The imaging speed of current mid-infrared photothermal (MIP) microscope is limited to tens of seconds per frame due to the long pixel dwell time and slow sample scanning process, which is insufficient for capturing dynamics inside living systems. In this work, we developed a video-rate MIP microscope by employing a lock-in free demodulation scheme to resolve single IR pulse induced contrast. We further developed a synchronous pump-probe Galvo scanning for reaching a line rate over 2kHz. With such scheme, the system is capable of resolving chemical dynamics of various biomolecules in living organisms at multiple scales.
Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.
We present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed by fingerprinting a single bacterium. A wide-field fluorescence-detected mid-infrared photothermal microscope developed allows video-rate bond-selective imaging of biological specimens.
Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Due to the limited spectral coverage of current SRS systems, most SRS applications utilized a narrow Raman band (<300 cm-1). Multi-window SRS imaging covering C-H, C-D and fingerprint regions offers richer chemical information. In this work, we present a multi-window SRS imaging system with a rapid widely tunable fiber laser. We implemented auto-balanced detection to enhance the signal-to-noise ratio of stimulated Raman loss by 23 times. We demonstrated SRS metabolic imaging of fungi, cancer cells, and Caenorhabditis elegans across the C-H, silent and fingerprint Raman windows. Our results showcase the potential of the multi-window SRS system for a broad range of applications.
Hyperspectral stimulated Raman scattering (hSRS) is a label-free microspectroscopic modality that enables live-cell metabolic imaging with chemical specificity. Yet, hSRS in the CH region has low throughput and poor chemical specificity, which limits its application to a broader range of metabolic studies. We propose a high-content, high-throughput hSRS imaging method by a sparsity-driven spectral unmixing and active spectral sub-sampling. We unprecedently generate chemical maps of four major metabolic species (lipid, protein, nucleic acid and carbohydrate) in a Mia PaCa-2 cell using seven spectral frames in the CH region, improving the acquisition speed by over an order of magnitude.
Stimulated Raman scattering (SRS) microscopy enables the imaging of molecular events on a human subject in vivo, such as filtration of topical drugs through the skin and intraoperative cancer detection. A typical approach for volumetric SRS imaging is through piezo scanning of an objective lens, which often disturbs the sample and offers a low axial scan rate. To address these challenges, we have developed a deformable mirror-based remote-focusing SRS microscope, which not only enables high-quality volumetric chemical imaging without mechanical scanning of the objective but also corrects the system aberrations simultaneously. Using the remote-focusing SRS microscope, we performed volumetric chemical imaging of living cells and captured in real time the dynamic diffusion of topical chemicals into human sweat pores.
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