Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering

TL;DR

This paper introduces a pupil engineering technique in mid-infrared photothermal microscopy that significantly suppresses background noise, enabling high-throughput, label-free chemical imaging of nanoparticles and bacteria with improved sensitivity and resolution.

Contribution

The authors develop a novel pupil engineering method for background suppression in MIP microscopy, achieving over three orders of magnitude noise reduction without losing lateral resolution.

Findings

Over three orders of magnitude background suppression achieved.

Six-fold improvement in signal-to-background noise ratio.

Successful hyperspectral imaging of bacteria and nanoparticles.

Abstract

Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from sub-wavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over three orders of magnitude background suppression by quasi-darkfield illumination in epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 um x 70 um. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500~nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria.