Biological analysis is dealing with a persistent gap in understanding: the tools used to reveal molecular chemistry rarely resolve it in space.
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For example, though infrared spectroscopy can identify functional defects without labels, its diffraction is limited to several micrometers, which is too coarse for subcellular detail. Raman spectroscopy, unlike infrared, reaches sub-micrometer resolution but is hindered by weak signals and fluorescence backgrounds in biological specimens.
Fluorescence offers resolution and sensitivity but relies on external labels and does not have intrinsic chemical specificity. When dealing with single cells, heterogeneous tissue, and organelles, these methods cannot deliver label-free, chemically specific imaging at the necessary scale.
In light of this, researchers at Monash University have leveraged Photothermal Spectroscopy's O-PTIR microscope to address these limitations.
The technique uses a tunable mid-IR pump to excite the sample, and a co-aligned visible probe in turn detects the photothermal response. As such, spatial resolution is governed by the visible wavelength rather than the IR wavelength.
This assembly yields non-contact, sub-micrometer, and label-free imaging on thin or heterogeneous living cells and specimens, with the option to simultaneously collect IR and Raman spectra from the same location.
The researchers were able to track how single-wavenumber mapping, hyperspectral imaging, and fluorescence-guided acquisition produce immense chemical detail that is not possible using conventional far-field IR.
The results were consistent across all applications analyzed by the team. O-PTIR is able to achieve lateral resolution near 0.4 µm, around 10–15 times finer than FTIR microscopy, with axial resolution nearing 1.7 µm. In an ovarian cancer study involving 78 patients, a convolutional neural network trained on O-PTIR bands achieved an AUC of 0.98 compared to 0.58 for a random forest model.
In other studies, researchers found that O-PTIR could differentiate related bacterial species at the single-cell level, resolve bone matrix-to-mineral transitions within 100–200 nm, localize polyethylene and polystyrene microplastics in fixed colon tissue, and map active-ingredient distribution in pharmaceutical powders.
The Monash team does acknowledge the limitations of their work, including a signal that strays from Beer–Lambert quantitation, point-by-point acquisition speed, cost of the instrumentation, and limited cross-laboratory standardization.
However, overall, the researchers were able to demonstrate that O-PTIR’s contribution is a viable platform for supporting label-free, sub-micrometer, chemically specific imaging suitable for native-state and living samples and is compatible with both Raman and fluorescence.
These capabilities position O-PTIR as the bridge between key molecular studies and diagnostic and translational work across cell biology, microbiology, pathology, and pharmaceutical analysis.
References
- Soni, T., Bedolla, D.E. and Wood, B.R. (2026) ‘Biological and biomedical applications of optical photothermal infrared spectroscopy (O-PTIR)’, Applied Spectroscopy, 80(7), pp. 659–691. DOI:10.1177/00037028261447532. https://journals.sagepub.com/doi/10.1177/00037028261447532
About Photothermal Spectroscopy Corp 
Photothermal Spectroscopy Corp (PSC) is a Santa Barbara, CA-based scientific equipment company that pioneered sub-micron IR microscopy and spectroscopy. PSC’s vision is to enable the power of sub-micron IR spectroscopy to be applied to high-value problems in both industry and academia via the adoption of O-PTIR and co-located multi-modal techniques.
O-PTIR is one of the highest growth IR microscopy techniques and addresses application areas in life science, microplastics, bioplastics, semiconductors, failure analysis, and chemical and polymeric materials.
mIRage-HSi IR Multimodal Microscope
mIRage-HSi combines sub-500 nm O-PTIR chemical imaging with laser-scanning speed, revealing label-free distributions of key macromolecules like lipids, proteins and nucleic acids while also resolving micro- and nanoplastic particles in complex biological samples.
Hyperspectral images are acquired in minutes and targeted single-wavenumber images in seconds, making detailed cellular studies and high-throughput particle workflows practical.
The mIRage-HSi supports multi-modal capabilities with simultaneous raman, co-located fluorescence microscopy and optical microscopy making it a powerful instrument for high resolution life science research.
Optical Photothermal IR Spectroscopy
Multimodal Optical Photothermal infrared spectroscopy (O-PTIR) used on the mIRage microscope platforms is a result of nearly 2 decades of expertise in photothermal based IR spectroscopy.
O-PTIR: How it works
O-PTIR overcomes the IR diffraction limit by combining a mid-IR pulsed, tunable laser that heats the sample. When the IR laser is at a wavelength that excites a molecular vibration in the sample, absorption occurs, thereby creating photothermal effects including photothermal expansion. A visible probe laser, focused to 0.5 µm spot size, measures the photothermal response via the scattered light, as shown in the illustration above.
The component of the reflected visible laser signal that is modulated at the IR pump laser repetition rate is directly proportional to the absorption coefficient of the sample at that wavenumber. The IR pump laser can be tuned through the entire fingerprint region in one second or less, to obtain an IR spectrum.
By operating in reflection mode, O-PTIR eliminates several longstanding limitations and has substantial benefits for the IR community, including sub-micron resolution using a non-contact optical method. The sub-micron resolution is demonstrated (right), showing reflection mode spectra on a multi-layer packaging film measured 0.5 µm apart with highly differentiated chemical fingerprints indicating different materials.
Measurements are collected quickly and easily without need for sample contact, unlike ATR spectroscopy. Additionally, O-PTIR provides spectra comparable to FTIR without the dispersive artifacts observed in ATR. By operating in reflection mode, the need for thin samples is also eliminated, leading to dramatically easier sample preparation and faster turnaround times.
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