An Introduction to Optical Photothermal Infrared (O-PTIR) Spectroscopy

Abstract

Optical photothermal infrared (O-PTIR) spectroscopy is an infrared super-resolution measurement technique where a shorter wavelength visible probe is used to measure and map infrared (IR) absorption with spatial resolution up to 30× better than conventional techniques such as Fourier transform infrared (FT-IR) and direct IR laser imaging systems.

This article introduces O-PTIR microscopy, explaining how it overcomes traditional IR spectroscopy limitations while enabling multimodal capabilities, including simultaneous IR+Raman spectroscopy and co-located fluorescence microscopy. O-PTIR overcomes key limitations of traditional FT-IR, such as poor spatial chemical resolution, time-consuming sample preparation, and spectral artifacts (1–4).

Introduction

Infrared and Raman spectroscopy are fundamental analytical techniques for probing molecular composition and structure via vibrational fingerprints. However, conventional FT-IR microscopy is hampered by diffraction-limited spatial resolution (typically ≥10 μm), time-consuming sample preparation, and spectral artifacts from poor contact or reflection geometries. Raman microscopy, while offering finer resolution, often yields weak signals and is plagued by fluorescence interference.

Direct mid-IR imaging of hydrated or irregular samples additionally suffers from strong water absorption and Mie scattering effects. These limitations impede rapid, high-resolution chemical mapping across materials science, life sciences, and failure analysis. Optical photothermal infrared (O-PTIR) microscopy overcomes these barriers by using a tunable IR pump to induce localized heating and a visible probe to detect the photothermal response. This approach delivers sub-micron resolution, minimal sample prep, and artifact-free, transmission-quality spectra in reflection mode—enabling truly multimodal IR, Raman, and fluorescence imaging.

How O-PTIR Works

The O-PTIR Technique

O-PTIR employs a pump-probe measurement scheme where a pulsed tunable IR laser (pump) induces localized heating in IR-absorbing regions, and a continuous-wave visible laser (probe) detects the resulting photothermal response. The sample is illuminated with an IR beam from a pulsed tunable source such as a quantum cascade laser (QCL) (1). When the IR wavelength matches a vibrational absorption band, energy conversion to heat occurs, causing localized temperature increases.

The temperature rise induces thermal expansion and refractive index changes within the heated volume. A coaxially aligned visible probe beam (typically 532 nm) focused to the same region detects these thermally-induced optical changes through variations in scattered light intensity. The detector signal undergoes lock-in detection at the IR pulse repetition frequency, providing high sensitivity to the photothermal modulation while rejecting background noise.

Signal collection occurs as a function of IR wavelength to generate absorption spectra, or as a function of spatial position to create chemical images. The technique relies on the correlation between IR absorption strength and the magnitude of the photothermal response, which follows from the linear relationship between absorbed IR power and temperature increase under typical measurement conditions.

O-PTIR systems typically employ Schwarzschild reflecting objectives that provide both IR illumination and visible light collection through the same optical path. Standard measurement parameters include 2 cm^-1 spectral resolution with spot sizes approaching 450 nm using 40× objectives. The non-contact nature of the measurement allows analysis of irregular surfaces and eliminates potential spectral artifacts from poor sample contact common in ATR methods.

Figure 1: O-PTIR uses a visible probe beam to detect infrared absorption with sub-500 nm spatial resolution via photothermal detection sensitive to thermal expansion and index of refraction changes in IR absorbing regions of a sample. (a) infrared pulse to sample, (b) visible light probe, (c) thermal expansion measured.

Sub-micron IR Spectroscopy and Imaging

The spatial resolution in O-PTIR (1,2) is determined by the probe wavelength rather than the IR wavelength, fundamentally overcoming the diffraction limit that constrains conventional IR microscopy. At 1000 cm^-1 (10 μm IR wavelength), conventional FT-IR microscopy with a 15×, 0.4 NA objective achieves ~15 μm lateral resolution. In contrast, O-PTIR using a 532 nm probe with 0.78 NA objective achieves ~416 nm resolution—a 36× improvement.

This wavelength-independent resolution enables consistent spatial performance across the entire mid-IR spectral range. The technique achieves true sub-micron analysis by detecting thermal effects rather than directly collecting IR photons, circumventing traditional diffraction limitations. Figure 2 provides a comparison(1).

Figure 2: Comparison of theoretical lateral spatial resolution of conventional IR microscopes (FT-IR and QCL-based) with the O-PTIR approach. Uniquely, O-PTIR’s spatial resolution is set by the probe wavelength and thus is constant over all IR wavelengths.

Figure 3: Sub-500nm resolution infrared chemical imaging with O-PTIR. The sample is an (a) IR chemical image of a PMMA bead at 1730 cm⁻¹ with 50 nm pixel size. Spatial dimensions shown in µm, (b) Cross section of IR absorption across the line indicated in panel (a) showing spatial resolution at the PMMA/epoxy boundary of around 350 nm. Dimensions for (b) are µm for the abscissa, and signal (mV) as ordinate.

Limited Sample Preparation

O-PTIR operates in reflection mode without requiring complex sample preparation stepstypical of traditional FT-IR methods (1). Samples can be analyzed directly in their native state, making the technique particularly suitable for analyzing irregular surfaces, thick samples, and materials that cannot be easily sectioned for transmission measurements.

Transmission Quality Spectra in Reflection Mode

O-PTIR spectra exhibit transmission-like quality while operating in reflection mode. The photothermal detection mechanism produces spectra that are directly comparable to conventional FT-IR transmission spectra, enabling the use of existing spectral libraries for compound identification.

Flexibility of Operation

O-PTIR systems typically employ Schwarzschild reflecting objectives that provide both IR illumination and visible light collection through the same optical path. Standard measurement parameters include 2 cm^-1 spectral resolution with spot sizes approaching 450 nm using 40× objectives. The non-contact nature of the measurement allows analysis of irregular surfaces and eliminates potential spectral artifacts from poor sample contact common in ATR methods.

Spectroscopy vs Imaging

O-PTIR operates in two primary modes: point spectroscopy for detailed chemical analysis of specific locations, and imaging for spatial mapping of chemical distributions. Spectroscopic measurements acquire full IR spectra by tuning the IR source across the desired wavelength range while monitoring the photothermal response. Imaging measurements fix the IR wavelength at specific absorption bands and raster the sample to map chemical distributions with sub-micron resolution.

Comparison to FT-IR

O-PTIR addresses fundamental limitations of conventional FT-IR microscopy, including diffraction-limited spatial resolution, sample preparation requirements, and spectral artifacts in reflection measurements. The technique maintains spectral fidelity while achieving sub-micron resolution through indirect photothermal detection rather than direct IR collection.

Multimodal Capabilities

Simultaneous IR+Raman

The pump-probe architecture enables simultaneous acquisition of IR and Raman spectra from identical sample locations (3). A continuous-wave probe laser serves dual purposes: detecting photothermal IR responses and exciting Raman scattering. This configuration provides complementary vibrational information, as IR and Raman spectroscopies probe different molecular vibrations based on distinct selection rules.

Simultaneous measurement eliminates spatial uncertainty between measurements and reduces total analysis time. The approach proves particularly valuable for fluorescent samples where conventional Raman suffers interference, as the O-PTIR signal remains unaffected by sample fluorescence. Multi-polymer studies have demonstrated successful identification using combined IR+Raman data even when individual techniques encounter limitations.

Figure 4: (a) The visible probe laser also generates Raman scattered light from the same measurement area, which, when collected, allows for the simultaneous acquisition of both IR and Raman spectra, with the same submicron spatial resolution, at the same location and at the same time. (b) top: optical image of various sizes of small PS beads. middle: the corresponding O-PTIR spectra from a 500nm PS bead. bottom: spectra from a 2 µm cluster of beads of approximately 4 µm in size. The IR spectra from a single 500 nm bead or a cluster of 2 µm beads all look the same. The spectra are raw and unfiltered.

Co-located Fluorescence and O-PTIR

Fluorescence microscopy integration with O-PTIR enables targeted chemical analysis guided by fluorescent labeling (4). Immunofluorescence staining or fluorescent proteins commonly used in life sciences can identify specific cellular structures, which then serve as targets for detailed O-PTIR molecular analysis. While fluorescence provides targeting specificity, it lacks information about molecular structure and conformation that O-PTIR supplies.

The technique also exploits intrinsic autofluorescence common in biological materials (aromatic amino acids, cross-linking molecules, photosynthetic compounds) for wide-field detection. This approach enables high-speed hyperspectral IR imaging with sub-500 nm resolution and 4 cm⁻¹ spectral resolution, demonstrated on samples ranging from diatoms to live microalgae.

Figure 5: The mIRage-LS provides an integrated Fluorescence camera in the optics path of the O-PTIR signal. This enables co-located O-PTIR and fluorescence microscopy. Fluorescence images can be obtained by exciting the region of interest (ROI) with different visible wavelengths and detecting the radiation re-emitted at longer wavelengths.

Applications

O-PTIR microscopy has proven invaluable across diverse fields(1). In life sciences, sub-micron IR imaging enables high-resolution mapping of proteins, lipids, and nucleic acids within individual cells and tissues with minimal preparation requirements. In pharmaceutical research, its multimodal approach delivers reliable detection and chemical identification of sub-micron particulates and biological contaminants, bolstering quality control and regulatory compliance. Environmental scientists leverage O-PTIR for rapid characterization of microplastics down to sub-10 μm sizes, producing FT-IR-quality spectra to distinguish polymer types in complex matrices. In semiconductor and materials research, sub-micron IR imaging and co-located fluorescence pinpoint organic residues, defects, and thin-film compositions unresolvable by traditional techniques. Across materials science, from 2D materials to cultural heritage samples, O-PTIR’s artifact-free, high-fidelity spectra unlock new insights into chemical heterogeneity at the nanoscale.

Conclusion

O-PTIR microscopy represents a significant advancement in vibrational spectroscopy by systematically addressing fundamental limitations of conventional IR techniques. The technique achieves sub-micron spatial resolution independent of IR wavelength, operates without complex sample preparation, and provides transmission-quality spectra in reflection mode. Multimodal capabilities, including simultaneous IR+Raman and co-located fluorescence, extend analytical capabilities beyond traditional single-technique approaches. These technical capabilities enable new research frontiers across diverse analytical challenges in materials science, life sciences, and industrial applications.

References

(1) Prater, C. B.; Kansiz, M.; Cheng, J.-X. A Tutorial on Optical Photothermal Infrared (O-PTIR) Microscopy. APL Photonics 2024, 9 (9). DOI: 10.1063/5.0219983

(2) O-PTIR Technique Description Page. Available at:https://www.photothermal.com/o-ptir-techniques-optical-photothermal-ir-spectroscopy (accessed 2025-09-09).

(3) Submicron IR and Raman Spectroscopy for Chemical Identification of Microplastics. Applications Note 107; Photothermal Spectroscopy Corp., February 2025. Available at: https://www.photothermal.com/applications/microplastics/microplastics-applications-note/ (accessed 2025-09-09).

(4) Prater, C.; Bai, Y.; Konings, S. C.; Martinsson, I.; Swaminathan, V. S.; Nordenfelt, P.; Gouras, G.; Borondics, F.; Klementieva, O. Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level. J. Med. Chem. 2023, 66 (4), 2542–2549. DOI: 10.1021/acs.jmedchem.2c01359

About the Authors

Craig Prater is Chief Technology Officer and co-founder of Photothermal Spectroscopy Corp. (PSC), where he led the development of Optical Photothermal Infrared (O-PTIR) spectroscopy and commercialization of the mIRage® infrared microscope. Previously, as CTO of Anasys Instruments, he co-invented AFM-based infrared spectroscopy (AFM-IR), enabling chemical imaging at ~10 nm spatial resolution. He spent 15 years at Digital Instruments/Veeco (now Bruker), advancing atomic force microscopy (AFM) and becoming the company’s first Technology Fellow. Dr. Prater has served as Principal Investigator on numerous federally funded projects (NIH, DOE, NSF, NIST) related to AFM-IR, O-PTIR, and related techniques. He earned his PhD in Physics from the University of California, Santa Barbara, and has authored over 140 scientific and trade publications and patents with more than 10,000 citations. He received the 2023 Williams-Wright Award for significant contributions to vibrational spectroscopy while working in industry.

Ji-Xin Cheng is a pioneering scientist, inventor, and entrepreneur whose work has significantly advanced the fields of optical imaging and molecular spectroscopy. He currently holds the Moustakas Chair Professorship in Optoelectronics and Photonics at Boston University. He also serves as Director of the BU Photonics Center Graduate Student Initiative. Cheng’s research has led to transformative innovations in chemical imaging, particularly through coherent Raman scattering microscopy, mid-infrared photothermal (MIP) imaging, and overtone photoacoustic microscopy. His work combines technology development with real-world applications in cancer diagnosis, neuromodulation, and phototherapy for infectious diseases. As an entrepreneur, Cheng is co-founder of Vibronix and Pulsethera, companies that aim to bring his laboratory innovations to commercial and clinical use. He also serves as a scientific advisor for Photothermal Spectroscopy Corp and Axorus, contributing to cutting-edge developments in imaging instrumentation and neuromodulation, respective.

Cheng has authored more than 350 peer reviewed publications and has received numerous accolades, including the Ellis R. Lippincott Award (Optica), the Pittsburgh Spectroscopy Award, and the SPIE Biophotonics Technology Innovator Award. Originally from China, Cheng earned both his BSc and PhD from the University of Science and Technology of China, followed by postdoctoral work at the University of Science and Technology of Hong Kong and Harvard University. His work has not only influenced scientific understanding but also shaped the future of optical and photothermal imaging technologies through both academic leadership and entrepreneurial vision.

Mustafa Kansiz is currently the Director of Product Management at Photothermal Spectroscopy Corp with responsibilities for new product development, marketing and applications development. He has over 25 years of experience working with FT-IR Microscopy and Imaging and Raman, spanning routine to research applications, in both industry and academia. Throughout his time, he has worked at Varian and Agilent Technologies serving in a range of technical and business development roles, including FT-IR Microscopy & Imaging Product Manager, Product Specialist, R&D Scientist and European FT-IR Sales Manager. He has a Ph.D. from Monash University on biotechnological application of FT-IR spectroscopy and multivariate statistics.