An official website of the United States government
This perspective highlights recent advances in super-resolution, mid-infrared imaging and spectroscopy. It provides an overview of the different near field microscopy techniques developed to address the problem of chemically imaging specimens in the mid-infrared “fingerprint” region of the spectrum with high spatial resolution. We focus on a recently developed far-field optical technique, called infrared photothermal heterodyne imaging (IR-PHI), and discusses the technique in detail. Its practical implementation in terms of equipment used, optical geometries employed, and underlying contrast mechanism are described. Milestones where IR-PHI has led to notable advances in bioscience and materials science are summarized. The perspective concludes with a future outlook for robust and readily accessible high spatial resolution, mid-infrared imaging and spectroscopy techniques.
more »
« less
Label-free imaging of subcellular features with mid-infrared photothermal microscopy
Mid-infrared photothermal imaging is a novel chemical imaging modality that combines high sensitivity with enhanced spatial resolution. Subcellular features in fibroblast cells and tissues are imaged and analyzed with regards to their molecular structures without the need of exogenous fluorophores at a resolution that overcomes the diffraction limited spot size of the mid-infrared excitation beam. With a phase-sensitive lock-in detection scheme, changes in the thermal diffusion properties can be detected and can provide a complementary sample characterization.
more »
« less
- Award ID(s):
- 1846659
- PAR ID:
- 10209052
- Editor(s):
- Liu, Zhiwen; Psaltis, Demetri; Shi, Kebin
- Date Published:
- Journal Name:
- SPIE Optical Engineering + Applications, Ultrafast Nonlinear Imaging and Spectroscopy VIII
- Issue:
- 114970Y
- Page Range / eLocation ID:
- 32
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
An approach is described for spectrally parallel hyperspectral mid-infrared imaging with spatial resolution dictated by fluorescence imaging. Quantum cascade laser (QCL)-based dual-comb mid-infrared spectroscopy enables the acquisition of infrared spectra at high speed (<1 millisecond) through the generation of optical beat patterns and radio-frequency detection. The high-speed nature of the spectral acquisition is shown to support spectral mapping in microscopy measurements. Direct detection of the transmitted infrared beam yields high signal-to-noise spectral information, but long infrared wavelengths impose low diffraction-limited spatial resolution. The use of fluorescence detected photothermal infrared (F-PTIR) imaging provides high spatial resolution tied directly to the integrated IR absorption. Computational imaging using a multi-agent consensus equilibrium (MACE) approach combines the high spatial resolution of F-PTIR and the high spectral information of dual-comb infrared transmission in a single optimized equilibrium hyperspectral data cube.more » « less
-
Mid-infrared microscopy is an important tool for biological analyses, allowing a direct probe of molecular bonds in their low energy landscape. In addition to the label-free extraction of spectroscopic information, the application of broadband sources can provide a third dimension of chemical specificity. However, to enable widespread deployment, mid-infrared microscopy platforms need to be compact and robust while offering high speed, broad bandwidth, and high signal-to-noise ratio. In this study, we experimentally showcase the integration of a broadband, high-repetition-rate dual-comb spectrometer (DCS) in the mid-infrared range with a scanning microscope. We employ a set of 1-GHz mid-infrared frequency combs, demonstrating their capability for high-speed and broadband hyperspectral imaging of polymers and ovarian tissue. The system covers 1000 cm−1 at νc = 2941 cm−1 with 12.86 kHz spectra acquisition rate and 5 µm spatial resolution. Taken together, our experiments and analysis elucidate the trade-off between bandwidth and speed in DCS as it relates to microscopy. This provides a roadmap for the future advancement and application of high-repetition-rate DCS hyperspectral imaging.more » « less
-
Collagen quantity and integrity play an important role in understanding diseases such as myelofibrosis (MF). Label-free mid-infrared spectroscopic imaging (MIRSI) has the potential to quantify collagen while minimizing the subjective variance observed with conventional histopathology. Infrared (IR) spectroscopy with polarization sensitivity provides chemical information while also estimating tissue dichroism. This can potentially aid MF grading by revealing the structure and orientation of collagen fibers. Simultaneous measurement of collagen structure and biochemical properties can translate clinically into improved diagnosis and enhance our understanding of disease progression. In this paper, we present the first report of polarization-dependent spectroscopic variations in collagen from human bone marrow samples. We build on prior work with animal models and extend it to human clinical biopsies with a practical method for high-resolution chemical and structural imaging of bone marrow on clinical glass slides. This is done using a new polarization-sensitive photothermal mid-infrared spectroscopic imaging scheme that enables sample and source independent polarization control. This technology provides 0.5 µm spatial resolution, enabling the identification of thin (≈1 µm) collagen fibers that were not separable using Fourier Transform Infrared (FT-IR) imaging in the fingerprint region at diffraction-limited resolution ( ≈ 5 µm). Finally, we propose quantitative metrics to identify fiber orientation from discrete band images (amide I and amide II) measured under three polarizations. Previous studies have used a pair of orthogonal polarization measurements, which is insufficient for clinical samples since human bone biopsies contain collagen fibers with multiple orientations. Here, we address this challenge and demonstrate that three polarization measurements are necessary to resolve orientation ambiguity in clinical bone marrow samples. This is also the first study to demonstrate the ability to spectroscopically identify thin collagen fibers (≈1 µm diameter) and their orientations, which is critical for accurate grading of human bone marrow fibrosis.more » « less
-
Mid-infrared Spectroscopic Imaging (MIRSI) provides spatially-resolved molecular specificity by measuring wavelength-dependent mid-infrared absorbance. Infrared microscopes use large numerical aperture objectives to obtain high-resolution images of heterogeneous samples. However, the optical resolution is fundamentally diffraction-limited, and therefore wavelength-dependent. This significantly limits resolution in infrared microscopy, which relies on long wavelengths (2.5 μm to 12.5 μm) for molecular specificity. The resolution is particularly restrictive in biomedical and materials applications, where molecular information is encoded in the fingerprint region (6 μm to 12 μm), limiting the maximum resolving power to between 3 μm and 6 μm. We present an unsupervised curvelet-based image fusion method that overcomes limitations in spatial resolution by augmenting infrared images with label-free visible microscopy. We demonstrate the effectiveness of this approach by fusing images of breast and ovarian tumor biopsies acquired using both infrared and dark-field microscopy. The proposed fusion algorithm generates a hyperspectral dataset that has both high spatial resolution and good molecular contrast. We validate this technique using multiple standard approaches and through comparisons to super-resolved experimentally measured photothermal spectroscopic images. We also propose a novel comparison method based on tissue classification accuracy.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.2568744
