Research · PhD, University of Edinburgh 2022 · Postdoc, IQS Barcelona 2022 to 2024
Singlet oxygen, from a mouse to a molecule
Photodynamic therapy kills a tumour with light, a drug and the oxygen already in the tissue. I spent five years measuring the molecule that does the killing, and the hardest part was seeing it at all.
Scroll to zoom · illustration of photodynamic therapy in general
Powers of ten
A photosensitiser is injected and collects in the tumour. Then light is aimed at it. Switch between one and two photons at every scale and watch where the action happens.
Light from outside
One-photon light lights the whole path through the skin. Two near-infrared photons only add up at the focus, which can sit inside the tumour. Near-infrared also travels further in tissue: 750 to 950 nm is the phototherapeutic window.
Skin, fat, muscle, tumour
Blood and scattering eat short wavelengths near the surface. One-photon light excites photosensitiser everywhere it reaches, healthy tissue included; the two-photon focus excites it only where the beam is squeezed.
The drug waits in the tumour
Photosensitisers accumulate more in tumour cells, over hours to days. They are inert in the dark, which is the whole point: the drug does nothing until light arrives.
A one-micrometre reach
Singlet oxygen lives about 3.5 µs in water, and in cells it acts within roughly 1 to 1.5 µm of where it is made. Wherever the photosensitiser sits, mitochondria or membranes, is what gets destroyed.
Absorb, cross, transfer
The photosensitiser absorbs, crosses to its long-lived triplet state, and on collision hands its energy to ground-state oxygen, itself a triplet. Spin is conserved, so only the triplet can do it. The oxygen leaves as singlet oxygen and oxidises the first lipid or protein it meets.
Damage where the light was
With two-photon excitation only the focal cell is damaged; its neighbours never saw a high enough photon density. That precision, plus deeper reach, is why two-photon PDT matters, and why measuring a molecule's two-photon singlet-oxygen yield matters even more.
01 · Seeing it
1270 nm photon counting
Detecting a molecule that barely glows
Singlet oxygen gives one faint infrared glow at 1270 nm as it decays. In water only about one in a million molecules emits a photon, and infrared detectors catch about 3% of those. Below, single photons arrive at a photomultiplier and build the time-resolved signal.
The rise is the photosensitiser's triplet handing over its energy; the decay is singlet oxygen's own lifetime, set by the solvent. Heavy water lets it live about twenty times longer, which is why many experiments use it, and why measuring in plain water, as cells require, is so much harder. In my postdoc in Light4Lungs I detected singlet oxygen inside bacteria with high-sensitivity photon detection, photodiodes and photomultipliers.
Simulation: emission time = triplet lifetime (2 µs, estimated for air-saturated water) + singlet-oxygen lifetime (thesis values), with 3% dark counts; lifetime fitted by maximum likelihood on the tail. The fit reads slightly long (about 3.8 µs and 71 µs against the thesis values of 3.5 and 68 µs) because the tail still carries some of the triplet rise and the dark counts; a real analysis would fit rise, decay and background together.
02 · The PhD
hollow-core photonic crystal fibre
A lab inside a fibre the width of a hair
Two-photon excitation only happens in a femtolitre focus, so in a cuvette the signal drowns in noise. My solution: fill the hollow core of a photonic crystal fibre with the photosensitiser solution, and guide the laser through it. The excitation is sustained along the whole fibre instead of one tiny spot.
How it guides. The thin glass walls of the ring of capillaries act as Fabry-Perot resonators. Away from their resonances they reflect light back into the core; at a resonance, λ = 2t·√(nglass² − ncore²)/m, light leaks out. Filling the core with water moves every resonance to about 0.55 times its wavelength in air, which is how the fibre can carry both a 470 nm probe and an 800 nm pump in water.
Why it wins. In the thesis's comparison for a 20 µm core and 15 cm length, the light-matter interaction figure is 4.18 for a 1 cm cuvette, 16.4 for a glass capillary and 14,720 for a hollow-core fibre. The fibre held about 2.8 nL per cm, around 30 nL in all: six orders of magnitude less sample than a cuvette.
Fibre drawing is schematic; the mode is the J₀² profile of a hollow waveguide. Membrane thickness is a free parameter here, not my fibre's measured value.
03 · The method
two-colour pump-probe
Counting singlet oxygen with a light switch
Direct 1270 nm detection is too weak in water inside a fibre, so I used a chemical reporter, Singlet Oxygen Sensor Green. It is dark until singlet oxygen adds across its anthracene, then it fluoresces about ten times brighter. Two lasers share the core: an 800 nm femtosecond pump excites the photosensitiser by two photons for 60 s, a 470 nm probe reads the reporter for 5 s, twenty cycles or more.
The reporter's growth rate gives the singlet oxygen made. Plotted against laser power on log-log axes, its slope must be 2 to prove a two-photon process. In the final design every photosensitiser gave a slope between 1.85 and 2.09. Divided by concentration, the rate gives the figure of merit for two-photon therapy: cross-section times singlet-oxygen yield.
The same log-log test is what the AI agent on my lab-agent page worked out for itself, unprompted.
04 · Seven designs
the engineering
From 350 mW to 10 mW, one problem at a time
| Design | Reporter / photosensitiser | Fibre | 800 nm power | What changed and what it fixed |
|---|---|---|---|---|
| 1 | 50–100 / 1–10 µM | 30 cm | 350 mW | Proof of concept. Weak signal, re-absorption, early plateau from oxygen running out. |
| 2 | 5 / 0.5 µM | 30 cm | 150–450 mW | Twenty times less reporter, more signal. First log-log proof of two photons (slope 2.03). |
| 3 | 1 / 0.05–0.5 µM | 30 cm | 150–450 mW | More photosensitisers. Found residues on the fibre walls skewing late measurements. |
| · | study | 30 cm | up to 300 mW | Photobleaching study: the reporter self-sensitises, its product bleaches, photosensitisers bleach above about 200 mW. |
| 4 | 1 / 0.05–0.1 µM | 30 cm | 50–150 mW | Light now in the core, not the cladding; both lasers in the same fundamental mode. |
| 5 | 1 / 0.05–0.1 µM | 15 cm | 50–150 mW | Shorter fibre, less micro-bending loss, cleaner spectra. |
| 6 | 1 / 0.1–1 µM | 15 cm | 50–150 mW | Probe down to 50 µW: ten times more counts, no oxygen plateau. |
| Final | 2 / 0.1–1 µM | 15 cm | 10–50 mW | Optical isolator (back-reflections were breaking the laser's mode-locking), telescopes matching both beams, autocorrelator checks. Best modes of all. |
05 · What was new
- The first quantitative measurement of two-photon photosensitised singlet oxygen in aqueous solution, in any optical system.
- The first use of an anti-resonant hollow-core fibre in photochemistry, and of the SOSG reporter ratiometrically inside a hollow-core fibre.
- Photosensitiser concentrations down to 10⁻⁸ M, two orders of magnitude below the previous fibre work, low enough to avoid aggregation: a copper phthalocyanine reported to make no singlet oxygen turned out to make plenty as a monomer.
- Two-photon cross-sections of clinical photosensitisers measured in water, many for the first time.
06 · The postdoc
Light4Lungs · IQS Barcelona
Breathing in the light
Light4Lungs, an EU Horizon 2020 FET Open project coordinated at IQS by Prof. Santi Nonell, whose singlet-oxygen probes I had used throughout my PhD, set out to treat drug-resistant lung infections with "a novel inhalable photodynamic therapy": persistent-luminescence particles delivered by aerosol, so the light source itself is breathed in. My work there was ultrasensitive photon detection, measuring singlet oxygen produced inside bacteria with photodiodes and photomultiplier tubes, and coordinating the optics, biochemistry and modelling teams.
Sources
- S. Adan Bermudez, Detection of Two-Photon Photosensitised Singlet Oxygen in Hollow-Core Photonic Crystal Fibres, PhD thesis, University of Edinburgh, 2022 (supervisor Prof. Anita C. Jones).
- G. O. S. Williams et al., ChemPhotoChem 2018: direct 1270 nm detection of singlet oxygen in a hollow-core fibre, the group's earlier work this thesis built on.
- light4lungs.eu: EU H2020 FET Open, grant 863102.