Non-invasive measurements of blood glucose levels by time-gating mid-infrared optoacoustic signals.

Non-invasive glucose monitoring (NIGM) represents an attractive alternative to finger pricking for blood glucose assessment and management of diabetes. Nevertheless, current NIGM techniques do not measure glucose concentrations in blood but rely on indirect bulk measurement of glucose in interstitial fluid, where glucose is diluted and glucose dynamics are different from those in the blood, which impairs NIGM accuracy. Here we introduce a new biosensor, termed depth-gated mid-infrared optoacoustic sensor (DIROS), which allows, for the first time, non-invasive glucose detection in blood-rich volumes in the skin. DIROS minimizes interference caused by the stratum corneum and other superficial skin layers by time-gating mid-infrared optoacoustic signals to enable depth-selective localization of glucose readings in skin. In measurements on the ears of (female) mice, DIROS displays improved accuracy over bulk-tissue glucose measurements. Our work demonstrates how signal localization can improve NIGM accuracy and positions DIROS as a holistic approach, with high translational potential, that addresses a key limitation of current NIGM methods.

Phase-shifting optothermal microscopy enables live-cell mid-infrared hyperspectral imaging of large cell populations at high confluency.

Rapid live-cell hyperspectral imaging at large fields of view (FOVs) and high cell confluency remains challenging for conventional vibrational spectroscopy-based microscopy technologies. At the same time, imaging at high cell confluency and large FOVs is important for proper cell function and statistical significance of measurements, respectively. Here, we introduce phase-shifting mid-infrared optothermal microscopy (PSOM), which interprets molecular-vibrational information as the optical path difference induced by mid-infrared absorption and can take snapshot vibrational images over broad excitation areas at high live-cell confluency. By means of phase-shifting, PSOM suppresses noise to a quarter of current optothermal microscopy modalities to allow capturing live-cell vibrational images at FOVs up to 50 times larger than state of the art. PSOM also reduces illumination power flux density (PFD) down to four orders of magnitude lower than other conventional vibrational microscopy methods, such as coherent anti-Stokes Raman scattering (CARS), thus considerably decreasing the risk of cell photodamage.

Wide-Field Mid-Infrared Hyperspectral Imaging by Snapshot Phase Contrast Measurement of Optothermal Excitation.

Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point scanning and impose a compromise between the imaging speed and field-of-view (FOV). Optothermal microscopy has been proposed as a promising imaging modality to avoid this compromise, although at restrictively small FOVs capable of imaging only few cells. Here, we present wide-field optothermal mid-infrared microscopy (WOMiM) for wide-field chemical-contrast imaging based on snapshot pump-probe detection of optothermal signal, using a custom-made condenser-free phase contrast microscopy to capture the phase change of samples after mid-infrared irradiation. We achieved chemical contrast for FOVs up to 180 µm in diameter, yielding 10-fold larger imaging areas than the state-of-the-art, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 µs in water, corresponding to a frame rate of ∼30 kHz. This proof-of-concept demonstrates that vibrational imaging can be achieved at an unprecedented imaging speed and large FOV with the potential to significantly facilitate label-free imaging of cellular dynamics.

Label-free metabolic imaging by mid-infrared optoacoustic microscopy in living cells.

We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.