific interactions with specific double stranded DNA target sequences) to identify the position of single genes in the mammalian cell nucleus (with Jackson, Manchester, and Giovannangeli, Paris). I will then use nanoparticles to study the binding of single transcription factor molecules to the gene. I will mark the gene by introducing protein binding sites into the RNA so that the early RNA produced by transcription can be also be visualised. This will for the first time allow the processes that switch genes on to be studied at single genes.

Technical Summary

The concept of signalling pathways is being replaced by a new paradigm of regulatory networks. The understanding of these networks doesn't require only the knowledge of the interactions between the elements but also the knowledge of the dynamics of these interactions on different time scales. Some crucial elements are present at low copy numbers in a single cell and current approaches based on fluorescence imaging lack single molecule sensitivity. Advances in nanosciences offer new tools to increase biological understanding. I propose to combine two recent breakthroughs in the fields of nanoparticle detection and nanoparticle preparation to effectively reach the goal of real-time single molecule imaging in living cells. The first breakthrough was the development of single gold nanoparticle detection down to 1.8 nm by photothermal heterodyne microscopy [Boyer et al., Science, 2002]. The second breakthrough was the preparation of highly versatile protein-like nanoparticles using peptides that spontaneously form a self-assembled monolayer on gold [Levy et al., J. Am. Chem. Soc., 2004]. The peptide monolayer can be tailored to obtain a variety of bioconjugates. The research programme proposes: 1) To build a photothermal microscope specially designed for long-term imaging of single nanoparticles in living cells; 2) To develop gold and silver nanoparticle probes that will target transcription factors, mRNA and specific genes within the nucleus; 3) To visualize these probes in real time to explore the dynamics of the transcription regulatory network. The combined analysis of transcription factor binding and transcription initiation at the single molecule level will give new insights into the dynamic and stochastic processes that regulate transcription at single genes in single cells. It will also provide valuable information and tools that are relevant for gene therapy, drug design and drug delivery.


10 25 50
Description During the five years of the Fellowship, my research has focused on the design and applications of nanoparticles for imaging and sensing in Biology.

One of the outcomes of this Fellowship is a powerful technological platform which has been expanded thanks to a successful EPSRC grant: we now have two photothermal microscopes which allow the direct observation of light-absorbing nanomaterials inside live cells with single particle sensitivity and very high pointing accuracy. The microscopes are within the Liverpool Centre for Cell Imaging and are open for collaborative work.

My main scientific achievements reside in our contributions to the understanding of the interaction and fate of nanoparticles in living cells and to the understanding of the structure of molecules attached to nanoparticles. These achievements have been published in peer reviewed journals and presented at international conferences.

I have directed the research of several PhD students, three of whom have obtained their PhDs during the Fellowship and one post-doctoral research fellow. Beyond my own career development as a group leader, the Fellowship has therefore significantly contributed to the training of scientists in the area of bionanotechnology.

The Fellowship has also enabled me to conduct a highly instructive experiment about the scientific process. Attempt at challenging and correcting the scientific record were met with enormous difficulties. The eventual publication at the end of 2012 of "Stripy Nanoparticles Revisited" (an article criticizing a body of work on nanoparticles), after three years of peer review, started a controversy which attracted interest well beyond the specialists of nanoparticles because of its implications for peer review, post-publication peer review, science communication, the role of social media, etc (see links at
Exploitation Route Our developments in terms of nanoparticles engineering have been both cited and used by other groups to develop imaging/sensing nanoprobes. Our results on uptake of nanoparticles in cells participate to an ongoing scientific discussion on controlling the access of nanoparticles to the cytosol of cells. This is important for applications of nanoparticles in drug delivery as well as intracellular sensing and imaging. "Put to use by others" should also include the value of negative results: challenging published but erroneous work has value, among other reasons, because it may help focusing research efforts and funding on sound science.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Alert 13
Amount £247,156 (GBP)
Funding ID BB/L014947/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2014 
End 12/2016
Title Photothermal microscope 
Description A microscope that enables detection of absorbing nanomaterials in live cells (e.g. gold) and tissue sections. 
Type Of Material Improvements to research infrastructure 
Year Produced 2011 
Provided To Others? Yes  
Impact Improvement to our understanding of non-fluorescent nanoparticles into cells (see publications 10.1371/journal.pone.0121683, 10.1098/rsos.140454) 
Description Outreach: schools, Nuffield, Scibar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talks sparked questions and discussion about nanotechnology (Scibar, Christmas lecture). Nuffield students were enthused about scientific research. Links were established with school teachers for future activities.

Example: a member of the audience at the Scibar event contacted me again recently; he has applied and secured funding to prepare an educational resource with us. He has now produced it (about nanoparticles applications in Stem cell biology) and it will become widely accessible for teachers in the next few weeks.
Year(s) Of Engagement Activity 2009,2010,2011