Infrared (IR) micro-spectroscopic imaging is a powerful technique which is gaining popularity in a wide range of fields, from biomedical and pharmaceutical research and development to materials science and analytical chemistry to industrial process and quality control. This vibrational spectroscopy technique uses the intrinsic molecular fingerprint of chemical species within the sample to produce chemical images without the need for staining, tagging or a priori knowledge of the sample.
The technique is especially effective in cytology, histology, microbiology, and stem cell research as it can be used to determine minute changes to the proteome, genome and metalobome without molecular tagging. There is a substantial body of scientific evidence now that shows that the combined chemical and morphological information obtained from this technique has the real potential to improve patient outcomes through earlier disease detection and classification.
However, in order for IR chemical imaging to reach the clinical diagnostic labs, it is essential that instruments be developed that have high sample throughput, excellent spectral fidelity and image quality with a small footprint and low cost of ownership.
Figure 1. High-resolution, wide-area, IR chemical imaging with the Spero microscope (right) of an unstained 5-10 um thick section of a cancerous colon tissue with a total area of about 6 cm2 collected in a matter of minutes demonstrating the high-throughput capability of the system. In comparison, an H&E stained parallel section of the same colon tissue is shown on the left. The IR image provides a rich chemical contrast without the need for molecular tagging or staining.
Figure 2. The spectral fidelity of the Spero microscope is tested rigorously against NIST traceable standards such as polystyrene sheets having spectral features throughout the fingerprint band. The above series of three plots show spectra collected from (top) 50 pixels (ca 10 um x 10 um area) and (middle) from a single representative pixel (1.36 um x 1.36 um area) of the Spero microscope. These spectra are then compared with (bottom) reference polystyrene spectra collected on an FTIR spectrometer.
The Spero Infrared Chemical Imaging Microscope
The Spero Infrared Chemical Imaging Microscope fits on a desktop and satisfies all the features required for industrial and clinical research applications. The instrument includes a broadly tunable quantum cascade laser illumination source to enable diffraction-limited, high signal-to-noise, micron resolution, wide field-of-view and spectral imaging over the full infrared fingerprint region. The Spero is capable of recording a full spectral image cube in minutes, without the need for liquid nitrogen cooling. The Spero is capable of measuring 650 x 650µm images at 1.4µm pixel resolution and 2x2mm images at 4.3µm pixel resolution at 30 fps video frame rates and in a single frame.
Figure 3. Spero enables label-free, high-resolution chemical imaging and identification of a wide range of materials and biomaterials including cells, tissues, and biofluids. The above example shows from left to right a visible image of an unstained skin tissue section, a high-definition IR chemical image collected on the Spero microscope of the same region, and a digitally stained image whereby the spectral response of each pixel is grouped into 1 of 10 spectral classes shown in the rightmost plot.
The Spero infrared chemical imaging microscope offers the user new data collection modalities not available on any other IR platform. The user now has the freedom to select anydiscrete IR frequency within the fingerprint band and perform live IR chemical imaging of the sample in real-time at 30 frames per second.
This modality is especially useful for isolating molecular contaminants and either molecular of physical defects within thick organic films such as PET.
Figure 4. Spero enables real-time inspection of materials to rapidly pinpoint locations of contaminants or defects on or below the surface. Buried defects such as microvoids often have a unique chemical, or spectral, signature in the IR enabling detailed analysis after detection.
The novel technology differentiating the Spero from other technology is the broadly tunable infrared laser source, which provides excellent imaging capability with rapid data acquisition times. At the same time, the Spero is able maintain or exceed the signal-to-noise ratio of standard infrared instrumentation without the requirement of cryocooled detectors.
This information has been sourced, reviewed and adapted from materials provided by Daylight Solutions Inc.
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