5-tips for the successful infrared chemical imaging of samples

The next-generation of label-free chemical imaging has arrived. The Spero-QT®, from Daylight Solutions, is the next breakthrough in high-performance mid-infrared microscopy offering high-resolution, real-time spectroscopic imaging for a wide range of applications from tablet and powder API monitoring to high-throughput digital histopathology.

The Spero-QT is powered by Daylight Solutions’ proven quantum cascade laser (QCL) technology and leverages key advances in uncooled FPA detectors and compound refraction based optics. As a photonics technology pioneer, Daylight Solutions has delivered more QCL-based mid-infrared systems to more applications world-wide than all others combined. With a highly-experienced team and field-proven, best-in-class family of products, we stand ready to help you with your most challenging of applications.  

Choice of substrate and sampling mode

Due to strong IR absorption in traditional glass slides, it is best to mount samples onto slides or substrates that are IR transmissive such as calcium fluoride or barium fluoride. Calcium fluoride is the most commonly used substrate due to its lower cost but does suffer from higher attenuation at wavelengths higher than about 10 µm (1000 cm-1).

An increasingly viable cost-effective alternative is to use thin polymer membranes made of polyethylene terephthalate (PET) that are attached to windowed metal carriers. For IR imaging in reflection or transflection (light transverses the sample thickness twice) sampling mode, reflective MirrIR Low-E slides from Kevley Technologies are often the best choice.

Sample preparation

For transmission and transflection analysis, some consideration should be given to the sample thickness in order to maximize the spectral data quality.  For any sample, the optimal sample thickness can be determined theoretically or experimentally by ensuring that the strongest recorded absorbance intensity does not exceed 4 AU.

For tissue analysis, a good rule of thumb is to cut microtomed sections between 8 – 12 microns for transmission experiments and approximately half this value for transflection experiments. For many polymers including PMMA films, samples up to 50 µm can achieve good results.

Selecting a Reference Background

Like any infrared absorbance spectroscopy technique, a reference background should be acquired under similar conditions as the sample acquisition.  This background is used to remove spectral contributions unrelated to the sample (e.g. contributions from the substrate, the atmosphere, or the instrument iteslf). This can be done easily by acquiring a ‘background’ image from a clean area, of the sample.  Ideally, the background should be recorded as close to the sample acquisition time as possible to remove minute contributions from the environment.

For liquid or live cell experiments, a background should account for the substrate and windows as well as the host solution which could be water or growth media.  It is important to keep the thickness of the sample holder and liquid the same as for the sample collection. For transflection, an image of the IR reflective slide surface can be used and for direct reflectance measurement a background with the sample removed can be used.

A new background should be taken for each substrate and objective lens combination and retaken regularly (typically once per hour) to account for water vapor and other atmospheric changes.

Optimize your data collection parameters

Consider the spectral range and resolution needed for your analysis. For solid samples including tissue specimens as well as liquid samples, 4 cm-1 spectral resolution with 2 cm-1 data spacing is more than adequate spectral resolution. In many cases, resolution of 8 cm-1 and even lower are perfectly suitable and may result in better SNR.

Furthermore, ask yourself if you need the full range of spectral bandwidth. If you are working with large samples and throughput is important to you, you may consider reducing the number of spectral samples to include only those clustered about key spectral bands, for example those peaks associated with an API. In the Spero-QT, the tunable laser source can be easily programmed to target just the frequencies desired, thereby reducing acquisition time and data file size proportionately.

Analyze your data

After collecting a spectral cube, export the data into either ENVI or MATLAB file formats so that it may be easily loaded into a number of chemometrics software tools. We routinely use and recommend ImageLab™ made by Epina (Vienna, Austria), an image-based multi-sensor (see note below) chemometrics package.

For exploratory analyses, unsupervised techniques such as PCA, VCA and k-means clustering are excellent starting points. Ultimately, it is best to build custom classifiers based on supervised algorithms such as PLS-DA, ANN, SVM and Random Forest techniques, Here are 5 specific tips when performing your analysis:

  • Optimize the signal-to-noise (SNR) ratio of the spectral data byemploying techniques such as smoothing filters such as Savitzy–Golay algorithm, PCA-reconstruction, or the minimum noise fraction (MNF) transform.
  • Employ quality (outlier) tests to ensure repeatability and reproducibility.  Define thresholds for SNR or water vapor lines to remove sub-optimal spectra.  
  • Spectral baselines can be distorted because of scattering.   The application of a derivative filter can minimize baseline offsets or slopes and in addition enhance the spectral resolution of infrared spectra, allowing overlapping bands to be resolved.
  • Normalize your data to minimize the effects of varying optical path lengths on the data by using techniques include Min-Max, Vector and Standard Normal Variate (SNV) normalization.

Reduce the dimensionality of the data and perform feature selection.  By encoding the chemical information into a smaller subset of spectral descriptors,  multivariate models can become more robust while also resulting in faster computations.

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: Mar 3, 2017 at 9:58 AM

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