Using Real-Time Infrared Chemical Imaging in Mass Spectrometry Tissue Analysis

Mass spectrometry (MS) is an analytical technique, which can be employed in studies involving the determination of the composition of complex biochemical samples having high degrees of specificity.

This technique has become the gold standard in the field of Life Sciences, which include proteomics, metabolomics, and toxicology. This is due to the specificity and ability of MS to quantify minute quantities of hormones, disease biomarkers, and metabolites.

Challenges to the adoption of mass spectrometry

Though MS is being widely accepted, there exist a significant number of challenges that hinder its adoption into clinical and research laboratories.

The most obvious barriers include low throughput and low spatial resolution while performing routine analysis of large heterogeneous samples like excised tissue, wherein the context of the anatomical structure of the sample, measuring from microns up to millimetres, holds major significance.

Mass spectrometry imaging (MSI)

Techniques using MSI like matrix-assisted laser desorption ionization (MALDI) and secondary ion mass spectrometry (SIMS) are being widely used in pharmaceutical histology and clinical laboratories. The simultaneous achievement of high-throughput and high-spatial resolution that may be required for the usual applications is not feasible.

Owing to the destructive and serial nature of the process of data collection, images of MS are created using low resolving power and this is followed by the degradation of the sample using high beam exposure.

In order to make MS imaging a more viable option, the local regions of concern are to be pre-selected by well-qualified personnel. The sliced tissue that is to be examined or its adjacent section that is parallel to it must be stained and analyzed using a standard microscope before conducting MS analysis.

However, the staining process using chemicals tends to change the sample’s biochemistry by eliminating important biochemical constituents that are targeted, and moreover the sections that are taken from the parallel tissues are never similar.

Further, techniques involving MS call for advanced skills, so that only staff with training at the doctoral level would be able to use the system and test the results. A tool that will facilitate non-destructive and high resolution screening is required.

This user-friendly tool must be able to scan large tissue areas to recognize the target areas for follow-on MSI without involving the necessity of a staining protocol. One instrument that can be used for such applications is the Spero microscope designed by Daylight Solutions.

IR and MS imaging are both providing a chemical view of tissue contents, the first being based on the IR light absorption from molecular bonds and the latter providing mass information from the same molecules. In principle, beyond their specificities and beyond little comparable technicalities, they should be considered as equivalent. In practice, they cannot be considered as equivalent.

MS imaging is clearly more difficult to operate and the interpretation of the data cannot be automated. Even worse, it usually requires isotopic labels to discriminate between mass-to-charge ratios and values and thus to identify chemical compounds in the sample. As a result, this technique is not still deployed for routines and remains the business of highly qualified staffs.

In comparison, IR microscopy was suffering from its slowness. Now, that ultrafast acquisitions have been made accessible, the next bottleneck to remove is the automation of data treatments; but, thanks to its robustness and to the uselessness to use reagents or labels, IR imaging will be soon more standardisable and turned into a routine chemical analytical technique. A technique that is now fast and simple is surely promised to great future in pharmacology and biosciences."

Dr. Petibois, head of research group “3D spectro-imaging” at Inserm U1029 and recent interview contributor to the Insights from Industry series

The Spero microscope

The Spero microscope facilitates the spatial distribution of functional chemical groups in tissues and cells in real-time. This paves the way for quick generation of whole-side quantitative maps of an array of biomolecules, which include lipids, proteins, nucleic acids, and collagen, without the need for the staining process.

Fresh frozen unstained sections of tissue as well as samples fixed with formalin can be analyzed using Spero. However, fresh frozen unstained tissue samples can preserve their lipid profile.

The detailed information given by Spero helps to easily identify the target regions for subsequent MSI or other similar molecular imaging methods automatically through pre-loaded algorithms or through a trained technician’s visual inspection. Since this is a non-destructive, label-free technique, the sample tissue retains its pristine condition for further analysis using standard staining procedures.

The Spero microscope employs mid-infrared absorption spectroscopy technique, which produces digitally stained, high-contrast images of cells, biofluids, and tissues. This is done by measuring the relative absorption of light at frequencies of resonant vibration of a variety of functional chemical groups like carbonyl (C=O) and ammonia (NH3) present in biological substances.

The speed and high resolution of the Spero microscope are attributed to a special kind of laser known as Quantum Cascade Laser (QCL), which was also designed by Daylight Solution. QCL’s optical frequency can be tuned rapidly to the frequency range for vibrational resonances of functional groups that are biochemically relevant. This frequency range is known as the “molecular fingerprint band”, which ranges from 900 to 1800 wavenumbers (cm-1).

Spero-Based Research

Spero is used in the following applications:

  • Chemical images of the amide I peak vibrational resonance relating to proteins for a 4.8cm2 sample area containing 200 needle biopsy cores of unstained breast tissue, can be produced within 9 minutes at a 1µm pixel resolution.
  • The high-throughput multiplexing capability of the Spero microscope has been found to be useful in rapid screening of serum droplet arrays along with TMAs and large sections of tissues. Spero can be applied to micro-well or pre-screen range of droplets prior to analysis using MS.
  • Spero can be used in histology laboratories as a workhorse tool to enhance the throughput and success rate of MSI analysis.

Conclusion

As Spero imaging technology is increasingly becoming a part of the workflow of standard analysis in clinical research and pharmaceutical laboratories, it can be anticipated that output and utilization of imaging technologies using mass spectrometer will become considerably greater, with increased saving of valuable time and resources.

Acknowledgements

Produced from materials originally authored by Dr Jeremy Rowlette, Director of Molecular Imaging, Daylight Solutions.

References

  1. P. Bassan, M. J. Weida, J. Rowlette and P. Gardner, Large scale infrared imaging of tissue micro arrays (TMAs) using a tunable Quantum Cascade Laser (QCL) based microscope, Analyst (RSC), 2014, DOI: 10.1039/c4an00638k
  2. G. Clemens, B. Bird, M.Weida, J. Rowlette and M. J. Baker, Quantum cascade laser-based mid-infrared spectrochemical imaging of tissues and biofluids, Spectroscopy Europe, 2014, Vol 26, no 4.
  3. C. Hughes, G. Clemens, B. Bird, T. Dawson, K. Ashton, M. Jenkinson, A. Brodbelt, M. Weida, E. Fotheringham, M. Barre, J. Rowlette, and M. Baker. Introducing discrete frequency infrared technology for high-throughput biofluid screening. Scientific Reports 6, 20172 (2016). DOI:10.1038/srep20173

About Daylight Solutions

Daylight Solutions’ molecular detection and imaging products consist primarily of lasers, sensors, and imaging systems, all of which leverage the company’s mid-infrared, quantum cascade laser (QCL) technology. This core technology provides a versatile platform from which new products are developed, allowing the company to serve markets that include Scientific Research, Life Sciences, Defense, and Commercial.

The company is committed to innovation and introduced the world’s first broadly tunable mid-infrared laser system for scientific research, the world’s first semiconductor-based laser for protecting aircraft against shoulder-fired missiles, and the world’s first mid-infrared laser-based microscope for real-time biochemical imaging and material analysis.

Daylight Solutions consists of two separate business units. In 2009 the company created a wholly owned subsidiary to address the specific requirements of the defense industry.  As a subsidiary, Daylight Defense developed the business and manufacturing infrastructure necessary to deliver classified, military-hardened products to the government. The Commercial business unit supports all other non-defense activities ranging from life sciences to industrial and consumer products.

Daylight Solutions and Daylight Defense are both ISO-9001 certified and possess advanced manufacturing capabilities.


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Last updated: Jan 11, 2019 at 8:34 AM

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