Fluorescence Lifetime Imaging Microscopy Provides Molecular Insights in Neurology
The technique can enable surgical precision, improve disease monitoring, and accelerate the development of new therapies.
By Ross Keyashian
Fluorescence lifetime imaging microscopy (FLIM) is rapidly becoming a transformative tool in neurology, enabling the visualization of molecular and metabolic processes in living brain tissue and other samples with unprecedented detail. FLIM provides contrast based on the local biochemical environment, independent of fluorophore concentration or photobleaching, by measuring the excited-state lifetimes of intrinsic or extrinsic fluorophores. FLIM is used in both current and emerging applications in neurology — including intraoperative tumor margin detection, neurodegenerative disease research, and monitoring of neuronal activity. However, technical and translational challenges must be addressed for broader adoption in clinics, such as building awareness within the scientific community and simplifying the relative complexity of system design and setup.
A fluorescence lifetime imaging microscopy (FLIM) image of a mouse brain. Courtesy of HORIBA.
Neurological diseases, including malignant brain tumors and neurodegenerative disorders, present unique challenges for diagnosis and treatment due to the difficulty of adequately visualizing these conditions. The need for real-time, high-resolution, and molecularly specific imaging in research and during surgery has driven the development of advanced optical techniques. FLIM stands out among these methods because it quantifies the decay time of fluorescence emission. This parameter is highly sensitive to the local chemical and physical environment of the fluorophore. Unlike conventional fluorescence intensity imaging, FLIM is unaffected by variations in dye concentration, excitation intensity, or photobleaching, making it a robust tool for both fundamental neuroscience research and clinical applications.
In recent years, FLIM has demonstrated particular promise in neurosurgery for intraoperative tumor margin detection, as well as in the study of neurodegenerative diseases and neuronal function. This review provides an in-depth look at FLIM’s operating principles, current and potential applications in neurology, and future directions for this powerful imaging modality.
FLIM instrumentation
During the past few decades, the development of FLIM instrumentation has significantly progressed, transitioning from bulky, complex laboratory systems to more compact and user-friendly setups. Early FLIM systems were primarily custom-built setups in research laboratories, requiring extensive expertise to operate and maintain. The core principle behind their use remained the same: measuring the average time a fluorophore remains in the excited state before emitting a photon, known as the fluorescence lifetime (τ). This property is influenced by a variety of factors, including pH, ion concentration, molecular binding, and the presence of quenchers, allowing FLIM to serve as a sensitive probe of the microenvironment. There are two principal FLIM modalities: time domain and frequency domain.
In time-domain FLIM, a pulsed laser excites the sample, and the time between excitation and emission is measured for each detected photon, typically using time-correlated single-photon counting (TCSPC). This approach, considered the gold standard in many scientific circles, is renowned for its high temporal resolution — often in the picosecond range — and its suitability for complex biological tissues, such as the intricate structure of tumors. Recent innovations in systems and components for time-domain FLIM, such as HORIBA’s FLIMera wide-field TCSPC camera, leverage single-photon avalanche photodiode (SPAD) arrays with in-pixel timing. This capability allows simultaneous recording of tens of thousands of fluorescence decays at video rates. This eliminates the need for scanning in certain applications, greatly increasing acquisition speed. Where higher spatial resolution is required, today’s scanning systems use galvanometers for precise laser scanning, capable of collecting images up to 4K × 4K spatial resolution while maintaining high temporal fidelity.
A FLIM image of a mouse colon. Courtesy of HORIBA.
Frequency-domain FLIM, on the other hand, uses a modulated excitation source and analyzes the phase shift and demodulation of the emitted fluorescence relative to the excitation. This approach enables rapid imaging and is particularly useful for high-speed applications, such as in live-cell research, though it may offer slightly lower temporal resolution compared to time-domain methods.
A typical FLIM setup for biomedical applications has evolved to include more compact and powerful laser sources (such as picosecond-pulsed diode or supercontinuum lasers); a laser scanning microscope (confocal or multiphoton); highly sensitive detectors (photomultiplier tubes or hybrid detectors); and specialized electronics for timing or phase measurements. Crucially, advanced FLIM systems are now being integrated with endoscopic probes or fiber bundles that are inherent to modern neurology. This represents a significant shift, enabling intraoperative or in vivo imaging in animal models and, increasingly, in human patients, moving FLIM out of the traditional lab setting and closer to clinical utility.
What is driving innovation?
FLIM’s ability to provide quantitative, label-free, and spatially resolved information has led to a range of impactful applications in neurology, often pioneered by leading academic and clinical research institutions. One of the most compelling possibilities is intraoperative tumor margin detection during brain surgery. Glioblastoma multiforme, for example, is notorious for its diffuse infiltration into healthy brain tissue, making complete resection challenging and contributing to its poor prognosis. Researchers at institutions such as the Icahn School of Medicine at Mount Sinai, among others, are actively exploring how FLIM can distinguish tumorous from normal tissue by exploiting differences in metabolic signatures, such as the fluorescence lifetimes of endogenous cofactors such as NADH and FAD.
Tumor cells, which rely heavily on glycolysis (the breakdown of sugar molecules into usable energy), exhibit distinct NADH lifetime profiles compared to healthy neurons and glia. Studies have shown that FLIM can detect these metabolic shifts at the tumor margin, offering surgeons real-time feedback and potentially improving resection accuracy and patient outcomes.
An on-screen analysis of FLIM images. Courtesy of HORIBA.
In addition to metabolic imaging, FLIM can be used to visualize the binding of targeted fluorescent probes to tumor-specific biomarkers. For example, lifetime-based contrast can differentiate between bound and unbound states of a probe, providing greater specificity than intensity measurements alone. This is particularly relevant in personalized medicine approaches. Recent clinical prototypes, including fiber optic FLIM endoscopes developed by groups at various university hospitals, have demonstrated the feasibility of intraoperative FLIM imaging in human patients, with some systems capable of imaging through small craniotomies and during minimally invasive procedures.
Beyond oncology, FLIM is increasingly used to study neurodegenerative diseases such as Alzheimer’s and Parkinson’s. In these conditions, FLIM can monitor changes in the lifetimes of intrinsic fluorophores, such as lipofuscin or advanced glycation end products, which accumulate in affected brain tissues. FLIM has also been used in retinal imaging — for example, fluorescence lifetime imaging ophthalmoscopy (FLIO) — to detect early metabolic changes associated with diseases such as macular telangiectasia and Stargardt disease, providing a noninvasive window into central nervous system health. Academic centers worldwide are investigating these applications, often in collaboration with instrument manufacturers to tailor systems for specific ophthalmic or neurological imaging needs.
Another emerging area is the use of FLIM to monitor neuronal activity. When combined with FLIM, voltage-sensitive fluorescent probes can resolve rapid changes in membrane potential with high temporal precision, surpassing the speed of traditional
intensity-based sensors. This capability opens new avenues for studying neuronal signaling, synaptic activity, and network dynamics in both basic and translational neuroscience research, with pioneering work often occurring in neuroscience departments at major research universities.
FLIM is also proving to be valuable for drug development and delivery studies. By tagging therapeutic agents or nanoparticles with lifetime-sensitive fluorophores, researchers can track drug distribution, binding, and clearance in real time, even distinguishing between free and bound drug states. This information is critical for optimizing drug design and assessing therapeutic efficacy in preclinical and clinical studies.
The role of fiber-based systems
While FLIM has demonstrated significant promise in research, its broader awareness and adoption within the general medical community are still evolving. Clinicians are primarily aware of established imaging modalities such as MRI, CT, and conventional fluorescence imaging. For FLIM to become a standard clinical tool, continued efforts in demonstrating its clear clinical benefit, ease of use, and integration into existing workflows are crucial. Publications in high-impact clinical journals, presentations at major medical conferences (neurosurgical, neurology, ophthalmology), and collaborative studies between engineers, scientists, and clinicians are vital for increasing this awareness.
A 5× magnified image of fish anatomy using FLIM. Courtesy of HORIBA.
The development of fiber-based FLIM systems directly addresses several limitations of traditional lab-based microscope systems, thereby enhancing their functionality for clinical use:
• Minimally Invasive Access: Lab-based FLIM systems often require direct optical access to the sample, typically through a microscope objective, which is excellent for ex vivo samples or in vitro cell cultures. Fiber-based systems, however, allow for imaging deep within tissues and organs, such as the brain, through small incisions or natural orifices, enabling truly minimally invasive in vivo imaging. This is particularly transformative for neurosurgery, allowing real-time feedback within the confines of a surgical cavity.
• Intraoperative Guidance: Unlike stationary lab systems, fiber optic probes can be integrated within surgical tools, endoscopes, or robotic platforms. This allows surgeons to receive real-time, molecularly specific information directly within the surgical field, aiding in precise tumor margin delineation or identifying critical anatomical structures that might otherwise be difficult to discern with the naked eye or conventional white-light imaging.
• Flexibility and Portability: While lab systems are powerful, they are typically large and fixed. Fiber-based systems, though still requiring complex control units, offer greater flexibility in deployment, potentially moving toward more portable systems that can be brought directly to the operating room or patient bedside.
• Imaging of Complex Geometries: Traditional microscopes are optimized for flat, thin samples. Fiber-based probes can conform to the irregular geometries of biological tissues and cavities, providing imaging capabilities in challenging anatomical locations not accessible by conventional microscopy. This is evident in applications such as intravascular imaging and imaging within the distal lung.
• Reduced Phototoxicity: While FLIM is inherently robust to photobleaching, fiber-based systems often allow for careful control of light delivery to specific tissue areas, potentially minimizing overall light exposure and potential damage to healthy tissues.
FLIM’s unique ability to provide quantitative, microenvironment-sensitive imaging makes it an invaluable tool in neurology. Its robustness against photobleaching and concentration artifacts, combined with its compatibility with both endogenous and exogenous fluorophores, allows for a wide range of applications — from guiding tumor resection to probing the molecular underpinnings of neurodegenerative diseases. Clinical studies have already demonstrated FLIM’s potential to improve surgical outcomes by enabling more precise tumor margin detection. In research settings, FLIM is helping to track the metabolic and molecular changes that drive neurological disease progression and responses to therapy.
Despite these advancements, several challenges remain. The integration of FLIM into routine clinical workflows requires further miniaturization of instrumentation, faster data acquisition and analysis, and the development of standardized protocols for data interpretation. Regulatory approval will also depend on the validation of FLIM-based biomarkers and demonstration of clinical benefit in large, multicenter trials.
Future outlook
Looking ahead, the future of FLIM in neurology seems assured. Advancements in detector technology, including higher-pixel-count SPAD arrays and faster readout; more compact and powerful laser sources; and sophisticated computational analytics, including machine learning for automated image analysis and real-time interpretation, are poised to make FLIM faster, more sensitive, and easier to use. The development of high-throughput, multispectral FLIM systems will enable the simultaneous monitoring of multiple molecular targets, further expanding the technique’s utility in both research and clinical settings.
And miniaturized, fiber-based FLIM probes are likely to become standard tools in neurosurgery and interventional neurology, providing real-time metabolic and molecular feedback during procedures. As protocols are standardized and clinical evidence accumulates, FLIM is well positioned to become a cornerstone technology in precision neuroimaging, supporting the next generation of diagnostics, therapeutics, and basic neuroscience discoveries.
Meet the author
Ross Keyashian, HORIBA’s U.S. fluorescence imaging product manager based in Dallas, has a diverse academic background, including an MBA and degrees in electrical engineering and physics. He is currently a researcher at Texas Christian University under professor Karol Gryczynski. Keyashian’s research focuses on intramolecular double-well coupling and the development of fluorescence lifetime imaging instrumentation; email: ross.keyashian@horiba.com.
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