Fluorescence-Detected Mid-Infrared Photothermal Microscopy

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 submicrometer spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependence of scattering on the 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 thermosensitive 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 interference, 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-detected mid-infrared photothermal microscope which allows video rate bond-selective imaging of biological specimens.

Photothermal Circular Dichroism of Single Nanoparticles Rejecting Linear Dichroism by Dual Modulation

“…Another strategy would be to enhance PCCD by choosing a surrounding medium with a larger circular birefringence. Alternatively, we might be able to improve the measurement sensitivity further by employing more efficient contrast mechanisms, as recently demonstrated in refs − . This would allow us to observe g -factors down to the 10 –4 range while at the same time substantially decreasing the heating and therefore avoiding reshaping.…”

Label-Free Autofluorescence-Detected Mid-Infrared Photothermal Microscopy of Pharmaceutical Materials

“…25 An alternative detection scheme based on measuring temperature-induced changes in fluorescence quantum efficiency has been recently independently developed by Simpson and co-workers 26 and Cheng and co-workers. 27 Fluorescencedetected photothermal mid-IR (F-PTIR) microscopy was shown to achieve at least an order of magnitude signal-tonoise improvement over conventional O-PTIR and was applied for living cell imaging 27 and characterization of phase separated microdomains in pharmaceutical amorphous solid dispersions. 26 Notably, the use of fluorescence enabled analysis of vibrational spectroscopy immediately adjacent to fluorescent regions of interest (e.g., targeted by selective chemical labeling with fluorophores).…”

Imaging approaches for monitoring three‐dimensional cell and tissue culture systems

“…However, some common limitations for PTI studies on tissues, especially quantitative biochemical PTI studies, include: the overlap of the water signal in the mid-IR region often resulting in low signal to noise values [189,215], and heat-induced photo-chemical reactions, which do not produce heat but rather may produce a new chemical species which may alter the photothermal properties of the sample [185]. The current ambitions of PTI imaging is focused towards overcoming these limitations, with studies focused towards heterodyne detection [216], digital holography and optical diffraction holography [17,191], VIPPS phase-sensitive lock-in detection Scheme [207,209,210] (using reconstruction methodologies from other fields such as high-order correlation reconstruction (where the thermal emitting processes are dominated by the thermal diffusion processes) from super-resolution microscopy [217], and machine learning [218,219]), and developing multimodal photothermal systems (such as epifluorescence using thermo-sensitive fluorescent probes [220], Raman [221,222], photoacoustic [223] and OCT [224,225]).…”