FT-IR Microscopy with High Spatial Resolution

Advances in spatial resolution for Fourier transform infrared (FT-IR) imaging historically have involved the use of a synchrotron source, but new optics have been developed that yield better spectral quality and spatial resolution than are provided by existing synchrotron sources. Kathleen Gough, Professor in the Department of Chemistry at the University of Manitoba, has been working with her group to conduct diagnostic tissue imaging with the new thermal source FT-IR system. She recently spoke to us about these efforts.

Your group has been working on an approach for imaging with FT-IR microscopy in which high numerical aperture objectives and new magnification optics, along with a thermal source spectrometer, provide imaging capabilities comparable to the highest synchrotron source imaging capability yet demonstrated (1). How does the spatial resolution obtained using this approach compare with that of previous FT-IR microscopy techniques, and what is the significance of this advancement (with respect to future practical applications, for example)?

FT-IR microscopy has a long history. The first IR microscopes were built over 60 years ago, and were designed for dispersive infrared spectrometers. These pioneers managed some amazing results but, as with most infrared work, nothing took off until FT-IR entered the mainstream. Detection limits for far-field infrared are governed by the wavelength-dependent Abbe limit, but the first instruments did not begin to approach this. Generally, there was a single-element detector, and the target region was set by adjusting a physical aperture. If the sample was situated on a motorized stage, an area could be mapped by raster scanning, collecting individual spectra at each pixel. The pixels gradually decreased in size as the technology improved. Eventually, a focal plane array (FPA) of microdetectors became commercially available, enabling the collection of a single image comprising 16 x 16, 32 x 32, 64 x 64 or 128 x 128 pixels. Motorized stages enabled collection of a mosaic of tiles for rapid acquisition of large-area images. An FPA-based instrument was installed at the (now decommissioned) Synchrotron Radiation Center in Madison, Wisconsin, at the IRENI beam line. The high numerical aperture optics and the brilliant synchrotron source combined to allow pixels with a nominal geometric resolution of 0.54 µm. This facility allowed extraordinary imaging capability (1,2), and thus a spectrochemical window onto subcellular chemistry of organelles, cancerous tissues, and stem cells, as well as the composition of layered polymers and other materials. Shortly after this, thermal source FT-IR microscopes, as opposed to synchrotron source FT-IR microscopes, achieved similar capability, by means of additional and modified optics.

To address the second part of your question, the significance is enormous. Infrared spectroscopy provides unique chemical information, in contrast to many other imaging modalities. For example, fluorescence imaging is typically restricted to identification of those specific targets that have been tagged with a reporter molecule, while infrared imaging provides a full chemical image of all the functional groups present. This is extremely important for basic research not just into disease, but also for any materials research. The rich chemical information in the IR spectra can be analyzed in myriad ways with statistical methods tailored to the problem, and it can be combined with results from other methodologies. Many groups are developing applications for spectropathology with the intent of achieving protocols that could be employed in a clinical setting or in a pathology laboratory. (See my answer to the final question.)

How does the modified FT-IR microscopy system differ from a standard system?

Objectives and condensers with high numerical aperture are essential. The thermal source IR microscope has a novel means of increasing the magnification (5x increase) by using extra mirrors within the microscope rather than relying solely on the objective for magnification. Motorized software-controls allow switching of mirrors between standard and high magnification without changing the condenser and objective setting, while maintaining a reasonable working distance of at least 12 mm. The end result is that spectrochemical images can be obtained with a thermal source-that is, in house-at a spatial resolution that is also compatible with the diffraction limit (1). We have even been able to develop 3D FT-IR tomographic imaging, previously achieved only at IRENI. The highest setting on our IR microscope involves 25x objective and condenser with 0.81 NA with which we can attain 0.66 µm pixels. As with the IRENI data, images are oversampled, to the point where spatial image restoration techniques may be applied.

Does the thermal source system have any limitations or disadvantages compared to a synchrotron-based system?

This is a completely subjective response, but I would have to say almost none. An advantage of being at a synchrotron is that there are many other beam lines, and that with some planning, correlative data can be acquired, which can be very powerful. Moreover, the IRENI end station offered the best far-field spatial resolution ever, with over-sampling even at the shortest wavelengths, and rapid data collection owing to the brilliance of the synchrotron illumination. My experience in conducting synchrotron IR imaging has been rewarding and productive. However, it has always required the investment of considerable funds and personnel for travel and data collection, both of which are important considerations in planning research projects. I was extremely fortunate to be able to collaborate with Professor Carol Hirschmugl at the IRENI beam line, where we obtained some fantastic images and were able to gain greater insight into the composition of plaques in Alzheimer-diseased (AD) brain tissue and into the substructure of Arctic sea ice diatoms, two important lines of research in my group. The disadvantage was that data were collected in a limited time frame, and it was not possible to run back into the lab the next week to double check a sample for which the data were noisy or replicates were needed, or different regions had to be imaged-all the usual things that are part of day to day research. The spatial resolution now achievable with a thermal source is essentially as good as at IRENI, given that a practical limit is on the order of 1 µm, with greater convenience for switching between magnification levels and much better working distance. Having the capability to do this kind of research in house, without the constraints of time and travel, is invaluable.

In another study, your group used FT-IR polarization contrast imaging to examine mechanically damaged mammalian tendon(3). What are the advantages of this approach compared with other approaches for analyzing the structure and organization of collagenous materials? How well does it detect mechanical damage and disorder within individual tendons?

Collagenous materials are incredibly important, as they comprise some 30–40% of all proteins in the human body. There are over 20 types of collagen, performing structurally critical roles in obvious body parts like skin, cartilage, tendon, and bone, and equally critical but less well known roles as basement membranes in many organs and interstitial tissues in muscles. The molecular unit of collagen Type I, known as tropocollagen, is a long, ordered polymeric-like unit, only 1.5 nm in diameter. These units are bundled into fibers that retain the molecular orientation properties at much greater length scales (micrometer and millimeter). The organization of these macroscopic units is variable and is determined by the roles that they play. Polarization contrast imaging with different microscopies is regularly used to assess the order, or lack thereof, in mammalian tissues. The gold standard is a long-used histochemical stain, picrosirius red; observation of stained tissue under polarized visible light allows for an assessment of the tissue organization. Polarization contrast with infrared is easily done by inserting a polarizer into the path of the light and is a standard technique for examining order in all sorts of materials, such as industrial polymers, for example. As described in our paper (3), we find that the beauty of using it to examine tendon sections, especially with our high magnification optics, is that we can rapidly detect the molecular order in the tendon at the typical fiber dimension of 1 µm. Our FT-IR imaging is done on snap-frozen, unfixed, desiccated tissue sections without any staining, one of the major advantages of the technique. Apart from drying the tissue, nothing has been added or removed and we see the entire spectrochemical signature. Light polarized parallel to the fiber excites chain modes, such as the C-N stretch, while light polarized perpendicular to the fiber excites the carbonyl groups that are roughly perpendicular to the chain direction. The degree of order can be read directly from the relative intensity of these absorption bands. The sample preparation is very simple, plus we see all the chemistry in the section. We can see location of cells within the tissue, another important feature, potentially very relevant to tissue inflammation and to tissue repair.

What is the potential of using the detailed information provided by this technique for spectropathology or even clinical applications?

I can give you a long answer, or I can direct you to a couple of extremely useful websites, namely those for The International Society for Clinical Spectroscopy (clirspec.org) and Raman4clinics (raman4clinics.eu). These two vibrant international research communities share a common goal of bringing vibrational spectroscopic techniques into the mainstream of spectropathology, clinical diagnostics, and even operating theaters. Suffice it to say that many scientists are working to bring the rich information available with this technology into the practical world of health care. As the web sites attest, they are well on the way and the future is very promising

What are the next steps in your research?

For my group, we are continuing to use the high-magnification thermal source FT-IR to study brain tissue from AD and transgenic mouse models, seeking molecular insight into this disease. Collagen studies are ongoing on several tissues, including mechanically damaged tendon, the basement membrane of organs such as kidney, and scar tissue in heart disease. We are pursuing the exciting opportunity to study autecology of diatoms to better understand community interactions of the important taxa in the Arctic marine food web, particularly relevant with regard to global climate change. The high spatial resolution thermal source system can be used to collect tomographic data sets for samples that are on the order of 10–20 µm in diameter; this includes diatoms, synthetic polymer threads, and a few other things. Finally, we are actively pursuing near-field IR techniques (3), by which we can probe regions as small as 20 nm-this is tremendously exciting as the research can be done in a correlative manner along with super-resolution fluorescence imaging.

References

• C.R. Findlay, R. Wiens, M. Rak, J. Sedlmair, C.J. Hirschmugl, J. Morrison, C.J. Mundy, M. Kansiz, and K.M. Gough, Analyst140, 2493–2503 (2015).

• C. J. Hirschmugl and K. M. Gough, Applied Spectrosc. 66, 475–491 (2012).

• R. Wiens, C.R. Findlay, S.G. Baldwin, L. Kreplak, J.M. Lee, S.P. Veres, and K.M. Gough, Faraday Discuss.187, 555–573 (2016).