Mid-infrared (mid-IR) spectroscopy and optoacoustic/optothermal (OA/OT) imaging are perfectly complementary technologies to each other. Vibrational molecular excitations by mid-IR absorption are utterly de-excited in the form of heat while efficient OA/OT signal generation primarily depends on heat deposition. This synergy allows overcoming the (otherwise) persistent limitations of traditional mid-IR spectroscopy and imaging in live-cell/fresh-tissue applications— i.e., sample opacity due to water absorption. Combination of mid-IR excitation and OA/OT detection has resulted in new tools for label-free live-cell, tissues, and in vivo metabolic research. Here we discuss basic principles on mid-IR detection for spectroscopy and imaging as well as the most recent developments on mid-IR OA and OT microscopy that overcome the limitations of conventional vibrational spectroscopy for biosensing and label-free metabolic microscopy.
The detection scheme of conventional mid-infrared (mid-IR) spectroscopy and imaging as well as the strong absorption of water restricts the application of mid-IR spectroscopy/imaging. We applied optoacoustic sensing for the non-invasive retrieval of mid-IR signals in biological tissues and developed Mid-IR Optoacoustic Microscopy (MiROM). This results in a label-free bond-selective microscopy that is able to monitor lipids, proteins, and carbohydrates down to a single-cell level with a lateral resolution of ~5 µm. We will discuss MiROM’s most recent developments for spatiotemporal metabolic monitoring in living cells and tissues as well as perspectives and recent advances for its application in vivo.
Imaging modalities based on vibrational spectroscopy (Raman or mid-IR imaging) have demonstrated high label-free chemical specificity for different biomolecules. Nevertheless, conventional mid-IR microscopy have been limited mostly to dry tissues and fixed cells due to the strong mid-IR absorption of water and due to the use of conventional negative-contrast detection. We introduce positive-contrast Mid-infraRed Optoacoustic Microscopy (MiROM) for label-free metabolic imaging in living cells. We showcase the unique capabilities of MiROM in living cells by monitoring the spatiotemporal distribution of carbohydrates, lipids, and proteins in adipocytes during lipogenesis as well as monitoring the lipid-protein dynamics in brown and white adipocytes during lipolysis. We discuss how MiROM yields unique label-free metabolic imaging abilities for a broader range of bioanalytical studies in living cells and its potential for analytical histology in unprocessed tissues.
Significance: Mid-infrared (IR) imaging based on the vibrational transition of biomolecules provides good chemical-specific contrast in label-free imaging of biology tissues, making it a popular tool in both biomedical studies and clinical applications. However, the current technology typically requires thin and dried or extremely flat samples, whose complicated processing limits this technology’s broader translation.
Aim: To address this issue, we report mid-IR photoacoustic microscopy (PAM), which can readily work with fresh and thick tissue samples, even when they have rough surfaces.
Approach: We developed a transmission-mode mid-IR PAM system employing an optical parametric oscillation laser operating in the wavelength range from 2.5 to 12 μm. Due to its high sensitivity to optical absorption and the low ultrasonic attenuation of tissue, our PAM achieved greater probing depth than Fourier transform IR spectroscopy, thus enabling imaging fresh and thick tissue samples with rough surfaces.
Results: In our spectroscopy study, the CH2 symmetric stretching at 2850 cm − 1 (3508 nm) was found to be an excellent source of endogenous contrast for lipids. At this wavenumber, we demonstrated label-free imaging of the lipid composition in fresh, manually cut, and unprocessed tissue sections of up to 3-mm thickness.
Conclusions: Our technology requires no time-consuming sample preparation procedure and has great potential in both fast clinical histological analysis and fundamental biological studies.
KEYWORDS: In vivo imaging, Optoacoustics, Spectroscopy, Signal to noise ratio, Short wave infrared radiation, Tissue optics, Light scattering, Pulsed laser operation, Laser sources, Biomedical optics
In this work we developed a novel near-infrared two-path optoacoustic spectrometer (NiR-TAOS) that could sense OA intensity changes due to metabolite concentration changes in-vivo. The main aim of dividing the optical path in two is 1) perform real time correction of the laser emission profile of the laser source at different wavelengths and, 2) perform pulse to pulse correction to remove laser beam fluctuation and instability to increase signal to noise ratio. Signal to noise ratio improvement was significant not only at spectral peaks, but also at all other wavelengths. The system can be used for broad applications in biomedical measurements such as various metabolites in the SWIR.
Conference Committee Involvement (3)
Advanced Chemical Microscopy for Life Science and Translational Medicine 2025
25 January 2025 | San Francisco, California, United States
Advanced Chemical Microscopy for Life Science and Translational Medicine 2024
27 January 2024 | San Francisco, California, United States
Advanced Chemical Microscopy for Life Science and Translational Medicine 2023
28 January 2023 | San Francisco, California, United States
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