An Aqueous Scanning Thermal Microscope for nanoscale thermal biology

The ability to measure and manipulate heat is a fundamental tool in any scientist's toolkit. Biological processes, chemical reactions and even fundamental physics are all intrinsically linked to temperature. By changing temperature, biological, chemical and physical processes can be sped-up, slowed-down or even stopped. Likewise, many processes result in the generation or consumption of thermal energy so can be monitored by measuring temperature. Modern science has responded to this by developing a wide range of heaters and thermometers that employ various underlying mechanisms, including optical, electrical and chemical. However, this shouldn't lead us to believe that temperature is a 'solved' problem that no longer requires innovation. A very clear example of this is the continuing push of scientists for tools that work at ever smaller length scales. Specifically, the accurate control and measurement of temperature at the micro- and nano-scale is very difficult, with no single approach offering a perfect solution.

This project is to build an accurate nano-scale heater/thermometer microscope that can operate in cell-friendly, water based environments. The technology behind this tool means that it can measure the temperature of (or heat) a nano-sized region of a sample. In addition to this, it can be positioned at any site on a sample and make a measurement before being moved to another site to repeat the process indefinitely. The tool is completely compatible with other forms of microscopy, allowing optical, topographic and thermal measurements to be made simultaneously. This flexibility has a wide range of applications in biology but two examples that will be explored during this project are given below:
In cell biology, control of temperature gives scientists a fascinating and flexible way to monitor or change cell behavior, even the ability to induce cell death. This last point has been exploited in a highly promising new approach to cancer treatment called 'nanoparticle-mediated photothermal therapy'. In this technique, gold nanoparticles specifically designed to target cancer cells absorb a light of specific wavelength and heat up. This heat can induce the death of the cancer cells directly or release pre-loaded therapeutic drugs. However, the mechanism of heat-induced death is poorly understood, mainly because of unknown nanoparticle temperature and their uncontrolled distribution in cells. Traditional methods would require a huge number of repeat experiments, together with indirect calculations of particle temperature to answer these questions. The tool we will develop can provide answers within one simple experiment by precisely locating its heater/thermometer at carefully chosen sites on different cells one at a time and delivering an exact quantity of heat to each.

Another example is measuring the temperature of different living cells. It should be no surprise that a cell's temperature is dependent upon its metabolism and its activity in response to the surrounding environment. Traditionally, biologists have used a range of optical tools to measure the temperature in and around cells. However, interpreting these measurements is fraught with difficulty, resulting in scientific disagreement and no consensus. The thermometer we will use in this project is based on a very well understood, unambiguous way of measuring temperature. This coupled with the ability to precisely locate the measurement on different regions of a single cell will offer valuable data to this lively scientific debate.

Ultimately, our instrument will provide a simple to use and flexible tool to measure and change temperature at length scales relevant to cutting edge research at the cellular and subcellular level.

Scanning Thermal Microscopy (SThM) is a form of scanned probe microscopy that permits sample temperature to be measured and manipulated with miliKelvin resolution on the nanoscale. It is based around Atomic Force Microscopy probes that have a thermal sensor located at their tip. SThM has proved to be a powerful tool in investigating nanoscale thermal phenomena in the physical sciences. However, it is limited to operation in electrically insulating environments, such as vacuum, air or under oils. Attempting to use it under aqueous solutions results in immediate failure of the SThM probe through electrochemical corrosion, making the instrument unsuited to the vast majority of biological studies.
In this research we propose to develop and evaluate a new SThM instrument (denoted as a-SThM) capable of operating in biologically benign aqueous solutions. The approach employed is based around electronic instrumentation that blocks electrochemical corrosion whilst still permitting electrical heating and interrogation of the SThM probe. As a consequence, commercially available SThM probes (or new a-SThM probes) can be employed in the system for biological studies.

The new instrument will be of great interest to biologists as temperature is fundamental to all aspects of life, regulating many cellular functions from gene expression to cell metabolism. Despite this importance, much of our knowledge of thermal effects on cells is based on macroscopic measurements of temperature, resulting in the mechanism of temperature-induced changes at the subcellular level being poorly understood.
Another obvious beneficiary will be research into nanoparticle-mediated thermal therapy, where localised hyperthermia offers great advantages in minimising damage to surrounding healthy tissues. However, a lack of suitable tools leaves determining the real temperature of individual nanoparticle heaters unsolved and identifying the appropriate dose for effective treatments extremely difficult.

The beneficiaries of an aqueous-Scanning Thermal Microscopy (a-SThM) instrument range from members of SThM and nano-thermal biology research communities through to those researching next-generation therapies and nano-materials. In order to ensure maximum impact in these diverse areas it is essential that the capability of the a-SThM be widely publicised during the project and that duplicates and derivatives of the instrument should be adopted after the project ends. For this reason the new instrument proposed here has been planned from the ground up in such a way as to maximise its exploitation. This planning encompasses the choice of commonly available underpinning technologies, through to the existing network of the applicants and the proposed communication strategy for academics, industry and the public.

The plan for technical sustainability is based around an open-access strategy for documenting the instrumentation developed during the project, combined with the explicit use of commercially available consumables to facilitate the propagation and long-term availability of the technique. In support of this the publications and presentations generated during the project will be used to drive adoption of the technique by exemplifying the remarkable measurements it enables.

To this end, the existing network of the applicants will be exploited to ensure that SThM and AFM instrument manufacturers, together with key members of the biology and bio-materials community are given every opportunity to engage with the project. This interaction will be used to inform instrument and procedure development during the 12-month course of the work. On the wider scale, scientific publications, presentations, industrial engagement events, public demonstrations and online media such as a blog with publicly hosted videos will all be employed. It is felt that this wide range of publicity activities will maximize visibility to other researchers, industry and the public.

Further impact will stem from exploitation of the unique measurements enabled by the proposed instrument. Ultimately, it is foreseeable that the measurements enabled by the instrument in its future will lead to clear societal benefits. These are particularly evident in the area of nanoparticle-mediated thermal therapy that promises to be an effective, targeted method of selectively killing cancer cells. This field is currently severely hampered by a lack of appropriate measurement technology, such as is being developed in this proposal. By bridging this gap, a-SThM offers the potential to accelerate the identification and characterization of powerful therapies that will not only benefit patients, but also provide economic benefits through the industries that bring the treatments to market.