Fluorescence-Enhanced Mid-Infrared Photothermal Microscopy

TL;DR

This paper introduces a fluorescence-enhanced mid-infrared photothermal microscopy technique that significantly improves sensitivity and image quality by using thermo-sensitive fluorescent probes, enabling high-resolution, specific, and real-time biological imaging.

Contribution

The authors develop a novel microscopy method that leverages fluorescence modulation to enhance sensitivity and image clarity in mid-infrared photothermal imaging.

Findings

Fluorescence intensity modulation is 100 times larger than scattering-based methods.

Image quality is improved by eliminating speckle artifacts.

The technique enables real-time, bond-selective imaging of biological specimens.

Abstract

Mid-infrared photothermal microscopy is a new chemical imaging technology in which a visible beam senses the photothermal effect induced by a pulsed infrared laser. This technology provides infrared spectroscopic information at sub-micron spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependance of scattering on temperature and the image quality is vulnerable to the speckles caused by scattering. Here, we present a novel version of mid-infrared photothermal microscopy in which thermo-sensitive 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 speckles, 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 through fingerprinting a single bacterium. Finally, the photobleaching issue is successfully addressed through the development of a wide-field fluorescence-enhanced mid-infrared photothermal microscope which allows video rate bond-selective imaging of biological specimens.