Attenuated total reflection Fourier transform infrared imaging with variable angles of incidence: a three-dimensional profiling of heterogeneous materials

Depth profiling in Fourier transform infrared (FT-IR) spectroscopic imaging has been demonstrated using a single reflection variable angle attenuated total reflection (ATR) accessory. Chemical information about samples can be obtained in three dimensions by acquiring ATR-FT-IR images at different angles of incidence through the ATR crystal. The image quality and field of view achieved at different angles of incidence has been discussed. A polymer film comprising two layers has been used as an example to demonstrate the principle of the measurement. The demonstrated approach is a promising tool to obtain depth profiles of heterogeneous materials. The extent of the measured depths is limited and ranges from approximately 0.3 to 4 microm, but the spatial resolution in the z-direction is not limited by diffraction. The development of this approach opens up the possibility to study the spatial heterogeneity of thin films including biological tissues, such as hair and skin, with high depth resolution.     

New opportunities in micro- and macro-attenuated total reflection infrared spectroscopic imaging: spatial resolution and sampling versatility

New opportunities exist to obtain chemical images using attenuated total reflection infrared (ATR-IR) spectroscopy. This paper shows the feasibility of obtaining FT-IR images with a spatial resolution of at least 3-4 microm using a Ge ATR objective coupled with an infrared microscope. The improved spatial resolution compared to FT-IR images obtained by the transmission method is due to the high refractive index of the ATR crystal, which gives a high numerical aperture and hence, a higher spatial resolution. FT-IR imaging with a conventional diamond ATR accessory has been investigated. This is the first time that FT-IR imaging is reported using such a versatile accessory based on a diamond ATR crystal. These results showed that a spatial resolution up to 13 microm can be achieved without the use of infrared microscope objectives. One advantage of the diamond element is that it allows pressure to be applied and hence, good contact to be obtained over the whole field of view.     

Attenuated total internal reflection infrared microspectroscopic imaging using a large-radius germanium internal reflection element and a linear array detector

The number of techniques and instruments available for Fourier transform infrared (FT-IR) microspectroscopic imaging has grown significantly over the past few years. Attenuated total internal reflectance (ATR) FT-IR microspectroscopy reduces sample preparation time and has simplified the analysis of many difficult samples. FT-IR imaging has become a powerful analytical tool using either a focal plane array or a linear array detector, especially when coupled with a chemometric analysis package. The field of view of the ATR-IR microspectroscopic imaging area can be greatly increased from 300 x 300 microm to 2500 x 2500 microm using a larger internal reflection element of 12.5 mm radius instead of the typical 1.5 mm radius. This gives an area increase of 70x before aberrant effects become too great. Parameters evaluated include the change in penetration depth as a function of beam displacement, measurements of the active area, magnification factor, and change in spatial resolution over the imaging area. Drawbacks such as large file size will also be discussed. This technique has been successfully applied to the FT-IR imaging of polydimethylsiloxane foam cross-sections, latent human fingerprints, and a model inorganic mixture, which demonstrates the usefulness of the method for pharmaceuticals.     

Applications of attenuated total reflection infrared spectroscopic imaging to pharmaceutical formulations

This paper demonstrates an approach to obtain chemical images of pharmaceutical tablets using attenuated total reflection infrared (ATR-IR) spectroscopy. FT-IR images with different fields of view and spatial resolution have been obtained using a combination of different ATR accessories. FT-IR imaging with the diamond ATR accessory and micro-ATR imaging technique have been compared. With the diamond ATR imaging accessory, compaction to a tablet can be performed and the chemical image measured in situ. It has been found that the diamond ATR imaging accessory gives information on the overall distribution of different components in a tablet while the micro-ATR imaging technique provides a closer look at the tablet with 4-microm spatial resolution. Low-concentration components down to 0.5% have been detected by the micro-ATR method. Both experimental and commercial systems are studied in this paper.     

Attenuated total internal reflection infrared microscopy of multilayer plastic packaging foils

Multilayer plastic foils are important packaging materials that are used to extend the shelf life of food products and drinks. Fourier transform infrared (FT-IR) spectroscopic imaging using attenuated total internal reflection (ATR) can be used for the identification and localization of different layers in multilayer foils. A new type of ATR crystal was used in combination with a linear array detector through which large sample areas (400 x 400 microm(2)) could be imaged with a pixel size of 1.6 microm. The method was tested on laminated plastic packing materials containing 5 to 12 layers. The results of the identification of the different materials using ATR-FT-IR were compared with differential scanning calorimetry (DSC) and the layer thickness of the individual layers measured by ATR-FT-IR was compared with polarized light microscopy (LM) and scanning electron microscopy (SEM). It has been demonstrated that individual layers with a thickness of about 3 microm could be identified in multilayer foils with a total thickness ranging from 100 to 150 microm. The results show a spatial resolution of about 4 microm (measured at wavenumbers ranging from 1000 to 1730 cm(-1)), which is about a factor of two better than can be obtained using transmission FT-IR imaging. An additional advantage of ATR is the ease of sample preparation. A good correspondence was found between visible and FT-IR images. The results of ATR-FT-IR imaging were in agreement with those obtained by LM, SEM, and DSC. ATR-FT-IR is superior to the combination of these techniques because it delivers both spatial and chemical information.     

Raman microspectroscopy: a comparison of point, line, and wide-field imaging methodologies

Three different Raman microspectroscopic imaging methodologies using a single experimental configuration are compared; namely, point and line mapping, as representatives of serial imaging approaches, and direct or wide-field Raman imaging employing liquid-crystalline tunable filters are surveyed. Raman imaging data acquired with equivalent low-power 514.5-nm laser excitation and a cooled CCD camera are analyzed with respect to acquisition times, image quality, spatial resolution, intensity profiles along spatial coordinates, and spectral signal-to-noise ratios (SNRs). Point and line mapping techniques provide similar SNRs and reconstructed Raman images at spatial resolutions of approximately 1.1 microm. In contrast, higher spatial resolution is obtained by direct, global imaging (approximately 313 nm), allowing subtle morphological features on test samples to be resolved.     

Fourier transform infrared imaging of human hair with a high spatial resolution without the use of a synchrotron

The cross-section of a human hair has been imaged for the first time using the micro attenuated total reflection (ATR) Fourier transformed infrared (FT-IR) method in combination with a focal plane array (FPA) detector. A rigorous approach was applied to determine the spatial resolution, namely, measuring the distance over which the band absorbance changes from 95 to 5% of the maximum absorbance when passing through a sharp interface. The measured value for IR transmission was approximately 16 microm, while the value obtained using ATR imaging was approximately 5 microm. The enhanced spatial resolution achieved by this method allows the medulla of the hair (approximately 8 microm in diameter) to be imaged clearly without the need for a synchrotron source. The spatial resolution of transmission and ATR imaging is compared, and advantages of ATR imaging are discussed.     

Characterization of a CCD-based digital x-ray imaging system for small-animal studies: properties of spatial resolution

A digital x-ray imaging system was designed for small-animal studies. This system is a fiber-optics taper-coupled imaging system with two CCD arrays uniquely jointed. The x-ray source of the system has a small focal spot of 20 microm. This digital imaging system contains specially designed shelves to provide magnification levels, ranging from 1.5x to 5x. The system is characterized in terms of its properties of spatial resolution. An observer-based spatial resolution measurement was conducted with a line-pair target and a sector test pattern. The modulation transfer function of the system, with different magnifications, was studied by use of a 10-microm lead slit. The average resolutions at 50% and 5% modulations at 1x magnification were measured as 3.9 and 8.4 lp/mm, respectively, where lp indicates line pairs. With 5x magnification, the 50% and the 5% modulations provided 13.2- and 29.9-lp/mm, respectively, average spatial resolutions. The measurements showed consistency between the two individual CCD arrays; the difference in resolution between the two CCDs is less than 1%, even at high magnifications.     

Computer-controlled optical scanning tile microscope

A new type of computer-controlled optical scanning, high-magnification imaging system with a large field of view is described that overcomes the commonly believed incompatibility of achieving both high magnification and a large field of view. The new system incorporates galvanometer scanners, a CCD camera, and a high-brightness LED source for the fast acquisition of a large number of a high-resolution segmented tile images with a magnification of 800x for each tile. The captured segmented tile images are combined to create an effective enlarged view of a target totaling 1.6 mm x 1.2 mm in area. The speed and sensitivity of the system make it suitable for high-resolution imaging and monitoring of a small segmented area of 320 microm x 240 microm with 4 microm resolution. Each tile segment of the target can be zoomed up without loss of the high resolution. This new microscope imaging system gives both high magnification and a large field of view. This microscope can be utilized in medicine, biology, semiconductor inspection, device analysis, and quality control.     

Multi-wavelength raman imaging using a small-diameter image guide with a dimension-reduction imaging array

A commercially available fiber-optic Raman probe was modified for high-resolution spectral Raman imaging using a 350 microm diameter optical fiber image guide coupled to a dimension-reduction imaging array (DRIA). The DRIA comprised 672 optical fibers, arranged as a square array (21 x 32 fibers) on one end and a linear array (672 x 1 fibers) on the other. An imaging spectrograph was used with the DRIA to acquire multi-wavelength Raman images from -250 to 1800 cm(-1) at a spectral resolution of approximately 5 cm(-1). The utility of this technique for in situ and remote Raman imaging is demonstrated by monitoring the polymerization of a model polymer, dibromostyrene (DBS), while simultaneously measuring the Raman Stokes/ anti-Stokes ratio as a function of sample heating time, over a sample area of approximately 4 x 1.6 mm.     

Spectroscopic imaging of latent fingermarks collected with the aid of a gelatin tape

This work introduces a new and nondestructive methodology for the collection and chemical identification of latent fingermarks. The main challenges of this work were (a) to find an appropriate medium to lift fingermarks from various surfaces and (b) to develop an analytical approach for the identification of small quantities of sample while avoiding spectroscopic interference from the lifting media. Two different lifting media were evaluated and analyzed by ATR-FT-IR spectroscopic imaging, which affords inherent chemical specificity with rapid acquisition of data. This is the first time that chemical images of latent fingermarks collected with gel lifters from different surfaces have been obtained. Spatially resolved chemical images from different depths within the same sample were obtained using ATR-FT-IR imaging with a variable angle ATR accessory to minimize interference from the substrate. The possibility of obtaining, through the developed methodology, three-dimensional depth profiles of surface contaminants collected with the lifting gel shows great potential for the investigation of samples for forensic interest.     

Combining the tape-lift method and Fourier transform infrared spectroscopic imaging for forensic applications

Conventional Fourier transform infrared (FT-IR) spectroscopy and microscopy have been widely used in forensic science. New opportunities exist to obtain chemical images and to enhance the spatial resolution using attenuated total reflection (ATR) FT-IR spectroscopy coupled with a focal-plane array (FPA) detector. In this paper, the sensitivity limits of FT-IR imaging using three different ATR crystals (Ge, ZnSe, and diamond) in three different optical arrangements for the detection of model particles is discussed. Model systems of ibuprofen and paracetamol particles having sizes below 32 mum were studied. The collection of drug particles was achieved with the aid of two different tapes: common adhesive tape and a film of polydimethylsiloxane (PDMS). The surface of the film with collected particles was measured directly via ATR-FT-IR imaging. Since the removal of tape from porous surfaces can be difficult, the application of micro ATR-FT-IR imaging directly to the surface of a newspaper contaminated with particles of model drugs is also discussed. In order to assess the feasibility of the chosen method in a forensic case study, the detection of diacetylmorphine hydrochloride traces in PDMS matrix and the finger surface is investigated. The scenarios considered were that of the detection of evidence collected at a crime scene with the tape lift method and the analysis of the finger of an individual after drug handling. The results show broad implications in the detection of drugs of abuse.     

Chemical imaging with variable angles of incidence using a diamond attenuated total reflection accessory

A new development in Fourier transform infrared (FT-IR) imaging using a diamond attenuated total reflection (ATR) imaging accessory in a novel manner that allows the angle of incidence to be varied in order to obtain images from subsurface layers of different thickness is introduced. Chemical images of samples from the same area but with different depths of penetration are obtained by changing the angle of incidence as well as using different spectral bands at different wavenumbers. Changes in the angle of incidence with this accessory were made possible by taking advantage of the relatively large numerical aperture employed by the original imaging optics. This arrangement allowed us to introduce an additional movable aperture in the optical design to restrict the angle of incidence to certain values. Two samples have been studied, one for the calibration of the angle of incidence while the other demonstrates the capability of obtaining three-dimensional (3D) information using this approach. Advantages of this new approach include the relatively high spatial resolution (it can spatially resolve features as small as 12 mum without a microscope) and no change in the imaging area and sampling area during manipulation of the angle of incidence.     

Contrast-enhanced imaging of cerebral vasculature with laser speckle

High-resolution cerebral vasculature imaging has applications ranging from intraoperative procedures to basic neuroscience research. Laser speckle, with spatial contrast processing, has recently been used to map cerebral blood flow. We present an application of the technique using temporal contrast processing to image cerebral vascular structures with a field of view a few millimeters across and approximately 20 microm resolution through a thinned skull. We validate the images using fluorescent imaging and demonstrate a factor of 2-4 enhancement in contrast-to-noise ratios over reflectance imaging using white or spectrally filtered green light. The contrast enhancement enables the perception of approximately 10%-30% more vascular structures without the introduction of any contrast agent.     

Two-dimensional photoacoustic imaging by use of Fourier-transform image reconstruction and a detector with an anisotropic response

Theoretical and experimental aspects of two-dimensional (2D) biomedical photoacoustic imaging have been investigated. A 2D Fourier-transform-based reconstruction algorithm that is significantly faster and produces fewer artifacts than simple radial backprojection methods is described. The image-reconstruction time for a 208 x 482 pixel image is approximately 1 s. For the practical implementation of 2D photoacoustic imaging, a rectangular detector geometry was used to obtain an anisotropic detection sensitivity in order to reject out-of-plane signals, thereby permitting a tomographic image slice to be reconstructed. This approach was investigated by the numerical modeling of the broadband directional response of a rectangular detector and imaging of various spatially calibrated absorbing targets immersed in a turbid phantom. The experimental setup was based on a Q-switched Nd:YAG excitation laser source and a mechanically line-scanned Fabry-Perot polymer-film ultrasound sensor. For a 800 microm x 200 microm rectangular detector, the reconstructed image slice thickness was 0.8 mm up to a vertical distance of z = 3.5 mm from the detector, increasing thereafter to 2 mm at z = 10 mm. Horizontal and vertical spatial resolutions within the reconstructed slice were approximately 200 and 60 microm, respectively.     

Analysis of counterfeit pharmaceutical tablet cores utilizing macroscopic infrared spectroscopy and infrared spectroscopic imaging

Advantages and limitations of analyzing authentic and counterfeit pharmaceutical tablets with both macro (nonimaging) attenuated total internal reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and micro ATR-FT-IR spectroscopic imaging have been evaluated. The results of this study demonstrated that micro ATR imaging was more effective for extracting formulation information (sourcing), whereas a macro ATR approach was better suited for counterfeit detection (screening). More importantly, this study demonstrated that a thorough analysis of the counterfeit core can be achieved by combining the results of both techniques.     

Full field of view super-resolution imaging based on two static gratings and white light illumination

The usage of two static gratings for obtaining super-resolved imaging dates back to the work by Bachl and Lukosz in 1967. However, in their approach a severe reduction in the field of view was the necessary condition for improving the resolution. We present an approach based on two static gratings without sacrificing the field of view. The key idea for not paying with the field of view is to use white light illumination to average the ghost images obtained outside the region of interest since the positions of those images are wavelength dependent. Moreover, large magnification is achieved by using a commercial microscope objective instead of a test system with a unity magnification as presented in previous works. Because of the large magnification, the second grating has a low spatial period. This allows us to create an intermediate image and use a second imaging lens with low resolution capability while still obtaining an imaging quality as good as that provided by the first imaging lens. This is an important improvement in comparison with the original super-resolving method with two fixed gratings.     

Improved monochromatic imaging spectrometer

Distortion inherent to a previously described system for acquisition of two-dimensional monochromatic spatial images is described. A solution is offered in the form of an improved instrument. The system uses a Czerny-Turner monochromator for spectral discrimination and a charge-coupled device (CCD) as the detector. A second Czerny-Turner monochromator, with identical dimensions, is added to correct the distortion, albeit with a slight loss in spatial resolution. With the earlier uncorrected arrangement, spatial resolution was on the order of 0.1 mm vertically and 0.3 mm horizontally, with a magnification of 0.52. With the same magnification, the new, corrected system offers spatial resolution of 0.1 mm vertically and 0.4 mm horizontally.     

Characterization of the spatial resolution of different high-frequency imaging systems using a novel anechoic-sphere phantom

The spatial resolution of high-frequency ultrasound (HFU, >20 MHz) imaging systems is usually determined using wires perpendicular to the beam. Recently, two tissue-mimicking phantoms (TMPs) were developed to estimate three-dimensional (3-D) resolution. Each TMP consists of nine 1-cm-wide slabs of tissue-mimicking material containing randomly distributed anechoic spheres. All anechoic spheres in one slab have the same dimensions, and their diameter is increased from 0.1 mm in the first slab to 1.09 mm in the last. The scattering background for one set of slabs was fabricated using 3.5-μm glass beads; the second set used 6.4-μm glass beads. The ability of a HFU system to detect these spheres against a speckle background provides a realistic estimation of its 3-D spatial resolution. In the present study, these TMPs were used with HFU systems using single-element transducers, linear arrays, and annular arrays. The TMPs were immersed in water and each slab was scanned using two commercial imaging systems and a custom HFU system based on a 5-element annular array. The annular array had a nominal center frequency of 40 MHz, a focal length of 12 mm, and a total aperture of 6 mm. A synthetic-focusing algorithm was used to form images with an increased depth-of-field. The penetration depth was increased by using a linear-chirp signal spanning 15 to 65 MHz over 4 μs. Results obtained with the custom system were compared with those of the commercial systems (40-MHz probes) in terms of sphere detection, i.e., 3-D spatial resolution, and contrast-to-noise ratio (CNR). Resulting B-mode images indicated that only the linear-array transducer failed to clearly resolve the 0.2-mm spheres, which showed that the 3-D spatial resolution of the single-element and annular-array transducers was superior to that of the linear array. The single-element transducer could only detect these spheres over a narrow 1.5 mm depth-of-field, whereas the annular array was able to detect them to depths of at least 7 mm. For any size of the anechoic spheres, the annular array excited by a chirp-coded signal provided images of the highest contrast, with a maximum CNR of 1.8 at the focus, compared with 1.3 when using impulse excitation and 1.6 with the single-element transducer and linear array. This imaging configuration also provided CNRs above 1.2 over a wide depth range of 8 mm, whereas CNRs would quickly drop below 1 outside the focal zone of the other configurations.     

Laser ablation coupled to a flowing atmospheric pressure afterglow for ambient mass spectral imaging

A plasma-based ambient desorption/ionization mass spectrometry (ADI-MS) source was used to perform molecular mass spectral imaging. A small amount of sample material was ablated by focusing 266 nm laser light onto a spot. The resulting aerosol was transferred by a nitrogen stream to the flowing afterglow of a helium atmospheric pressure glow discharge ionization source; the ionized sample material was analyzed by a Leco Unique time-of-flight mass spectrometer. Two-dimensional mass spectral images were generated by scanning the laser beam across a sample surface. The total analysis time for a 6 mm (2) surface, which is limited by the washout of the ablation chamber, was less than 30 min. With this technique, a spatial resolution of approximately 20 microm has been achieved. Additionally, the laser ablation configuration was used to obtain depth information of over 2 mm with a resolution of approximately 40 microm. The combination of laser ablation with the flowing atmospheric pressure afterglow source was used to analyze several sample surfaces for a wide variety of analytes and with high sensitivity (LOD of 5 fmol for caffeine).