Light comes in various forms and spans a wide spectrum of wavelengths. The act of seeing involves the interaction of matter and light. The lens in a human eye refracts visible light to be focused onto the retina and provides vision, but not all light can be seen. Infrared (IR) light consists of electromagnetic radiation with wavelengths longer than visible light, making it invisible to the human eye. Microscopy generally describes the use of microscopes to view samples far too small to be seen with the naked eye. IR microscopy studies the interaction of matter with IR light and is commonly utilized for chemical analysis and imaging of samples.
An IR microscopy system is composed of three major components: an optical microscope, an infrared detector, and a Fourier Transform Infrared (FTIR) spectrometer. FTIR enables IR spectroscopy where a collimated beam of IR light is directed at the sample.
The sample absorbs this light at specific frequencies matching the functional groups it is composed of and indicating its chemical composition. FTIR provides this data as an IR spectrum displaying the magnitudes of absorption at various frequencies. The IR detector and optical microscope work in conjunction to spatially resolve this chemical analysis on different regions of the sample.
IR microscopy is a powerful tool for chemical analysis and impurity detection, lending to diverse applications in the fields of forensics, environmental analysis, medical diagnostics, materials characterization, biological analysis, and mineralogy, to name a few. FTIR microscopy is accompanied by its fair share of advantages including:
- Near real-time data collection and analysis
- Ease of use on almost any solid samples
- Non-destructive, non-altering sample analysis
- High signal-to-noise ratios with short scan times across all frequencies
- Wide scan ranges (typically at least 4,000 to 600 cm-1) with high wavelength accuracy (better than 0.005 cm-1)
- Extensive reduction of stray light
Yet, despite these advantages, FTIR microscopy also has its limitations:
- Signal reduction via beam divergence
- Atmospheric absorption due to water and IR scattering from particulates
- Limited to about 1 minute per analysis for areas of interest near the diffraction limit, making imaging of large-area samples too slow to be practical
QUANTUM CASCADE LASER (QCL) SPECTROSCOPY
FTIR observes sample responses for all wavelengths in the IR range simultaneously. For high spatial resolution image analysis via FTIR microscopy, this can translate to scans of several hours for large-area samples. Assuming a large number of samples have to be analyzed in a typical laboratory, the FTIR microscopy approach would inevitably lead to a massive backlog of samples.
An alternative approach to efficiency is available in Infrared Laser Imaging (ILIM) microscopy where sample responses are analyzed corresponding to individual and specific IR wavelengths. ILIM is facilitated by the use of quantum cascade lasers (QCL), semiconductor lasers that emit light in the mid- to far-IR regions of the electromagnetic spectrum, and is also called QCL spectroscopy. By focusing on specific and optimal IR range wavelengths, QCL spectroscopy enables the generation of high contrast images at a much faster rate. For a sample area of about a hundred square millimeters, FTIR would take two and half hours while QCL would achieve the same in about 25 seconds.
Nonetheless, for all its speed advantage and quick scans, QCL only examines specific parts of the IR spectrum. This advantage diminishes once further spectral information, beyond a specific range, is required. Therefore, the ideal solution would be a combination of FTIR and QCL technology.
INTRODUCING THE HYPERION II
Combining FTIR and ILIM microscopy in a single device, the Hyperion II offers an innovative approach to IR microscopy. Featuring QCLs and an optimized beam path for fast IR laser imaging, the Hyperion II system can be equipped with different accessories for variable magnification, temperature-controlled stages, and extending the overall spectral range from UV to the far-IR (250 nm – 100 cm-1). Capitalizing simultaneously on the speed and sensitivity of QCL (much greater brightness than traditional SiC sources) and FTIR microscopy, the Hyperion II system offers rapid spectral data collection over the full spectral-domain (SiC source-based) to determine wavenumbers of interest for fast chemical imaging (QCL based) of samples of varying sizes. Switching between modes of acquisition is readily accomplished with a single click in the OPUS control software.
The use of the SiC broadband thermal emitter with the low spectral power density and incoherent emission characteristics in FTIR means coherence artifacts are negligible for traditional IR microscopy. In contrast, laser-based high-brightness sources utilized in QCL spectroscopy result in significant coherence. As a result, coherence artifacts are often observed in sample images collected utilizing laser-based sources.
To provide for spatial coherence reduction, the Hyperion II incorporates an optimized and patented mirror design that results in artifact-free images requiring no digital post-processing of images, a time-consuming process that can also introduce artifacts. This is enabled through effective, homogeneous, and broadened laser illumination resulting in a combination of high-quality QCL based hyperspectral imaging and live IR viewing allowing sample analysis in the IR at single wavelengths, in real-time, and at standard video framerates. This is crucial for examining large sample areas in very little time.
As such, the Hyperion II facilitates an innovative workspace where the user can flexibly switch between FTIR and QCL spectroscopy, capitalizing on their strengths while working around their limitations in a complementary fashion.
CASE STUDIES IN BIOLOGICAL TISSUE ANALYSIS AND OTHER IMPORTANT APPLICATIONS
A relatable real-life application demonstrating the scope of the Hyperion II system involves biological tissue analysis. The analysis of tissue samples via IR microscopy can be important in the characterization of tissue suspected to be diseased. Because IR microscopy is highly specific, the identification and classification of a disease can be readily accomplished using fast imaging. The use of IR imaging can eliminate the need for sample staining and counter staining, and greatly streamline the pathological analysis of the tissue. Additionally, IR microscopy can be most effective in monitoring the metabolic efficiency of drug intake. Many active drugs exhibit fluorescence upon UV illumination, where fluorescence illumination can be used to see where the drug has been metabolized. The subsequent effect on the tissue can then be well characterized using IR microscopy imaging of the tissue. Typically, these fluorescing drugs and drug markers are in such low concentration as to be undetectable via IR microscopy, but readily observed via fluorescence imaging.
Another important example is the analysis of microplastics, which refers to polymer particles of diameters less than 5 mm often found in water sources such as riverbeds, arctic ice, natural fertilizers, and soils. Microplastics have also found their way into drinking water, with current efforts to battle microplastic contamination of the environment and human food chain, a major initiative.
The detection of microplastics is vital in efforts to investigate and analyze the impact of these particulates. Chemical analysis of liquid samples using IR microscopy, specifically FTIR, has been the standard for several years. The Hyperion II incorporates FTIR and ILIM in a single device while offering all three measurement modes: transmission, reflection, and ATR. This effectively helps scale the FTIR approach to cases where larger sample populations are common, as in microplastics analysis.
In this context, the flexibility to switch between FTIR and QCL modes allows the user to generate detailed chemical maps of liquid samples on IR-transparent substrates. This can be achieved either over a continuous spectral range or sweep scans over discrete spectral ranges when sampling for specific microplastics. In this manner, the Hyperion II effectively allows the user to balance between high speed, narrow band QCL imaging, and broadband FTIR imaging. Together, this combination allows the user to identify regions of interest, perform measurements, and extract relevant characterization parameters such as the identity, size, and number of microplastic particles.
The advantages of the Hyperion II also translate toward solutions in applications such as pharmaceutical tablet analysis, materials identification, forensic sciences, and many others. Adaptive K-means clustering enabled by the OPUS software assists in the autonomous evaluation of raw chemical imaging data. This is of great use in biological tissue analysis where spectra on the order of millions can be analyzed within a few minutes. Supplemented by Hyperion II’s coherence reduction technology, these images are free of unwanted coherence artifacts, speckles, and fringes without the need for extensive post-processing.
Similarly, the Hyperion II overcomes limitations in full mid-IR imaging which is essential to reliably identify inorganic pigments and filler materials (whose spectra occur below the 1000 cm-1 range) for car paint chip analysis, especially in hit-and-run car accidents. Here, the Hyperion II’s broadband FTIR imaging can be used to unambiguously identify car paint chips while the QCL mode can provide for increased contrast analysis, making it easy to assess multiple layers and determine the cause of an accident.
The ease of flexibility between using QCL and FTIR spectroscopy is also evident in high-throughput screening of surface contamination on metals. A representative case considers the identification of silicone oil residues in a pocket watch. As metal has no IR signature, its surface reflects IR light easily. The presence of impurities and residues would then be observed by IR light absorption in the reflected light. Armed with this knowledge, QCL spectroscopy can be used to selectively survey specific bands on the sample and identify the corresponding stains. With a simple click, the user can then switch to the FTIR mode and obtain a full mid-IR spectrum on the contaminant area, and confirm the nature of the residue.
A NEW FRONTIER IN IR MICROSCOPY
Combining FTIR and QCL technology enables the collection of high-resolution chemical images for well-defined spectral ranges at incredible speeds. The Hyperion II platform highlights a single device pairing FTIR and QCL capabilities in a comprehensive and innovative solution for detailed chemical imaging and analysis, setting a new benchmark for IR microscopy.
To learn more visit: www.bruker.com/HYPERION