What goes on deep within the brain? This age-old question has long eluded clear answers—yet NADH is unexpectedly revealing some of its profound secrets.
As a pioneer in cellular energy enhancement and antioxidant processes, NADH is not only a critical player in maintaining health but also a key tool in scientific research. Its concentration changes are closely linked to cellular energy levels and redox status, reflecting a variety of intracellular physiological changes. Combined with its intrinsic fluorescent properties, NADH has become an ideal marker for label-free intracellular imaging: it enables real-time monitoring of cellular metabolic states and detection of early signals for neurological disorders such as Alzheimer’s disease and epilepsy.
 
Recently, A research team led by Professor Osaki from Japan has unveiled their newest research outcomes in Light: Science & Applications, a leading international journal in the field. It has opened a window into the deep brain which thrusting NADH back into the spotlight of scientific attention.

To understand the challenge of visualizing the deep brain, one must recognize that brain tissue is akin to a dense sponge: light scatters and is absorbed rapidly upon entry, making clear observation of internal structures extremely difficult with traditional optical methods.
Scientists previously used two-photon fluorescence imaging to observe NADH luminescence in tissues and cells. While this technique offers greater penetration depth than conventional one-photon fluorescence imaging, it only reaches 100-200 micrometers into the brain’s superficial layers—failing to access the cerebral cortex, let alone critical deep structures like the hippocampus and midbrain.

Ultrasound and magnetic resonance imaging (MRI) serve as alternative imaging modalities, yet they lack the high resolution of optical imaging. Thus, the major technical challenge today is achieving both sufficient penetration depth and high-resolution imaging—until the development of a revolutionary technology: label-free multiphoton photoacoustic microscopy (LF-MP-PAM).
The core principle of this novel technology is exquisitely designed: it uses light to excite NADH, then captures the corresponding signals via sound, overcoming the limitations of traditional optical imaging.

Near-Infrared Laser: The High-Penetration Probe
A 1300 nm near-infrared femtosecond laser acts as the probe—its penetration capacity far surpasses ordinary visible light, easily passing through brain tissue to reach deep regions. Since infrared light has relatively low energy, a pulsed three-photon excitation technique (with a pulse width of only 20-30 femtoseconds) is used to excite NADH fluorescence. This approach efficiently captures signals while avoiding cellular damage.

Ultrasound Transducer: The High-Sensitivity Stethoscope
NADH excited by the laser emits faint ultrasonic waves, as if cells are "communicating". A high-sensitivity ultrasonic transducer integrated into the device accurately captures these acoustic signals and converts them into high-resolution images. Notably, ultrasonic waves experience far less attenuation in biological tissues than light waves—effectively solving the long-standing "penetration depth" problem.
 
The device further verifies signal accuracy by synchronously collecting optical, photoacoustic, and third-harmonic signals to reconstruct cellular morphology, with a high degree of overlap among the three signal types ensuring reliable imaging results.
The performance of LF-MP-PAM is truly remarkable:

• It achieves an imaging depth of 700 micrometers in mouse brain slices—over three times the depth of traditional optical imaging—and clearly resolves the subcellular distribution of NADH in individual neurons. It even detects subtle differences such as higher NADH levels in the midbrain compared to the cerebral cortex.
• In brain organoid models (laboratory models simulating human brain structure), its detection depth reaches an astonishing 1100 micrometers.

• It boasts excellent dynamic imaging capabilities: 256×256 pixel high-definition imaging at 0.76 frames per second, enabling real-time monitoring of dynamic changes in cellular metabolism. For example, it rapidly captures the elevation of NADH levels after 24 hours of cellular hypoxia—consistent with known pathological changes, fully validating signal reliability.

While currently a research tool seemingly far from daily life, this technology is poised to reshape future health management models in unexpected ways:
Enabling Early Detection of Neurological Diseases
Neurodegenerative diseases often exhibit early pathological features such as energy impairment, oxidative stress dysregulation, and inflammation—all leading to abnormal NADH metabolism. With this label-free imaging technology, clinicians can capture pathological signals from the deep brain before symptoms appear, enabling early intervention and treatment.
Providing a New Tool for Brain Development Research
NADH plays a pivotal role in fetal and infant brain development. This non-invasive technique allows observation of metabolic changes in deep brain tissues, helping scientists unravel the mysteries of "how the brain develops" and providing a scientific basis for the prevention and treatment of pediatric brain disorders.
Driving Precision in Health Management
In the future, monitoring brain NADH status may enable assessment of how lifestyle factors (e.g., staying up late, stress) impact brain health by inducing metabolic disorders—laying the groundwork for personalized health recommendations and targeted interventions.
 
This study marks the first time we have "seen" NADH in the deep brain clearly, blazing a new trail for brain health research. As technology advances, this hidden "energy code" in the brain is sure to bring more surprising breakthroughs for human health in the near future.
 
References
Osaki T, Lee W D, Zhang X, et al. Multi-photon, label-free photoacoustic and optical imaging of NADH in brain cells[J]. Light: Science & Applications, 2025, 14(1): 264.