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

Abstract

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.

Figure 1

Operational principle and characterization of WOMiM. (a) Schematic of WOMiM. MIR pulses are focused on the sample by a parabolic mirror. The phase change due to MIR absorption is obtained by capturing MIR-ON and MIR-OFF micrographs with a CPCM. (b) Chemical-contrast image is obtained by subtracting a MIR-OFF image from a MIR-ON image. (c) MIR-ON image (MIR wavenumber at 2850 cm^–1) and (d) MIR-OFF image of TAG drops. (e) WOMiM micrograph by subtracting (d) from (c). (f) Subtraction of two MIR-OFF images. (g) Zoom-in FOVs of the smallest observed TAG drop, as marked by the red dash rectangles in (e,f). (h) Line profiles of the smallest observed TAG drop in (g). (i–n) Illustrations of WOMiM micrographs (subtraction images) at varying MIR excitation flux density (0.039–1.97 μW/μm²). (o) Intensity plot of a TAG drop center and an arbitrary point in the background as indicated by red and blue arrows in (n). (p–r) Intensity variation plots as the MIR was turned on at 1 s (q) and turned off at 1 s (r), where the intensity is acquired at the location of the two arrows in (p).

Figure 2

Imaging of optothermal signal in water and hyperspectral imaging of TAG drops. (a) Schematic diagram of a pulse train for single-pulse-level synchronization. In this synchronization mode, each single MIR-ON image is captured as a MIR pulse arrives. The corresponding MIR-OFF image is later captured when the OPO trigger stops. (b–g) Illustration of subtraction images with the exposure delay varying from 181.2 to 222.2 μs. Each image is a subtraction of 50-averaged MIR-ON and 50-averaged MIR-OFF phase images. (h) Line profile across the 2D optothermal signal (hot spot) for characterization of the MIR excitation area in (d). Line profile suggests a full width at half-maximum of 186.2 μm for the diameter of MIR excitation area. (i) Plot of “OPO trigger in” and the optothermal signal, where the optothermal signal is the z-profile of a pixel [indicated in (c)] from an image stack (exposure delay varies from 9 to 238.6 μs with a step size of 8.2 μs). Dots (black or green): original data from the image stack. Red dashed line: cubic spline interpolation. The green dots in (i) correspond to values from six images in (b–g) labeled “(b)” to “(g)”. (j–l) Three selected images from a hyperspectral image stack. Scale bar = 50 μm. (m) Hyperspectral image stack. The wavenumber varies from 2950 to 2830 cm^–1 with a step size of 10 cm^–1. (n) WOMiM spectra of selected points as indicated by the arrows in (j), and the corresponding TAG spectrum measured by Fourier transform infrared (FTIR) spectroscopy. In the WOMiM spectra, smooth lines are obtained using cubic spline interpolation. In (b–g) and (j–l), dark current noise of camera was filtered out by Gaussian blurry.