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

Summary

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.

Abstract

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's ability to measure real-time changes in mitochondrial redox state– 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.  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.

Introduction

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 diseases^2,3. Despite the crucial role mitochondria play in disease progression, evaluating mitochondrial function can be invasive, costly, and limited.

Protocol

All animals for the experiments were maintained in accordance with National Research Council guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (Boston, MA, USA).

1. Setting up and utilizing RRS

1. Device description:
   NOTE: The portable RRS system (Pendar Technologies; product is for research purposes only) uses a 441 nm laser due to a resonant enhancement of the Raman spectrum of mitochondrial cytochromes with this wavelength. The laser runs between 4 mW and 8 mW.
   1. Connect a laser excitation source to a custom-built probe via a ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16 G Catheter	BD Insyte Authoguard	381454	Used to cannulate the portal vein
Activated Charcoral	Vet Equip Vapor Guard Activated Charcoal	931401	Used to filter excess vapor during anasthesia
BSA	Sigma-Aldrich	A7906	Perfusate composition
Bubble Trap	Radnoti	130149
Dexamethasone	Sigma-Aldrich	D2915	Perfusate composition
Glutamax	Thermo Fisher Scientific	35050061	Perfusate composition
Masterflex L/S Precision Pump Tubing (24 G)	Fisher Scientific	13-200-292	Used to flow perfusate through the liver
Masterflex peristaltic pumps	Cole Parmer, Vernon Hill, IL	7528-30	Used to flow perfusate through the liver
Membrane Oxygenator	Radnoti	130144	Used to oxygenate perfusate.
Penicillin-streptomycin	Sigma-Aldrich	P4458	Perfusate composition
RAPIDPoint 500 Blood Gas System	Siemens Healthineers	41115805	For blood-gas analysis
Resonance Raman System	Pendar Technologies	A4D441	Contact Pendar Technologies on inquiries about this product