In a study published in Nature Communications, the team of Professor CHENG Jixin from Boston University and Researcher ZHU Hongbo from Changchun Institute of Optics, Fine Mechanics and Physics (CIOMP) of the Chinese Academy of Sciences (CAS) reported the progress of the research on the realization of high-resolution, high-speed volumetric quantitative chemical imaging of biomolecular information using a three-dimensional bond-selective and computational mid-infrared photothermal microscope.

Optical microscopy plays a pivotal role in modern biological research and clinical practice1. Its capabilities of visualizing and quantifying subcellular structures provide deep insights into cell. Among many optical microscopic imaging schemes, the emerging mid-infrared photothermal (MIP) microscopy inherits IR absorption spectroscopy's advantages but circumvents conventional IR micro spectroscopy's low-resolution and slow speed limit, so it has been widely concerned by the industry. However, the existing MIP microscopes have problems such as slow volume imaging speed and low depth resolution, which are the main bottlenecks restricting the development of this technology.

Dr. ZHAO Jian of Boston University has presented a non-interferometric computational MIP microscopy scheme for 3D bond-selective label-free imaging. The method synergistically integrates the time-gated pump-probe MIP microscopy with the pulsed laser-based Intensity Diffraction Tomography (IDT), termed Bond-Selective Intensity Diffraction Tomography (BS-IDT).

The principle of BS-IDT is that each absorption peak area in the mid-IR fingerprint region (≈5 μm to ≈20μm) corresponds to a unique molecular vibrational bond that, if harnessed, can differentiate distinct biochemical compounds in the sample. When a mid-IR laser pump beam illuminates a sample, the radiation absorbed by the molecular vibrational bond causes a transient and localized temperature increase, which leads to the sample's scattering cross-section variations. The high-speed pump mid-IR pulse laser can create periodic mid-IR light absorption in the sample. This oscillation creates “Hot” and “Cold” states, respectively, where the chemical-specific RI variations are present or absent in the sample. The sufficiently fast and sensitive pulsed IDT imaging system with multiple off-resonant probe beams can capture this information to recover the chemical-specific RI variations of the object quantitatively by subtraction between “Hot” and “Cold” states.

The blue diode laser ring array developed by CIOMP plays an important role in realizing BS-IDT as a detection laser beam. This laser ring array containing 16 continuous wave (CW) diode lasers with a 450 nm central wavelength. They work at a tunable repetition rate (0 to 10 kHz) and pulse duration (≈0.6 μs to ≈1 μs), so that they can synchronize with the high speed of the pulsed mid-IR beam for detecting the MIP-induced RI variations. The oblique illumination angle of each laser is set to match the microscope’s objective numerical aperture (NA), which maximizes the spatial frequency coverage allowed by the system. This spatial frequency enhancement follows synthetic aperture principles and expands the accessible bandwidth to achieve the diffraction-limited resolution of incoherent imaging systems. Compared with the most advanced MIP microscope based on optical diffraction tomography, BS-IDT can realize high-speed (≈0.05Hz, up to ≈6Hz) and high-resolution (≈350 nm laterally, ≈1.1 μm axially) 3D chemical-specific, quantitative computational imaging over a large FOV (≈100 μm×100μm) and mid-IR fingerprint spectroscopy on cells and multicellular C. elegans with a simple system design. Furthermore, it has improved the chemical volumetric imaging speed by ≈40 times, the depth resolution, and the FOV by ≈3 times.

BS-IDT technology is a simple and low-cost biological cell imaging technology solution, which can non-destructively quantify the volume and mass of different chemical components distribution or organelles inside a single cell without needing contrast agents. BS-IDT will not perturb the cell functions. It can be applied to IR metabolic dynamic imaging of living organisms. This technology will show great application potential in the field of three-dimensional biological imaging.

This is an important breakthrough made by the high-power semiconductor laser team led by Academician WANG Lijun and Researcher NING Yongqiang of CIOMP in the application of visible light semiconductor lasers, which not only promotes the application of blue light semiconductor lasers in the biomedical field.