Conventional mid-infrared (mid-IR) Fourier transform infrared (FT-IR) spectroscopic imaging systems employ an incoherent globar source and achieve spectral contrast through interferometry. While this approach is suitable for many general applications, recent advancements in broadly tunable external cavity Quantum Cascade Lasers (QCL) offer new approaches to and new possibilities for mid-IR micro-spectroscopic imaging. While QCL-based devices have yet to achieve the wide spectral range generally employed by spectroscopists for molecular analyses, they are starting to be used for microscopy at discrete frequencies. Here, we present a discrete frequency IR (DFIR) microscope based on a QCL source and explore its utility for mid-IR imaging. In our prototype instrument, spectral contrast is achieved by tuning the QCL to bands in a narrow spectral region of interest. We demonstrate wide-field imaging employing a 128x128 pixel liquid nitrogen cooled mercury cadmium telluride (MCT) focal plane array (FPA) detector. The resulting images demonstrate successful imaging as well as several unique features due to coherence effects from the laser source. Here we discuss the effects of this coherence and compare our instrument to conventional mid-IR imaging instrumentation.
Fourier transform infrared (FT-IR) spectroscopic imaging is a powerful tool to obtain chemical information from
images of heterogeneous, chemically diverse samples. Significant advances in instrumentation and data processing
in the recent past have led to improved instrument design and relatively widespread use of FT-IR imaging, in a
variety of systems ranging from biomedical tissue to polymer composites. Various techniques for improving signal
to noise ratio (SNR), data collection time and spatial resolution have been proposed previously. In this paper
we present an integrated framework that addresses all these factors comprehensively. We utilize the low-rank
nature of the data and model the instrument point spread function to denoise data, and then simultaneously
deblurr and estimate unknown information from images, using a Bayesian variational approach. We show that
more spatial detail and improved image quality can be obtained using the proposed framework. The proposed
technique is validated through experiments on a standard USAF target and on prostate tissue specimens.
Raman spectroscopy of bone is complicated by fluorescence background and spectral contributions from other tissues. Full utilization of Raman spectroscopy in bone studies requires rapid and accurate calibration and preprocessing methods. We have taken a step-wise approach to optimize and automate calibrations, preprocessing and background correction. Improvements to manual spike removal, white light correction, software image rotation and slit image curvature correction are described. Our approach is concisely described with a minimum of mathematical detail.
The effect of optical clearing with glycerol on the Raman spectra of bone tissue acquired transcutaneously on right and left tibiae from four mice is studied. Multiple transcutaneous measurements are obtained from each limb; glycerol is then applied as an optical clearing agent, and additional transcutaneous measurements are taken. Glycerol reduces the noise in the raw spectra (p=0.0037) and significantly improves the cross-correlation between the recovered bone factor and the exposed bone measurement in a low signal-to-noise region of the bone spectra (p=0.0245).
Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images.
We report tomographic reconstruction of objects located several millimeters below the surface of highly scattering
media. For this purpose we adapted proven software developed for fluorescence tomography with and without the use of
spatial priors<sup>1</sup>. For this first demonstration we acquired Raman spectra using an existing ring/disk fiber optic probe with
fifty collection fibers<sup>2</sup>. Several illumination ring diameters were employed to generate multiple angles of incidence.
Tomographic reconstruction from Raman scatter was tested using a 9.5 mm diameter Teflon® sphere embedded in a gel
of agarose and 1% Intralipid. Blind reconstruction of the sphere using the 732 cm<sup>-1</sup> C-F stretch yielded an accurate shape
but an inaccurate depth. Using the known shape and position of the object as spatial priors, a more accurate
reconstruction was obtained. We also demonstrated a reconstruction of the tibial diaphysis of an intact canine hind limb
using spatial priors generated from micro-computed tomography. In this first demonstration of Raman tomography in
animal tissue, the P-O stretch of the bone mineral at 958 cm<sup>-1</sup> was used for the reconstruction. An accurate shape and
depth were recovered.
We have used fiber optic probes with global illumination/collection (PhAT probe, Kaiser Optical Systems) and ring
illumination/disk collection configurations for transcutaneous Raman spectroscopy of bone tissue. Both
illumination/collection schemes can be used for recovery of spectra of subsurface components. In this paper the global
illumination configuration provides minimum local power density and so minimizes the probability of damage to
specimens, animals or human subjects. It also allows non-destructive subsurface mapping under certain conditions. The
ring/disk probe utilizes a ring of laser light and collects Raman scatter from within the diameter of the ring. This design
distributes the laser power for efficient heat dissipation and provides a better collection ratio of subsurface to surface
components than the global illumination design. For non-invasive tissue spectroscopy the ring/disk design also provides
better rejection of fluorescence from melanocytes. We have tested the performance of these Raman probes on polymer
model systems and chicken tibiae.
Transcutaneous bone Raman spectroscopy with an exciting annulus of 785-nm laser light surrounding the field of view of a circular array of collection fibers is demonstrated. The configuration provides distributed laser light. The annulus is located 2 to 3 mm beyond the edge of the field of view of the collection fibers to reject contributions from skin and other overlying tissues. Data are presented for rat and chicken tissue. For rat tibia, the carbonate/phosphate ratio measured at a depth of 1 mm below the skin is in error by 2.3% at an integration time of 120 s and within 10% at a 30-s integration time. For chicken tibia 4 mm below the skin surface, the error is less than 8% with a 120-s integration time.
We demonstrate the first transcutaneous Raman spectroscopic measurements of bone tissue employing a fiber optic probe with a uniformly illuminated array of collection fibers. Uniform illumination reduces local power density to avoid damage to specimens. Non-confocal operation provides efficient signal collection, and together with NIR laser excitation (785 nm diode laser) allows good depth penetration enabling recovery of spectra from beneath the skin. Multivariate data reduction is used to resolve Raman spectra of bone tissue from the spectra generated from overlying tissue. The probe utilizes non-confocal optics and
uniform illumination allowing the system to collect spectra from above and below the range of best focus while applying a low power density. Despite extensive photon migration in the tissue specimens, the system can resolve transcutaneous signals because the collection cone of each fiber is asymmetric with respect to the center of illumination. Here we report preliminary results of tissue specimens taken from chicken tibia as well as from a human elbow.