Reviewed by Lexie CornerApr 15 2025
According to a study published in Advanced Photonics, researchers at Zhejiang University in China, led by Prof. Delong Zhang, have developed a new approach that represents a significant advancement in vibrational imaging. This approach enables further exploration of nanoscale chemical and biological studies.
SIMIP enables high-resolution images rich in both chemical and spatial information. A quantum cascade laser (QCL) excites molecular vibrations, while a spatial light modulator (SLM) generates striped light patterns that are projected onto the sample. A scientific CMOS (sCMOS) camera captures the modulated fluorescence signals, which are processed using Hessian SIM and sparse deconvolution algorithms to generate high-resolution chemical and structural images. Subtracting the hot image from the cold image yields the hybrid SIMIP image. Credit: P. Fu, B. Chen, et al., doi 10.1117/1.AP.7.3.036003.
Super-resolution microscopes have enabled detailed observation of the nanoscale environment, but they require fluorescent tags, which provide limited chemical information and primarily offer structural characteristics. In response to this limitation, vibrational imaging methods were developed to identify molecules based on their unique chemical bonds without altering the material.
These techniques detect physical changes, such as temperature-induced acoustic signals or changes in refractive index due to heat absorption, when samples absorb mid-infrared (MIR) light. However, existing methods often face challenges with low signal levels, making it difficult to achieve both high chemical contrast and resolution.
The new method, structured illumination midinfrared photothermal microscopy (SIMIP), addresses these limitations by offering double the resolution of traditional microscopy.
SIMIP microscopy integrates the principles of structured illumination microscopy with midinfrared photothermal detection. Mid-infrared photodetection provides chemical specificity, while structured illumination microscopy enhances the spatial resolution of the sample.
Delong Zhang, Professor, Zhejiang University, China
The system utilizes a quantum cascade laser (QCL) to excite specific chemical bonds, leading to localized heating that reduces the brightness of nearby fluorescent molecules. A 488-nm continuous-wave laser and a spatial light modulator (SLM) generate striped light patterns, which are projected onto the sample at various angles.
These patterns create Moiré fringes, encoding high-frequency features into observable low-frequency signals detectable by a scientific CMOS (sCMOS) camera. By comparing images captured with and without vibrational absorption, SIMIP reconstructs high-resolution images that provide both spatial and chemical information.
In a proof of concept, the team used Hessian SIM and sparse deconvolution methods to achieve a spatial resolution of approximately 60 nm and an imaging speed of over 24 frames per second, surpassing traditional MIR photothermal imaging. The accuracy of SIMIP was validated by testing 200-nm polymethyl methacrylate beads with thermosensitive fluorescent dyes.
SIMIP successfully recreated the vibrational spectra by sweeping the QCL across the 1420–1778 cm–1 range, which closely matched the data from Fourier transform infrared (FTIR) spectroscopy.
SIMIP achieved 1.5 times the resolution of traditional MIR photothermal imaging, with a full width at half-maximum (FWHM) of 335 nm, compared to 444 nm in conventional methods. It also demonstrated the ability to distinguish between polystyrene and polymethyl methacrylate beads within sub-diffraction aggregates, a feat impossible with conventional fluorescence microscopy.
An additional advantage of SIMIP is its ability to detect autofluorescence, the natural fluorescence emitted by certain biological molecules. This is achieved by switching from widefield SIM to point-scanning SIM for structured autofluorescence excitation or by using a shorter-wavelength probe beam for widefield photothermal detection, enhancing compatibility with existing optical setups.
Combining SIM and MIP, SIMIP provides high-speed, super-resolution chemical imaging beyond the diffraction limit, enabling new possibilities in materials science, biological research, and chemical analysis. Future applications include identifying small-molecule metabolites and studying their interactions with cellular structures.
The team plans to enhance SIMIP’s temporal synchronization to further improve imaging speed and accuracy, as well as explore the use of temperature-sensitive dyes to increase sensitivity. With minimal hardware adjustments, SIMIP is ready for deployment in laboratories worldwide.
Journal Reference:
Fu, P., et al. (2025) Breaking the diffraction limit in molecular imaging by structured illumination mid-infrared photothermal microscopy. Advanced Photonics. doi.org/10.1117/1.AP.7.3.036003