Flexible Miniaturized Photometer Probe for Real-Time Metabolic Intrinsic Fluorescence Imaging

Clinical Sig: Ischemic heart disease remains the leading cause of mortality worldwide, driven by an imbalance between myocardial oxygen supply and demand. While current diagnostics, such as coronary angiography, are effective at detecting coronary artery blockages, they often fail to identify conditions that elevate oxygen demand without arterial obstruction, like aortic valve stenosis or hypertrophic cardiomyopathy. Detection of local metabolic products, such as NADH, could offer a more comprehensive diagnostic approach for these scenarios.
In tumor surgery, accurately differentiating tumor margins from healthy tissue is crucial for complete resection. Traditional approaches, such as intraoperative frozen section analysis, are slow and lack real-time feedback.
Localized NADH autofluorescence imaging offers a direct approach to measuring the tissue metabolic state. NADH fluorescence has shown promise in diagnosing myocardial ischemia in preclinical studies and could provide real-time surgical guidance during tumor resection by distinguishing the metabolic profiles of cancerous versus normal cells. Despite its potential, current NADH autofluorescence imaging techniques face significant challenges due to NADH's weak quantum efficiency and the limited tissue penetration of UV excitation light, which is prone to high scattering and absorption. These limitations restrict its use to surface-level detection when relying on benchtop optical setups. We aim to develop a bio-integrated optoelectronic device for NADH autofluorescence imaging that enables high spatial resolution while utilizing materials that provide a compatible bio-electronic interface.
Materials and Methods: We developed a flexible, miniaturized photometer probe for real-time metabolic imaging. It includes an indium gallium nitride (InGaN) UV microscale LED and an NPN silicon aluminum (SiAl) microscale phototransistor, housed in a custom epoxy chamber on a four-layer flexible PCB substrate with copper and polyimide dielectric layers, and a tungsten stiffening layer, all encapsulated in parylene. The epoxy provides mechanical protection for wire bonds and enhances uniform illumination by acting as a light diffuser. The InGaN micro-LED, with its compact size, offers a low forward voltage compatible with a 3.3V power supply, and enables a penetrating form factor for small-animal hearts. To enhance the signal-to-noise ratio for the weak intrinsic fluorescence signal of NADH, we employed a Si phototransistor, which offers higher signal gain compared to a conventional Si photodiode. We further reduced noise by incorporating a bandpass absorbance filter made from photodefined epoxy doped with three organic dyes, blocking the excitation light while allowing the emission signal to pass.
Results: In vitro tests demonstrated a linear fluorescence range within clinically relevant levels for both cardiac and brain tumor applications. The absorbance filter reduced the LED's external quantum efficiency to less than 1% transmittance at peak intensity, and our optimized design improved the fluorescence dynamic range by 630% compared to the previous unoptimized setup.
Discussion and Future Work: Upcoming studies will test the probe in glioblastoma models and living rat hearts using a single-site, injectable format. Plans include multi-site metabolic imaging and integration with physiological parameters like cardiomyocyte action potentials and calcium imaging. We will explore replacing the SiAl phototransistor with a III-V photodiode, as it offers better responsivity in the UV range and lower dark current compared to a phototransistor, resulting in an improved signal-to-noise ratio.