Thermally Activated Delayed Fluorescence: A New Tool for Time-Resolved Imaging

Abstract

Time-resolved fluorescence imaging is limited by cellular autofluorescence, which shares similar lifetimes to many synthetic fluorophores and degrades signal-to-noise ratio; heavy-metal phosphors can extend lifetimes enough to separate signals via time-gating but carry cytotoxicity and cost drawbacks. Thermally activated delayed fluorescence (TADF) materials achieve comparable microsecond-to-millisecond lifetimes using purely organic molecules, via a small singlet-triplet energy gap that enables reverse intersystem crossing. Key challenges remain, including oxygen quenching, poor water solubility, and broad emission spectra, but strategies like nanoparticle encapsulation and multiple-resonance TADF design are addressing these issues, with proof-of-concept studies already showing successful, low-toxicity cellular imaging. TADF materials are therefore emerging as a promising tool for time-gated and multi-channel imaging in living cells.

Issues With Time-Resolved Fluorescence Imaging

Fluorescence spectroscopy is an indispensable tool in biological research used for imaging, tracking, and marking structures and molecules within living cells. It is a fast, non-invasive technique, suitable for studying both static and dynamic processes. These techniques rely on exciting a target fluorophore and detecting its emitted light, and time-resolved imaging techniques are often characterized by the molecule's fluorescence lifetime.¹

In practice, this detection can be complicated by the intrinsic fluorescence of the cell itself. This phenomenon is known as autofluorescence.

Autofluorescence can be observed from a range of biomolecules. For example, two of the most significant contributors are the redox cofactors NADH and NADPH, which are essential participants in cellular metabolism. Both absorb near 340 nm with emission peaking around 460 nm.² Their fluorescence lifetimes fall in the nanosecond regime (approximately 1–4 ns³ depending on which protein they bind to). This emission overlaps with many common synthetic fluorophores, introducing a background signal that reduces the signal-to-noise ratio and obscures weak target signals.

Several strategies exist to separate target fluorescence from this background. In flow cytometry, non-specific antibodies with the same fluorescent label (isotypes) can establish an autofluorescence baseline, which can be subtracted from the labeled fluorescence during analysis. Fluorescence lifetime imaging microscopy (FLIM) exploits the fact that bound and free forms of fluorophores have different lifetimes, so individual contributions can be unmixed using multi-exponential decay fitting. However, this mathematical approach becomes unreliable when the target fluorophore has a lifetime similar to that of the background (for example, a probe emitting at 5 ns against cellular autofluorescence at 2–4 ns).

An alternative approach to lifetime measurements is to use gating detection methods, such as in time-gated luminescence microscopy or time-resolved luminescence imaging (TRLI). Here, a delay is introduced between the excitation pulse and the detection window, so that the short-lived autofluorescence has decayed before measurement begins (see Figure 1). However, the effectiveness of this approach scales with the difference in lifetime between the background and the probe. The greater this difference, the cleaner the measurement.

Figure 1. Luminescence measurements over time, and measurement timeline for time-gated fluorescence spectroscopy.

This is where traditional fluorescent materials start to struggle, as they will have lifetimes on the same scale as the autofluorescence. There is therefore a need to find long-lifetime, biocompatible fluorophores.

Phosphorescent materials containing heavy metals such as iridium and platinum⁴ can achieve the microsecond to millisecond lifetimes required for effective time-gating. However, metal-containing phosphors raise concerns about cytotoxicity and present barriers to cellular uptake, since cell membranes actively limit the passage of metallic or charged species.^5,6 The scarcity and cost of platinum-group metals are also practical limitations.

The ideal solution to these issues would be purely organic material capable of the same extended emission lifetimes as metal phosphors.

Thermally Activated Delayed Fluorescence

Thermally activated delayed fluorescence (TADF) offers a way to achieve those extended emission lifetimes in purely organic molecules. The mechanism relies on the energy landscape between the lowest excited singlet state (S₁) and the lowest excited triplet state (T₁) of the fluorophore (see Figure 2). In conventional fluorescent molecules, these two states are separated by a relatively large energy gap, and intersystem crossing (ISC) from S₁ to T₁ can populate the triplet state. As fluorescence cannot occur for T₁ → S₁ , this energy will be non-radiatively lost, reducing the efficiency of the fluorophore.

Figure 2. Jablonski diagrams showing emission mechanisms in traditional fluorophores and TADF materials.

TADF molecules are engineered with a small singlet–triplet energy gap (ΔE_ST), typically less than 0.2 eV. With such a small gap, the ambient thermal energy can drive reverse intersystem crossing (rISC) from T₁ back to S₁, from which radiative decay produces the delayed fluorescence emission.

Because of this, TADF materials have a two-component emission: a prompt fluorescence component with a nanosecond lifetime arising from direct S₁ decay, and a delayed fluorescence component spanning microseconds to milliseconds arising from the ISC–rISC cycling.

It is this delayed component that is valuable for time-gated imaging. The cycling also allows TADF molecules to harvest triplet excitons that would otherwise be lost, giving a theoretical internal quantum efficiency (IQE) approaching 100%. Utilizing both prompt fluorescence and the delayed fluorescence increases its photoluminescent quantum yield (PLQY).

Figure 3. Image and steady-state photoluminescence spectrum of 4CzIPN. Data captured using ⁠an Ossila Spectrofluorometer. Image adapted here with permission.

As well as having strong, well-defined emission (see Figure 3), TADF materials offer practical benefits that make them well-suited to biomedical applications. Because they contain no heavy metals, they avoid the cytotoxicity and biocompatibility concerns associated with metal-based phosphors. Their material properties such as their emission wavelength, PLQY, and lifetime can all be tuned by adjusting the donor and acceptor components. TADF compounds can also be produced at lower cost than rare-metal-containing alternatives and deposited using relatively simple solution-processing techniques.

Current Issues with TADF

Quenching and Solubility

One practical barrier to using TADF materials in biological systems is that they can be easily quenched by surrounding oxygen. The long-lived triplet states that allow delayed fluorescence can be readily deactivated through energy transfer to nearby oxygen. However, encapsulating TADF molecules in nanoparticles or matrices can substantially reduce oxygen contact, preserving the optical properties.

Unfortunately, this can introduce another issue. Some of the polymers that can resist oxygen quenching are often not soluble in water, which is an issue for most biological samples. However, this issue can be overcome by clever encapsulation and nanoparticle design.

In one example of this, Z. Zhu et al. showed that classic TADF materials including 4CzIPN, NAI-DPAC, and BTZ-DMAC combined with the peptide F6G6(rR)₃R₂ self-assemble into nanoparticles which dissolve in aqueous solution and can penetrate the cell membrane easily.⁷

Reducing Spectral Broadening

Conventional TADF molecules often exhibit broad, featureless emission spectra arising from their charge-transfer excited states. This poor color purity limits their suitability for multi-channel (or multiplex) imaging, which is the simultaneous labeling of multiple cellular targets with spectrally distinct probes.

However, over the past 5 years, multiple-resonance TADF (MR-TADF) molecules have presented a promising solution to this issue. Their specific molecular structure produces a much narrower emission bandwidth, while retaining the small ΔE_ST and high PLQY of conventional TADF. This narrower emission spectrum could enable multi-channel fluorescence imaging with clearly separated spectral windows for different biomarkers within the same cell.⁸

Examples of TADF in Biofluorescent Probes

Research into TADF probes for biological imaging is still in its early stages, but several proof-of-concept experiments have demonstrated its potential.

In conventional fluorescence microscopy, the TADF material 2,6-bis[4-(diphenylamino)phenyl]anthraquinone (TPAAQ) has been formulated into nanoparticles that demonstrate strong emission and superior photostability compared to some standard fluorophores. These nanoparticles have successfully imaged A549 human lung cancer cells across multiple cell generations.^9,10

Ensuring water stability is critical for TADF molecules used in any biological context, as many of the best-performing emitters are hydrophobic small molecules. The donor–carbazole derivative 4CzIPN, for example, initially showed limited performance in aqueous media. Encapsulation within glassy host matrices has provided a solution. When embedded in a glassy polymer matrix to form “organic dots” (O-dots), the 4CzIPN nanoparticles achieved a PLQY of up to 94%, while the matrix protected the TADF molecule from oxygen quenching. These O-dots can be dispersed in water, and successful uptake into HEK293 cells was demonstrated with evidence of low cytotoxicity.¹¹

Another example of TADF's suitability for time-resolved imaging comes from the development of CPy-Odots. The TADF dye 2,3,5,6-tetracarbazole-4-cyanopyridine (CPy) was encapsulated in an amphiphilic DSPE-PEG2000 polymer matrix to create these organic quantum dots. These CPy-Odots exhibited a solution-phase fluorescence lifetime of 9.3 μs, an absolute PLQY of 38.3%, low cytotoxicity, and excellent biocompatibility. Their longer lifetime easily distinguished them from cellular autofluorescence in time-resolved imaging, and they performed better than commercial membrane markers in long-term tracking.¹²

In 2019, F. Ni et al showed that polymer dots attaching TADF molecules to a triphenylphosphonium (TPP⁺) group (known to accumulate in mitochondria), enabled selective mitochondrial imaging. The targeting efficiency of these polymer dots was comparable to the commercial dye MitoTracker Green.¹³

Outlook

TADF materials are only at the beginning of their journey towards routine biomedical use. Current research is still focused on identifying and characterizing TADF materials using techniques like steady-state fluorescence spectroscopy and subsequent biocompatibility experiments such as measuring cytotoxicity, molecular uptake into the cell, and proof-of-concept imaging experiments.

However, these thermally activated delayed fluorescent materials could improve the applications of techniques like time-gated lifetime imaging, improving the signal-to-noise ratio of fluorescence techniques and allowing for more sophisticated multi-channel analysis.

With the right formulation advances, these metal-free, tunable, highly efficient organic emitters have the potential to significantly improve imaging techniques and expand the range of biological processes that can be visualized within the living cell.

References

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About the Author

Dr. Mary O'Kane is an Application Scientist at Ossila, where she creates, sources, and reviews resources on a wide range of topics to help researchers get the most out of their work, including in the field of fluorescence imaging. She has a master's degree in physics from the University of Sheffield, where she went on to complete a PhD specializing in perovskite solar cells and perovskite precursor inks. Since moving into science communication, Mary has written extensively for the research community, translating complex technical concepts into accessible, practical guides. Her work has appeared in several respected online publications, including IEEE Spectrum, Electronic Design, and the American Solar Energy Society, reflecting her broad expertise across photovoltaics, materials science, and related fluorescence technologies.