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This work was supported by generous funding to S.N.T. from the US National Institutes of Health (R01DK134590). We also gratefully acknowledge funding to S.N.T from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R24OD034189), National Science Foundation (EEC 1941543), Polsky Family Foundation, and Shriners Children&#39;s Boston (Grant #BOS-85115). In addition, we acknowledge funding to R.J. by Grant #LIFER23-263034 from the American Association for the Study of Liver Diseases Foundation. The authors express deep gratitude for expert support from the Pathology Core at Harvard Medical School for histology studies. We would like to extend our gratitude to Dr. Robert Balaban and Dr. Armel Femnou from the NIH National Heart, Lung, and Blood Institute for their expertise in mitochondrial energetics and their important assistance in developing the RRS library of individual mitochondrial complexes.

<ol>
	<li>San-Mill&#225;n I. The key role of mitochondrial function in health and disease. Antioxidants (Basel). 2023;12(4):782.</li>
	<li>Rossmann MP, et al. Mitochondrial function in development and disease. Dis Model Mech. 2021;14(6):dmm048912.</li>
	<li>Chen W, Zhao H, Li Y. Mitochondrial dynamics in health and disease: mechanisms and potential targets. Signal Transduct Target Ther. 2023;8:333.</li>
	<li>Acin-Perez R, et al. A novel approach to measure mitochondrial respiration in frozen biological samples. EMBO J. 2020;39(13):e104073.</li>
	<li>Sharma E, et al. Overview of methods that determine mitochondrial function in human disease. Metabolism. 2025;170:156300.</li>
	<li>Kowaltowski AJ, Abdulkader F. How and when to measure mitochondrial inner membrane potentials. Biophys J. 2024;123(24):4150-4157.</li>
	<li>Quinn KP, et al. Characterization of metabolic changes associated with the functional development of 3D engineered tissues by noninvasive, dynamic measurement of individual cell redox ratios. Biomaterials. 2012;33(21):5341-5348.</li>
	<li>Perry DA, et al. Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest. Sci Transl Med. 2017;9(408):ean0117.</li>
	<li>Nguyen KT, et al. Mitochondrial hyperoxidation contributes to warm ischemia-reperfusion injury in rat and pig livers. Commun Med. 2026;6:307.</li>
	<li>de Vries RJ, et al. Noninvasive quantification of the mitochondrial redox state in livers during machine perfusion. PLoS One. 2021;16(10):e0258833.</li>
	<li>Ajenu EO, et al. Advancing assessment methods for human cardiac grafts during normothermic machine perfusion. Transplantation. 2025;110(2):e374-e384.</li>
	<li>Thomas AR, et al. Resonance Raman spectroscopy detects ischemia in experimental coarctation of the aorta in a rodent model. J Cardiovasc Transl Res. 2025;18:566-573.</li>
	<li>Cakir B, et al. In vivo noninvasive mitochondrial redox assessment of the optic nerve head to predict disease. PNAS Nexus. 2023;2(5):pgad148.</li>
	<li>Jain R, et al. Real-time monitoring of mitochondrial oxygenation during machine perfusion using resonance Raman spectroscopy predicts organ function. Sci Rep. 2024;14:7328.</li>
	<li>Covian R, Edwards LO, Balaban RS. Effect of the mitochondrial membrane potential on the absorbance of the reduced form of cytochrome c oxidase. Biochim Biophys Acta Bioenerg. 2024;1865(3):149048.</li>
	<li>Ljungdahl PO, Pennoyer JD, Robertson DE, Trumpower BL. Purification of highly active cytochrome bc1 complexes from phylogenetically diverse species by a single chromatographic procedure. Biochim Biophys Acta. 1987;891(3):227-241.</li>
	<li>Covian R, Chess D, Balaban RS. Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes. Anal Biochem. 2012;431(1):30-39.</li>
	<li>Haque O, et al. Twenty-four hour ex-vivo normothermic machine perfusion in rat livers. Technology. 2020;8(1-2):27-36.</li>
</ol>

The authors declare competing interests. S.N.T and P.R. have provisional patent applications relevant to this study. P.R. is an employee and shareholders of Pendar Technologies. S.N.T&#39;s are managed by MGH and Partners HealthCare in accordance with their conflict-of-interest policies. J.N.K&#39;s competing interests are managed by BCH&#39;s conflict-of-interest policies. The following patented technologies have been used in this study: US2020/0281474A1 In vivo monitoring of cellular energetics with Raman spectroscopy (application). Additional patent applications for use in ophthalmology, tissue viability, and burn injury assessment using Resonance Raman Spectroscopy have been submitted, where R.J. is also an inventor.&#160;&#160;

This study highlights a noninvasive, real-time, and highly sensitive method of quantifying mitochondrial health using a portable Resonance Raman Spectroscopy system that is comprised of a laser in a compact probe, connected with a fiber optic cable to a laser pump. This method used a 441 nm laser to excite mitochondria, which produce unique spectral signals depending on their oxidation state. Thus, RRS is able to quantify each state of mitochondria through their spectral signatures and compare the emitted spectra with pr...

Mitochondria play a crucial role in cellular function and homeostasis. Mitochondria are the main drivers of cellular metabolism, regulating ATP generation via oxidative phosphorylation. They also serve as signaling organelles that dictate cell fate by regulating apoptotic pathways, as well as ROS and Ca+2 signaling1. Thus, mitochondrial dysfunction can lead to various neurodegenerative, muscular, and cardiovascular diseases2,3. Despite the crucial role mitochondria play in disease progression, evaluating mitochondrial function can be invasive, costly, and limited.
Key indicators of mitochondrial function, such as oxygen consumption rate, oxidative phosphorylation, and the activity of various complexes within the electron transport chain (ETC), are measured through respirometry. The ETC is composed of four complexes (I&#8211;IV) embedded in the inner mitochondrial membrane (IMM). These complexes, with the help of other co-enzymes, transfer electrons from reduced substrates, such as NADH and FADH2, to the final electron acceptor, O2, which is then reduced to water. Most contemporary respirometry assays, such as the Seahorse and Orobros O2k, require tissue processing, thereby restricting their application for translational research4,5. Measuring mitochondrial membrane potential (MMP) is another method of evaluating mitochondrial dysfunction, which utilizes cationic lipophilic fluorophores, such as JC-1, along with microscopic imaging, where coupled (healthy) and uncoupled (damaged) mitochondria fluoresce with different wavelengths6. However, these assays can have low sensitivity5. The dye itself can also have high cytotoxicity at high concentrations5. Lastly, similarly to respirometry measurements, this technique also requires tissue processing. Spectroscopy-based techniques have recently been used as noninvasive methods of evaluating mitochondrial health. For instance, Phosphorus Magnetic Resonance Spectroscopy (P-MRS), a method that evaluates cellular metabolism by measuring the concentrations of phosphorus-containing metabolites, has been used to characterize engineered adipogenic tissue undergoing differentiation7. However, this technique also requires specialized equipment and training.
Our group recently demonstrated a real-time, noninvasive, and highly specific technique for measuring mitochondrial redox state as a marker of organ viability using Resonance Raman Spectroscopy (RRS). RRS uses inelastic scattering of photons for the quantification of redox states of mitochondrial cytochromes. Porphyrin rings in heme moieties of molecules such as mitochondrial cytochromes, hemoglobin, and myoglobin produce a characteristic Raman spectrum depending on their oxidation state when excited with a 441 nm wavelength laser8. Though the RRS system utilizes a 441 nm wavelength, lasers with 420 nm and 405 nm wavelengths can also be used, each of which preferentially enhances a different cytochrome complex. The spectrum obtained from an unknown sample of interest is cross-referenced with pre-recorded libraries of fully oxidized or reduced intact mitochondria, yielding the ratio of reduced to total mitochondria or the average redox state of the in situ ETC cytochromes. This ratio is defined as the Resonance Raman Reduced Mitochondrial Ratio (3RMR).
There are 3 distinct calculations/types of 3RMR: mitochondrial 3RMR (mito-3RMR), complex III 3RMR (cIII-3RMR), and complex IV 3RMR (cIV-3RMR). Each of these indices is calculated through the fraction of reduced to the total number of a given component. For instance, the 3RMR of cIII would be the ratio of reduced cIII to the total number of cIII in the tissue (cIII-R/(cIII-O+CIII-R). The same pattern applies to cIV. Thus, 3RMR-cIII would refer to the ratio of reduced cIII to the total amount of cIII, and 3RMR-cIV would refer to the ratio of reduced cIV to the total amount of cIV. Through regression analysis, either mitochondrial redox states (mito-3RMR) are calculated utilizing spectral libraries of intact mitochondria, or individual complex redox states (cIII-3RMR and cIV-3RMR) are calculated utilizing cIII and cIV spectral libraries. A recent publication9 demonstrated that mito-3RMR is representative of the weighted average of cIII and cIV redox states (or cIII-3RMR and cIV-3RMR). It is important to note that cI and cII are not Raman-active or resonantly enhanced through the 441 nm wavelength. Consequently, they are not computed when calculating the mito-3RMR.
Leveraging these types of 3RMR, we have been defining the optimal range of 3RMR. In &#34;healthy&#34; organs, mitochondria are intact and have sufficient oxygen supply. In contrast, damaged organs may have insufficient delivery or utilization of oxygen, thereby causing ETC complexes to accumulate electrons. Electron accumulation causes &#34;reductive stress,&#34; shifting the mitochondria from oxidized to reduced state and increasing 3RMR values. Several studies8,9,10,11,12 ranging from hearts and livers in rodent and porcine models, have begun to shed light on the optimal &#34;healthy&#34; range for 3RMR, which is proposed to be between 10 and 30. For example, Perry et al., subjected mammalian hearts to hypoxia, where a 3RMR of 30% was shown to yield the optimal balance for sensitivity and specificity to detect hypoxic tissue8. This trend was further confirmed in additional studies with cardiac tissue11,12, and liver grafts10. Additionally, Nyugen et al.9, demonstrated that 3RMR values drop to very low (oxidized) values for tissues in which metabolism has halted, with&#160;3RMR levels (7.667 &#177;&#177; 4.926) of non-transplantable warm-ischemic livers after 3 h of normothermic machine perfusion, compared to 3RMR values above 10 (~12&#8211;15) for fresh and transplantable warm-ischemic livers9.
Above and beyond these demonstrations of how 3RMR can measure tissue viability, the sensitivity, specificity and ease-of-use of RRS make it highly applicable to many other types of research where cellular metabolism and mitochondrial function are affected. In previous studies, RRS has been utilized to detect macular degeneration and glaucoma in mice through evaluating the metabolic activity of unmyelinated axons of retinal ganglion cells at the optic nerve head13. Additionally, it was used to detect coarctation of the aorta (CoA) in a rat model of congenital heart disease, a diagnosis that is often missed in newborn cardiac screenings12. This technology was leveraged to understand organ function and viability under various experimental conditions that are relevant in the field of organ transplantation, including studies that assess organ viability on ex vivo machine perfusion10,11, evaluate reoxygenation post-static cold storage14, or mitigate reperfusion injury of warm-ischemic livers9.
This paper seeks to establish RRS as a real-time, sensitive tool for assessing mitochondrial health. Whole livers from a rat model were subjected to varying levels of ischemia and oxygen reperfusion as a stress test to assess mitochondrial response. In this oxygen stress test, fresh livers are subjected to 5 min of initial perfusion flow, 5 min of ischemia, and 5 min of reperfusion. 3RMR, as well as lactate, K+, partial pressure of oxygen (pO2) measurements was taken throughout the stress test. A faster response to changes in oxygenation was observed with 3RMR compared with traditional blood-gas analytes. With these experiments, the use of RRS is highlighted not just for mechanistic studies, but also as a diagnostic method as well as a therapeutic testbed. Thus, the shared protocol may be useful to a variety of researchers working with mitochondria. Overall, RRS, with its noninvasive, real-time, and highly specific nature, could have a substantial impact on cellular metabolism.

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real-time, noninvasive evaluation of mitochondrial function using resonance raman spectroscopy

Real-Time, Noninvasive Evaluation of Mitochondrial Function Using Resonance Raman Spectroscopy

Although mitochondria are central to pathogenesis and disease progression, mechanistic insight into mitochondrial health dynamics remains costly and inaccessible. To address this gap, this study employs Resonance Raman Spectroscopy (RRS) to assess mitochondrial function, using a portable system that delivers real-time, noninvasive, and quantitative measurements of mitochondrial cytochrome redox states in rat livers. In this protocol, we demonstrate the use of this technology, including setup, data acquisition, and data processing. This study presents a proof-of-concept experiment that highlights RRS&#39;s ability to measure real-time changes in mitochondrial redox state&#8211; and, by extension, mitochondrial function. Briefly, the RRS device was connected to a laser pump as well as a data acquisition computer and placed 1 cm away from the rat liver.&#160; Acquisition parameters were selected in accordance with the rat liver protocol; redox states were measured in oxygenated and ischemic conditions utilizing an oxygen stress test. Changes in mitochondrial redox states were tracked throughout the oxygen stress test.

Fresh livers were subjected to periods of ischemia and reperfusion utilizing an oxygen stress test. Then, the real-time changes in 3RMR were compared throughout the stress test to oxygen consumption rate, outflow lactate, and potassium obtained from perfusate analysis at T0, T5, T10, T15. Under healthy conditions, 3RMR values are expected to be between 10% and 30% (Figure 5A). As expected, the 3RMR values at T0 and T5 fall wit...

We present a noninvasive, real-time method for monitoring mitochondrial health using Resonance Raman Spectroscopy (RRS). We used RRS to quantify mitochondrial redox states and developed a metric for mitochondrial health, the Resonance Raman reduced mitochondrial ratio (3RMR), which shows a faster response to changes in oxygenation than traditional blood-gas analytes.

Although mitochondria are central to pathogenesis and disease progression, mechanistic insight into mitochondrial health dynamics remains costly and ...

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Research

JoVE Journal

Medicine

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Massachusetts General Hospital, Harvard Medical School.  We present a noninvasive, real-time method for monitoring mitochondrial health using Resonance Raman Spectroscopy (RRS). We used RRS to quantify mitochondrial redox states and developed a metric for mitochondrial health, the Resonance Raman reduced mitochondrial ratio (3RMR), which shows a faster response to changes in oxygenation than traditional blood-gas analytes.

Real-Time, Noninvasive Evaluation of Mitochondrial Function Using Resonance Raman Spectroscopy

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