Mid-Infrared Spectroscopic Imaging (MIRSI)

Fundamental molecular vibrational modes of organic molecules occur in the mid-infrared region of the electromagnetic spectrum. These vibrational modes result in sharp (resonance) peaks in the mid-infrared and have been used to identify molecules in homogeneous bulk materials for more than four decades. The chemical significance of peaks in the “fingerprint region” of the mid-infrared spectrum has also been tabulated extensively in spectroscopy literature. The chemical composition of substances can be ascertained by analyzing their mid-infrared spectrum. Combining spectroscopy with microscopy has been a relatively recent development and this has resulted in spatially resolved spectroscopic information. With this innovation, it is possible to obtain an image of a chemically diverse sample and discover the chemical composition at every pixel in that image. This chemical information has been used in a variety of fields from forensics to art restoration to performing completely automated histopathology in tissue sections. In tissue, the molecular composition and chemical detail at every pixel can be mapped over the entire imaging area and tissue function can be studied.

Mid-infrared spectroscopic imaging is one of the most promising functional imaging tools available today. It is capable of chemical identification via pattern recognition and has been demonstrated to be successful in prostate, breast and colon cancer detection. Moreover, the biochemical basis for disease detection is also a byproduct of the analysis. Chemical or molecular changes often occur earlier than morphological changes detected by older microscopy techniques and can not only help understand disease, but can also provide an earlier detection. Reliable early detection is the key to solving several diseases including breast, colon and prostate cancer.

Photothermal Mid-infrared Spectroscopic Imaging

The Reddy lab specializes in obtaining actionable insights into chemically complex samples using spectroscopic imaging instrumentation and machine learning techniques. Our focus is primarily on technological innovations and data analytics for investigating objects’ microscopic and macroscale chemical properties. Photothermal MIRSI is a new and exciting MIRSI technology that has revolutionized biomedical sample analysis. It provides five distinct advantages over previous MIRSI techniques:

(1) The spatial resolution is 0.5 μm, which is 10X better than FTIR imaging (~5 μm). Previously inaccessible sub-micron spatial details are visualized and quantified while simultaneously enabling microscopic chemical identification from IR spectra.

(2) It can perform imaging reliably in the presence of moisture and liquids. Routine live-cell spectroscopic imaging is possible wherein cells are living in culture media.

(3) Photothermal MIRSI can obtain IR spectra and images of the surface of samples of any thickness in back-reflection mode without reflective substrates, making sample preparation easy. We are not restricted to thin slices to obtain IR transmission or reflection spectra.

(4) The field of view can be a centimeter or larger. The measurement process is non-contact and we can obtain spectra and images in a versatile, dynamic manner on the fly.

(5) We can obtain three-dimensional (3D) spectral data from up to 100 μm below the surface of samples. This enables subsurface assessment.    

Polarization-sensitive photothermal MIRSI

The Reddy lab has developed a new generation of MIRSI instrumentation with source and sample-independent polarization control that is especially useful in studying dichroic samples such as collagen fibers. We have formulated a novel data processing pipeline to reliably ascertain fiber locations and orientations on challenging clinical biopsy samples. This technique facilitates analysis of fibrous structures that exhibit dichroism such as reticulin fibers. We have also developed techniques to quantify the orientation of fibers and obtain detailed maps of fiber orientation and distribution. This work utilizes dichroic properties, tensor calculus, and polarization-sensitive photothermal MIRSI measurements for fiber analysis. Moreover, our multi-modal image processing algorithms enable the alignment and registration of MIRSI data with microscopy and other imaging modalities.

Optical Coherence Tomography

Optical Coherence tomography (OCT) is a technique that makes it possible to see deep inside an object using light without cutting the object. It is one of the most promising biomedical diagnostic tools available today. It is a non-invasive tomography technique that provides three dimensional images of tissue without sectioning or puncturing with a needle. We can use OCT for non-invasive disease diagnosis in a variety of diseases including glaucoma, Barrett’s esophagus, Celiac Disease, etc. In one of my projects as a post-doc, we are developing an optical device that is in the shape and size of a Tylenol pill or capsule. This tethered capsule is swallowed by a patient, then goes down the esophagus into the stomach. As it goes through the esophagus it performs video rate imaging of the entire esophagus at a high (~15μm) resolution. Unlike current endoscopies, it not only provides images of the surface of the esophagus, but also from deep inside the tissue. This is a central aspect of OCT. The entire procedure takes 2 to 3 minutes and does not require sedation or anesthesia. Data is processed in real time and can be seen by the doctor immediately. The doctor can examine the data and ask the patients relevant questions straightaway so as to narrow down the potential disease possibilities and arrive at more reliable conclusions.

In the diagnosis of celiac disease for example, the current standard of care is to sedate the patient, insert a needle-based device and collect small pieces of tissue from several points in the esophagus. The tissue is frozen, sliced, stained chemically and observed under a microscope. The diagnosis process takes several days to weeks. In contrast, OCT provides real time results without sedation and is significantly more reliable since we image the entire esophagus as opposed to taking samples at a small number of point (quasi “random” sampling). Such techniques are likely to improve patient care dramatically in the future.

High-Definition Imaging

Image quality in infrared spectroscopic imaging can be improved dramatically by an appropriate redesign of instruments. Theoretical insights obtained from modeling light propagation through the entire instrument have guided a design that has resulted in high-definition imaging wherein the important image details are now accessible. This has improved the accuracy of cancer detection using infrared spectroscopic imaging.

High-resolution mid-infrared spectroscopic imaging was demonstrated recently in our paper that received recognition from various scientific communities. We presented a new way of understanding and designing infrared imaging instruments based on a modular approach that utilizes an operator formalism. This technique of instrument construction provides an intimate connection between theory and experiment. There is a direct, one-to-one correspondence between an optical component (like a lens) in an experiment and an operator in the theory. Replacing one component in an experiment is equivalent to replacing one operator and vice versa. Therefore, changes to an instrument design can be understood and analyzed easily and this rapid feedback loop between theory and experiment enables the design of significantly better instruments. We demonstrated the power of these ideas by developing a high-definition mid-infrared spectroscopic imaging instrument that provides significantly higher image detail than in current commercial instruments. The figure on the left is an example of data from such an instrument where a chemical map of tissue is obtained using our proposed instrument. This provides excellent tissue detail with the press of a button without the laborious chemical stains that are normally required.

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