Microscopy breaks the barriers to nanoscale chemical imaging

Today’s super-resolution microscopy allows us to observe the nanoscale world with unprecedented details. However, the need for fluorescent tags reveals structural details, but provides little chemical information about the sample being studied.

This drawback facilitates the development of vibration imaging techniques, allowing molecules to be identified based on their own chemical bonds without modifying the sample. These methods detect physical changes in the sample when absorbing mid-infrared (miR) light, such as shifts in refractive index caused by heat absorption and temperature-induced acoustic signals. Still, existing methods often struggle with weak signal levels, making it difficult to achieve both high resolution (how we can see the details) and strong chemical contrast (how well the molecules can distinguish).

As reported in Advanced Photonics, a newly developed technology, Structured Lighting Mid-Level Photothermal Microscope (SIMIP), addresses this limitation at twice the better resolution than traditional microscopes.

Developed by researchers at Z Jiang University in China, led by Professor DeLong Chang, this new approach represents important advances in vibrational imaging, opening up new possibilities for nanoscale chemical and biological analyses.

Zhang said, “SIMIP microscopy integrates the principles of a structured illumination microscope with mid-layer photothermal detection. Mid-infrared light detection provides chemical specificity, while structured illumination microscopes increase the spatial resolution of the sample.”

The system consists of quantum cascade lasers (QCLs) that excite specific molecular bonds, causing local heating to reduce the brightness of adjacent fluorescent molecules. At the same time, a SIM system consisting of a 488 nm continuous wave laser and a spatial light modulator (SLM) produces striped light patterns projected onto the sample at different angles.

These patterns create Moiré Fringes by encoding previously unresolved high-frequency details into detectable low-frequency signals captured by scientific CMO (SCMOS) cameras. By comparing images taken with or without vibrational absorption, Simip reconstructs high-resolution images rich in both chemical and spatial information.

The team applied Hessian SIM and sparse deconvolution algorithms to achieve higher spatial resolutions of up to about 60 nm, surpassing traditional miR photothermal imaging at imaging speeds of over 24 frames per second.

To verify the accuracy of SIMIP, the researchers tested it with 200 nm polymethyl methacrylate beads embedded with a thermosensitive fluorescent dye. By sweeping QCl in the range of 1,420-1,778 cm-1, SIMIP successfully reconstructed the vibrational spectra and closely matched the results of Fourier transform infrared (FTIR) spectroscopy.

From a resolution perspective, SIMIP was 444 nm vs. 335 nm hemisemia (FWHM) in the standard method, achieving a 1.5-fold improvement over traditional miR light temperature imaging. Furthermore, we were able to distinguish between polystyrene and methacrylate polymethyl within sub-separated aggregates, which was not possible with standard fluorescent microscopy.

An additional benefit of SIMIP is its ability to detect autofluorescence, that is, the natural fluorescence released by a particular biological molecule. This can be achieved by switching from widefield SIM to point-scanning SIM for structured excitation of autofluorescence or by using short-wavelength probe beams in widefield photothermal detection methods to improve compatibility with existing optical setups.

By integrating SIM with MIP, SIMIP enables high speed and super-resolution chemical imaging beyond the diffraction limits. This method opens up new possibilities for observation in materials science, biomedical research, and chemical analysis. For example, researchers would use SIMIP to detect small molecule metabolites and analyze their interactions with cell structures.

The team is currently planning to enhance temporal synchronization of the simip to further improve imaging speed and accuracy, and to examine temperature-sensitive dyes to increase sensitivity. With hardware changes to existing SIM systems, SIMIP is ready for adoption in laboratories around the world.