Widefield Super-Resolution Infrared Spectroscopy and Imaging of Autofluorescent Biological Materials and Photosynthetic Microorganisms Using Fluorescence Detected Photothermal Infrared (FL-PTIR).

We have demonstrated high-speed, super-resolution infrared (IR) spectroscopy and chemical imaging of autofluorescent biomaterials and organisms using camera-based widefield photothermal detection that takes advantage of temperature-dependent modulations of autofluorescent emission. A variety of biological materials and photosynthetic organisms exhibit strong autofluorescence emission under ultraviolet excitation and the autofluorescent emission has a very strong temperature dependence, of order 1%/K. Illuminating a sample with pulses of IR light from a wavelength-tunable laser source causes periodic localized sample temperature increases that result in a corresponding transient decrease in autofluorescent emission. A low-cost light-emitting diode-based fluorescence excitation source was used in combination with a conventional fluorescence microscopy camera to detect localized variations in autofluorescent emission over a wide area as an indicator of localized IR absorption. IR absorption image stacks were acquired over a range of IR wavelengths, including the fingerprint spectral range, enabling extraction of localized IR absorption spectra. We have applied widefield fluorescence detected photothermal IR (FL-PTIR) to an analysis of autofluorescent biological materials including collagen, leaf tissue, and photosynthetic organisms including diatoms and green microalgae cells. We have also demonstrated the FL-PTIR on live microalgae in water, demonstrating the potential for label-free dynamic chemical imaging of autofluorescent cells.

Phase separation in surfactant-containing amorphous solid dispersions: Orthogonal analytical methods to probe the effects of surfactants on morphology and phase composition.

Amorphous-amorphous phase separation (AAPS) is an important phase transition process for amorphous solid dispersion (ASD) performance both in terms of drug release as well as physical and chemical stability during storage. Addition of surfactants to ASD systems can impact both of these processes. One possible mechanism through which surfactants affect ASD performance is via their impact on AAPS. Unfortunately, despite their increasing usage in ASD formulations, the effect of surfactant on AAPS is still poorly understood, and there are limited analytical techniques that provide microstructural and composition information about phase separated ASDs. In this study, the impact of four surfactants (sodium dodecyl sulfate, Tween 80, Span 20 and Span 85) on water-induced phase separation in ASDs formulated with ritonavir and polyvinylpyrrolidone/vinyl acetate (PVPVA) was investigated using a variety of orthogonal analytical methods. Transparent films of ASDs with different compositions were prepared by spin coating. Fluorescence confocal microscopy in combination with an in situ humidity chamber was used to monitor the kinetics and morphology of phase separation following exposure to high relative humidity. Optical photothermal IR analysis of phase separated films enabled characterization of domain composition and surfactant distribution. Liquid-liquid phase separation concentration, zeta potential and solution nuclear magnetic resonance spectroscopy measurements enabled interpretation of interaction with and partition of surfactants into the drug-rich phase. It was found that phase separation kinetics and morphology were notably changed by the surfactants. Further, the surfactants showed different affinities for the drug-rich versus the aqueous/polymer-rich phases. The employed analytical techniques were found to be complementary in providing insight into surfactant location in phase separated systems. This study highlights the complexity of phase separation, especially in the presence of surfactants, and provides a foundation to understand the impact of AAPS on ASD performance.

Micro to Nano: Multiscale IR Analyses Reveal Zinc Soap Heterogeneity in a 19th-Century Painting by Corot.

Formation and aggregation of metal carboxylates (metal soaps) can degrade the appearance and integrity of oil paints, challenging efforts to conserve painted works of art. Endeavors to understand the root cause of metal soap formation have been hampered by the limited spatial resolution of Fourier transform infrared microscopy (µ-FTIR). We overcome this limitation using optical photothermal infrared spectroscopy (O-PTIR) and photothermal-induced resonance (PTIR), two novel methods that provide IR spectra with ≈500 and ≈10 nm spatial resolutions, respectively. The distribution of chemical phases in thin sections from the top layer of a 19th-century painting is investigated at multiple scales (µ-FTIR ≈ 102 µm3, O-PTIR ≈ 10-1 µm3, PTIR ≈ 10-5 µm3). The paint samples analyzed here are found to be mixtures of pigments (cobalt green, lead white), cured oil, and a rich array of intermixed, small (often ≪ 0.1 µm3) zinc soap domains. We identify Zn stearate and Zn oleate crystalline soaps with characteristic narrow IR peaks (≈1530-1558 cm-1) and a heterogeneous, disordered, water-permeable, tetrahedral zinc soap phase, with a characteristic broad peak centered at ≈1596 cm-1. We show that the high signal-to-noise ratio and spatial resolution afforded by O-PTIR are ideal for identifying phase-separated (or locally concentrated) species with low average concentration, while PTIR provides an unprecedented nanoscale view of distributions and associations of species in paint. This newly accessible nanocompositional information will advance our knowledge of chemical processes in oil paint and will stimulate new art conservation practices.

How to unravel the chemical structure and component localization of individual drug-loaded polymeric nanoparticles by using tapping AFM-IR.

AFM-IR is a photothermal technique that combines AFM and infrared (IR) spectroscopy to unambiguously identify the chemical composition of a sample with tens of nanometer spatial resolution. So far, it has been successfully used in contact mode in a variety of applications. However, the contact mode is unsuitable for soft or loosely adhesive samples such as polymeric nanoparticles (NPs) of less than 200 nm of wide interest for biomedical applications. We describe here the theoretical basis of the innovative tapping AFMIR mode that can address novel challenges in imaging and chemical mapping. The new method enables gaining information not only on NP morphology and composition, but also reveals drug location and core-shell structures. Whereas up to now the locations of NP components could only be hypothesized, tapping AFM-IR allows accurately visualizing both the location of the NPs' shells and that of the incorporated drug, pipemidic acid. The preferential accumulation of the drug in the NPs' top layers was proved, despite its low concentration (<1 wt%). These studies pave the way towards the use of tapping AFM-IR as a powerful tool to control the quality of NP formulations based on individual NP detection and component quantification.

Relationships between chemical structure, mechanical properties and materials processing in nanopatterned organosilicate fins.

The exploitation of nanoscale size effects to create new nanostructured materials necessitates the development of an understanding of relationships between molecular structure, physical properties and material processing at the nanoscale. Numerous metrologies capable of thermal, mechanical, and electrical characterization at the nanoscale have been demonstrated over the past two decades. However, the ability to perform nanoscale molecular/chemical structure characterization has only been recently demonstrated with the advent of atomic-force-microscopy-based infrared spectroscopy (AFM-IR) and related techniques. Therefore, we have combined measurements of chemical structures with AFM-IR and of mechanical properties with contact resonance AFM (CR-AFM) to investigate the fabrication of 20-500 nm wide fin structures in a nanoporous organosilicate material. We show that by combining these two techniques, one can clearly observe variations of chemical structure and mechanical properties that correlate with the fabrication process and the feature size of the organosilicate fins. Specifically, we have observed an inverse correlation between the concentration of terminal organic groups and the stiffness of nanopatterned organosilicate fins. The selective removal of the organic component during etching results in a stiffness increase and reinsertion via chemical silylation results in a stiffness decrease. Examination of this effect as a function of fin width indicates that the loss of terminal organic groups and stiffness increase occur primarily at the exposed surfaces of the fins over a length scale of 10-20 nm. While the observed structure-property relationships are specific to organosilicates, we believe the combined demonstration of AFM-IR with CR-AFM should pave the way for a similar nanoscale characterization of other materials where the understanding of such relationships is essential.

The importance of correcting for variable probe-sample interactions in AFM-IR spectroscopy: AFM-IR of dried bacteria on a polyurethane film.

AFM-IR is a combined atomic force microscopy-infrared spectroscopy method that shows promise for nanoscale chemical characterization of biological-materials interactions. In an effort to apply this method to quantitatively probe mechanisms of microbiologically induced polyurethane degradation, we have investigated monolayer clusters of ∼200 nm thick Pseudomonas protegens Pf-5 bacteria (Pf) on a 300 nm thick polyether-polyurethane (PU) film. Here, the impact of the different biological and polymer mechanical properties on the thermomechanical AFM-IR detection mechanism was first assessed without the additional complication of polymer degradation. AFM-IR spectra of Pf and PU were compared with FTIR and showed good agreement. Local AFM-IR spectra of Pf on PU (Pf-PU) exhibited bands from both constituents, showing that AFM-IR is sensitive to chemical composition both at and below the surface. One distinct difference in local AFM-IR spectra on Pf-PU was an anomalous ∼4× increase in IR peak intensities for the probe in contact with Pf versus PU. This was attributed to differences in probe-sample interactions. In particular, significantly higher cantilever damping was observed for probe contact with PU, with a ∼10× smaller Q factor. AFM-IR chemical mapping at single wavelengths was also affected. We demonstrate ratioing of mapping data for chemical analysis as a simple method to cancel the extreme effects of the variable probe-sample interactions.

Absorption spectroscopy and imaging from the visible through mid-infrared with 20 nm resolution.

Absorption spectroscopy and mapping from visible through mid-IR wavelengths has been achieved with spatial resolution exceeding the limit imposed by diffraction via the photothermal induced resonance technique. Correlated vibrational (chemical), and electronic properties are obtained simultaneously with topography with a wavelength-independent resolution of ≈20 nm using a single laboratory-scale instrument. This marks the highest resolution reported for PTIR, as determined by comparing height and PTIR images, and its first extension to near-IR and visible wavelengths.

Using 2D Correlation Analysis to Enhance Spectral Information Available from Highly Spatially Resolved AFM-IR Spectra.

The recent combination of atomic force microscopy and infrared spectroscopy (AFM-IR) has led to the ability to obtain IR spectra with nanoscale spatial resolution, nearly two orders-of-magnitude better than conventional Fourier transform infrared (FT-IR) microspectroscopy. This advanced methodology can lead to significantly sharper spectral features than are typically seen in conventional IR spectra of inhomogeneous materials, where a wider range of molecular environments are coaveraged by the larger sample cross section being probed. In this work, two-dimensional (2D) correlation analysis is used to examine position sensitive spectral variations in datasets of closely spaced AFM-IR spectra. This analysis can reveal new key insights, providing a better understanding of the new spectral information that was previously hidden under broader overlapped spectral features. Two examples of the utility of this new approach are presented. Two-dimensional correlation analysis of a set of AFM-IR spectra were collected at 200-nm increments along a line through a nucleation site generated by remelting a small spot on a thin film of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). There are two different crystalline carbonyl band components near 1720 cm-1 that sequentially disappear before a band at 1740 cm-1 due to more disordered material appears. In the second example, 2D correlation analysis of a series of AFM-IR spectra spaced every 1 micrometer of a thin cross section of a bone sample measured outward from an osteon center of bone growth. There are many changes in the amide I and phosphate band contours, suggesting changes in the bone structure are occurring as the bone matures.

Localization of human hair structural lipids using nanoscale infrared spectroscopy and imaging.

Atomic force microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument (AFM-IR) capable of producing IR spectra and absorption images at a sub-micrometer spatial resolution. This new device enables human hair to be spectroscopically characterized at levels not previously possible. In particular, it was possible to determine the location of structural lipids in the cuticle and cortex of hair. Samples of human hair were embedded, cross-sectioned, and mounted on ZnSe prisms. A tunable IR laser generating pulses of the order of 10 ns was used to excite sample films. Short duration thermomechanical waves, due to infrared absorption and resulting thermal expansion, were studied by monitoring the resulting excitation of the contact resonance modes of the AFM cantilever. Differences are observed in the IR absorbance intensity of long-chain methylene-containing functional groups between the outer cuticle, middle cortex, and inner medulla of the hair. An accumulation of structural lipids is clearly observed at the individual cuticle layer boundaries. This method should prove useful in the future for understanding the penetration mechanism of substances into hair as well as elucidating the chemical nature of alteration or possible damage according to depth and hair morphology.

Atomic force microscope controlled topographical imaging and proximal probe thermal desorption/ionization mass spectrometry imaging.

This paper reports on the development of a hybrid atmospheric pressure atomic force microscopy/mass spectrometry imaging system utilizing nanothermal analysis probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to correlate topographic images of a surface with atomic force microscopy and a mass spectral chemical image of the same surface, utilizing the same probe without moving the sample from the system, was demonstrated. Co-registered mass spectral chemical images and atomic force microscopy topographical images were obtained from inked patterns on paper as well as from a living bacterial colony on an agar gel. Spatial resolution of the topography images based on pixel size (0.2 µm × 0.8 µm) was better than the resolution of the mass spectral images (2.5 µm × 2.0 µm), which were limited by current mass spectral data acquisition rate and system detection levels.

Nanoscale infrared (IR) spectroscopy and imaging of structural lipids in human stratum corneum using an atomic force microscope to directly detect absorbed light from a tunable IR laser source.

An atomic force microscope (AFM) and a tunable infrared (IR) laser source have been combined in a single instrument (AFM-IR) capable of producing ~200-nm spatial resolution IR spectra and absorption images. This new capability enables IR spectroscopic characterization of human stratum corneum at unprecendented levels. Samples of normal and delipidized stratum corneum were embedded, cross-sectioned and mounted on ZnSe prisms. A pulsed tunable IR laser source produces thermomechanical expansion upon absorption, which is detected through excitation of contact resonance modes in the AFM cantilever. In addition to reducing the total lipid content, the delipidization process damages the stratum corneum morphological structure. The delipidized stratum corneum shows substantially less long-chain CH2 -stretching IR absorption band intensity than normal skin. AFM-IR images that compare absorbances at 2930/cm (lipid) and 3290/cm (keratin) suggest that regions of higher lipid concentration are located at the perimeter of corneocytes in the normal stratum corneum.

Nanometer-scale infrared spectroscopy of heterogeneous polymer nanostructures fabricated by tip-based nanofabrication.

There is a significant need for chemical identification and chemical imaging of nanofabricated structures and devices, especially for multiple materials integrated at the nanometer scale. Here we present nanofabrication, chemical identification, and nanometer-scale chemical imaging of polymer nanostructures with better than 100 nm spatial resolution. Polymer nanostructures of polyethylene, polystyrene, and poly(3-dodecylthiophene-2,5-diyl) were fabricated by tip-based nanofabrication. Nanometer-scale infrared measurements using atomic force microscopy infrared spectroscopy (AFM-IR) obtained quantitative chemical spectra of these nanostructures. We show chemical imaging of intersecting patterns of nanometer-scale polymer lines of different chemical compositions. The results indicate that for closely packed heterogeneous nanostructures, the spatial resolution of AFM-IR is not limited by nanometer-scale thermal diffusion, but is instead limited by the cantilever sensitivity and the signal-to-noise ratio of the AFM-IR system.

Nanoscale mid-infrared evaluation of the miscibility behavior of blends of dextran or maltodextrin with poly(vinylpyrrolidone).

Determining the extent of miscibility of amorphous components is of great importance for certain pharmaceutical systems, in particular for polymer-polymer and polymer-small molecule blends. In this study, the application of standard atomic force microscopy (AFM) measurements combined with nanoscale mid-infrared (mid-IR) spectroscopy was explored to evaluate miscibility in binary polymer blends. The miscibility characteristics of a set of 50/50 (w/w) polymer blends comprising of poly(vinylpyrrolidone) (PVP) with dextran or maltodextrin (DEX) of varying molecular weights (MWs) were investigated. Standard AFM characterization results show good agreement with inferences drawn from differential scanning calorimetry (DSC) analysis in terms of forming either single or two phase systems. AFM analysis also provided insight into the microstructure of the two phase systems and how domain sizes varied as a function of polymer MWs. Nanoscale mid-IR evaluation of the blends, performed by collecting local mid-IR spectra or spectral maps, provided an extra dimension of information about the dependence of polymer MWs on chemical composition of the different phases. AFM, combined with nanoscale mid-infrared analysis, thus appears to be a promising technique for the evaluation of miscibility in certain pharmaceutical blends.

Nanoscale mid-infrared imaging of phase separation in a drug-polymer blend.

The applicability of nanoscale mid-infrared (mid-IR) spectroscopy for the study of the micro- and nanostructure of pharmaceutical drug-polymer systems was explored. Felodipine-poly(acrylic acid) (PAA) blends were used as model systems. Standard atomic force microscopy evaluation as a function of drug-polymer composition suggested limited miscibility, in line with previous findings. Localized spectra on a 50:50 (w/w) felodipine-PAA dispersion revealed that the discrete submicrometer domains formed corresponded to an amorphous felodipine-rich phase while the continuous phase tended to be rich in PAA. Further, spectroscopic imaging at selected wavenumbers, enabling discrimination between both constituents, confirmed this finding and made it possible to chemically image differences in composition between each phase with submicrometer resolution.

Spatial differentiation of sub-micrometer domains in a poly(hydroxyalkanoate) copolymer using instrumentation that combines atomic force microscopy (AFM) and infrared (IR) spectroscopy.

Atomic force microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument (AFM-IR) capable of producing sub-micrometer spatial resolution IR spectra and absorption images. This new capability enables the spectroscopic characterization of microdomain-forming polymers at levels not previously possible. Films of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) were solution cast on ZnSe prisms, followed by melting and annealing to generate crystalline microdomains of different sizes. A tunable IR laser generating pulses of the order of 10 ns was used for excitation of the sample films. Short duration thermomechanical waves, due to infrared absorption and resulting thermal expansion, were studied by monitoring the resulting excitation of the contact resonance modes of the AFM cantilever. Dramatic differences in the room-temperature IR spectra are observed in the 1200-1300 cm(-1) range as a function of position on a spatial scale of less than one micrometer. This spectral region is particularly sensitive to the polymer backbone conformation. Such dramatic spectral differences have also been observed previously in bulk IR measurements, but only by comparing room-temperature spectra with ones collected at higher temperatures. Less dramatic, but significant, AFM-IR spectral differences are observed in the carbonyl stretching region around 1720 cm(-1) as a function of location on the sample. Two overlapping, but relatively sharp, carbonyl bands are observed near 1720 cm(-1) in more crystalline regions of the polymer, while a broader carbonyl stretching band appears centered at 1740 cm(-1) in the more amorphous regions. Using this spectral region, it is possible to monitor the development of polymer crystalline structures at varying distances from a nucleation site, where the site was generated by bringing a heated AFM tip close to a specific location to locally anneal the sample.

Direct measurement of tapping force with a cantilever deflection force sensor.

An experimental set up dedicated to the measurement of atomic force microscope tapping force was developed. In the set-up, a standard TappingMode probe cantilever was used to tap another cantilever equipped with its own low noise and high sensitivity deflection detection system for force measurement. The amplitude and phase change of the tapping lever as well as the deflection of the sensing lever were simultaneously recorded as a function of tip/surface separation. Since the deflection of the sensing cantilever reflects the average force over one interaction cycle, we measured the total average force quantitatively after calibrating the spring constant and deflection sensitivity of the sensing lever. Considerable effort was made to achieve the same force curve in the tapping force measurement as occur during imaging of conventional samples such that the detected tapping force reflects the same interaction of the imaging process.

Studies of tip wear processes in tapping mode atomic force microscopy.

Tip integrity is crucial to atomic force microscope image quality. Tip wear not only compromises image resolution but also introduces artifacts. However, the factors that govern wearing have not been systematically studied. The results presented here of tip wearing on a rough titanium surface were determined by monitoring changes in tip shape and the evolution of histograms of complex surface curvatures under different control parameters. In contrast with the common assumption that operating at a low set point (the ratio of tapping amplitude to free oscillation amplitude) wears the tip quickly, we observed that a low set point actually minimizes tip wear on a hard surface regardless of the free amplitude. The results can be interpreted qualitatively with theoretical calculations based on momentum exchange at tapping impact. Operating at a low set point allows more robust scanning than with a high set point (tapping near free amplitude), providing a method to slow down tip wear. Another advantage of a low set point is that amplitude error grows faster than with a high set point by nearly an order of magnitude, permitting an increase in scanning speed.