Circularly Polarised Luminescence Laser Scanning Confocal Microscopy

Marginal organelle specific intra-cellular temperature changes often signal the early onset of life-altering diseases, such as cancer or acute mitochondrial disorder. These currently cannot be monitored and there is also no optical microscopy technique that enables the bio-imaging community to study chiral molecular interactions based on chiroptical (chiral optical) activity. With our unique expertise, we seek to address this by combining lanthanide coordination chemistry and a versatile new instrumental design. Once fully developed and validated it could open new horizons towards disease progression studies.

Temperature variations occur in cell division, gene expression, enzymatic reactions and natural cell metabolism. They may also signal the onset of pathological states and dysfunctions. Cancer tissue typically has a higher temperature (T) than surrounding healthy tissue, associated with higher cellular metabolic rates. The accurate monitoring of intracellular temperature may enable a better understanding of complex cellular events and aid the detection of diseases at the cellular level, allowing new methods of early diagnosis and therapy to be developed. Temperature monitoring is also of fundamental relevance in thermal therapies, such as hyperthermia and thermal ablation, explored as minimally invasive alternatives to surgical procedures.
The proposed project will harness the phenomenon of Circularly Polarised Luminescence (CPL), where different enantiomers of the same chemical entity produce different handedness (left or right) of light. Luminescent lanthanide complexes have been shown to possess a remarkably unique photophysical property, where the ratio of their left- and right-handed luminescence is extremely sensitive to the temperature of their surroundings. Therefore, these could be exploited as high precision intracellular temperature probes.

Our newly developed all solid-state CPL spectrometer is a paradigm shift in CPL spectroscopy, and due to its small size and versatility, it enables truly widespread application. Adapting this technique into a high spatial (optical precision) and temporal (acquisition speed) resolution microscope setup is straightforward due to our expertise and track record in instrument development. Therefore, we propose constructing and validating the world's first Confocal Laser Scanning CPL Microscope (CPL-LSCM). We are fully aware that deep tissue imaging using conventional optical microscopy is challenging. Hence, we also plan to incorporate low energy, biologically safe Near Infra-Red (NIR) multiphoton activation (MP) to increase the observable depth of tissue. It will enable unprecedented live-cell enantioselective chiroptical microscopy and provides immense scientific value, opening new horizons for studying and tracking emissive chiral molecules, both endogenous and engineered bio-probes.

With our pioneering work in rapid CPL spectroscopy it is within our grasp to achieve. Our proposal has great potential beyond the benefits associated with the wide and diverse multidisciplinary scientific community. It could also generate immense commercial interest, initiate a new chapter in modern-day live-cell optical microscopy, and shed light on the previously unexplored biochemical processes that fundamentally underpin life and life-threatening disease progressions.
Our overarching aim is to establish it as the go to 'research tool' in helping answer fundamental questions, such as why life has been founded on one particular (L) enantiomer of its chiral amino acid building blocks but needs the opposite (D) enantiomer of glucose. It can also expand our understanding of complex bio-molecules such as enzymes and elementary biochemical process such cell division, unearthing the explicit role and driving force of chirality in them.

The key innovation is to provide the multidisciplinary bio-imaging research community with a novel versatile research tool for chiroptical enantioselective imaging and fill the void in live-cell intracellular thermometry. To achieve this, we have set out to accomplish the following work packages:

1) Adaptation of our existing CPL instruments to facilitate time-resolved multiphoton CPL spectroscopy to study compounds synthesised in WP3. We have advanced our instrument build to a stage where a simple 'plug and play' approach will yield the upgrade within a few weeks after sourcing the required components.

2) Construction and validation of the world's first CPL laser scanning confocal microscope (CPL-LSCM) to facilitate enantioselective chiroptical imaging using single and multiphoton excitation. Due to the small size and versatility of our newly developed all solid-state CPL spectrometer, adaptation of this technique into a microscopical setup is straightforward. We also plan to incorporate multiphoton activation to increase the observable depth of tissue.

3) Synthesis of bright CPL active lanthanide complexes as live-cell colourimetric thermometry probes. Over the past decade, we have led the way in the development of responsive optical bio-imaging probes based on lanthanide luminescence and demonstrated that several of them undergo thermally induced racemisation or on/off switching of emission, rendering them to be suitable candidates for thermometric assays. We will also generate solid-state encapsulated (polymer-based) calibration standards (test slides) to be used with the CPL-LSCM constructed.

This field of research will allow the broad life sciences community to harness the unexploited benefits of CPL spectroscopy and microscopy. This will initiate a new chapter in modern-day live-cell optical microscopy and shed light on the previously unexplored biochemical processes that fundamentally underpin life and life-threatening disease progression.