Discrete Frequency Infrared Microspectroscopy and Imaging with a Tunable Quantum Cascade Laser

Kole, Matthew R.; Reddy, Rohith; Schulmerich, Matthew V.; Gelber, Matthew K.; Bhargava, Rohit

doi:10.1021/ac302513f

Cited by 103 publications

(105 citation statements)

References 42 publications

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“…At present, wide-field scanning of a sample is possible in seconds, providing tens of thousands of spectra. A variety of choices are available for the IR source, including globar 7 , synchrotron 8-12 and quantum-cascade lasers (QCLs) 13 , as well as for the detector (2D FPA, linear array or single element) 14 . The three major IR-spectroscopic sampling modes (Fig.…”

Section: Introductionmentioning

confidence: 99%

Using Fourier transform IR spectroscopy to analyze biological materials

et al. 2014

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IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

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Section: Introductionmentioning

confidence: 99%

Using Fourier transform IR spectroscopy to analyze biological materials

et al. 2014

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“…43 In the future, we plan to deconvolute the 2D IR images, but for now we experimentally scramble the spatial coherence using a rotating diffuser, as reported previously. 35,44 …”

Section: Resultsmentioning

confidence: 99%

Spatially Resolved Two-Dimensional Infrared Spectroscopy via Wide-Field Microscopy

et al. 2016

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We report the first wide-field microscope for measuring two-dimensional infrared (2D IR) spectroscopic images. We concurrently collect more than 16 000 2D IR spectra, made possible by a new focal plane array detector and mid-IR pulse shaping, to generate hyperspectral images with multiple frequency dimensions and diffraction-limited spatial resolution. Both frequency axes of the spectra are collected in the time domain by scanning two pairs of femtosecond pulses using a dual acousto-optic modulator pulse shaper. The technique is demonstrated by imaging a mixture of metal carbonyl absorbed polystyrene beads. The differences in image formation between FTIR and 2D IR microscopy are also explored by imaging a patterned USAF test target. We find that our 2D IR microscope has diffraction-limited spatial resolution and enhanced contrast compared to FTIR microscopy because of the nonlinear scaling of the 2D IR signal to the absorptivity coefficient for the vibrational modes. Images generated using off-diagonal peaks, created from vibrational anharmonicities, improve the molecular discrimination and eliminate noise. Two-dimensional wide-field IR microscopy provides information on vibrational lifetimes, molecular couplings, transition dipole orientations, and many other quantities that can be used for creating image contrast to help disentangle and interpret complex and heterogeneous samples. Such experiments made possible could include the study of amyloid proteins in tissues, protein folding in heterogeneous environments, and structural dynamics in devices employing mid-IR materials.

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“…The kidney tissue example presented in this paper demonstrates the importance of higher spatial resolutions in order to extract biochemical information from glomerular structures (Figure 3 and Figure 5). In the future, new advances in Quantum Cascade Lasers as very bright IR light sources [54][55][56][57] , 3D spectral imaging 58 , and breakthroughs in the field of nanoscale IR technologies 52,53,59,60 hold exciting new avenues of research that may have huge implications in the future of tissue imaging.…”

Section: Representative Resultsmentioning

confidence: 99%

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Sreedhar

Varma

Nguyen

et al. 2015

JoVE

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High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.

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Discrete Frequency Infrared Microspectroscopy and Imaging with a Tunable Quantum Cascade Laser

Cited by 103 publications

References 42 publications

Using Fourier transform IR spectroscopy to analyze biological materials

Using Fourier transform IR spectroscopy to analyze biological materials

Spatially Resolved Two-Dimensional Infrared Spectroscopy via Wide-Field Microscopy

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

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