Label-free IR cell imaging faces two long-standing limitations: the diffraction limit averages away subcellular heterogeneity, and overlapping vibrational bands blur precise molecular assignment.
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For scientists using vibrational spectroscopy for cell characterization, this makes the nucleus, cytoplasm, and glycogen- and lipid-rich regions hard to consistently identify and analyze across cell types. This has proven to be a key barrier to the implementation of IR as a standardized tool for diagnostics.
To investigate and address this gap, a research team at Jagiellonian University in Krakow has leveraged the combined power of FPA-based FTIR with Photothermal Spectroscopy's submicron-resolution O-PTIR (∼0.3 µm) and fuzzy cluster analysis for chemically meaningful segmentation.
Using this setup, they analyzed a number of human cell lines, such as endothelial cells, urinary bladder tumor cells, pericytes, and astrocytes. The team found that O-PTIR was able to produce single-frequency chemical maps at the amide I protein band (1660 cm-1), and ratio maps at 1240/1740 cm-1 (phosphate/lipid contrast) and 1740/2850 cm-1 (triacyl glyceride/total lipid contrast), all of which were captured at 0.3 µm step size.
Segmentation was able to resolve a nucleic acid-rich nucleus, a lipid-rich class encompassing membranous structures and lipid droplets, and, in RT4 urothelial cells, a specific glycogen-rich class.
The 1240/1740 ratio enhanced the clarity of the nuclear region by limiting the influence of local path-length variation, and the 1740/2850 ratio allowed for detailed mapping of triacylglycerols within lipid-rich domains. The RNA marker at 996 cm-1 was fully resolved in the O-PTIR spectra.
The experimental signatures were reproduced using in silico linear-combination modeling of reference biomolecules, which in turn supported a distinct “IR barcode” for each compartment.
The researchers have identified O-PTIR as a tool compatible for use with traditional FTIR owing to its submicron resolution and sensitivity capabilities. Moreover, it is well-suited for subcellular, label-free analysis, and isolating assignments that band overlap and diffraction would otherwise obscure.
O-PTIR’s non-destructive functionality, combined with the capacity to image hydrated specimens, makes it more feasible to characterize cells in their native state, extending the study framework to metabolic shifts, drug responses, and pathological remodeling.
References
- Antolak, A., Pragnaca, A., Augustyniak, K., Zahra, S.T., Pięta, Ł., Wislocka-Orlowska, A., Majzner, K., Baranska, M. and Malek, K. (2026) ‘IR map of the human cell’, Analytical Chemistry, 98(26), pp. 19545–19555. DOI:10.1021/acs.analchem.6c00513. https://pubs.acs.org/ancham/article/98/26/19545/5167074/IR-Map-of-the-Human-Cell
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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