Mid-infrared, label-free spectroscopic imaging
In order to quantitatively observe the chemical and morphological structure of a heterogeneous sample without destroying it, mid-infrared (mid-IR) spectroscopic imaging can be used. Also known as chemical imaging, mid-IR is a strong label-free technique which uses no fluorescent tags, dyes or stains. Through a single, swift measurement, mid-IR reveals the existence of chemical species, as well as their exact location and quantity.
During the last 20 years, mid-IR chemical imaging has been frequently and successfully used in a variety of applications in both industry and research. Despite the success, it was not until fairly recently that technology has advanced enough to enable the technique to produce chemical images which are both precise and fast enough for the robust analysis of biological tissue1.
Disease diagnosis using chemical imaging
In order to label certain biomolecules or to determine different cell types for diagnostic purposes, traditional histopathology requires the staining of tissue samples. This process is destructive because it deeply changes the specimen’s chemical structure, which then impedes the subsequent molecular analyses of the tissue area. This is exceptionally important during the pre-clinical phase of drug development, when it is imperative to submit a single tissue section to a number of analyses. Additionally, in situations such as surgery it can be too time consuming for a pathologist to stain a biopsied section and then visually inspect it using a traditional microscope.
Distinct biomolecules in a sample absorb parts of the infrared spectrum in diverse ways, and mid-IR chemical imaging uses this to its advantage. Different chemical signatures can be observed in healthy compared to diseased tissues. Consequently, the spatial differentiation between healthy and unhealthy tissue is feasible without any staining. As a result, the specimen is both chemically and structurally untouched for subsequent genomic or proteomic analyses.
In addition, mid-IR chemical imaging has been utilized to discern grade and disease sub-type. Thus, the preparation and examination of a sample can be performed faster, which allows a diagnosis to be reached more speedily—in an operating theater, for example.
Chemometric analysis software can be used in order to automate the analysis. Such software is easily accessible, and it helps the pathologist to establish a diagnosis 2. Additionally, the technique gives more data with regard to the metabolic state of cells, which may not be accessible using conventional staining techniques.
Pathologists still face challenges when interpreting the stained images of liver biopsies in order to diagnose possible liver disease and its extent. In the process of imaging, it is not always simple to make the distinction between healthy liver cells and those affected by scarring (fibrosis) due to recurrent damage caused by certain diseases (alcohol, hepatitis or toxins, for example). However, it is of great importance to differentiate between the two because fibrosis can cause the loss of liver function (cirrhosis) or even cancer. Cirrhosis can occur in different ways and is thus connected with numerous distinct chemical signatures, this makes it hard to ascertain the seriousness of the damage and make a likely prognosis.
Diabetes and other comorbid conditions make the process of obtaining chemical signatures even more complex. As a result, a number of variables need to be taken into account when extracting useful diagnostic or prognostic data from chemical imaging or liver biopsies. There is hope that recent improvements in the field of quantum cascade laser-based infrared spectroscopic imaging can help.
Quantum cascade laser infrared spectroscopic imaging
As the source of light, quantum cascade laser infrared spectroscopic imaging utilizes a quantum cascade laser (QCL) that can be tuned broadly. Such a setup makes high-SNR discrete-frequency imaging at video rates possible 3.
The fact that the precise targeting of a particular spectral frequency can be made means that the imaging can be focused on the desired tissue features, which can then be identified more readily in real-time. This becomes especially interesting when attempting diagnosis from a liver biopsy, because it makes it possible to more easily draw a distinction between the various structural features of the liver.
In addition, being able to choose the frequency used to analyze a sample makes it possible to obtain manageable sets of data from spectroscopic imaging of large samples. In the past, the size of the datasets acquired from the spectroscopy of a large sample was too unwieldy to make an analysis feasible. In the case of a 192 mm2 colon tissue section that is obtained using QCL infrared spectroscopy, the resulting image is composed of 11 million pixel spectra and is smaller than 1GB in size3.
Contemporary research on liver biopsies that used this new technique was able to distinguish between healthy hepatocytes and fibrotic tissue4. Furthermore, it has shown how the chemical signatures of these cells are altered based on the patient’s diabetic status4. In addition, QCL-infrared spectroscopic imaging determined specific chemical signatures correlating with different severities of liver disease. This new technique might be viewed as a breakthrough in the diagnosis and research of liver disease.
The Spero® QCL imaging system
Spero® (Daylight Solutions) is the first laser-based infrared microscope in the world, and the only one that is commercially available. The small desktop microscope comes equipped with the advanced QCL-infrared spectroscopy technology5. The tunable laser light is ultra-bright, providing simultaneous high-resolution and a wide field of view. The combination of these features makes it possible to obtain a rapid through-put of samples, which can drastically increase the speed of sample screening. Furthermore, Spero includes a live mode that makes it possible to obtain real-time imaging. A full spectral scan can be obtained in seconds using the full data collection mode.
Infrared spectroscopy based on QCL has extended the potential of using the imaging technique in pathological analysis. Compared to conventional infrared spectroscopy, it has allowed greater detail to be acquired in only a fraction of the time. In a field as dynamic as this one, the described state-of-the-art imaging technology will most certainly become a priceless asset in a variety of applications.
- Lyra da Cunha MM, Trepout S, Messaoudi C, et al. Overview of chemical imaging methods to address biological questions. Micron 2016;84:23–36.
- Fernandez DC, Bhargava R, Hewitt SM, et al. Infrared spectroscopic imaging for histopathologic recognition. Nature Biotechnology 2005;23:469‑474
- Daylight Solutions Application Note 201602. Available at http://www.daylightsolutions.com/assets/006/5629.pdf
- Sreedhar H, Varma VK, Gambacorta FV, et al. Infrared spectroscopic imaging detects chemical modifications in liver fibrosis due to diabetes and disease. Biomed Opt Express 2016; 7(6):2419‑2424.
- Daylight Solutions website. http://www.daylightsolutions.com/life-sciences/spero.htm. Accessed 29 November 2016