In situ quantification of metabolic function using fluorescence lifetime imaging

Lead Research Organisation: University College London
Department Name: Cell and Developmental Biology

Abstract

Metabolism is the set of chemical transformations taking place inside a cell that keep an organism alive, such as breaking of bonds between atoms in nutrient molecules to provide energy or building complex molecules from simpler units for growth. These processes are fundamental to life, and their failure is associated with diseases such as cancer, diabetes and neurodegeneration. To understand the role of metabolism in particular processes, we require tools that provide quantitative measures of metabolic properties in living tissues. However, current techniques are limited, leaving gaps in our knowledge. We propose to develop new tools that will provide accurate and reliable quantification of metabolic properties, using the technique of fluorescence lifetime imaging (FLIM).
In FLIM, a laser is scanned across a living tissue to excite fluorescent molecules added into the sample or naturally present. The laser beam is a train of up to 80 million light pulses every second. When a particle of light (a photon) is absorbed by a fluorescent molecule, FLIM measures the time taken for the light to be re-emitted. The average time taken for this to occur, the fluorescence lifetime, is extremely sensitive to the immediate environment of the molecule. The reactions of metabolism can, in principle, be studied in living systems by choosing appropriate fluorescent probes and measuring these delay times.
Pooling the expertise of research groups working in fast laser spectroscopy, photophysics, metabolism and fluorescence imaging of live cells, the objective of this proposal is to create methods to measure three fundamental metabolic properties using FLIM.
First, we will study two fluorescent molecules that are naturally present in all tissues; NADH and NADPH. The fluorescence lifetime of these molecules changes as disease progresses, and instruments have been designed for diagnosis based on this observation. However, their clinical use has been limited by a lack of understanding of the biochemical changes that cause this variation. We will determine which metabolic pathways dictate the fluorescence lifetime of NADH and NADPH by activating and inhibiting particular pathways in live cells and observing how the lifetimes of these molecules change. This will lay foundations for the application of FLIM to diagnose disease, benefitting patients by obviating the need for invasive biopsy.
Second, we propose a method to measure the voltage produced by the "cell powerhouses", the mitochondria, using FLIM to observe the behaviour of a fluorescent dye (TMRM) when it is taken up by cells. Mitochondria are responsible for releasing the energy stored in carbohydrates, fats and proteins. Akin to a battery, the voltage across the mitochondrial membrane governs this process, but it also determines cell death and the production of free radicals, implicated in ageing. The membrane potential is central to mitochondrial function and the ability to make accurate measurements of it in live tissues will be key to understanding its role in disease.
Finally, we will develop a method for measuring levels of ATP, regarded as life's "universal energy currency", using FLIM. ATP is produced by the mitochondria and powers the majority of cellular processes, from DNA production to cell motion. To measure this quantity in live tissues, we will introduce DNA coding for two fluorescent proteins attached to an ATP binding site. The fluorescent proteins are brought into proximity when ATP attaches, allowing energy to be transferred between them. FLIM can be used to measure how many proteins are undergoing this process, known as Förster resonance energy transfer (FRET). The ATP concentration can then be calculated from this fraction. As ATP is so central to life's chemistry, creating a method to accurately measure its production and consumption inside living samples will make a major contribution to the ultimate aim of finding a cure for some of the world's most severe diseases.

Technical Summary

Correct metabolic function is pivotal to cell health, while defects in metabolism are implicated in a wide range of major diseases. In order to understand the metabolism of specific tissues, the availability of reliable, unambiguous and accurate tools that report metabolic state in live samples is essential. Over the past 20 years, this role has been played by fluorescence imaging, providing important information about the metabolic state of living cells. However, the standard measurements of fluorescence intensity in current use yield, at best, only semi-quantitative measurements. We therefore propose to develop novel approaches, employing fluorescence lifetime imaging microscopy (FLIM) to make accurate quantifications of three variables fundamental to cell metabolism, extending the range of approaches available to study the metabolic state of live biological models. FLIM measures the fluorescence lifetime of a target fluorophore at each pixel of an image. As the lifetime of a probe is highly sensitive to its immediate environment, FLIM is potentially a rich source of biochemical data. However, to clarify and optimise the information content of these measurements, it is necessary to understand the relationship between probe photophysics and the underlying biochemistry and physiology. Our proposal therefore combines two research laboratories with expertise in molecular physics and ultrafast spectroscopy, and in metabolism and fluorescence imaging of live samples, in order to (A) develop FLIM of NAD(P)H as a label-free technique for monitoring redox state in complex tissues, (B) establish novel protocols for the absolute measurement of mitochondrial membrane potentials inside live cells and (C) accurately quantify ATP concentrations in subcompartments of intact tissue using genetically-encoded FRET probes. Such techniques will find widespread use in quantifying metabolic function in situ, placing FLIM at the forefront of investigations into health and disease.

Planned Impact

Metabolism plays a major role in health and disease. This project aims to provide biomedical researchers with new tools to study metabolic properties of fundamental importance - NAD(P)H levels, the mitochondrial membrane potential and ATP production and consumption - by the application of fluorescence lifetime imaging microscopy (FLIM). For this reason, the primary beneficiary of this proposal is clearly the wider academic community, and these links are outlined in the Academic Beneficiaries section. However, there are a number of other non-academic areas in which this proposal will provide impact.
Firstly, a number of systems are in development for high-throughput screening of live cells using fluorescence lifetime measurements. Our protocols will be scaleable to these systems, allowing their use in the pharmaceutical industry for large-scale assays of treatments developed to target metabolism. As altered metabolic states in pathophysiology are so ubiquitous, the application of these techniques for drug discovery would have a clear impact on economic wealth creation. This is particularly true of our approach to quantify ATP concentrations using FRET. The acceptor rise amplitude technique that we will apply in our protocols will allow determination of the concentration of a target molecule with unprecedented accuracy. Similar fluorescent protein based FRET probes are now routinely developed for a given target of interest. As such, proving the principle of acceptor rise amplitude determination in the high-throughput measurement of ATP concentrations will open the door for large-scale yet accurate determination of concentrations of a desired molecular species in live biological samples.
Secondly, as our proposal crucially depends on the collaboration between biological and physical science laboratories, it represents a clear demonstration of the increasing need for interdisciplinarity in modern scientific research. Thus, given proper engagement, the public understanding of science will be increased by using our research as a prime example of the context in which 21st century biology is beginning to take place. In particular, our work will demonstrate to students that the borders between disciplines introduced at school cease to exist at the cutting edge, so those considering a career in science need not "give up" their interest in two of the three core subjects to pursue the other. The early exposure to collaboration between disciplines will inform the paths of these students through higher education. This will increase the likelihood that the next generation of scientists will be produced with a wide knowledge and skills range, having a positive impact on science in the United Kingdom and thus the economic prosperity of the nation. Such impact will also be felt locally and in the shorter term, with the junior postdoctoral researcher involved in this project gaining experience in a diverse array of experimental approaches in both the life and physical sciences, priming him for a career at the interface between disciplines.
Finally, there is obvious potential for the techniques developed in this proposal to increase wellbeing. The new tools proposed in this work will open up avenues for the fundamental understanding of metabolism in health and disease. In particular, their ability to quantitatively probe the function of mitochondria in situ will enhance the understanding of ageing in specific tissues, as these organelles are responsible for the majority of free radical species produced in the cell. With the nation's rapidly ageing population set to be a significant factor to social welfare in the coming decades, an increased understanding of the biology of ageing and neurodegenerative disease, facilitated by our techniques, will have a huge impact on the quality of life of the nation.
The means by which we will facilitate impact to potential beneficiaries are discussed in the attached Pathways to Impact document.

Publications

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Blacker TS (2019) Polarized Two-Photon Absorption and Heterogeneous Fluorescence Dynamics in NAD(P)H. in The journal of physical chemistry. B

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Blacker TS (2019) Metabolic Profiling of Live Cancer Tissues Using NAD(P)H Fluorescence Lifetime Imaging. in Methods in molecular biology (Clifton, N.J.)

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Blacker TS (2016) Investigating mitochondrial redox state using NADH and NADPH autofluorescence. in Free radical biology & medicine

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Blacker TS (2017) Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy. in The journal of physical chemistry. C, Nanomaterials and interfaces

 
Description Our proposal combined two research laboratories with expertise in molecular physics and ultrafast spectroscopy, and in metabolism and fluorescence imaging of live samples, in order to

(A) develop fluorescence lifetime imaging of NAD(P)H as a label-free technique for monitoring metabolic state in complex tissues

(B) establish protocols for the absolute measurement of mitochondrial membrane potentials inside live cells

(C) accurately quantify ATP concentrations in intact tissue using genetically-encoded FRET probes. Such techniques will find widespread use in quantifying metabolic function in situ, placing FLIM at the forefront of investigations into health and disease.
Exploitation Route the use of FLIM to differentiate between NADH and NADPH has proven invaluable and novel approach to explore cell metabolism at the level of the single cell. This has been applied in a number of collaborations leading to multiple high impact publications. techniques and interpretations, including development of a mathematical model are all in the public domain to be freely used by others.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046109/
 
Description response mode grant
Amount £574,160 (GBP)
Funding ID BB/P018726/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description collaboration with Prof Christof Maack 
Organisation University Hospital Saarland (UKS)
Country Germany 
Sector Hospitals 
PI Contribution We applied advanced FLIM based imaging to address metabolic changes in the heart associated with altered transhydrogenase activity.
Collaborator Contribution We were approached by the collaborating group to help interpret changes in metabolism associated with altered outcomes in cardiac ischaemia attributable to altered expression of the mitochondrial transhydrogenase
Impact Reversal of Mitochondrial Transhydrogenase Causes Oxidative Stress in Heart Failure. Nickel AG, von Hardenberg A, Hohl M, Löffler JR, Kohlhaas M, Becker J, Reil JC, Kazakov A, Bonnekoh J, Stadelmaier M, Puhl SL, Wagner M, Bogeski I, Cortassa S, Kappl R, Pasieka B, Lafontaine M, Lancaster CR, Blacker TS, Hall AR, Duchen MR, Kästner L, Lipp P, Zeller T, Müller C, Knopp A, Laufs U, Böhm M, Hoth M, Maack C. Cell Metab. 2015 Sep 1;22(3):472-84.
Start Year 2014
 
Description collaboration with Prof Rosario Rizzuto, University of Padova, Italy 
Organisation University of Padova
Country Italy 
Sector Academic/University 
PI Contribution We used the advanced FLIM based imaging techniques to characterise metabolic changes associated with cancer phenotype and altered calcium signaling,
Collaborator Contribution Our partners are engaged in cancer cell biology and approached us for help in characterising and interpreting metabolic changes in cells associated with neoplastic transformation.
Impact The collaboration led to a publication: The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1a. Tosatto A, Sommaggio R, Kummerow C, Bentham RB, Blacker TS, Berecz T, Duchen MR, Rosato A, Bogeski I, Szabadkai G, Rizzuto R, Mammucari C. EMBO Mol Med. 2016 May 2;8(5):569-85. The collaboration is multidisciplinary - Bentham is involved in mathematical modelling, the Italian lab are involved in cancer cell biology, our contribution was in advanced imaging microscopy.
Start Year 2015
 
Description October 2015, Invited Speaker 9th Workshop on TCSPC in Biomedical Sciences, Bethesda, USA 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk at an international meeting related to applications of fluorescence lifetime imaging
Year(s) Of Engagement Activity 2015
 
Description 1st and 2nd Workshops on Advanced Time-Resolved Imaging Techniques, Prague, Czech Republic 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact talk about applications of multi photon lifetime imaging in interpretations of cell metabolism.
Year(s) Of Engagement Activity 2015,2016
 
Description Experimental workshops on time-resolved fluorescence, In2Science outreach programme, University College London, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact talk for school children from disadvantaged backgrounds to introduce them to the potential of science
Year(s) Of Engagement Activity 2017,2018
 
Description June 2018, Invited Speaker "Fluorescence lifetime imaging of NADH and NADPH", 1st UK Redox Biology Discussion Forum, Kings College London, UK 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Study participants or study members
Results and Impact Workshop on approaches to study of redox biology for mixed group of graduate students, Industrial partners and academic staff
Year(s) Of Engagement Activity 2018
 
Description Talk to a general audience at Milan Expo 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact this will happen in a few weeks' time.

this will happen in a few weeks' time - this entry will be updated after the event.
Year(s) Of Engagement Activity 2015
URL http://www.padiglioneitaliaexpo2015.com/en/appointments/2015-09-19/meetings-exhibitions/good-food-an...
 
Description Talks to courses in advanced imaging techniques 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Both myself and the post doctoral research fellow involved in teh work have given several talks to graduate and professional groups in advanced imaging techniques and in the use and interpretation of FLIM
Year(s) Of Engagement Activity 2014,2015,2016,2017
 
Description school visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I have given several talks to schools 6th form science societies or to 6th formers who attend a course run at UCL. We have also had visits from children ranging from 10 -18 years of age to see demonstrations of microscopy and to talk about energy and mitochondria.

I was approached by students who wanted to come to the lab, and we now routinely take 6th formers into the lab for Nuffield placement projects.
Year(s) Of Engagement Activity 2013,2014,2015