Fig. 1: BS-IDT principle, instrumentation, and image reconstruction.
a MIP-based chemical imaging principle. Created with BioRender.com. The top inset in “a” shows a mid-IR spectrum in the fingerprint region of ~900 cm−1−1900 cm−1. Each crest in the spectrum corresponds to a unique chemical compound. The sample under mid-IR laser illumination: “Hot” state; sample without mid-IR laser illumination: “Cold” state. Pump-probe detection: pulsed mid-IR laser illumination (pump beam) on the sample causes transient local RI change. RI variations for hot and cold states are probed by another visible oblique beam. 3D chemical morphology maps with different biomolecular distributions: (1) subtract hot reconstruction from the cold; (2) tune wavenumber to obtain a different chemical compound of interest. Biomolecule icons in “a” are created with ref. 65. Cancer Cell icon in “a” is adapted from ref. 65. b System design. BS-IDT is based on a wide-field transmission microscope (Methods). Probe beam: oblique laser illumination (~450 nm) from a laser array that consists of 16 diode lasers. Pump beam: off-axis gold parabolic mirror focuses mid-IR (MIR) laser beam from a quantum cascade laser (QCL) onto the sample. c 3D RI map reconstruction. Created with BioRender.com. For each oblique illumination, the collected 2D intensity data can be mapped into the 3D frequency domain. Using data from all 16 oblique illuminations, the 3D cold RI map can be recovered by an inverse Fourier transform of the synthesized Ewald’s sphere. Cancer Cell icon in “c” is adapted from ref. 65. d 3D chemical imaging workflow. The leftmost inset of “d” shows a time synchronization scheme. The pulse duration for both probe and MIR laser is ~1 µs. The probe pulse is ~0.5 µs delayed relative to the MIR pulse. For the MIR laser, an additional 50 Hz on/off duty-cycle modulation is imposed to generate “Hot” and “Cold” states. For each probe laser illumination, paired “Hot” and “Cold” 2D raw imaging data are collected as indicated by dashed-line square. “Hot” or “Cold” 3D RI maps are reconstructed using all the 16 “Hot” or “Cold” raw images based on the IDT forward model, illustrated as the angle-dependent depth-resolved phase transfer function. By subtraction operation, the 3D chemical image illustrating RI variations is extracted.
