Fourier-transform infrared imaging (FT-IR) is a well-established modality but requires the acquisition of a spectrum over a large bandwidth, even in cases where only a few spectral features may be of interest. Discrete frequency infrared (DF-IR) methods are now emerging in which a small number of measurements may provide all the analytical information needed. The DF-IR approach is enabled by the development of new sources integrating frequency selection, in particular of tunable, narrow-bandwidth sources with enough power at each wavelength to successfully make absorption measurements. Here, we describe a DF-IR imaging microscope that uses an external cavity quantum cascade laser (QCL) as a source. We present two configurations, one with an uncooled bolometer as a detector and another with a liquid nitrogen cooled Mercury Cadmium Telluride (MCT) detector and compare their performance to a commercial FT-IR imaging instrument. We examine the consequences of the coherent properties of the beam with respect to imaging and compare these observations to simulations. Additionally, we demonstrate that the use of a tunable laser source represents a distinct advantage over broadband sources when using a small aperture (narrower than the wavelength of light) to perform high-quality point mapping. The two advances highlight the potential application areas for these emerging sources in IR microscopy and imaging.

Abstract. The ability to probe fresh tissue is a key feature to biomedical Raman spectroscopy. However, it is unclear how Raman spectra of calcified tissues are affected by freezing. In this study, six transverse sections of femoral cortical bone were subjected to multiple freeze/thaw cycles and probed using a custom Raman microscope. Significant decreases were observed in the amide I and amide III bands starting after two freeze thaw cycles. Raman band intensities arising from proline residues of frozen tissue appeared consistent with fresh tissue after four cycles. Crystallinity values of bone mineral diminished slightly with freezing and were noticeable after only one freezing. Mineral carbonate levels did not deviate significantly with freezing and thawing. The authors recommend freezing and thawing bone tissue only once to maintain accurate results. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

The soybean industry requires rapid, accurate, and precise technologies for the analyses of seed/grain constituents. While the current gold standard for nondestructive quantification of economically and nutritionally important soybean components is near-infrared spectroscopy (NIRS), emerging technology may provide viable alternatives and lead to next generation instrumentation for grain compositional analysis. In principle, Raman spectroscopy provides the necessary chemical information to generate models for predicting the concentration of soybean constituents. In this communication, we explore the use of transmission Raman spectroscopy (TRS) for nondestructive soybean measurements. We show that TRS uses the light scattering properties of soybeans to effectively homogenize the heterogeneous bulk of a soybean for representative sampling. Working with over 1000 individual intact soybean seeds, we developed a simple partial least-squares model for predicting oil and protein content nondestructively. We find TRS to have a root-mean-standard error of prediction (RMSEP) of 0.89% for oil measurements and 0.92% for protein measurements. In both calibration and validation sets, the predicative capabilities of the model were similar to the error in the reference methods.

To support the translation of Raman spectroscopy into clinical applications, synthetic models are needed to accurately test, optimize and validate prototype fiber optic instrumentation. Synthetic models (also called tissue phantoms) are widely used for developing and testing optical instrumentation for diffuse reflectance, fluorescence, and Raman spectroscopies. While existing tissue phantoms accurately model tissue optical scattering and absorption, they do not typically model the anatomic shapes and chemical composition of tissue. Because Raman spectroscopy is sensitive to molecular composition, Raman tissue phantoms should also approximate the bulk tissue composition. We describe the fabrication and characterization of tissue phantoms for Raman tomography and spectroscopy. These phantoms have controlled chemical and optical properties, and also multilayer morphologies which approximate the appropriate anatomic shapes. Tissue phantoms were fabricated to support on-going Raman studies by simulating human wrist and rat leg. Surface meshes (triangle patch models) were generated from computed tomography (CT) images of a human arm and rat leg. Rapid prototyping was used to print mold templates with complex geometric patterns. Plastic casting techniques used for movie special effects were adapted to fabricate molds from the rapid prototypes, and finally to cast multilayer gelatin tissue phantoms. The gelatin base was enriched with additives to model the approximate chemistry and optical properties of individual tissue layers. Additional studies were performed to determine optimal casting conditions, phantom stability, layer delamination and chemical diffusion between layers. Recovery of diffuse reflectance and Raman spectra in tissue phantoms varied with probe placement. These phantoms enable optimization of probe placement for human or rat studies. These multilayer tissue phantoms with complex geometries are shown to be stable, with minimal layer delamination and chemical diffusion.

Confocal and multiphoton optical imaging techniques have been powerful tools for evaluating the performance of and monitoring experiments within microfluidic devices, but this application suffers from two pitfalls. The first is that obtaining the necessary imaging contrast often requires the introduction of an optical label which can potentially change the behavior of the system. The emerging analytical technique stimulated Raman scattering (SRS) microscopy promises a solution, as it can rapidly measure 3D concentration maps based on vibrational spectra, label-free; however, when using any optical imaging technique, including SRS, there is an additional problem of optical aberration due to refractive index mismatch between the fluid and the device walls. New approaches such as 3D printing are extending the range of materials from which microfluidic devices can be fabricated; thus, the problem of aberration can be obviated simply by selecting a chip material that matches the refractive index of the desired fluid. To demonstrate complete chemical imaging of a geometrically complex device, we first use sacrificial molding of a freeform 3D printed template to create a round-channel, 3D helical micromixer in a low-refractive-index polymer. We then use SRS to image the mixing of aqueous glucose and salt solutions throughout the entire helix volume. This fabrication approach enables truly nonperturbative 3D chemical imaging with low aberration, and the concentration profiles measured within the device agree closely with numerical simulations.