Spectral imaging is an extremely powerful analytical tool that combines chemical and morphological information to identify and quantify the distribution of molecular species. A wide range of fields, including biology, forensics, and materials science, regularly use infrared (IR) microscopes to generate spectral images without labeling. The origin of the IR microscope dates back to 1949, when a reflection-based microscope was fitted with a single-beam dispersive IR spectrometer.1 For much of the next 30 years, IR microscopes were little more than a scientific novelty. It wasn’t until 1977 that scientists paired a Fourier Transform Infrared Spectrometer (FTIR) with a microscope.2 that they have started to produce analytical quality data.

Over the following decades, FTIR microscopy quickly became the method of choice for microchemical analysis. Although there have been many incremental changes to the technology, until recently there has been little change to the fundamental methodology behind IR microscopes.

Fundamentals of IR Microscopy Design

Most FTIR microscopes are designed to operate in the mid-infrared region (mid-IR) of the spectrum, from 2.5 to 25 m (4000 to 400 cm-1) because it directly corresponds to the fundamental vibrational frequencies of covalently bound molecules. In many ways, this makes Medium IR absorption spectroscopy the simplest molecular spectroscopy since it is a direct measurement technique. At the same time, the mid-IR region presents significant optical challenges because the wavelength range is so wide and the photon energies are so low.

The traditional FTIR approach uses an off-axis parabolic mirror to collimate a broadband thermal emission source, called a globar, and send it through a scanning interferometer. The sample branch of the interferometer includes a reflective condenser to illuminate the sample and a Schwarzschild (reflective) objective to recollify the transmitted light. Finally, the light is measured with an IR photodetector, and Fourier transform to reconstruct the transmission spectrum (see Fig. 1). Despite all this complexity, the robust nature of this technique always fueled the commercial success of FTIR microscopy until the early 2000s. However, as other microspectroscopy techniques, such as confocal Raman, became more common. readily available, the comparative performance of IR microscopy began to decline.

Now it looks like IR microscopy is in the midst of yet another revolutionary due to the commercial availability of QCL between 3 and 13 m (3300–770 cm-1). Not only are modern QCLs tunable over a frequency range of several hundred wavenumbers with linewidths of -1, but they are also inherently spatial single-mode and linearly polarized. As a result, replacing the globar with one QCL bank (usually four) improves the optical performance of the microscope and completely eliminates the need for an interferometer. Hence, this led to the development of a whole new form of IR microscopy, often referred to as Discrete Frequency Infrared (DFIR) or Direct Infrared Laser (LDIR) microscopy. Although this technology is relatively new, there are already a handful of commercially available QCL-based microscopes from companies such as DRS Daylight Solutions (San Diego, CA) and Agilent (Santa Clara, CA).

In April 2021, Dr. Rohit Bhargava, IR microscopy expert at the University of Illinois at Urbana-Champaign (Urbana, IL), along with two of his graduate students, Yasmua Phal and Kevin Yeh, published an extremely detailed review of QCL – IR microscopes enabled.4 In this article, they discussed the technical considerations that should be taken into account when choosing the right laser, detector, and optics for a DFIR microscope.

The advantage of QCL imaging

Perhaps the most important benefit of using QCL instead of broadband light sources for IR microscopy is the spectral irradiance at the sample level. Even though typical silicon nitride globes produce several watts of optical power, it is distributed over a relatively large solid angle and an even wider spectral range. In contrast, typical QCLs produce hundreds of milliwatts of average power at a divergence angle of a few milliradians. Therefore, it’s no wonder that QCL-based IR microscopes can acquire data exponentially faster than traditional microscopes. FTIR microscopes. In a typical DFIR system, the limiting factor on sample acquisition is defined by the sample damage threshold.

A vivid example of the difference between QCL and FTIR microscopes was demonstrated by Claus Kuepper, Angela Kallenbach-Thieltges and others at the Ruhr University in Bochum (Bochum, Germany) – they evaluated the use of the IR microscopy based on QCL as a means of cancer diagnosis. .5 In their study of 110 colorectal cancer patients, published in Nature‘s Scientific reports, they managed to show a sensitivity of 96% and a specificity of 100% with respect to histopathology. But according to the authors, “the main obstacle to the clinical translation of IR imaging is now overcome by the short acquisition time for high quality diagnostic images”.

In the additional material,6 they showed a comparison of a gold standard to hematoxylin and eosin (see Fig. 2a), an image taken with a Spero QT QCL IR microscope from DRS Daylight Solutions (see Fig. 2b) and a microscope. FTIR (see Fig. 2c). The two images made it possible to identify tumor regions and infiltrating inflammatory cells; what is important to emphasize is that the QCL-based system was able to measure the sample in 34 minutes. In contrast, the FTIR image took approximately 5,400 minutes.FIGURE 2. Comparison of IR-based tissue analysis. Analysis of colorectal cancer tissue using hematoxylin and eosin stain (a), QCL-based microscope (b), and FTIR-based microscope (c). [6]

In a 2017 article published in Analyst, Bhargava’s group showed similar results when studying polymer samples using IR microscopes based on FTIR and QCL.7 Using an Agilent QCL-based prototype microscope, they measured polyethylene glycol (PEG) films using both s– and p-polarized light to determine macromolecular orientation successfully. For comparison, the FTIR system took 1620 minutes to collect the spectral image, while the QCL-based system took only 9 minutes.

To look forward

Bhargava, Phal and Yeh summarized it best in the conclusion of their recent review article: “… technology has pushed IR spectroscopic imaging beyond previously predicted limits. While there are still significant financial hurdles associated with integrating a bank of four QCLs into an IR microscope, the benefits of IR spectral imaging are clear. As QCL’s production continues to improve over time, there is no doubt that prices will continue to decline as capacity increases.

RECOGNITION

The author would like to thank Ellen Miseo, Chief Scientist of TeakOrigin and current President of the Coblentz Society, for her contribution on the current state of QCL-based IR microscopy.

THE REFERENCES

1. R. Barer, A. Cole and HW Thompson, Nature, 163, 4136, 198-201 (1949).

2. R. Cournoyer, JC Shearer and DH Anderson, Anal. Chem., 49, 14, 2275-2277 (1977).

3. See http://bit.ly/IRMicrospectroscopy.

4. R. Bhargava, YD Phal and K. Yeh, Appl. Spectrosque., 00037028211013372 (2021).

5. C. Kuepper et al., Sci. representative, 8, 1, 1-10 (2018).

6. C. Kuepper et al., Sci. representative, 8, 1, supplement (2018).

7. TP Wrobel, P. Mukherjee and R. Bhargava, Analyst, 142, 1, 75-79 (2017).



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